Production and Properties of Microbial Inulinases

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Production and Properties of Microbial Inulinases: Recent Advances a

Naveen Kango & Sumat Chand Jain

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Department of Applied Microbiology and Biotechnology, Dr. Hari Singh Gour University, Sagar, (M.P.), India Available online: 26 Jul 2011

To cite this article: Naveen Kango & Sumat Chand Jain (2011): Production and Properties of Microbial Inulinases: Recent Advances, Food Biotechnology, 25:3, 165-212 To link to this article: http://dx.doi.org/10.1080/08905436.2011.590763

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Production and Properties of Microbial Inulinases: Recent Advances Naveen Kango and Sumat Chand Jain Department of Applied Microbiology and Biotechnology, Dr. Hari Singh Gour University, Sagar (M.P.), India Microbial inulinases find application in enzymatic hydrolysis of inulin for production of fructose (sweetener) and inulooligosaccharides (prebiotics). Inulin is one of the most abundant nonstructural polysaccharides found widely dispersed in plants and awaits judicious utilization. Demands for an alternative healthy sweetener and multifunctional fructooligosaccharides have prompted investigators to explore microorganisms for inulinase production and to develop bioprocesses for production of high-fructose syrup and oligosaccharides based on enzymatic hydrolysis of raw inulin. Inulinases have been characterized in several molds, yeasts and a few bacteria. Recently, there has been a spurt of interest in finding novel inulinase producers, cloning and expression of inulinase gene in heterologous hosts and use of crude plant inulin and agro-industrial media in both submerged and solid state fermentation for inulinase production. The review discusses current knowledge on production, properties and applications of microbial inulinases and looks into the recent advances in the field. Key Words:

inulinase; inulin; fructose; prebiotics; fructooligosaccharides

INTRODUCTION After starch, fructans are the most abundant nonstructural polysaccharides found in a wide range of plants. Inulin is a polydispersed fructan consisting mainly of β-(2→1)-D-fructosyl-fructose links terminated by a sucrose residue (De Leenheer, 1996). It serves as a storage polysaccharide in many plants of compositae and gramineae and is accumulated in the underground roots and tubers of several plants including Vernonia herbacea, Jerusalem artichoke (Helianthus tuberosus), chicory (Cichorium intibus, Cichorium endivia), and dahlia (Dahlia pinnata) (Gupta and Kaur, 1997). The degree of polymerization

Address correspondence to Dr. Naveen Kango, Assistant Professor, Department of Applied Microbiology and Biotechnology, Dr. Hari Singh Gour University, Sagar (M.P.) India 470003; E-mail: [email protected]

166

N. Kango and S. Chand Jain HOCH2

HOCH2 O

O

O G

F CH2


O HOCH2

O n = Ca. 30

F CH2

n O HOCH2

β, 2–1 fructosylfructose bond

O F

CH2OH

Figure 1: Structure of inulin (Vandamme and Derycke, 1983).

in inulin (MW 60,000) of plant origin is < 200 (Fig. 1) and varies according to plant species, weather conditions and age. Some well known plant sources of inulin are listed in Table 1. Major sources of inulin for industrial scale production are chicory, Jerusalem artichoke (topinambur), and dahlia. The inulin content differs between the plant species. The world production of inulin is currently estimated to be about 350,000 tons. Main producers are Belgium, France, the Netherlands, and Chile. Chicory (Cichorium intybus) is a temperate climate biennial root crop. Crop requirements, harvesting, and processing are similar to sugar beet production. Jerusalem artichoke (Helianthus tuberosus) is a perennial tuberous plant. The yield is higher if harvested annually. Dahlia (Dahlia spp.) is a tuberous plant mainly cultivated for its flowers. Dahlia tubers are used as inulin source but the content is lower than that of chicory (Franck and De Leenheer, 2002; Peters, 2007). Inulinases are fructofuranosyl hydrolases produced by a wide range of organisms including plants, bacteria, molds, and yeasts. The general reaction

Production and Properties of Microbial Inulinases Table 1: Inulin content of some plants (Modified from Van Loo et al.,

1995).


Source

Onion Jerusalem artichoke Dahlia Chicory Leek Garlic Artichoke Banana Rye Barley Dandelion Burdock Camas Murnong Yacon Salsify

Plant part

Inulin content (% of fresh weight)

Bulb Tuber Tuber Root Bulb Bulb Leaves-heart Fruit Cereal Cereal Leaves Root Bulb Root Root Root

2–6 14 – 19 9 – 12.5 15 – 20 3 – 10 9 – 16 3 – 10 0.3 – 0.7 0.5 – 1 0.5 – 1.5 12 – 15 3.5 – 4.0 12 – 22 8 – 13 3 – 19 4 – 11

mainly involves action of two enzymes: (1) exoinulinase (E.C. 3.8.1.80) which splits the terminal fructose units from inulin and (2) endoinulinase (E.C. 3.2.1.7) that breaks down inulin into inulooligosaccharides (IOS). The former can be used for production of high fructose syrup from natural inulins (saccharification), and the latter can be used for producing inulooligosaccharides of varying lengths (Fig. 2). Many of these fructosyl-hydrolytic enzymes possess a wide substrate specificity; the classification of which type depends on whether the sucrose to inulin hydrolytic ratio (S/I ratio) is higher or less than 1. Many microbial preparations of inulinase also possess invertase activity accompanying the inulinase activity. Their catalytic activity is described in terms of I/S (inulinase/sucrase) ratios (Vandamme and Derycke, 1983; Laloux et al., 1991; Pessoni et al., 1999). IOS produced from inulin are reported to have similar physiological functions to fructooligosaccharides (FOS) (Hidaka et al., 1987; Roberfroid, 1993; Kaur and Gupta, 2002). In response to increasing demand from consumers for healthier and lower calorie foods, a number of oligosaccharides are being explored for their functionalities. Inulooligosaccharides have been reported to enhance population of Bifidobacteria in intestine (bifidogenic). Predominance of Bifidobacteria in the large intestine has potential to contribute to the prevention of many diseases and maintenance of overall health. Several investigators have reported that fermentation of fructans in humans and animals is selectively bifidogenic (Gibson and Wang, 1994; Roberfroid et al., 1995; Hussein et al., 1999; Cummings et al., 2001). The intestinal microflora of the human subjects fed on sucrose, oligofructose, and inulin (15 g day−1 ) was

167

168

N. Kango and S. Chand Jain , 2-1 fructosyl linkage G

F


Exo-inulinase

Hi-fructose syrup

Inulin

Endo-inulinase

Inulo-oligosaccharides

Low calorie sweetener

Neutraceutical (Dietary fiber)

Fermentable substrate

Functional food ingredient

Figure 2: Hypothetical inulin being acted upon by microbial exo- and endoinulinase enzymes. Action of exoinulinase liberates fructose from the macromolecule while endoinulinase produces inulooligosaccharides.

found to contain 17%, 82%, and 71% Bifidobacteria (% of total microflora), respectively. Population of Bacteroids was much lower in case of subjects fed on oligofructose (16%) and inulin (26%) compared with those on sucrose (72%). The counts of Fusobacteria were much lower (1%) in case of oligofructose diet compared with 9% observed in sucrose fed subjects (Gibson et al., 1995). It has been suggested that the ability of Bifidobacteria to change intestinal microbiota lies in its ability to produce bacteriocins that inhibit detrimental bacteria and compete for substrates and adhesion sites on intestinal epithelium along with immune stimulatory activities (Gibson et al., 1995). Inulooligosaccharides also help in synthesis of B-vitamins and absorption of certain ions (Gibson and Roberfroid, 1995). The low-caloric oligofructose are also advised for obese people. Their energy content is only 40–50% that of digestive carbohydrates, giving them a caloric value of 1–2 Kcal g−1 (Kaur and Gupta, 2002). Positive effects of oligofructose intake are also demonstrated in cases of constipation (Hidaka et al., 1991; Hond et al., 2000) and absorption of calcium and magnesium (Ohta et al., 1994; Delzenne et al., 1995; Van den Heuvel et al., 1999). Benefits of oligofructose intake are also indicated in hyperglycemia (Oku et al., 1984), and oligofructoses are also reported to be hypolipidemic. Systemic effects of dietary supplementation of 10% (w/w) oligofructose (OFS) on male Wistar rats revealed lowering of serum triacylglycerols (TAG) by 39%, 73%, and 57% in standard, fiber-free, and high fat diets, respectively, compared to


Production and Properties of Microbial Inulinases

controls (Delzenne and Kok, 1999). Ingestion of FOS has been reported to suppress aberrant crypt foci (ACF) in the colon, which are preneoplastic markers of adenomas and carcinomas of colon (Wargowich et al., 1996). It is evident that oligofructose have many applied functionalities and are of increasing interest as nutraceutical supplements for their use as prebiotics. Inulooligosaccharides are resistant to digestion of enzymes of the GI tract because of the presence of fructose in β-configuration. These can, however, be fermented selectively by beneficial commensals such as Bifidobacteria. Fructosans having β-(2,1) linkages are common in almost all cereals, making up 1–4%, and are referred as inulin type fructans. Escriva and Martinez-Anaya (2000) have suggested use of inulinases in the release of fructosans from fructans in sourdough processes. There is much interest in adding FOS to dairy products because these prebiotics enhance the absorption of calcium (Ohta et al., 1994; Van den Heuvel et al., 1999). The use of prebiotic ingredients in combination with probiotics (synbiotic) offers an exciting possibility to enhance the health benefits. Fructose is the sweetest natural sweetener and is 1.5–2 times sweeter than sucrose (Flemming and GrootWassink, 1979). It is less cariogenic than sucrose and has no bitter aftertaste of saccharin and hence can be used as an alternative sweetener for diabetics (Vandamme and Derycke, 1983). For any normal person, fructose also is emerging as a safe alternative sweetener to sucrose, which is suggested to contribute to corpulence and atherosclerosis. Moreover, it has better solubility, less viscosity, and its low levels can be metabolized without insulin (Flemming and GrootWassink, 1979). It is already being used as a sweetening agent in food and drink industry (Vandamme and Derycke, 1983). Conventional method of fructose preparation from starch (fructose corn syrups) needs at least three enzymatic steps involving amylolysis with αamylase, amyloglucosidase and isomerisation with glucose isomerase and maximal yields are reported to be 45% fructose solutions (Vandamme and Derycke, 1983). This process yields only 45% fructose along with glucose (50%) and oligosaccharides (5–8%). Recovery of fructose from this mixture is done using expensive ion exchange chromatographies (Zittan, 1981). An easier, more direct, cheaper, and quicker alternative could be enzymatic hydrolysis of polydispersed reserve fructan, inulin using microbial inulinases. The yield in such process can be up to 90–95% fructose solution (Gupta et al., 1994; Vranesic et al., 2002). Acid hydrolysis of inulin is not recommended because of undesirable coloring of inulin hydrolysate and formation of tasteless difructose anhydride (Vandamme and Derycke, 1983). The sugar syrups thus obtained can be used for production of ethanol as well (Guiraud et al., 1981a; Guiraud et al., 1982; Ohta et al., 1993). Since inulin accumulates as a reserve polymer in several agricultural cash crops such as chicory, Jerusalem artichoke, and dahlia and therefore can serve as an abundant and rapidly renewable substrate for enzymatic hydrolysis.

169

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N. Kango and S. Chand Jain


SCREENING AND INULINASE ASSAY Rhizosphere soils of inulin rich plants and naturally decaying inulin rich material have been used for isolation of inulinase producing microorganisms (Vandamme and Derycke, 1983, Viswanathan and Kulkarni, 1996; Gern et al., 2001; Kango, 2008). Presence of microbial hydrolases can be detected in solid media by zone of clearing surrounding the colony as result of hydrolysis of insoluble polymeric substrate or by staining of soluble substrates after microbial growth to visualize a distinct hydrolysis zone. Colonies growing on medium containing inulin as the sole C-source can be regarded as inulinase producers. Screening medium as suggested by Kim (1975) containing inulin as the only carbon source had following composition (% w/w): inulin 2.0, K2 HPO4 0.1, MgSO4 .7H2 O 0.05, NaNO3 0.15, (NH4 )H2 PO4 0.20, KCl 0.05, FeSO4 .7H2 O 0.01, and agar 1.8 (initial pH 6.0). According to Vandamme and Derycke (1983), these organisms are able to dissolve solid inulin particles contained in the medium and often form a distinct surrounding zone of clearing which can be seen without using any stain. However, Gern et al. (2001), while screening 16 DSM fungal strains and three bacterial isolates, did not notice any such zone formation by any of the inulinase producing strains and thus made selection on the basis of faster growth of microorganisms on medium containing inulin as sole C-source. They used screening medium as prescribed above for fungal strains. However, the medium used for screening bacterial strains contained (grams per liter) inulin 5.0, Na2 HPO4 .12H2 O 0.9, K2 HPO4 0.7, MgSO4 .7H2 O 0.1, CaCl2 .2H2 O 0.2, (NH4 )2 SO4 0.5, succinic acid 1.0, yeast extract 0.5, agar 18, and trace element solution 1 mL. More recently, inulin agar medium used to isolate inulinase hyperproducing Kluyveromyces strains based on inulin hydrolysis zones contained 0.25% w/v inulin (from dahlia tubers), yeast nitrogen base, (NH4 )2 SO4 (CruzGurrero et al., 2006). Inulin was added in the manner described by Tsang and GrootWassink (1985) to develop turbidity in the plates. Passador-Gurgel et al. (1996) have reported use of a microtitre reader system for screening inulinase producing yeasts. Cell growth in ELISA plates was carried out by inoculating 200 μL of medium with 10% (v/v) preculture of the test strain with an absorbance of 4.0. These plates were shaken at 800 rpm upto 48 h at 37◦ C and absorbance was recorded at 620 nm after each hour. Among the strains screened, K. marxianus ATCC 36097 showed best growth reaching an absorbance of 9.5 after 25 h accompanied with maximum inulinase activity (2.60 U mL−1 ). Increasing importance of inulooligosaccharides has prompted researchers to focus upon strains that produce endoinulinase exclusively. This needs determination of action pattern of inulinase and analysis of end products using TLC, HPTLC, or HPLC (Gern et al., 2001; Cho and Yun, 2002). The fructans, inulin, and



oligofructose are known to possess many of the physiological properties of dietary fibers (DF), and their determination in food samples can be done by treating samples with amyloglucosidase and inulinase and analyzing the sugars released by ion-exchange chromatography (Prosky and Hoebregs, 1999). Ronkart et al. (2007) have demonstrated the use of high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) in analysis of inulooligosaccharides resulting from hydrolysis of globe artichoke inulin (DP = 80) using commercial endoinulinase (Beldem, Belgium). Hydrolysis was conducted for 24 h at 50◦ C and pH 5.0 by adding 125 μL of endoinulinase (30001800 UI mL−1 ) to 50 mL of 5% w/v globe artichoke inulin. The reaction was stopped by boiling in microwave and the reaction product was lyophilized. The product was injected into a dionex DX500 column and sugar profiles of both globe artichoke extract and hydrolysate were analyzed using HPAEC-PAD. Action of inulinase produced fructose and fructooligosaccharides (DP 2-4) with F4 as the major product. Alternative methods for identifying inulinase producers are based on hydrolysis of substrates coupled with dyes and use of enzymes for indirect assay. Castro et al. (1995) used Remazol Brilliant Blue-inulin (RBB-inulin) as substrate for screening of 186 South American soil isolates and could identify six promising strains resulting in extracellular activities up to 757.8 U L−1 (Isolate no. VIR 64). Dye labeled substrate was prepared by covalently linking RBB with inulin based on method described by Rinderknecht et al. (1967). Inulinase activity can also be detected and quantified using the hexokinase/glucose-6-phosphate dehydrogenase (HK/G6PDH) method (Beutler, 1984). This method determines the rate of appearance of fructose from 2% (w/v) inulin (Passador-Gurgel et al., 1996). Fructose liberated by inulinase action is phosphorylated by hexokinase to yield fructose-6-phosphate which is then converted to glucose-6-phosphate by phosphoglucose isomerase. Glucose-6-phosphate is then oxidized to 6-phosphogluconate in the presence of NAD by G6PDH. Simultaneous to this an equimolar amount of NAD is reduced to NADH leading to increase in absorbance at 340 nm which is directly proportional to fructose concentration. Most common assay of inulinase involves incubation of enzyme sample with solution of pure commercial inulin followed by quantification of reducing sugar (fructose-equivalents) liberated from the substrate. One unit of inulinase is defined as the amount of enzyme that liberates 1 μmol of fructose per minute (Vandamme and Derycke, 1983). Standard substrates used for assay are purified chicory inulin or Dahlia inulin (Sigma Chemical Co., St. Louis, MO, USA). Usually, hydrolysis of 1% (w/v) solution of inulin is carried out at pH 5.0 and 50◦ C (Kango, 2008). The qualitative analysis of end products of inulin hydrolysis is done using TLC, HPLC or HPAEC-PAD. Fructose (F), 1-kestose (GF2 ; β-D-fructofuranosyl(2-1)-β-D-fructofuranosyl-(2-1)-a-D-glucopyranoside), and nystose (GF3 ,

171

172


β-D-fructofuranosyl-(2-1)-β-D-fructofuranosyl-(2-1)-β-D-fructofuranosyl-aD-glucopyra-noside) and other fructooligosaccharides (DP 2-5) are used as suitable standards for analyzing the end products of inulinase activity (Ronkart et al., 2007; Kango, 2008).


MICROBIAL SOURCES OF INULINASE A number of microorganisms including molds, yeasts, actinomycetes, and other bacteria are known to produce inulin hydrolyzing enzymes (Vandamme and Derycke, 1983; Pandey et al., 1999; Singh and Gill, 2006). A list of inulinase producers along with enzyme yield and fermentation conditions is given in Table 2. Among molds, Aspergillus and Penicillium are the prominent genera while Kluyveromyces is the most explored yeast for inulinase production. Recent reports indicate some of the promising producers among bacteria including some species of Xanthomonas and Pseudomonas.

Molds A number of molds have been reported to produce inulin hydrolyzing enzymes (Table 2). Among molds most prominent inulinase producers belong to genera Aspergillus and Penicillium (Barthomeuf et al., 1991; Viswanathan and Kulkarni, 1995a, b; Balayan et al., 1996). Aspergillus niger is one of the most explored fungus for production of inulinase. Several strains of this fungus have been reported to produce high levels of inulinase. Wild type strains of A. niger have been successfully mutagenised by UV resulting in high yielding mutants (Derycke and Vandamme, 1984; Ongen-Baysal et al., 1994; Viswanathan and Kulkarni, 1995a; Cruz et al., 1998; Nguyen et al., 1999; Skowronek and Fiedurek, 2003). A wide range of enzyme titres from Aspergillii have been reported by different investigators. Gupta et al. (1994) while working with A. aureus, A. fischeri, A. flavus, A. nidulans and A. niger obtained activities in the range of 1.0– 1.2 U mL−1 after 9 days of incubation while Cruz et al. (1998) obtained 2.47 U mL−1 of inulinase produced by A. niger 245 after 60 h. Gupta et al. (1998) obtained 1.2 U mL−1 of inulinase from A. oryzae after 9 days and Kochhar et al. (1999) who examined inulinase production by four species of Aspergillus have reported 0.379 U mL−1 from A. oryzae NCIM 631 after 6 days, 10.02 U mL−1 from A. candidus NCIM 88, 0.053 U mL−1 for A. chevalieri NCIM 940 after 9 days and 0.113 U mL−1 from A. terreus NCIM 653 after 15 days. Ongen-Baysal et al. (1994) observed higher inulinase activity of A. niger A42 (54 U mL−1 ) in medium in which carbon source was 10 g L−1 crude Jerusalem artichoke extract (total sugar content 16% w/v) after 8 days of incubation. Aspergillus ficuum JNSP5-06, isolated from soil samples, produced 25 U mL−1 in 5 days at 30◦ C on inulin containing medium (Jing et al., 2003a). de Souza-Motta

173

Yeasts Kluyveromyces marxianus NRRL Y-7571

Rhizoctonia solani

Penicillium sp.TN88 Trichoderma viride

Aspergillus ficuum JNSP 5–06 Penicillium spinulosum

Aspergillus niger SL 09 Aspergillus parasiticus

Molds Aspergillus nigerNK-126 Aspergillus niger SL 09 Aspergillus niger AUP19 Aspergillus niger strain 13/36

Microorganism

30

1,317

2.9

4.0

40

25

6.0

5.0

1.6

30

0.94

6.5

25

30

30

5.0

2.9

30

30

9.9

6.0

118

80

6.5

176

28

30

6.0

54

T ◦C

30

pH

54

Activity (U ml−1 )

Molasses

Jerusalem artichoke powder Jerusalem artichoke powder

Jerusalem artichoke powder Inulin

Jerusalem artichoke powder Inulin

Sucrose

Sucrose

Inulin, galactose

Dandelion tap root extract Sucrose

Substrate

Table 2: Production of inulinase by microorganisms.

Yeast extract, CSL

Yeast extract NH4 NO3 , (NH4 )2 HPO4 (NH4 )2 HPO4 , NH4 NO3

Yeast extract NH4 NO3 , (NH4 )2 HP04

Yeast extract NH4 NO3 , NH4 H2 PO4 Yeast extract, (NH4 )2 HPO4

Corn steep liquor, NH4 H2 PO4 Yeast extract, NaNO3 , (NH4 ) 2 HPO4 NH4 H2 PO4

NH4 H2 PO4

Yeast extract

Nitrogen source

96

200

300∗

96

96

48

120

24

120

96

72

96

96

Incubation Period (h)

200

200

200

140

220

220

140

150

Rev min−1


(Continued)

Treichel et al., 2009

Ertan et al., 2003

Nakamura et al., 1997 Ertan et al., 2002

Ertan et al., 2002

Jing et al., 2003b

Ertan et al., 2002

Zhang et al., 2004b

Skowronek and Fiedurek, 2004

Ge and Zhang, 2005 Kumar et al., 2005

Kango, 2008

Reference

174

722

K marxianus NRRL Y-7571 K marxianus NRRL Y-7571

30 30

27.24b

18.89b

6.0

5.0

8.0

5.0

5.0

463c

212

60

39.56

62.85

K. marxianus ATCC 52466 Kluyveromyces marxianus Pichia guilliermondii Pichia guilliermondii Cryptococcus aureus

6.8

8.6–11.6

K. bulgaricus

3.5

176

K. marxianus

28

28

28

28

30

30

30

30 30

K. marxianus CDBB-L-278 K. lactis var. lactis

6.5 6.5

55.4 47.7

K. marxianus YS-1 K. marxianus

30

30

36

T ◦C

50.2

6.5

5.5

pH

K. marxianus YS-1

436.7a

1,139

Activity (U ml−1 )

K. marxianus

Microorganism

Table 2: (Continued).

Inulin

Inulin

Inulin

Inulin

Inulin

Sucrose

Sucrose

Inulin

Sugarcane bagasse and molasses Asparagus officinalis tubers Dahlia tubers Asparagus racemosus tubers Inulin

Sugarcane molasses Molasses

Substrate

Yeast extract

Yeast extract

Yeast extract

Yeast nitrogen base, (NH4 )2 SO4 Yeast nitrogen base, (NH4 )2 SO4 Peptone, yeast extract Peptone, yeast extract Yeast extract, bacto-peptone Yeast extract

Beef extract Beef extract

Beef extract

Soybean bran, CSL

Yeast hydrolysate, CSL Yeast extract, CSL

Nitrogen source

60 60

200∗ 200∗

170

170

170

220

150

72

72

48

18

30

24–48

72

450∗ 150

24

200

24

60

200∗

200

24

72

Incubation Period (h)

SSF

150

Rev min−1


Gao et al., 2007

Gao et al., 2007

Selvakumar and Pandey, 1999a Parekh and Margaritis, 1985 Gong et al., 2007

Cruz-Guerrero et al., 2006 Cruz-Guerrero et al., 2006 Bernardoo et al., 2005 Vranesic et al.,2002

Singh and Bhermi, 2008 Singh et al., 2007b Singh et al., 2006

Mazutti et al., 2010

Sguarezi et al., 2009 Makino et al., 2009

Reference

175

5.0

5.0

5.0

7.0

7.0

7.0

6.5

7.5

85

52.53

52.38

15

22.09

135.2

621c

0.552

b units

aU

gds−1 : Units per grams dry substrate. mg−1 . c units l−1 . ∗ fermenter cultivation.

Staphylococcus strain Streptomyces sp. GNDU-1

Cryptococcus aureus Debaryomyces hansenii Yarrowia lipolytica Bacteria Xanthomonas sp. Xanthomonas campestris pv. phaseoli Bacillus smithi T7

46

37

50

37

37

28

28

28

Inulin

Inulin

Inulin

Sucrose

Chicory roots

Inulin

Inulin

Inulin

Yeast extract, NH4 H2 PO4 Yeast extract, bacto-peptone Yeast extract

Tryptone

Yeast extract

Yeast extract

Yeast extract

Yeast extract

72

200

200

24

30

18

150∗

150

22

72

72

42

100

170

170

170


Selvakumar and Pandey, 1999a Gill et al., 2003

Gao et al., 2008

Naidoo et al., 2009

Park and Yun 2001

Gao et al., 2007

Gao et al., 2007

Sheng et al., 2007


176


et al. (2005) have obtained 11 U mL−1 of inulinase by A. niveus 4128URM in medium containing 20 g L−1 inulin after 72 h of incubation. Skowronek and Fiedurek (2004) have optimized inulinase production by A. niger using Simplex method, a modification of efficient sequential optimization technique. Software MultiSimplex AB (v.2.1.1) 1998 was used for the purpose of optimizing five variables (sucrose, yeast extract, NaNO3 , MgSO4 ·7H2 O, K2 HPO4 ) for enhancing inulinase production up to 79.8 U mL−1 . A mixture of endo and exo acting inulinases is recommended in fructose production from natural inulins as their synergistic action leads to higher yields (Fig. 2). Nakamura and co-workers (1978 a) were the first to report that a strain of Aspergillus niger excreted these two distinct inulin-hydrolyzing enzymes. Thereafter, Penicillium purpurogenum and Chrysosporium pannorum were reported to produce both endo- and exoinulinase (Onodera and Shiomi, 1988; Xiao et al., 1989a, b). Moussa and Jacques (1987, 1990) reported production of exo and endoinulinase from A. ficuum while Uhm et al. (1999) purified endoinulinases upto homogeneity from the fungus. The enzyme hydrolyzed inulin and inulooligosaccharides into inulotetraose (DP4 ) and inulotriose (DP3 ). Enzyme-based technologies can only be realized by developing cost-effective methods of enzyme production. Use of low-value, naturally occurring substrates in enzyme production can bring about significant cost reduction in overall process (Kango et al., 2003). The enzyme sample obtained from culture of A. niger NK-126, a wild type isolate from onion peels, grown on dandelion tap root extract was able to produce fructose, inulobiose and inulooligosaccharides from chicory inulin. The fungus produced a maximum of 54 U mL−1 of inulinase enzyme on the same medium (Kango, 2008). Temperature range for maximum growth and inulinase production by fungi has been reported to be 28–30◦ C (Vandamme and Derycke, 1983). Considerable variations in incubation periods due to strain, medium composition and culture conditions have been suggested, for example, 12 days for Fusarium oxysporum grown on the aqueous extract of chicory root, 60 h for A. niger van Tieghem grown on kuth root extract (30◦ C, 150 rpm, 120 U mL−1 ), and 96 h for A. niger grown on dandelion root extract (30◦ C, 150 rpm, 54 U mL−1 ) (Gupta et al., 1988; Viswanathan and Kulkarni, 1995a; Kango, 2008). Glucose, fructose, and sucrose, alone or in combination with inulin, have been found to repress inulinase formation (Vandamme and Derycke, 1983; Allais et al., 1987; McKellar and Modler, 1989). Media low in free sugars or high in slowly metabolized polysaccharides favours inulinase production (Derycke and Vandamme, 1984), but low concentrations (0.1%) of free sugars appear to support inulinase synthesis by stimulating rapid growth of organisms during initial phase. Thus, inulinase synthesis by fungi is subject to catabolite repression by rapidly utilizable free sugars. Viswanathan and Kulkarni (1995a) achieved 120 U mL−1 inulinase production with A. niger van Tieghem in an optimized medium containing fructose, inulin and sucrose as



C-sources and mixed N-sources, corn steep liquor (CSL), yeast extract, ammonium sulphate, and sodium nitrate at pH 6.0. Nakamura et al. (1997) screened 120 fungal colonies on agar plates containing inulin and found Penicillium sp. TN-88 to produce 2.25 U mL−1 of inulinase (I/S: 9.0). The strain TN-88 produced 5.9 U mL−1 (I/S: 4.7) when grown in presence of 0.1M inorganic nitrogen (0.1 M NH4 Cl) while almost a similar quantity (5.8 U mL−1 ) but with a higher I/S ratio of 9.5 was found when yeast extract (10 g L−1 ) was used. Carbohydrates including most free sugars (fructose, glucose, and galactose) and starch resulted in much lower inulinase yield (0.1–0.6 U mL−1 ) while 1.5% w/v pure inulin derived from Dahlia tubers (Sigma Chem. Co.) supported a yield of 9.0 U/mL. Strains that produce endoinulinase exclusively are of special interest since the enzyme action yields inulooligosaccharides (Gern et al., 2001). Endoinulinases have been characterized from several molds including A. niger (Nakamura et al., 1994), Penicillium purpureogenum (Onodera and Shiomi, 1988), Chrysosporium pannorum (Xiao et al., 1989a), and Penicillium sp. TN88 (Nakamura et al., 1997). Further, it is important to characterize the product of endoinulinase action in terms degree of polymerization (DP). The action of inulinase of A. niger, C. pannorum, and P. purpureogenum results in production of F3 , F4 , and F5 inulooligosachrides while Penicillium sp. TN-88 produced exclusively F3 or inulotriose. Inulooligosaccahrides are nondigestible oligosaccharides, much the same as fructooligosaccharides that confer prebiotic advantages to the consumer. These have bifidogenic, hypolipidimic and macrophage activating activities (Yun et al., 1997). Rhizoctonia solani produced maximal levels of inulinase, that is, 3.02 and 2.87 U mL−1 on inulin and artichoke powder (3% w/v), respectively. Very low activity was observed in media containing soluble starch, pectin and sucrose. R. solani inulinase appeared to be an inducible enzyme (Ertan et al., 2003). Similar results have been reported for C. pannorum inulinase production (Xiao et al., 1988). Root powder of Saussurea lappa (Kuth or Costus) has been utilized for high fructose syrup production by chemical means (Kulkarni et al., 1969). Use of crude inulin makes the process cost-effective since extraction of pure inulin involving repeated diffusion with steam is tedious and costly (Vogel, 1993). The inulin content in the kuth root powder was 30% (dry weight) and total carbon and nitrogen contents were 45.6% and 0.34%, respectively (Viswanathan and Kulkarni, 1995b). Manzoni and Cavazzoni (1992) reported that the temperature and pressure involved during autoclaving were sufficient to extract inulin from Jerusalem artichoke root powders. Following this, several workers have used plant roots and tubers (e.g., yacon, asparagus, dandelion) as sources of crude inulin for inulinase production. Among the substrates, inulin and sucrose are the preferred carbon sources for inulinase production by molds. For the microbial strain showing only inulinase activity, inulin serves as the best substrate but if the microorganism exhibits

177

178


inulinase activity coupled with invertase activity, sucrose can be used for enzyme production.


Yeasts Fructofuranosidic bonds splitting enzymes have been described in various microorganisms including yeasts (Demeulle et al., 1981; Vandamme and Derycke, 1983). Among yeasts, inulinases have been characterized from different strains of Kluyveromyces (Tables 2 and 3). Sheng et al. (2007) have reported inulinase activity (85 U mL−1 ) by Cryptococcus aureus G7a in medium containing 4% w/v inulin and 0.5% w/v yeast extract in artificial sea water at 28◦ C, pH 5.0 and 170 rpm in 42 h. Yeasts produce inulinases that are capable of exo-hydrolysis of inulin resulting in fructose as the major product (Passador-Gurgel et al., 1996). Hensing et al. (1995) have demonstrated production of inulinase by K. marxianus in a high cell-density fed-batch process. High cell density cultivations where biomass concentration exceeds 100 kg m−3 are carried out for production of yeast biomass (Fieschko et al., 1987). Using data obtained from fed-batch cultivation, they could determine process kinetics and optimize key parameters needed for modeling. They observed a 20% increase in the extracellular inulinase (measured as Cp-product concentration Kg inulinase m−3 ) in fed batch cultures on sucrose in presence of elevated pressure (2.5 bar) and high concentration of EDTA (0.75 g L−1 ). Yuan and Bai (2008) observed inulinase activity (54 U mL−1 ) of Kluyveromyces marxianus Y1 in the medium containing inulin (40 g L−1 ) after 120 h of incubation at shake flask level at 38◦ C, pH 5.0 and agitation rate 150 rpm. Solid state fermentations (SSF) are carried out in near absence or absence of free-flowing water and microbial growth and product formation occurs at or near the surfaces of solid materials (Pandey, 1992). These employ particulate low-value substrates (e.g., wheat bran, corn cobs, sugarcane bagasse) and offer several benefits being easier and cost effective. Mazutti et al. (2010a) used sugarcane bagasse supplemented with corn steep liquor, cane molasses and soybean bran for production of inulinase by K. marxianus NRRL-Y7571 in SSF in a packed bed bioreactor and found maximum inulinase activity as 436.7 U gds−1 after 24 h at 30◦ C. Xiong et al. (2007) studied the effect of nutrient level for production of inulinase. They reported maximum inulinase (409.8 U gds−1 ) production on 12.72% w/v inulin mixed with wheat bran as a solid substrate by a newly isolated Kluyveromyces S120 in SSF. Singh et al. (2007c) observed higher inulinase activity of K. marxianus YS1 (55.4 U mL−1 ) in medium in which carbon source was dahlia tuber extract (2% w/v) at an agitation rate of 200 rpm and aeration of 0.75 vvm in bioreactor after 60 h. In another study, this strain utilized raw inulin extract (4% w/v) from root tubers of Asparagus officinalis to produce 50.2 U mL−1 of inulinase

179

Exo II Exo II

Aspergillus ficuum JNSP5-06 Exo I

A. niger A. niger 12 II III A. niger 319 A. fumigatus I II

A. niger M89

Molds Aspergillus niveus Blochwitz Aspergillus awamori var. 2250 Aspergillus niger

Microorganism

6

68.1

200 62

40 46

4.5 4.5

4.5

5.5 6.0

28

70

45 55 60

5.0 5.3 5.0

45 45

45

60 60

55

55–60

50

4.35

4.0–5.0

4.5

102.6 97.9 62.5 36.5

T (◦ C)

4.0, 4.8 45

pH

69

M(r) kDa I/S ratio

Optimum

Table 3: Sources and properties of some microbial inulinases.

31.5 25.3

43.1 mg ml−1

0.25 mM 1.25 mM

1.87 mM 1.25 mM

3.53 mM

0.003 mM

Km

32.7 mg min−1 ml−1 217 46.3

526 μmol min−1 mg−1

666.7 μmol min−1 ml−1

Vmax

8.8 4.5

5.4

4.15 4.24 4.48 4.15

4.4

pI


Exo Exo

Exo

Exo Exo

Endo

Action

(Continued)

Chen et. al, 2009

Gill et al., 2004 Gill et al., 2006

Chen et al., 1997

Derycke, 1981 Nakamura et al., 1978 a,b,c

Mutanda et al., 2008 Ji et al., 1998

de-Souza-motta et al. 2005 Arand et al., 2002

Reference

180

II III Rhizopus sp. TN-96 Yeasts Cryptococcus aureus G7a Debaryomyces phaffii

60

83

48, 66 81

300 300 80

Cladosporium cladosporioides Fusarium oxysporum I (Mycelial) II (Extracellular) Penicillium janczewskii

Penicillium janczewskii Penicillium sp. strain TN-88 Penicillium sp. I

54.8 115 66

34 31 63

53

0.31 0.36

1.0 0.50 20

M(r) kDa I/S ratio

Aspergillus ficuum Alternaria alternata Chaetomium sp. C34

Endo IV Endo V Aspergillus ficuum

Microorganism

Table 3: (Continued).

50 50

5 4.0

55 45 40

45

4.5 5.0 4.0 5.5

55 55

30 37 60

55 55

45 45 50

T (◦ C)

5 4.0

5.8 6.2 4 – 5.5

4.5 6.0

5.0 5.0 5.4

pH

Optimum

115 μmol min−1 mg−1

40.8 53.8 833.3 μmol min−1 ml−1

Vmax

Demeulle et al., 1981

Sheng et al., 2008

Exo

Nakamura and Nakatsu, 1977

Pessoni et al., 1999 Moriyama et al., 2002

Pessoni et al., 2007

Gupta et al., 1988

Ferreira et al., 1991

Mutanda et al., 2009 Uhm et al., 1999 Hamdy, 2002 Zhang et al., 2004a

Reference

20.06 mg ml−1

0.0085 mg min−1

Exo

Exo

Endo

Endo

Endo Endo Exo

Action

Ohta et al., 2002

pI

0.233 mM 0.157 mM 9.0 mM

0.173 mM

16.7 μmol dm3 20 μmol dm3 6.3 × 10−2 M 2.09 × 10−2 μmol min−1 ml−1 0.43 mM

8.1 mM 0.066 M 0.199 mM

14.8 25.6 4.75 mM

Km


181

Xanthomonas oryzae No. 5

Pichia guilliermondii Bacteria Arthrobacter ureafaciens Bacillus polymyxa MGL 21 Bacillus subtilis Bacillus subtilis 430A Bifidobacterium infantis ATCC 15697 Streptomyces sp.

Kluyveromyces marxianus Y1 Kluyveromyces sp. Kluyveromyces sp. Y-85

7.5

6

45

139

6.0

70

5.5

0.84 0.62

75

50

70

37

55

37

50

6.0 7

52 55 60

4.5 4.6 6

55 52

4.4 4.6

55.5

65 57

42

55

5.5

16.7 g l−1

1.63 mM

6.8 mM 8.0 mM

0.7 mM

13.3 mM

450 μmol min−1 mg-1 12.1 g l−1 h−1

2.17 nmol s−1 4.3


Endo

Exo

Exo

Exo

Exo

Exo

Wanker et al., 1995 Vullo et al., 1991 Warchol et al., 2002 Sharma and Gill, 2007 Cho and Yun, 2002

Uchiyama et al., 1975 Kwon et al., 2003

Gong et al., 2007

Wang et al., 2000 Wei et al., 1997

Yuan and Bai, 2008


182


under similar conditions (Singh and Bhermi, 2008). Gong et al. (2007) have described inulinase production (60 U mL−1 ) by Pichia guilliermondii within 48 h of fermentation in shake flasks. Passador-Gurgel et al. (1996) screened 14 strains of the genera Kluyveromyces, Candida, Debaromyces, and Schizosaccharomyces and found K. marxianus ATCC 36907 to be the best strain producing 2.60 U mL−1 inulinase in 24 h cultivation time. The enzyme was stable at 50◦ C and pH 4.0. Similar stability profiles of K. marxianus CBS6556 inulinase have been reported by Rouwenhorst et al. (1988). K. marxianus CDBB-L-278, a wild hyper-producing strain has been reported to produce inulinase in media containing inulin and glycerol as sole C-sources. The strain produced total (extracellular plus intracellular) and extracellular inulinase activities equivalent to 68.4 U mL−1 and 34.78 U mL−1 , respectively. The strain was able to produce inulinase in presence of 2-deoxyglucose in both solid and liquid media. 2-Deoxyglucose, a glucose analog is incorporated in the medium to examine whether the enzyme synthesis in the test strain is de-repressed. However, the inulinase synthesis receded sharply in the presence of higher concentration of 2-deoxyglucose (0.05 and 0.1% w/v) and also glucose and fructose (0.25% w/v) indicating that the strain was not de-repressed (Cruz-Guerrero et al., 1995). Other K. marxianus strains and yeasts have also been examined for de-repressed synthesis of inulinase by incorporating 2-deoxyglucose in solid media (Thonart et al., 1988; Tsang and GrootWassink, 1988). The general idea regarding inulinase synthesis is that they are inducible (Vandamme and Derycke, 1983). However, CDBB-L278 could produce inulinase on glycerol as sole C-source and hence can be considered partially constitutive for synthesis of the enzyme. The kinetic values and activation energy of enzymatic reaction of K. marxianus inulinase suggest that the enzyme has much higher affinity for inulin than sucrose. According to Vandamme and Derycke (1983), the affinity of inulinase towards inulin depends upon the molecular weight of the polymer i.e. the degree of polymerization of inulin in context. The inulinase of K. marxianus has been reported to have high affinity for inulin (Km : 3.04 mM) than sucrose (Km : 40.18 mM) (Cruz-Guerrero et al., 1995) and has good thermal stability at 50◦ C. The highest achieved value of enzyme activity with inulin as a substrate by Kluyveromyces marxianus var. bulgaricus was 8.6 U mL−1 after 48 h of fermentation at the optimal pH 3.4 and temperature 29◦ C (Vranesic et al., 2002). The cost of enzymes remains a bottleneck in realizing their application at large scale. Use of inexpensive substrates, such as byproducts of agroindustries, can effectively subsidize the recurring cost of enzyme production. Makino et al. (2009) have optimized agro-industrial medium containing (g L−1 ) molasses 90.0, corn steep liquor (CSL) 45.0, and yeast extract 4.0 for inulinases production by Kluyveromyces strains. Inulinase activity 735 ± 26



U mL−1 was achieved using strain K. marxianus NCYC 587. Their results showed that about seven times greater activity can be produced using such low-priced agro-industrial residues as compared to synthetic medium. Mazutti et al. (2010a) demonstrated the use of packed-bed bioreactor (PBR) in solid state fermentation for inulinase production using K. marxianus NRRL Y-7571. The PBR consisted of a cylindrical vessel connected to humidifier at the bottom for supplying moist air (90–95% relative humidity). Medium contained 3 kg of dry sugarcane bagasse supplemented with (% w/w) sugarcane molasses 15.0, corn steep liquor 30.0, and soybean bran 20.0. After optimization, inulinase yields as high as 436.7 ± 36.3 U gds−1 and productivity of 18.2 U gds−1 hr−1 were achieved. Medium formulation based on agro-industrial residues was optimized using experimental design and response surface method and activities as high as 1139 U mL−1 were obtained on medium containing sugar cane molasses 100 g L−1 , corn steep liquor 100 g L−1 , and yeast hydrolysate 6 g L−1 with K. marxianus (Sguarezi et al., 2009). Mazutti et al. (2010 b) studied kinetics of PBR and observed that inulinase production is reduced with the bioreactor height as a consequence of deactivation due to high temperature (36◦ C) in these zones. Treichel et al. (2009) have obtained 1,317 ± 65 U mL−1 of inulinase activity by K. marxianus NRRL Y-7571 in medium containing 250 g L−1 of molasses, 80 g L−1 of corn steep liquor (CSL), 6 g L−1 of yeast extract at 300 rpm of agitation, and 1.5 vvm aeration rate in a bioreactor employing a sequential strategy of experimental design. Mazutti et al. (2010c) have demarcated major differences in pH and thermal stability among inulinase obtained from submerged and solid state cultures of K. marxianus. Inulinases produced in submerged conditions were less susceptible towards pH change while those obtained using SSF were more thermostable.

Bacteria Literature indicates that the preferred choice for inulinase production have been molds and yeasts. However, desirability of inulinases stable at low pH and high temperature has prompted several workers to explore bacteria for inulinase production. Synthesis of inulinase by Clostridium thermosuccinogenes sp. nov., a strictly anaerobic bacterium, was found to be growth dependent and the enzyme was completely cell bound (Drent et al., 1991). Studies on inulinase synthesis by Bacillus sp. viz. Bacillus polymyxa 29, B. polymyxa 722, and B. subtilis 68 by Zherebtsov et al. (2002) suggested the optimal parameters for the growth to be pH 7.0 and 33–35◦ C. Sucrose was suitable inducer in case of B. subtilis 68 while other two produced higher inulinase in presence of starch. Uzunova et al. (2001) used Bacillus sp. 11 strain isolated from a thermal Bulgarian spring (with temperature 102◦ C and pH 8.0–8.8) and immobilized it on membrane for production of thermostable inulinase.

183


184


Immobilization resulted in a two-fold increase in the enzyme production in comparison to free cells and displayed high operational stability. Selvakumar and Pandey (1999a) compared Staphylococcus sp. RRL1 and Kluyveromyces marxianus ATCC 52466 for production of extracellular inulinase. In a liquid medium containing inulin as the sole carbon source, the bacterium produced 30% more inulinase activity (618 U L−1 ) at initial pH, temperature, agitation and the inoculum size being 6.5, 37◦ C, 150 rpm and 4%, respectively. Strain RRL1 could also utilize wheat bran under solid state conditions to produce 107.64 U of inulinase per gram dry fermented substrate in 48 h while K. marxianus ATCC 52466 produced 122.88 U g−1 dry substrate in 72 h (Selvakumar and Pandey, 1999b). The study demonstrated use of SSF for production of inulinase by both bacteria and yeast. Cho and Yun (2002) have studied endoinulinase produced by Xanthomonas oryzae no. 5 in a 5 L stirred tank fermenter and recorded highest growth and inulinase activity (77 U mL−1 ) during the 30th hour of fermentation. The enzyme was stable in the pH range of 6.0–9.0 with 7.5 and 50◦ C as optimum pH and temperature, respectively. Park and Yun (2001) have suggested use of chicory root powder (5 g L−1 ) to achieve maximum endoinulinase production (15 U mL−1 ) by Xanthomonas sp. after 22 h of fermentation. The choice of substrate can make the enzyme cost-effective since use of chicory root powder reduced production cost nearly by 760 times as compared to pure commercial inulin. Ayyachamy et al. (2007) have reported inulinase production equivalent to 117 U gds−1 by Xanthomonas campestris in solid state fermentation of garlic peels. Similarly, Sharma et al. (2006) have obtained inulinase production (524 U L−1 ) by Streptomyces sp. using garlic powder. The activity was about 1.6 fold higher than that observed with inulin (321 U L−1 ) as carbon source. Xanthomonas campestris var. phaseoli has been improved using random mutagenesis to produce 22.9 U mL−1 inulinase (Naidoo et al., 2009).

PROPERTIES OF MICROBIAL INULINASES Properties of inulinases have been characterized from several microorganisms (Table 3). Multiple forms of inulinases (both exo and endo) are reported from molds and the enzymes are typically different. Pertaining to industrial conditions and better solubility of inulin at higher temperatures, thermostability is one of the desirable qualities. Among molds, Penicillium sp. 1 has been reported to produce mixture of three inulinases, TI, II, and III, and their optimal temperatures were 45–50◦ C (Nakamura and Nakatsu, 1977) while A. niger inulinases have been shown to be optimally active at 45–55◦ C (Nakamura et al., 1978a, b; Zittan, 1981). A. niger 12 inulinase (Type 2) when incubated at 60◦ C for 30 min had 54% residual activity (Nakamura et al., 1978c). More recent reports indicate presence of thermostable inulinase in A. niger and A. fumigatus showing optimal activity at 60◦ C (Chen et al.,



1997; Gill et al., 2006). Fig. 3 shows homology among some fungal inulinase sequences obtained from Swiss Prot and EMBL databases and aligned using CLUSTAL W software for multiple alignments. Enzyme sequences belonged to Aspergillus niger endoinulinase (Q0ZR34_ASPNG), Aspergillus ficuum inulinase (INU2_ASPFI), Penicillium sp. TN-88 endoinulinase (Q9HFA5_9EURO), Aspergillus fumigatus inulinase (Q4WDS9_ASPFU), and Aspergillus awamori exoinulinase (Q96TU3_ASPAW). The identity score (∗ ) among these fungal inulin hydrolyzing enzymes was found to be 22.58%. Multiple alignments of five inulinases of different fungal origin revealed presence of several conserved motifs. Some of the conserved motifs (e.g., WMNEPNGL, TWHLF, WGN, WGHATS, RDPKVF, SVEVFGGQGE) were similar to those observed in different bacterial and fungal inulinases by Singh and Gill (2006). The multiple sequence alignment of fructosyl transferases, inulinases, invertases, and levanases revealed the presence of Arg-Asp-Pro (RDP) motif, which is presumed to have a common functional role (Sprenger et al., 1995; Chavez et al., 1998). This RDP motif participates in substrate binding and is responsible for specificity of the enzyme towards fructopyranosyl residues (Nagem et al., 2004). Most of the yeast inulinases have been reported to be optimally active near 50 or 55◦ C. The optimal temperature for inulinase of Candida kefyr, Candida salmenticensis, Kluyveromyces fragilis, and D. phaffii was 50, 46, 55, and 50◦ C, respectively, while that of Debaryomyces cantarellii was 30◦ C (Negoro and Kito, 1973; Negoro, 1978; Guiraud et al., 1981b; Demeulle et al., 1981). The pH optimum of most inulinases lies between 4.0 and 5.5 (Vandamme and Derycke, 1983). Optimal pH 4.0 has also been reported for inulinase activity from Kluyveromyces marxianus var. bulgaricus (Cazetta et al., 2005). Similar values between 4.0 and 4.5 were observed for purified inulinase of A. niger 245 (Cruz et al., 1998). The maximum activity was observed at pH 4.5 by Kluyveromyces sp. Y-85 inulinase (Wenling et al., 1999). Pessoa and Vitolo (1999) have found optimum pH between 3.2 and 5.0 for inulinase from K. marxianus while Kushi et al. (2000) have reported pH optimum to be 4.7 for K. marxianus var. bulgaricus inulinase. Ettalibi and Baratti (2001) have reported optimum pH 4.7 for A. ficcum inulinase while optimum pH between 4.0 and 5.0 has been reported for A. niger inulinases (Kango, 2008). Most fungal inulinases are found optimally active in the pH range of 4.0–6.0 (Table 3).

PURIFICATION AND MOLECULAR BIOLOGY OF INULINASES Techniques enabling the separation of enzymes directly from the bulk liquid, without prior removal of particulate matter are of much importance. Fluidized or expanded bed adsorption can be an effective intervention in this regard (Draeger and Chase, 1991). Efforts of direct adsorption of inulinases on beds have been made using ion-exchange adsorbents. Pessoa and Vitolo (1999) demonstrated that inulinase can be efficiently recovered and purified

185

186


Q0ZR34_ASPNG INU2_ASPFI Q9HFA5_9EURO Q4WDS9_ASPFU Q96TU3_ASPAW


Prim.cons.

Q0ZR34_ASPNG INU2_ASPFI Q9HFA5_9EURO Q4WDS9_ASPFU Q96TU3_ASPAW Prim.cons.





10 20 30 40 50 60 | | | | | | -------------MLNPKVAYMVWMTCLGLTLPSQAQSNDYRPSYHFTPDQYWMNEPNGL -------------MLNPKVAYMVWMTCLGLTLPSQAQSNDYRPSYHFTPDQYWMNEPNGL -------------MISQGLTGALKALPLVCALVARAVADDYRPAFHFCPAENWMNEPNGL MAACFVYALAFGLFLPISSTTATSTRASTTVQPLDTVPVDFRPVYHFVPEQNWMNEPNGL ---------------MAPLSKALSVFMLMGITYAFNYDQPYRGQYHFSPQKNWMNDPNGL : :* :** * : ***:**** MAACFVYALAFGLMLNPKVAY2VWMTCLGLTLPSQAQSNDYRPSYHFTPDQ2WMNEPNGL 70 80 90 100 110 120 | | | | | | IKIGSTWHLFFQHNPTANVWGNICWGHATSTDLMHWAHKPTAIAD-----ENGVEAFTGT IKIGSTWHLFFQHNPTANVWGNICWGHATSTDLMHWAHKPTAIAD-----ENGVEAFTGT IQINSTWHLFYQADPAANVWGNECWGHATSSDLLHWDHLPVAIPV-----ENGIESFTGT IKIGSTWHLFFQHNPTGNFWGNLSWGHATSTDLVSWTHQPIAISS-----GDGIQAFTGT LYHNGTYHLFFQYNPGGIEWGNISWGHAISEDLTHWEEKPVALLARGFGSDVTEMYFSGS : ..*:***:* :* . *** .**** * ** * . * *: *:*: IKIGSTWHLFFQHNPTANVWGNICWGHATSTDLMHWAHKPTAIADRGFGSENGVEAFTGT 130 140 150 160 170 180 | | | | | | AYYDPNNASGLGDSANPPYLAWFTGYTVSSQ-----------TQDQRLAFSVDNGATWTK AYYDPNNTSGLGDSANPPYLAWFTGYTTSSQ-----------TQDQRLAFSVDNGATWTK SYYDSNNTSGLGTSTNPPYLAFFTGYTESNK-----------TQDQRLAYSTDLGQTWVK AYFDSENLSGLGSPSNAPYLAFYTGYFPSTG-----------VQDQRLAYSLDHGTTWIK AVADVNNTSGFGKDGKTPLVAMYTSYYPVAQTLPSGQTVQEDQQSQSIAYSLDDGLTWTT : * :* **:* :.* :* :*.* *.* :*:* * * ** . AYYDPNNTSGLGDSANPPYLAWFTGYT2SSQTLPSGQTVQEDTQDQRLA2SVDNGATWTK 190 200 210 220 230 240 | | | | | | FQGNPIISTSQEAPHDITGGLESRDPKVFFHRQSGNWIMVLAHGGQDKLSFWTSADTINW FQGNPIISTSQEAPHDITGGLESRDPKVFFHRQSGNWIMVLAHGGQDKLSFWTSADTINW FAGNPIIGAAQEAPQDISGGLESRDPKVFFHAPSGKWVMVLAHGGQDKLTFWTSLDAKNW YAGNPIISKTQEEPHDITKGLETRDPKVFYHTPSGRWVMILAHGGQNKVTFWTSSDAESW YDAANPVIPNPPSPYEAEYQN-FRDPFVFWHDESQKWVVVTSIAELHKLAIYTSDNLKDW : . : * : *** **:* * .*::: : . .*::::** : .* FQGNPIISTSQEAPHDITGGLESRDPKVFFHRQSGNW2MVLAHGGQDKLSFWTSADTINW 250 260 270 280 290 300 | | | | | | TWQSDLKSTSINGLSSDITGWEVPDMFELPVEGTEETTWVVMMTPAEGSPAG--GNGVLA TWQSDLKSTSINGLSSDITGWEVPDMFELPVEGTEETTWVVMMTPAEGSPAG--GNGVLA TWVSDLSSSQIEGFPSSITGWEVPDMFQLPIQGIKKTTWVLIFTPAQGSPPG--GNGVVA TWRSDFNANSIPNLPGGINGWEVPDFFELAIKGTTQKKWVMIITPATGSPAG--GNGVFA KLVSEFGPYNAQGG-----VWECPGLVKLPLDSGNSTKWVITSGLNPGGPPGTVGSGTQY . *:: . . . ** *.:.:*.:.. ...**: *.*.* *.*. TWQSDLKSTSINGLSSDITGWEVPDMFELPVEGTEETTWVVMMTPAEGSPAGTVGNGVLA 310 320 330 340 350 360 | | | | | | ITGSFDGKSFTAD-----PVDASTMWLNNGRDFDGALSWVNVPASDGRRIIAAVMNSYGS ITGSFDGKSFTAD-----PVDASTMWLDNGRDFDGALSWVNVPASDGRRIIAAVMNSYGS LTGSFDGETFVAD-----PVDPSTLWLDYGRDFDGALSWENVPASDGRRIIAAVMNSYGS VVGSFDGAVFTAD-----PVDPSAFWLDYGRDFDGALSWENVPASDGRRILASVMNSYGG FVGEFDGTTFTPDADTVYPGNSTANWMDWGPDFYAAAGYNGLSLNDHVHIGWMNNWQYGA ..*.*** *..* * :.:: *:: * ** .* .: .:. .* :* .**. ITGSFDGKSFTADADTVYPVDASTMWLDNGRDFDGALSWVNVPASDGRRIIAAVMNSYGS

Figure 3: Multiple alignment of some fungal inulinase amino acid sequences. Sequences

have been selected from Swiss Prot and EMBL databases and aligned using CLUSTAL W software for multiple alignment (Thompson et al., 1994). Sources of fungal inulinases: Aspergillus niger endoinulinase (Q0ZR34_ASPNG); Aspergillus ficuum inulinase EC 3.2.1.7 (INU2_ASPFI); Penicillium sp. TN-88 Endoinulinase precursor (Q9HFA5_9EURO); Aspergillus fumigatus Inulinase (Q4WDS9_ASPFU); Aspergillus awamori Exoinulinase (EC 3.2.1.80) (Q96TU3_ASPAW). Identity (∗ ): 126 is 22.58%; Strongly similar (:): 99 is 17.74%; Weakly similar (.): 70 is 12.54%; Different: 263 is 47.13% (color figure available online).


Q0ZR34_ASPNG INU2_ASPFI Q9HFA5_9EURO Q4WDS9_ASPFU Q96TU3_ASPAW


Prim.cons.




370 380 390 400 410 420 | | | | | | NPPTTTWKGMLSFPRTLSLKKVGTQQHFVQQPITELDTISTSLQTLENQTITPG-QTLLS NPPTTTWKGMLSFPRTLSLKKVGTQQHFVQQPITELDTISTSLQILANQTITPG-QTLLS NPPTTTWKGMLSFPRTLALKQIGSKQYFLQQPVAELSTIDGSLTSIQNQTITPN-QTLLS NPPTNTWKGMLSFPRTLELQQFNSKLRFLQLPVAELSAYTWLIANITNQTIAPG-QTLLS NIPTYPWRSAMAIPRHMALKTIGSKATLVQQPQEAWSSISNKRPIYSRTFKTLSEGSTNT * ** .*:. :::** : *: ..:: ::* * .: . : . : : NPPTTTWKGMLSFPRTLSLKKVG22QHFVQQPITEL2TISTSLQ2LENQTITPGEQTLLS 430 440 450 460 470 480 | | | | | | SIRGTALDVRVAFYPD-AGSVLSLAVRKGA--SEQTVIKYTQSDATLSVDRTESGDTSYD SIRGTALDVRVAFYPD-AGSVLSLAVRKGA--SEQTVIKYTQSDATLSVDRTESGDISYD SIHGTSLDIRMAFVID-SGATLSLAVRKGG--SEQTVIRYFQSNSTLSVDRTASGDISYD DIHSRTLDIEMSFTPS-PGATLSLSVRKGG--SQQTFIRYAESAQQLSVDRNASGNISYD TTTGETFKVDLSFSAKSKASTFAIALRASANFTEQTLVGYDFAKQQIFLDRTHSGDVSFD . ::.: ::* . .:.:::::* .. ::**.: * : : :**. **: *:* SIRGTALDVRVAFYPDSAGS2LSLAVRKGANFSEQTVIKYTQSDATLSVDRTESGDISYD 490 500 510 520 530 540 | | | | | | PAASGVHTAKLEEDDTGLVSIRVLVDTCSVEVFGGQGEAVISDLIFPSDSSDGLALEVTG PAAGGVHTAKLEEDGTGLVSIRVLVDTCSVEVFGGQGEAVISDLIFPSDSSDGLALEVTG PAAGGVHTAQLAQDNTELVHIWALIDTCSVEVFGGEGEAVISDLIFPSNSSDGLSLEVSG PAAAGVHTATVQPDASGEMHLRVLVDTCSLEVFGGQGEAVISNLIFPDVSADGVSLEVSG ETFASVYHGPLTPDSTGVVKLSIFVDRSSVEVFGGQGETTLTAQIFPSSDAVHARLASTG : ..*: . : * : : : ::* .*:*****:**:.:: *:*. .: * :* PAAGGVHTAKLEEDDTGLVSIRVLVDTCSVEVFGGQGEAVISDLIFPSDSSDGLALEVTG 550 | GNAVLQSVDVRSVSLE-GNAVLQSVDVRSVSLE-GTAMLRSVNVSSVSL--GTVALRSVEVREVLL--GTTEDVRADIYKIASTWN *.. .:: .: G2AVLQSVDVRSVSLEWN

Figure 3: (Continued)

directly from the cultivation medium without prior removal of cells using Streamline-DEAE in an expanded-bed column recovering 93% of inulinase. Ettalibi and Baratti (1987) have purified one invertase, five exoinulinases, and three endoinulinases from a commercial inulinase preparation, Novozym 230 (Novo Enzymes, Denmark) produced from A. ficuum. Purification involved ammonium sulfate precipitation and a combination of ion exchange chromatographies yielding eight fractions exhibiting aforesaid enzyme activities. Inulinase from Rhizopus sp. strain TN-96 was purified by DEAE-cellulofine A-500 and sephacryl S-200 chromatography, and the enzyme had a molecular weight of 83 kDa and specific activity of 17 U mg−1 (Ohta et al., 2002). A thermostable inulinase was purified from acidophilic fungus, Scytalidium acidophilum (Kim et al., 1994). The pH optimum of the enzyme was acidic (3.0–3.5) and retained about 95% and 85% of activities at 60 and 65◦ C, respectively, after 6 h of incubation.

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188


It is difficult to separate the multiple inulinases produced by a single organism completely by conventional methods owing to similarities in their properties. Jing et al. (2003b) could separate A. ficuum inulinases by native polyacrylamide gel electrophoresis (PAGE) resulting in five protein bands with inulinase activity. Out of these, three bands were identified as exoinulinase and two bands as endoinulinases. Wang et al. (2003) isolated Aspergillus ficuum JNSP5-06 producing both exo- and endoinulinase. Five inulinases, including three exoinulinases and two endoinulinases, have been purified to homogeneity using DEAE cellulose and Sepharose CL-6B and characterized (Table 3). The optimum temperature for all the purified inulinases was around 45◦ C. Optimum pH for exoinulinases was 4.5 and for enodinulinases, 5.0 (Chen et al., 2009). The extracellular inulinase of Cryptococcus aureus G7a was purified by Sephadex G-75 gel-filtration and DEAE-sepharose anion exchange. The molecular weight of the purified enzyme was estimated to be 60.0 kDa (Sheng et al., 2008). Three different extracellular β-fructofuranosidases (two inulinases and one invertase) of Penicillium janczewskii were purified by anion exchange, hydrophobic interaction, and gel filtration. Apparent molecular mass of purified inulinase was estimated to be 80.0 kDa (Pessoni et al., 2007). An endoinulinase (Mr 66.0 kDa) from Chaetomium sp. C34 was purified by DEAE-cellulose, Q-sepharose, Sephacryl S-200 chromatography techniques (Zhang et al., 2004a). Purification and characterization of specific inulinases from commercial preparations, and their application has drawn considerable attention from investigators. An endoinulinase with molecular weight of 68.1 kDa from A. niger has been purified using sephacryl S-200 (Mutanda et al., 2008). A 63 kDa exoinulinase was purified from commercial exoinulinase preparation of Aspergillus ficuum using sephacryl S-200 size exclusion and ion exchange chromatography (Mutanda et al., 2009). After purification, these enzymes were examined for production of FOS and fructose from commercial inulin. Conditions for purification of inulinase from K. marxianus var. bulgaricus ATCC 16045 using ion exchange fixed bed were evaluated and a maximum recovery of 67.5% with 6.6 purification fold was obtained using a NaCl gradient (0–1 M), at a flow rate of 100 cm h−1 and pH 4.1. (Kalil et al., 2010). Cho and Yun (2002) purified an endoinulinase from Xanthomonas oryzae No.5 by column chromatography using phenyl-sepharose and DEAE-sephacel (Table 3). The specific activity of the purified enzyme was 1372 U mg−1 and high concentration of inulin (125 mg mL−1 ) was found to be inhibitory for their activity. Both crude and purified preparations had similar reaction patterns producing inulooligosaccharides with DP 5, 6 and 7. Time dependent end product analysis showed that with increase in time, higher chain oligos were further degraded into shorter chain oligos of DP5. Among other bacterial endoinulinases, enzymes from Pseudomonas (170 kDa and 270 kDa) and



Athrobacter sp. (75 kDa) have also been characterized (Lee et al., 1998; Kang et al., 1998). Substrate specificity is an important feature of inulinase since most of them have associated activities against raffinose, levan and most commonly, sucrose. All the three inulinase types of Penicillium sp. were found to hydrolyse sucrose, raffinose and levan (Nakamura and Nakatsu, 1977). A. niger 12 inulinase (type 2) could also hydrolyze these, while its type 3 inulinase selectively hydrolyzed inulin (Nakamura et al., 1978a, b, c). Zittan (1981) has also found inulinases from Aspergillus sp. to be highly specific for inulin. Almost all yeast inulinases possess invertase activity and can also hydrolyze raffinose. Inulinase of Arthrobacter ureafaciens had levanase activity (Tanaka et al., 1982) while inulinase obtained from Bacillus polymyxa could hydrolyze sucrose, levan and raffinose (Kwon et al., 2003). Manganese ions are shown to enhance the activity of A. niger 12 and Penicillium sp. inulinase significantly. Relative activity of the Penicillium sp. type 3 inulinase was enhanced by 166 times by addition of manganese ion (10−3 M). Up to 33% of relative activity enhancement was observed in A. niger 12 inulinase in presence of these ions (Nakamura and Nakatsu, 1977; Nakamura et al., 1978a). Cobalt has also been found to be an activator that brings about improvement in the Penicillium sp. inulinase activity (Nakamura and Nakatsu, 1977). However, most metal ions are either ineffective or act as inhibitor in case of yeast inulinases (Demeulle et al., 1981). Ca+2 , K+ , Na+ , Fe2+ , and Zn2+ activated and Mg2+ , Hg2+ , and Ag+ inhibited the activity of the purified enzyme of Cryptococcus aureus G7a (Sheng et al., 2008). Singh et al. (2007a) have reported Mn2+ and Ca2+ increase the enzyme activity by 2.4- and 1.2-fold, respectively, while Hg2+ and Ag2+ completely inhibited the activity of K. marxianus YS-1 inulinase. Hg2+ is reported to completely inhibit enzyme activity (Warchol et al., 2002; Kwon et al., 2003). Heavy metal ions of Hg2+ , Pb2+ , and Cu2+ are strong inhibitors of A. niger 319 inulinase activity (Chen et al., 1997). Aspergillus fumigatus exoinulinase (type II) was completely inhibited in presence of 5 mM Hg2+ and Fe2+ whereas K+ and Cu2+ enhanced its activity (Gill et al., 2004). Ba2+ and Ca2+ were found to stimulate the enzyme activity while Cu2+ , Fe3+ , and Hg2+ and iodoacetate were recorded as strong inhibitors in case of Alternaria alternata inulinase (Hamdy, 2002). Over the last decade, several successful attempts have been made to fish out, clone and express inulinase gene from distinct hosts including molds, yeasts, and bacteria (Table 4). The inuA1 of Aspergillus niger AF10 had open reading frame of 1551 bp encoding 516 amino acids and a conserved sequence WMNEPN. The gene was cloned using pUC118 and the gene inuA1 was integrated into the genomic DNA of Pichia pastoris GS115 by inserting into a single site for recombination, yielding the recombinant GS115/inuA1. Inulinase expressed by the recombinant yeast was inducible by methanol and produced 50.6 U mL−1 in fermentation broth in 72 h which was 11 times that

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190


of the wild type A. niger AF10. The sequence of inuA1 showed some homology to that of other inulinase genes (nucleotide sequence EMBL accession nos. ABO12772, ABO12771, AJ006951, and E08802) at the highest homology of 98% (Zhang et al., 2003, 2004b). Analysis of the recombinant showed integration of inu1 gene with the genome of P. pastoris. In another study, gene Inu1 of Pichia guilliermondii strain1 was expressed in Pichia pastoris X-33 and the resulting recombinant pPICZaA-Inu1 showed production of high amounts of recombinant inulinase 286.8 ± 5.4 U mL−1 with specific activity 8873 ± 55.3 U mg−1 in 120 h. The recombinant inulinase purified by affinity chromatography had molecular weight of 57.6 kDa (Zhang et al., 2009a). Inulinase gene INU1 from K. marxianus KW02 was inserted into P. pastoris GS115, yielding the recombinant yeast. The enzyme activity of recombinant P. pastoris in the fermentation broth was 52 U mL−1 , 12 times more than that of the wild type and the enzyme distribution in the recombinant was significantly different as it secreted 65% extracellularly as compared to 39% of the wild type (Zhang et al., 2005). The INU1 gene encoding an exoinulinase from K. marxianus var. marxianus ATCC 12424 has been expressed in S. cerevisiae (Laloux et al., 1991). The transgenic yeast produced 0.36–0.4 U mg−1 of cells and had significantly better distribution of enzyme (extracellular: cell bound) than the donor strain. A citric acid producing marine yeast, Yarrowia lipolytica SWJ-1b was transformed to express cell bound inulinase using a surface display vector containing Inu1 gene of K. marxianus CBS 6556 (Yue et al., 2008). Inulinase yield in the transformant was 22.6 Umg−1 of cell dry weight in 96 h. Inulin hydrolysis was optimum at 12% (w/v) inulin concentration and 181.6 Ug−1 inulinase added as dry cells and released mono- and disaccharides from inulin. This yeast could also utilize inulin to produce 77.9 gL−1 of citric acid using fructose generated in the medium containing 10% (w/v) inulin (Liu et al., 2010). Inulinase gene (Kcinu) from Kluyveromyces cicerisporus was expressed in Kluyveromyces lactis using an episomal vector directed by Kcinu promoter. The enzyme activity of recombinant Kluyveromyces lactis was 391 U mL−1 after 120 h, which was 2.2-fold that of the wild type host (Yu et al., 2010). Structure elucidation for understanding enzyme action and protein engineering is a recent approach in biocatalysis. The structure of A. awamori exoinulinase at 1.55Å was similar to that of another member of glycoside hydrolase family 32 invertase (β-fructosidase) from Thermotoga maritima. The enzyme is a glycoprotein containing five N-linked oligosaccharides and under similar crystallization conditions resulting two crystal forms are obtained which differ by the degree of protein glycosylation. The X-ray structure of the inulinase-fructose complex, at a resolution of 1.87 Å revealed two catalytically important residues, Asp41 and Glu241. The enzyme seems to have a double displacement mechanism for its action on inulin (Nagem et al., 2004). Kulminskaya et al. (2003) have described substrate binding characteristics and regioselectivity of hydrolysis of Aspergillus awamori 2250 exoinulinase

191

Kwon et al., 2003

Escherichia coli Escherichia coli

Bacillus subtilis Bacteroides fragilis BF1

1455 nucleotides, 55.5 kD, exoinulinase Exoinulinase (levanase) 1,866 bp

Wanker et al., 1995 Zherebtsov et al., 2003

Zhang et al., 2009b

Zhang et al., 2005 Laloux et al., 1991 Brevnova et al., 1998 Zhang et al., 2009a

Liu et al., 2010

Moriyama et al., 2002 Onodera et al., 1996 Yu et al., 2010

Escherichia coli

Pichia guilliermondii

K. marxianus KW 02 K. marxianus ATCC 12424 K. marxianus Pichia guilliermondii

K. marxianus CBS 6556

Penicillium sp. strain TN-88 P. purpurogenum. Kluyveromyces cicerisporus

Zhang et al., (2002, 2003, 2004a) Ohta et al., 1998

Reference

Liebl et al., 1998 Tsujimoto et al., 2003

Characteristics

Thermotoga maritima Geobacillus stearothermophilus KP1289 Bacillus polymyxa MGL21

Host

E. coli JM109 Pichia pastoris 1551 bp, endoinulinase inuA, inuB (1,548 bp each), endoinulinase InuD, 2,106 bp – 1548-bp endoinulinase Kluyveromyces lactis Kcinu, inulinase activity 391 U ml−1 Yarrowia lipolytica INU1,Specific activity 22.6 U mg−1 Pichia pastoris Inu-1, 52 U ml−1 Saccharomyces cerevisiae 59. 6 kDa S. cerevisiae Pichia pastoris X-33 pPICZaA-Inu1, 286.8 ± 5.4 U ml−1 and 8873 ± 55.3 U/mg, 57.6 kDa Pichia pastoris X-33 pPICZalphaA, 58.7 ± 0.12 U ml−1 , 60 kD E. coli Stable at 90–95◦ C, 50 kD E. coli HB101 Inu A, 56.744 kD

Aspergillus niger AF10 Aspergillus niger 12

Source

Table 4: Genes and cloning of microbial inulinases in suitable hosts.



192


using 1 H-NMR analysis. The signals of 1 H -NMR spectra of the reaction mixture at the initial and intermediate stages and complete reaction indicated that fructose is the only major product and that the exoinulinase had no transglycosylating activity. In a recent study, homology modeling of the exoinulinase from Bacilllus stearothermophilus and endoinulinase from Aspergillus niger was studied (Basso et al., 2010). The structure of Aspergillus awamori exoinulinase (Nagem et al., 2004), the only one to be resolved so far, has been used as a template (PDB ID: 1Y4W). Docking and molecular dynamics simulations and analysis of the structural differences between the enzymes provided the basis of their different regio-selectivity and action. B. stearothermophilus exoinulinase was found to be 37% homologous with A. awamori exoinulinase and 34% with A. niger endoinulinase. Active site of B. stearothermophilus exoinulinase had hole-shaped binding pockets while A. niger endoinulinase had funnel shaped active site leading to difference in the regio-selectivity (Basso et al., 2010). Atomic force microscopy of K. marxianus inulinase revealed that the enzyme is made up of two subunits of different sizes. Thermal inactivation of the enzyme was attributed to dissociation of monomers by loss of intersubunit contacts at 60◦ C (Artyukhov et al., 2010). Protein engineering by insertion of an entire exoinulinase domain into a thermophilic scaffold of maltodextrin binding protein from Pyrococcus furiosus (PfMBP) resulted in enhanced kinetic stability of the enzyme during fructose production from inulin at 37◦ C (Kim et al., 2009).

IMMOBILIZATION The immobilization of inulinase of K. marxianus var. bulgaricus was carried out using gelatin support by gel entrapment and then cross-linking by treatment with glutaraldehyde with an immobilization yield of 82.60%. The optimum pH for both free and immobilized inulinase was 3.5, and the optimum temperatures were 55◦ C for the free and 60◦ C for the immobilized enzyme. Vmax and Km were 37.60 U mg−1 protein and 61.83 mM for the free inulinase and 31.45 U mg−1 protein and 149.28 mM for the immobilized enzyme, respectively. This indicated a significant shift in temperature optimum and Km values of the enzyme (de-Paula et al., 2008). The effect of hardening agents such as glutaraldehyde on inulinase activity of Kluyveromyces marxianus cells immobilized in calcium alginate beads has been studied by Bajpai and Margaritis (1985). The immobilization of K. marxianus cells was carried out in barium alginate hardened by treatment with 0.6 M glutaraldehyde for 30 min. Ettalibi and Baratti (2001) have immobilized commercial inulinase preparation, 230 (Novo Industries, France) onto glass beads of different porosities (80–3000 Å) by amination of glass beads with 3-aminopropyl triethanoxysilane, activation with glutarldehyde and incubation with inulinase solution (47.0 mg protein equivalent to 7560 units). A shift in optimum temperature by 10◦ C



was recorded and the half life of the immobilized enzyme was 350 d at 50◦ C with 2M sucrose. The immobilized cell system had up to 85% residual activity and immobilized enzyme was thermostable at 65◦ C (Barranco-Florido et al., 2001). The residual activity in glutaraldehyde treated beads after five cycles was up to 83%, which was much higher than that of untreated beads (39%). Enhanced recovery of K. marxianus inulinase was made using anionic resin Streamline DEAE and maximum adsorption of about 1428 U mL−1 was recorded (Makino et al., 2005). Recently, continuous production of oligofructose syrup from Jerusalem artichoke juice using immobilized A. niger endoinulinase on chitin has been reported (Nguyen et al., 2010). The enzyme was covalently bound to the carrier chitosan using glutaraldehyde with a recovery of 66%. Immobilization leads to a shift in pH optima from 4.5–5.0 to 5.5–6.0. Such shifts in pH optima after immobilization have been reported in other inulinases as well (Gupta et al., 1992; Kochhar et al., 1999; Ettalibi and Barratti, 2001; de Paula et al., 2008). Immobilized endoinulinase also showed a higher temperature optimum of 65◦ C than free enzyme (60◦ C). Such increase has also been noticed in temperature optima of immobilized preparation of endoinulinase from Pseudomonas 200 sp. and A. ficuum (Novozym 230) by 2.5◦ C and 10◦ C, respectively (Yun et al., 2000; Ettalibi and Barratti, 2001). The residual activity of 90% and 95% of free and immobilized enzymes was recovered after 5 d of incubation without substrate at 60◦ C and pH between 4.5 and 6.5. Km and Vmax for free and immobilized endoinulinase were 2.04% (w/v) and 80.88 Umg−1 protein, and 2.19% (w/v) and 291.58 Ug−1 support, respectively. Immobilized enzyme (10 g) was used in a packed-bed column reactor (1 cm × 30 cm, 30 mL) and Jerusalem artichoke juice was fed at (25 mL h−1 ) from the bottom using a peristaltic pump. Resulting hydrolysis as analyzed by HPLC suggested that fructan molecules of higher degree of polymerization (DP8 and more) were hydrolyzed while the oligofructose (DP7–DP3) was increased significantly to approximately 15% (from 51% to 65%) while the fructose concentration remained unaffected (Nguyen et al., 2010). Kovaleva et al. (2009) have shown that inulinase immobilized on macroporous anion-exchange resin (AV-16-GS) had higher optimal temperature and pH stability than free inulinase.

INDUCED MUTAGENESIS A mutant KM 24 was generated from X. campestris pv. phaseoli by treatment with ethyl methane sulfonate (EMS) that produced 22.09 ± 0.03 U mL−1 inulinase on 3% (w/v) sucrose and 2.5 % (w/v) tryptone in 18 h, which was 2.4-fold higher than that of the wild strain (Naidoo et al., 2009). Wei et al. (1998) treated Kluyveromyces sp. Y-85 with EMS and isolated mutants resistant to catabolite repression using 2-deoxy-D-glucose in growth medium. The mutant Kluyveromyces sp. Y-85 K6 was found to exhibit 2-fold higher expression of

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inulinase than the wild-type in glucose-containing medium indicating resistance to catabolite repression. The Pichia guilliermondii mutant M-30 was generated by UV and LiCl treatment. Inulinase activity of the wild strain 48.1 U mL−1 was enhanced up to 127.7 U mL−1 in mutant M 30 using response surface method (Yu et. al, 2009). The same mutant produced 455.9 U gds−1 of inulinase activity under optimized conditions (initial moisture 60.5%, inoculum 2.5%, ratio of wheat bran to rice bran 0.42, temperature 30◦ C pH 6.0) in SSF (Guo et al., 2009). Viswanathan and Kulkarni (1995b) have used crude kuth root powder for production of inulinase by A. niger mutant UV 11 in 10l fermenter and obtained 290 U mL−1 of inulinase in media containing 1% (w/v) Kuth root powder and 1% (w/v) CSL (pH 5.4; 30◦ C) at 1.5 vvm aeration and 300 rpm. Geotrichum candidum was mutagenised using methyl methane sulphonate (MMS) and a mutant with inulinase activity of 32.06 U mL−1 was obtained. Further exposure to EMS and UV radiations yielded a mutant exhibiting improved activity of 39.34 U mL−1 . The potential mutant was selected by imposing antibiotic selection pressure by culturing mutants on 5fc–YPR agar medium containing (mg mL−1 ): yeast extract 3.0, peptone 5.0, raffinose 20, agar 20 and 5-fluorocytosine 0.02-0.010. Over 50-fold enhancement in enzyme production (71.85 U mL−1 ) was achieved when the process parameters including incubation period (48 h), sucrose concentration (5.0 g L−1 ), pH (6.0), inoculum size (2.0% v/v), and urea (0.2% w/v) were optimized using Plackett-Burman design (Mughal et al., 2009).

APPLICATIONS OF MICROBIAL INULINASES Fructose Production High-fructose syrup makes a suitable low-calorie alternative sweetener as fructose is twice as sweet as sucrose and has better organoleptic properties (Byun and Nahm, 1978). General scheme of fructose production from natural sources involves shredding of inulin containing tubers followed by enzymatic hydrolysis of inulin, retrieval of fructose as precipitate and its decoloration using activated carbon. GrootWassink and Flemming (1980) proposed a scheme that undertakes prior inulin extraction from Jerusalem artichokes and its subsequent hydrolysis with microbial inulinases. Decoloration of the fructose syrup is done by passing it through anion and cation exchangers at different pH. Further, Kierstan (1980) proposed refinement of inulin after its extraction from source and use of Ca(OH)2 followed by carbonation for precipitation of noninulin contaminants such as gums, organic acids, and colored compounds. Here onwards the refined inulin can be hydrolysed and the impurities are less enough to be sorted out in a single step filtration over activated carbon (Vandamme and Derycke, 1983). Different types of packed



bed column reactors with immobilized inulinases from K. fragilis (Kim et al., 1982), Aspergillus ficuum (Kim and Rhee, 1989), and entrapped whole cells of K. marxianus (Bajpai and Margaritis, 1985) using amino-ethyl cellulose, chitin and gelatin, respectively, have been reported for production of fructose syrups. Among these, maximum volumetric productivity of 72 g L−1 h−1 was obtained with immobilized entrapped whole cells of K. marxianus. Sirisansaneeyakul et al. (2007) have reported production of fructose by inulinases of A. niger TISTR 3570 and Candida guilliermondii TISTR 5844 isolated from Jerusalem artichoke. A. niger inulinase liberated 37.5 g L−1 of fructose from 100 g L−1 inulin in 20 h at 40◦ C while the Candida inulinase resulted in formation of 35.3 g L−1 of fructose in 25 h under similar conditions. Singh et al. (2007b) have reported fructose yield of 39.2 and 40.2 g L−1 from Asparagus racemosus raw inulin extract and pure inulin in 4 h, respectively, using exoinulinase from K. marxianus YS-1. Nakamura et al. (1995) immobilized inulinase preparation from A. niger mutant 817 with high specific activity (71 U mg−1 ) on amino-cellulofine to achieve 160 U g−1 enzyme immobilization and observed 410 g L−1 h−1 of volumetric productivity for 5% (w/v) inulin solution at 40◦ C over 45 d of continuous operation. Inulinases accompanied with invertase activity result in liberation of glucose from sucrose. Glucose being inhibitory to inulinase, use of purified inulin or plant extract after removal of sucrose is advisable. Synergistic effect of exoinulinase and endoinulinase can also be used to avoid such inhibition (Zhang et al., 2004b). Wenling et al. (1999) have demonstrated continuous production of fructose syrup using fructan solution (2.25–4.5% w/v; pH 5.0) of Jerusalem artichoke, in a continuous bed column reactor carrying immobilized inulinase (from Kluyveromyces sp. Y-85) beads and achieved maximum volumetric productivity of 234.9 g L−1 h−1 . The product was a mixture of 85% D-fructose and 15% D-glucose. In this case, the optimum temperature (50◦ C) for hydrolysis in continuous operation was slightly lower than the batch reaction (55◦ C) and the column reactor had a half-life of 32 days. Ricca et al. (2009) have proposed a kinetic model of complete hydrolysis of chicory inulin by Fructozyme L, a commercial inulinase preparation from A. ficuum, and have analyzed its Michaelis–Menten kinetics. The kinetic parameters for inulinase at 40◦ C were Km = 24.2 g L−1 and Vmax = 0.108 g L−1 min−1 . Kinetic models of enzymatic inulin hydrolysis at three different temperatures, 40, 50, and 60◦ C, were validated. This demonstrated successful monitoring of the progress of inulin hydrolysis by following fructose production rather than inulin consumption. Garcia-Aguirre et al. (2009) isolated a K. marxianus strain from naturally fermented sugary sap of agave plants (aguamiel) from traditional rural producers of pulque. Inulinase synthesized by the strain was used to hydrolyze agave sap under optimized conditions at 31◦ C, 50 rpm and pH 6.2 in bioreactor resulting in generation of fructoserich syrups from agave fructooligosaccharides containing 95% of fructose and

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5% of glucose. This high-fructose syrup can be used as additive in food and beverage industries. The ability of inulinases to invert sugar can also be utilized for production of fructose from sucrose. Ettalibi and Baratti (2001) have utilized a thermostable inulinase from A. ficuum immobilized on porous glass beads for this purpose. The resulting continuous packed bed reactor showed 90% coversion with volumetric productivity of 27g L−1 h−1 with 1 M sucrose at 45◦ C. The reactor had a high operational stability with a half life of 350 d at 50◦ C. An exoinulinase purified from A. ficuum produced 98 mg mL−1 fructose from 150% (w/v) inulin after 12 h at 50◦ C and pH 5.0 (Mutanda et al., 2009).

Production of Inulooligosaccharides (IOS) Inulin can be selectively hydrolysed by the action of endoinulinase into inulooligosaccharides such as inulotriose and inulotetraose. These are functionally similar to other oligosaccharides and can be used as soluble dietary fiber, a functional sweetener or a prebiotic (Hidaka et al., 1987; Roberfroid, 1993). Chicory, Jerusalem artichoke, and dahlia are the common inulin-rich crops targeted as source of inulin for industrial applications (Bacon and Edelman, 1951). These can be used as abundant substrates for oligosaccharide production due to their high inulin content. Efforts are underway to develop a practicable enzymatic process for production of IOS using raw inulin of such plants. Literature has several reports about enzymatic systems employing microbial extracellular endoinulinase for producing inulooligosaccharides either in immobilized or free form. Naidoo et al. (2009) have reported production of fructooligosaccharides by endoinulinase of X. campestris pv. phaseoli KM 24 mutant. Their results indicated maximum FOS production of 11.9 g L−1 h−1 and specific productivity of 72 g g−1 h−1 when the mutant was grown on medium containing 3% (w/v) inulin and 2.5% (w/v) tryptone in a fermenter. Yun et al. (2000) have reported production of inulooligosaccharides by endoinulinase of Pseudomonas sp. immobilized onto various matrices including chitin, chitosan, activated alumina, anionic ion exchange resin and polyaminomethyl styrene. Resulting hydrolysates consisted of more than 80% of total oligosaccharides from inulin. The inulooligosacharides were identified to be inulo-biose DP2 (40–44.3%), and others DP3 (22–24.4%), DP4 (4.9–6.1%), and DP>4 (6.4–9.4%). Kim et al. (1997) have reported production of inulooligosaccharides by endoinulinase of Pseudomonas sp. Their results indicated maximum production of inulooligosacharides (75.6%) when 50 g L−1 inulin and 15 U g−1 inulinase were used. The inulooligosaccharides were identified to be ranging from DP2–DP7, where the major oligosaccharides were DP2–29.8%, DP3–21.4%, and DP4–8.1% oligomers. Yun et al. (1997) have reported production of high-content inulooligosaccharides by endoinulinase from a commercial inulinase preparation. The maximum yield of



oligosaccharides achieved was around 96% irrespective of substrate concentration ranging from 50 g L−1 to 150 g L−1 . Mutanda et al. (2008) have reported production of inulooligosaccharides by endoinulinase from A. niger. The resulting hydrolysate after 48 h consisted of inulotrioses (70.3 mM), inulotetraoses (38.8 mM), and inulopentaoses (3.5 mM) when inulin concentration, 150 mg mL−1 and enzyme dosage, 60 U mL−1 were used at pH 6.0 and 50◦ C. Process for production of oligosaccharides using two endoinulinases from Pseudomonas sp. (liberating DP2 and DP3 IOS) and Xanthomonas sp. (liberating DP5 and higher IOS) has been described by Cho et al. (2001). The dual endoinulinase system utilizes appropriate proportions of these enzymes to dictate the product distribution and the degree of polymerization in the final IOS preparation. Mutanda et al. (2008) subjected 15% (w/v) pure commercial inulin for hydrolysis by an endoinulinase purified from A. niger. IOS of DP 3, DP 4, and DP 5 were produced in varying proportions viz. 3.17% w/v, 1.17% w/v, and 0.72% w/v, respectively, after 3 h incubation at 60◦ C. Risso et al. (2009) have demonstrated use of inulinase for generation of fructooligosaccharides (FOS) from sucrose. Inulinase (4 U mL−1 ) from Kluyveromyces marxianus NRRL Y-7571 generated maximum FOS (16.7% weight) from sucrose solution (55% w/v) at 40◦ C, pH 6.0 and organic: aqueous solvent ratio of 25:100.

Production of Ethanol The idea of direct ethanol production from raw inulin collected from inexpensive agricultural crops using yeasts with inulin hydrolyzing and inulin fermenting abilities is of much interest. This results in a direct process and extraction of inulin, and its separate hydrolysis is avoided. Guiraud et al. (1981a) studied ethanol production by 15 yeast strains under anaerobic conditions at 28◦ C for 7 d in medium containing up to 125 g L−1 of inulin and achieved 5–6% ethanol with Candida kefyr, C. pseudotropicalis, C. macedoniensis, Kluyveromyces fragilis, K. marxianus, and Torulopsis colliculosa. Guiraud et al. (1982) could achieve total fermentation of juice of Jerusalem artichoke tubers by ethanolic fermentation under anaerobic conditions by K. marxianus and K. fragilis. Using Jerusalem artichoke tubers, ethanol production of 2500– 7500 L ha−1 can be obtained along with by products such as dry pulp, protein residues, and spent-liquid that can be used as manure. Margaritis and Bajpai (1982) have estimated 1400–2700 kg acre−1 year−1 ethanol yield and up to 250 kg acre−1 year−1 of single cell protein (SCP) production using Jerusalem artichoke tubers. Duvnjak et al. (1981) have suggested raw Jerusalem artichoke juice as a complete substrate to support ethanolic fermentation with K. marxianus. Kim and Rhee (1990) used inulinase and Zymomonas mobilis for ethanol production on Jerusalem artichoke juice. Nakamura et al. (1996) have used mixed cultures of A. niger and S. cerevisiae for simultaneous saccharification and fermentation to produce ethanol from Jerusalem artichoke juice.

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Zhang et al. (2010a) have suggested use of tuber meal of Jerusalem artichoke for ethanol production using a recombinant Saccharomyces sp. W0. This high ethanol producing yeast was transformed to carry inulinase gene (Inu1) of P. guilliermondii strain 1 (Zhang et al., 2010b). Fermentation medium (1500 mL) containing 50% (w/v) tuber meal and 0.1% w/v (NH4 )2 SO4 was inoculated with recombinant Saccharomyces sp. W0 (10% v/v), and fermentation was carried out at 28◦ C for 120 h under static condition without any aeration. Recombinant yeast could produce 12.05 mL of ethanol per 100 mL of medium in 144 h and the productivity was 0.319 ± 0.9 g ethanol per gram of sugar. Some recent reports suggest increasing interest in application of inulinases in production of a Mexican alcoholic beverage, tequila, from fructan rich agave juices. Enzyme application can be successfully used to replace additional acidic thermal hydrolysis step in tequila manufacture (Avila-Fernández et al., 2009). Blue agave (Agave tequilana) is a desert succulent plant used for the tequila industry. Tequila manufacturing process involves harvesting of the stem of the agave plant and removing the leaves. Processing involves cooking and hydrolysis of the core of the agave to convert the inulin into a mixture of simple carbohydrates with fructose (about 70%) and glucose (about 20–25%) as main components (Peters, 2007). Waleckx et al. (2011) have demonstrated hydrolysis of more than 90% of fructan content in the juice extract (cooking honey) of blue agave using commercial inulinase (Fructozyme L). Under optimum conditions at 60◦ C and a pH of 4.0–5.0, more than 90% of fructan hydrolysis in the cooking honey could be achieved after a 12 h treatment. Enzyme application was also validated by two industrial trials and complete process, including enzymatic hydrolysis of raw juice; fermentation and distillation were successfully done; and aroma and sensory qualities of the tequila were assured.

CONCLUSION Inulinases constitute an important class of enzymes with various applications based on inulin hydrolysis, including those in production of high-fructose syrups, fructooligosaccharides, and feedstock generation. They are produced by myriad microorganisms but Kluyveromyces marxianus and Aspergillus niger are most commonly exploited for commercial reasons. GRAS status of these organisms makes them more suitable for applications in food industries. The market share for food, nuutraceutical and pharmaceutical preparations incorporating FOS (prebiotic) and fructose has increased in recent years. Cost of the enzyme remains the bottleneck in realizing its large scale application. Search for novel inulinase producers, optimization of process parameters for production and application, enzyme immobilization, and cloning of inulinase gene in suitable hosts are some of the targets currently being explored. Agroindustrial media made from low-value substrates such as sugarcane bagasse,


molasses, and corn steep liquor are being examined for inulinase production in submerged and solid state fermentations. Recombinant yeasts containing inulinase gene have potential applications in generation of bioethanol from inulin. More knowledge of hitherto commercially unexplored sources of inulin will augment substrate availability and product development. Concerted efforts for development of cost-effective strategies suiting to requirements of industries using inulooligosachrides and fructose in commercial preparations should be made.


ACKNOWLEDGMENT Author (NK) acknowledges the financial support received from University Grants Commission, New Delhi F. No. 32(SR)-580/2006, and M.P. Council of Science and Technology, Bhopal. SCJ acknowledges financial support received from MPCST, Bhopal in project BAC 2499.

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