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Amylase enzyme from Bacillus subtilis S8-18: A potential desizing agent from the marine environment

Balu Jancy Kalpana Muthukrishnan Sindhulakshmi Shunmugiah Karutha Pandian∗

Department of Biotechnology, Alagappa University, Karaikudi, Tamil Nadu, India

Abstract The present study is aimed at developing an economical medium for the production of α-amylase from Bacillus subtilis S8-18, a marine sediment isolate from Palk Bay, with various agricultural by-products that are cheap and rich in starch. These products include wheat bran, wheat husk, rice bran, rice husk, and potato peel and are used to replace soluble starch present in the Luria Bertani (LB) broth (synthetic medium). The rice husk was found to be the best to influence enzyme production significantly (61,186 IU mL−1 ) when compared with the yield of 30,026 IU mL−1 obtained by commercial starch. Hence, LB broth containing rice husk was considered an economical medium. In addition, the effect of various nutritional and physiological factors on enzyme production

was also investigated. Furthermore, the desizing efficiency of α-amylases produced by synthetic and economical media was evaluated through various assays like reducing sugar estimation, weight loss assay, drop absorbency assay, scanning electron microscopy, and Fourier transform infrared analyses. In addition, a commercial α-amylase from B. subtilis was also used in desizing analyses for comparative purposes. It revealed that the α-amylase from the economical medium was as effective in desizing the cotton fabrics as that of the commercial enzyme and much superior to the enzyme produced through the synthetic medium. C 2013 International Union of Biochemistry and Molecular Biology, Inc. Volume 61, Number 2, Pages 134–144, 2014

Keywords: amylase, Bacillus subtilis S8-18, desizing, economical medium, scanning electron microscopy (SEM)

1. Introduction Amylases, which are acting on starch components, constitute a class of “the most important industrial enzymes” as they constitute approximately 30% of the enzyme market [1]. They have great significance owing to their potential application in a number of industrial processes such as food, textiles, paper, sugar, leather, and detergents. They also play a vital role in clinical, medical, and analytical chemistry [2]. Although the amylases are derived from several bacteria, yeasts, and fungi, bacterial amylases, especially strains of Bacillus sp., are found to dominate the enzyme industry and are widely used for the production of α-amylase [3, 4]. As amylases are

Abbreviations: SDS, sodium dodecyl sulfate; FTIR, Fourier transform infrared; OD, optical density. ∗ Address

for correspondence: Shunmugiah Karutha Pandian, MSc, PhD, Department of Biotechnology, Alagappa University, Karaikudi-630 003, Tamil Nadu, India. Tel.: +91 4565 225215; Fax: +91 4565 225202; e-mail: [email protected]. Received 12 December 2012; accepted 4 May 2013 DOI: 10.1002/bab.1122 Published online 18 October 2013 in Wiley Online Library (wileyonlinelibrary.com)

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in huge demand in industrial applications, any improvement in enzyme production and its activity and stability can lead to better process performance, economics, and feasibility. In this light, to meet the demand of the above-mentioned industries, production of α-amylase in an economically feasible manner is very essential [5]. Use of a low-cost medium rather than the expensive synthetic medium is the best choice for achieving this. The components of synthetic media such as nutrient broth and Luria Bertani (LB) broth as well as the substrate like soluble starch are very expensive. Hence, to reduce the cost of the medium, these contents have to be replaced with the cheapest and easily available agricultural by-products [6]. In the present study, an economical medium with various agricultural byproducts for amylase production has been developed. In the case of textile industries, during the manufacturing process, to prevent the yarn from breaking, a removable protective layer is applied to the threads (sizing). Starch is a very popular sizing agent because it is economical, provides satisfactory weaving performance, and it can be removed quite easily [7]. The sizing agent has to be removed before dyeing and finishing by the process called desizing. For this desizing purpose, generally α-amylase enzyme has been used preferably over the chemical-desizing agents [8], and there are only a few reports that have dealt with the desizing

application of α-amylase [9–11]. The present investigation intensively explores the application of amylase enzyme in desizing the cotton fabrics and differs from other reports by comparatively analyzing the enzymes obtained through synthetic and economical media. Though a number of amylases were reported in the past few decades, their origins are mainly terrestrial environments such as soil and reports on amylases from the marine origin are still very few [12–14]. Hence, we are reporting here the amylase enzyme with desizing potential from B. subtilis, which was isolated from the marine sediment sample.

2. Materials and Methods 2.1. Bacterial strain and submerged fermentation The strain B. subtilis S8-18 was isolated from a marine sediment sample collected from Thondi coastal area of Palk Bay. The strain identification was accomplished by 16S rRNA gene analysis, and the sequence was submitted to NCBI with the GenBank Accession Number EU624423 [15]. The strain was maintained on a Zobell Marine Agar (Himedia Laboratories, Mumbai, India) plate and was found to be a good producer of amylase enzyme with the use of production medium containing (g L−1 ) tryptone-10; yeast extract-5; NaCl-10 and starch of soluble-10, and the pH was 7.0 ± 0.1. The seed preparation for enzyme production was carried out as per Kalpana et al. [16]. An overnight culture of B. subtilis S8-18 was raised in Zobell marine broth (HiMedia Laboratories, India) at 37 ◦ C at 170 rpm. The seed preparation was done by adding 1% inoculum of overnight culture to the production medium and incubating at 37 ◦ C for 8 H at 170 rpm. This fresh seed with a CFU of 11.66 × 108 cells mL−1 was used (1%) to inoculate 50 mL of production medium in the 250-mL conical flask and incubated at 37 ◦ C for 24 H on an orbital shaker at 170 rpm. The cells were pelleted at 6,708g for 15 Min at 15 ◦ C, and the resulting supernatant was collected and used as crude amylase enzyme. The enzyme activity was determined based on the blue color intensity of starch–iodine complex; one unit of enzyme activity is defined as the amount of enzyme that causes 0.01% color reduction in starch–iodine complex in 1 Min [17]. The growth of bacterial cells in production medium was measured by UV–Vis Spectrophotometer (Shimadzu UV-2450) at OD600 nm using sterile uninoculated medium as a blank.

2.2. Optimization of production conditions The optimization studies were carried out through submerged fermentation with synthetic production medium, viz., LB broth containing soluble starch. For finding the suitable substrate that supports maximum enzyme secretion, the starch present in the production medium was replaced with various soluble carbon sources such as glucose, lactose, maltose, mannitol, and sucrose at 1% concentration. The enzyme production was also checked without the addition of any carbon source. For the nitrogen sources, various organic and inorganic nitrogen supplements (1%) such as tryptose, beef extract, yeast

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extract, tryptone, malt extract, peptone, ammonium chloride, ammonium bicarbonate, ammonium sulfate, ammonium nitrate, casein, and urea were used to replace both tryptone and yeast extract. Here also, the production medium was provided with none of the nitrogen sources [18]. To determine the effect of pH on growth and enzyme production, the initial pH of the medium was adjusted to a wide range, that is, from 5 to 12, using 1 N NaOH and HCl [3]. For determining the influence of temperature on bacterial growth and enzyme production, the production medium inoculated with S8-18 was incubated at 30, 35, 40, 45, 50, 55, 60, and 65 ◦ C for 24 H. Similarly, for the optimization of incubation time, the enzyme production was checked from 6 to 96 H with regular intervals of 6 H [17]. To determine the effect of cell density on enzyme production, the medium was inoculated with 0.5%, 1%, 1.5%, 2%, 2.5%, and 3% of 8 H inoculum. The synthetic production medium was added with different metal ions in three different concentrations (1, 5, and 10 mM). The metal ions used for the analysis were Al3+ , Ca2+ , Cd2+ , Co2+ , Mg2+ , Mn2+ , Cu2+ , Fe2+ , Hg2+ , Pb2+ , and Zn2+ [19]. The medium with none of the ions served as the control. Moreover, detergents such as SDS, Tween 20, and Triton X were also applied in the production medium with 1% concentration. To check the impact of salt on enzyme production and bacterial growth, NaCl was added to the production medium to a final concentration of 1%–10%. Similarly, for determining the influence of glucose on substrate utilization and enzyme production, the medium was provided with 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, and 3.0% of glucose together with soluble starch [19].

2.3. Production of amylase by economical medium To reduce the cost of enzyme production resulting from the usage of synthetic media containing expensive starch substrate, cheap and easily available starch rich sources in the form of agricultural by-products were applied at a 1% level to replace the soluble starch in production medium [6]. These products included rice, wheat, corn, rice bran, rice husk, wheat bran, potato pulp, and potato peels and were purchased from the local market. They were dried, ground to fine powder, sieved through a 1-mm mesh, and stored in dry, airtight containers until use. Enzyme production was accomplished through submerged fermentation using these substrates. For measuring the bacterial growth (OD600 nm ), the sterile uninoculated production media containing the respective sources served as blank.

2.4. Evaluation of amylase for desizing of cotton fabrics The capability of α-amylase enzyme to desize the cotton fabrics was evaluated using the starch sized, 100% cotton gray fabric. The fabric, with the size of 10 × 10 cm2 , was treated with α-amylase at 60 ◦ C at various time intervals and with various enzyme concentrations. The enzyme concentration is expressed in % of total weight of the fabric. The fabric treated

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Biotechnology and Applied Biochemistry with water served as the control. The desizing efficiency of the enzyme was detected by weight loss assay, reducing sugar test, starch–iodine assay, and drop absorbency test. Scanning electron microscopic (SEM) analysis and Fourier transform infrared (FTIR) spectroscopy analysis were also carried out to observe the changes in surface morphology of the fabric and to determine the functional groups, respectively. In all these assays and analyses, both enzymes obtained through synthetic medium and economical medium were applied for evaluating their desizing capability. Furthermore, a commercial α-amylase enzyme obtained from B. subtilis in the form of lyophilized powder (Sigma Cat. No. 10069) with the specific activity of 435 U mg−1 was also used at the concentration of 1 mg mL−1 in desizing experiments for comparative purposes.

2.4.1. Determination of weight loss (%) The weight loss assay was performed as per Sahinbaskan and Kahraman [20] with slight modification. Before enzymatic treatment at 60 ◦ C, the fabrics were weighed and their mass weight was documented. After incubation, both the control and treated fabric samples were washed with water, oven dried at 125 ◦ C for 5–10 Min, and again weighed. The reduction in sample weight (weight loss) after enzyme treatment indicates that the fabric was being desized by the enzyme. The weight loss (%) was determined by using the following formula: WL = ([W1 − W2]/W1) × 100, where WL is the weight loss, W1 represents the mass weight of fabric before enzyme treatment, and W2 is the weight of fabric after treatment.

2.4.2. Reducing sugar assay and iodine assay The reducing sugars that were liberated from the fabric due to enzyme treatment were estimated by the DNSA method [21] with glucose as the standard at OD540 nm (Shimadzu UV-2450, Japan) and expressed in µg mL−1 . For the iodine test, pieces of control and treated samples were boiled in water for 30 Min. The contents were then cooled and filtered, and 0.25 mL of iodine solution (1% KI and 0.1% I2 ) was added. The treated fabric samples were observed visually for discoloration, and the absorbance was measured at 690 nm. Discoloration and/or reduction in absorbance values could be the indication of samples being desized.

2.4.3. Drop absorbency test A drop absorbency test for the control and enzyme-treated fabrics was performed by AATCC Test method 79-2000 [22]. According to this method, a droplet of water was dropped on the surface of fabric samples from a height of 1.0 cm and the time required for the specular reflection of the water droplet to disappear was recorded using a stopwatch. Ten readings were taken at different areas of the fabric samples, and the mean absorbency time (seconds) was expressed.

2.4.4. SEM and FTIR analyses The SEM (Vega 3, TESCAN) analysis was carried out to check the removal of sizing agent (starch) by observing the changes in surface morphology of the treated sample [20]. Further, the

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control and treated fabric samples were subjected to FTIR analysis using BRUKER Optik GmbH, TENSOR 27 spectrophotometer to determine the associated functional groups. Twenty scans were carried out for every fabric sample to reduce the influence of noise, and the spectra were obtained between the wave numbers 4,000 and 400 cm−1 [11].

2.5. Statistical analysis All assays corresponding to optimization, production, and desizing were carried out three times and values were expressed as mean ± SD. The statistical analyses were performed using the SPSS software program (Version 17.0). Dunnet and oneway analysis of variance tests were used for comparison study. Differences with P < 0.001***, P < 0.005**, and P < 0.05* were considered as significant against the control.

3. Results 3.1. Effect of carbon and nitrogen sources Among the various carbon sources tested, the soluble starch was found to be best for α-amylase enzyme production, which gave rise to 39,266 IU mL−1 (Fig. 1A). Next to starch, maltose played a key role for higher enzyme production that was followed by lactose, sucrose, glucose, and mannitol. Thus, the enzyme was synthesized not only with starch but also with other carbon sources and the yield was similar in all these sources. Although the maximum production was observed with starch, highest growth (2.152 OD600 nm ) was recorded with glucose, revealing that enzyme production was not growth associated. Regarding the substrate concentration, 1% was suitable, yielding maximum enzyme production. Further increase in starch concentration resulted in lower enzyme production (Fig. 1B). A combination of tryptone (1%) and yeast extract (0.5%) served as a good organic nitrogen source. Similarly, it was the ammonium chloride that yielded maximum enzyme production among the inorganic nitrogen sources (Fig. 1C).

3.2. Effect of pH, temperature, incubation time, and inoculum size The analysis of pH profile showed that the bacterium S8-18 could grow at high alkaline conditions and produce α-amylase enzyme (Fig. 2A). On raising the pH gradually, the enzyme production increased from 25,038 IU mL−1 at pH 6.0 to 33,435 IU mL−1 at pH 10.0, beyond which the production started to decrease but not significantly. Although pH 10.0 supported maximum enzyme production, it was pH 11.0 that allowed maximum bacterial growth (1.799). Figure 2B indicates 45 ◦ C as the optimum temperature for α-amylase enzyme production (39,525 IU mL−1 ) and growth of organism. A significant level of enzyme (36,480 IU mL−1 ) was synthesized even at elevated temperatures such as 60 ◦ C; thereafter, it declined. The optimum incubation time for enzyme production was 24 H, beyond which the enzyme production began to decline gradually (Fig. 3A). With the different concentrations of seed (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, and 3.0%) as inoculum

Desizing Action of B. subtilis Amylase

FIG. 2

pH (A) and temperature (B) profile of S8-18 α-amylase (IU mL−1 ) and bacterial growth (OD600 nm ).

size, 1% was the best to yield maximum enzyme production (33,650 IU mL−1 ) (Fig. 3B).

3.3. Effect of NaCl, detergents, metal ions, and glucose on enzyme production

FIG. 1

Effect of nutritional parameters on α-amylase enzyme production and growth of B. subtilis S8-18. (A) Carbon sources (1%), (B) substrate concentration, and (C) nitrogen sources (1%).


Analysis of the effect of NaCl at different concentrations (0%– 10%) on enzyme production and growth (Fig. 4A) showed that although the S8-18 strain was able to grow in up to 10% NaCl, the enzyme production was decreased at above 2% salt. Thus, maximum enzyme production (34,777 IU mL−1 ) occurred at 2% of NaCl, whereas in the absence of salt it was 33,214 IU mL−1 . In the present study, the amylase production was suppressed by glucose (Fig. 4B). In the absence of glucose and when the starch was the sole carbon source, the enzyme production reached a maximum level of 31,321 IU mL−1 . In contrast, upon the addition of glucose from 0.2% to 1.5% concentration along

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Biotechnology and Applied Biochemistry

FIG. 3

Optimization of incubation time (A) and inoculum size (B) for S8-18 α-enzyme production (IU mL−1 ).

with starch, the enzyme production began to fall and at 1.5% glucose, only 59% of enzyme production was observed. External factors such as cations and additives have been known to affect the production of enzyme. Figure 4C shows the stronger inhibitory effect of detergents (1%) such as SDS, Triton X-100, Tween 20, and glycerol on enzyme production. All these components greatly suppressed enzyme production but not growth. Although Triton X-100 completely inhibited enzyme production, 25%, 18%, and 15% of enzyme production was recovered in the presence of glycerol, SDS, and Tween 20, respectively. Various metal ions in different concentrations (1, 3, and 5 mM) were applied in the production medium to determine their influence on enzyme production (Table 1). The calcium and magnesium ions induced, but not significantly, α-amylase enzyme production at all three concentrations over the control (37,141 IU mL−1 ). At 1 mM concentration, while

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FIG. 4

Effect of NaCl (A) and glucose (B) and influence of detergents/additives (C) on enzyme production and growth. The additives were added to the production medium to the final concentration of 1%.

the Ca2+ ion marginally induced the enzyme production to 37,728 IU mL−1 , the Mg2+ ion resulted in 39,174 IU mL−1 . Furthermore, at elevated concentrations of 5 and 10 mM, these ions enhance enzyme production. Similarly, Fe2+ (1 and 5 mM), Cu2+ , and Mn2+ (1 mM) ions played an important role in accelerating amylase production. In contrast to this, Cd2+ ,


Effect of various metal ions on α-amylase enzyme production and S8-18 bacterial growth

TABLE 1

S. No.

Metal

1

Control

2

Al3+

3

4

5

6

7

8

9

10

11

Ca

2+

Cd2+

Co2+

Cu

2+

Fe2+

Hg

2+

Mg2+

Mn

2+

Pb2+

Concentration (mM)

Continued

TABLE 1 Concentration (mM)

Enzyme production (IU mL−1 )

Growth (OD600 nm )

1

–

–

–

5

–

–

–

–

10

–

–

10

–

–

1

37,728 ± 346

1.75 ± 0.87

5

39,038 ± 472

1.48 ± 0.76

10

39,536 ± 290

1.76 ± 0.54

1

–

–

5

–

–

10

–

–

1

–

–

5

–

–

10

–

–

1

37,140 ± 501

1.86 ± 0.09

5

–

–

10

–

–

1

39,243 ± 174

1.62 ± 0.34

5

39,516 ± 356

2.46 ± 0.29

10

3,937 ± 60

1.74 ± 0.71

1

–

–

5

–

–

10

–

–

1

39,174 ± 225

2.07 ± 0.06

5

39,775 ± 356

2.09 ± 0.79

10

39,684 ± 498

2.32 ± 0.33

1

39,024 ± 325

0.28 ± 0.46

5

9,874 ± 123

0.67 ± 0.02

10

–

–

1

34,875 ± 653

1.74 ± 0.08

5

26,093 ± 532

1.60 ± 0.14

10

15,312 ± 583

2.00 ± 0.35


Growth (OD600 nm )

37,141 ± 412

2.15 ± 0.22

1

–

5

(Continued)


S. No. 12

Metal Zn2+

Each value is expressed as the mean value derived from three independent experiments with standard deviation. The symbol – indicates no growth and no enzyme activity in respective columns.

Co2+ , Hg2+ , and Zn2+ ions completely inhibited the growth of B. subtilis S8-18 even at the 1 mM concentration.

3.4. Production of α-amylase using agricultural products The rice husk induced enzyme production to its maximum level compared with the other agricultural products tested (Table 2). It induced the enzyme secretion significantly (P < 0.005) to 61,186 IU mL−1 , whereas the soluble starch gave rise to only 30,026 IU mL−1 . Hence, LB broth containing rice husk was considered an economical medium and the amylase obtained through this medium termed an economical enzyme. Considering the cost of commercial starch substrate (US$ 29.82 per kg), these agricultural by-products, especially the rice husk, were inexpensive, that is, US$ 0.07 per kg, which accounts for 0.24% of the total cost of commercial starch. Hence, employing these products as substrates can lead to a huge reduction in the cost of enzyme production. In addition to rice husk, cheap sources such as potato peel and wheat husk were also used for enzyme production at various concentrations (0.25%– 3.0%) and yielded significant enzyme production (P < 0.05). The optimum concentration for rice husk and wheat husk was found to be 0.5% and for potato peel it was 2.5%, after which no improvement was detected. This could be due to the thickness of the fermentation medium, which resulted in the decreased agitation and poor mixing of air, which are essential for the growth of organism and production of enzyme [23].

3.5. Desizing of cotton fabric by amylase Based on the greater yield of enzyme secretion by rice husk over other agricultural by-products, the enzyme obtained through rice husk was subjected to desizing analysis. Thus, the desizing activity of the enzyme obtained from synthetic medium (LB+ starch) and economical medium (LB+ rice husk) was evaluated on cotton fabrics in comparison with commercial α-amylase enzyme simultaneously. The optimum enzyme concentration (%) and incubation time (Min) were determined as 1.5% (31,286 IU mL−1 ) of the total weight of the fabric and 60 Min, respectively, for efficient desizing activity by synthetic enzyme

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4

Rice

0.50b

47,010 ± 679*

1.78 ± 0.02

5

Wheat

0.72b

40,301 ± 72

1.60 ± 0.1

6

Raggi

0.81b

42,343 ± 488

1.85 ± 0.02

7

Corn

0.90b

34,866 ± 217

b

11

Wheat husk

0.13b

41,439 ± 347*

1.71 ± 0.01

a Source:

Himedia Laboratories, Mumbai, India.

b Source:

Tamilnadu State Agricultural Marketing Board, India.

Each value of enzyme production and growth OD is expressed as the mean value derived from three independent experiments with standard deviation with the statistical significance of P < 0.005** and P < 0.05* .

(Tables 3a and 3b). For economical and commercial enzymes, 60 Min incubation time with 1% enzyme concentration was optimal, whereas increased time and enzyme concentration beyond these optimum levels ended up with no improvement or adverse effects in the desizing process. This optimization study was accomplished by weight loss assay, drop absorbency assay, and reducing sugar estimation; the results of these three tests correlated with each other and hence led to confirmation. Table 4 depicts the cumulative analysis of desizing activity of commercial, synthetic, and economical α-amylase enzymes. It is obvious that the economical enzyme played a better role in desizing (P < 0.005) than the synthetic α-amylase enzyme (P < 0.05) and it was comparable and near equal to the level of commercial B. subtilis α-amylase enzyme. This revealed 31% of weight loss, 4,331 µg mL−1 of liberated reducing sugars, and 2.71 Sec as drop absorbency time, whereas the control fabrics accounted for 1% weight loss, 5 µg mL−1 of reducing sugars, and 218 Sec of absorption time. Almost instantaneous

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180 120

6 ± 1 218 ± 6

1.39 ± 0.09

Drop absorbency time (Sec)

32,527 ± 231

547.86 ± 15.12

0.13b

5.32 ± 1.75

Wheat bran

Reducing sugar (µg mL−1 )

10

12.17 ± 1.21

1.81 ± 0.04

1.07 ± 0.26

24,314 ± 256

Weight loss (%)

0.07b

60

Rice bran

45

9

30

1.64 ± 0.07

15

61,186 ± 236

Control

0.07

Parameter

Rice husk

Influence of incubation time on desizing activity of synthetic α-amylase enzyme

8

Incubation time (Min)

1.37 ± 0.07 **

3 ± 1

1.43 ± 0.04

3 ± 1

39,897 ± 413

2 ± 1

0.32b

3 ± 1

Potato pulp

8 ± 1

3

11 ± 3

2.19 ± 0.02

158.16 ± 25.34

44,445 ± 438*

413.44 ± 13.49

–

498.90 ± 12.21

Potato peel

794.34 ± 9.53

2

739.97 ± 10.12

1.21 ± 0.05

674.15 ± 7.62

30,026 ± 187

11.93 ± 1.38

29.82a

12.90 ± 1.41

Soluble starch

13.24 ± 0.82

1

S. No.

Growth OD (600 nm)


11.58 ± 0.70

Cost of the substrates (US$/kg)

11.85 ± 0.39

Starchy substrates (1%)

240

Effect of low-cost substrates on α-amylase enzyme production and bacterial growth

TABLE 3a

TABLE 2

10.58 ± 0.89



TABLE 3b

Influence of enzyme concentration on desizing activity of synthetic α-amylase enzyme with 60 min as constant incubation time

Enzyme concentration (%) Parameter

Control

1.0

1.5

2.0

2.5

3.0

Weight loss (%)

1.07 ± 0.26

9.75 ± 0.87

12.95 ± 0.45

11.88 ± 0.33

11.17 ± 1.23

10.68 ± 1.38


5.32 ± 1.75

46.32 ± 3.15

547.86 ± 1.27

129.05 ± 1.74

58.22 ± 0.60


218 ± 6

10 ± 2

11 ± 3

13 ± 1

3 ± 1

163.09 ± 2.4 5 ± 2

Each value is expressed as the mean value derived from three independent experiments with standard deviation. The total protein content present in 1% of enzyme was 140 mg mL−1 .

TABLE 4

Comparative analysis of desizing potential of α-amylase enzyme obtained through synthetic and economical production media with commercial α-amylase

Parameter

Control

Commercial enzyme treated

Weight loss (%)

1.07 ± 0.26

34.45 ± 0.74**


5.32 ± 1.75

5,101.61 ± 15.75***


218 ± 6

Synthetic enzyme treated 13.095 ± 0.43* 671.1 ± 5.724*

2 ± 1**

4 ± 1*

Economical enzyme treated 31.01 ± 0.65** 4,330.51 ± 43.21** 3 ± 1**

Each value is expressed as the mean value derived from three independent experiments. ± indicates the standard deviation from mean value with the statistical significance of P < 0.001 *** , P < 0.005** and P < 0.05* . The low-cost enzyme represents the enzyme obtained by using rice husk as the substrate.

water droplet absorption was observed in the fabric desized with the economical enzyme. The desizing efficiency of the commercial enzyme and the enzymes obtained through synthetic and economical media was further evaluated through SEM and FTIR analyses. The SEM images (Fig. 5A) clearly evidenced the desizing potential of all the three α-amylase enzymes. On observing the surface of the control fabric sample, the starch layer, the sizing agent, was seen as a coating over the fabric, whereas in samples treated with α-amylase enzymes, the layer of starch was removed and hence the vertical and horizontal arrangements of yarn in the fabrics were clearly visible. The FTIR analysis (Fig. 5B) revealed a significant difference in the peak absorbance values of control and treated fabric samples, particularly in the fabrics treated with economical and commercial enzymes. In all the samples, the sharp peaks at 1,700–1,600 and 2,900–2,800 cm−1 indicate the presence of pectin substances and alkyl ester chain of cotton wax, respectively. Similarly, the N–H wagging of protein substances results in major peaks at 620–800 cm−1 . The low-cost enzyme-treated sample revealed notable reduction in the absorbance values pertaining to these functional groups, whereas the synthetic enzyme-treated sample did not show any significant differences. Compared with control, the peaks in the range of 3,250–3,400 reduce considerably in the sample treated with low-cost enzyme,


whereas the synthetic enzyme caused slight variation in peak absorbance.

4. Discussion The marine sediment bacterium B. subtilis S8-18 was subjected to various optimization conditions for maximum production of amylase enzyme and the secreted enzyme was proved to be a suitable candidate for enzymatic desizing. The bacterium secreted a maximum level of amylase by utilizing starch as the substrate. In earlier studies also, starch was reported as a potential substrate for amylase production [24,25]. However, in the presence of other carbon sources and even in the absence of carbon source, a considerable amount of amylase production was detected and it indicates the constitutive nature of S8-18 amylase. Such constitutive synthesis has been observed in Halobacillus sp. MA-2 [26] and in B. licheniformis [27], where α-amylase production took place even in the absence of carbohydrates and with other carbon sources such as maltose, glucose, and fructose. In contrast to this, amylase synthesis has been found to be inductive in Chromohalobacter sp. [19], Bacillus sp. [18], and in Micrococcus sp. [28], where the enzyme was produced in the presence of starch and other starch-containing substrates only. The suitable starch concentration for maximum amylase production was 1% and the reduced enzyme production beyond this optimum level was due to the high

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FIG. 5

(A) SEM images showing the desizing efficiency of commercial α-amylase (a), α-amylase from synthetic medium (b), and α-amylase from economical medium (c). Control represents the fabric samples treated with water and treated represents the fabric samples treated with respective α-amylase enzymes. (B) FTIR spectrograph of control, commercial enzyme, synthetic enzyme, and economical (rice husk) enzyme-treated fabric samples.

viscosity developed in production medium at such elevated starch concentrations, which prevents the free oxygen transfer essential for growth of bacteria [29]. Regarding the nitrogen source, a complex supplement involving a combination of two organic nitrogen sources, that is, tryptone (1%) together with yeast extract (0.5%), served as the best source for maximum amylase production (38,569 IU mL−1 ) compared with other simple organic and inorganic nitrogen sources. By using yeast extract in conjunction with other organic sources such as bactopeptone and peptone, higher amylase production has been achieved in many other Bacillus sp. too [3, 30]. Whereas all other inorganic nitrogen sources inhibited both cell growth and enzyme production, ammonium chloride yielded higher

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amylase production that was equal to that of tryptone + yeast extract. Amylases produced by most of the microorganisms are subjected to catabolic repression by readily metabolizable substrates like glucose. Thus, when glucose is provided along with substrate it was found to diminish amylase synthesis in many Bacillus [18, 31] and Chromohalobacter spp. [19] and in Halomonas meridiana [32]. In the present investigation too, amylase production was suppressed by glucose. In contrast to this, α-amylase production induced by glucose has also been reported in Aspergillus oryzae DSM [33]. pH and temperature are the few factors responsible for causing changes in cell membrane and cell wall, which can ultimately affect the release of extracellular enzymes into the culture medium [34, 35]. The strain S8-18 was also under the influence of pH and temperature changes, which affected its growth and amylase production. However, it was able to grow in both acidic and alkaline conditions and produce amylase enzyme. The production was found to increase with increasing pH, especially in the pH range of 9–11 with pH 10 as optimum. This is in agreement with α-amylase produced by Bacillus sp. PN5 at pH 10 [3]. The temperature profile suggested the mesophilic nature of the strain S8-18, which exhibited maximum enzyme production as well as growth at 45 ◦ C. In contrast to this, Bacillus spp. that showed a major difference in temperature optima for bacterial growth and enzyme production have been reported in previous studies [3, 36]. Although the majority of amylases were produced at stationary phase, that is, at 48 and 60 H [26, 36], in the present study, S8-18 produced maximum amylase at the 24th hour itself, which is suitable for the prompt recovery of enzyme. Among the metal ions, Ca2+ and Mg2+ were found to enhance enzyme synthesis almost equally in all three test concentrations, whereas the other ions exerted their suppressive action. Also, S8-18 amylase production was strongly inhibited by detergents like SDS, which is in total agreement with earlier reports [37, 38]. In the present study, the soluble starch in the synthetic production medium was replaced with the naturally occurring low-cost starchy substrates and it was observed that enzyme production was doubled with the addition of rice husk. Amylase production has been attempted through a variety of agricultural by-products and has succeeded worldwide. Wheat bran (1.25%) along with pearl millet (1%) has been reported to increase α-amylase production twofold in B. licheniformis [6]. A 10-fold increase in α-amylase synthesis was achieved with 1.5% of pearl millet starch in the mutant strain of B. licheniformis [39]. In the present investigation, rice husk (0.5%) was found to be a suitable substrate for S8-18 strain, which induced a twofold increase in amylase production compared to the soluble starch. Rice husk, which is the protective layer of rice grains, represents about 20% of the weight of the rice harvested and about 80% is made of organic components [40]. It consists of cellulose and lignin as major components. Nevertheless, the bacterium B. subtilis S8-18 was able to utilize the rice husk and produced a significant level of amylase


enzyme. To date, rice husk has been identified and reported as a substrate for the production of protease [41–43] and cellulase enzymes [44, 45]. There are only few reports on α-amylase enzyme production through rice husk [46, 47] and they found no significant increase in enzyme secretion. This is the first study finding rice husk (0.5%) as an effective substrate for αamylase production. This may possibly be due to the presence of residual starches obtained from rice, which sufficed to induce amylase production. As rice husk is inexpensive, it can be used to replace soluble starch and for large-scale (commercial) production of α-amylase enzyme. In spite of few reports on the desizing application of α-amylase enzyme in recent years [9, 11], the present investigation strongly evidenced the desizing activity of S8-18 amylase by including a comparison with commercial α-amylase of B. subtilis, through various reliable assays. The study of the effect of α-amylase enzyme concentration on desizing revealed that desizing activity is directly proportional to the enzyme concentration but to a limited extent. However, when the concentration of the enzyme was increased beyond the optimum level (1.5%), adverse effects were observed the desizing activity. An α-amylase from Aspergillus sp. has been reported as an effective desizer for cotton fabrics [48]; the optimum enzyme concentration was found to be 300 U mL−1 Min−1 and further increase in the concentration had no significant effect on the desizing of the cloth. This might be because the appropriate enzyme to substrate ratio is essential to obtain optimal results [49]. Determination of weight loss is the most common method used for evaluating the desizing process in which higher weight loss values indicate better removal of size mix from the treated fabrics. Estimation of reducing sugars that are formed as a result of starch hydrolysis also determines the desizing efficiency as the amount of reducing sugars is directly proportional to the amount of starch being hydrolyzed. Saravanan et al. [11] reported that there was no correlation between the reducing sugars released and weight loss (%). In contrast to this, in the present investigation, weight loss and liberated reducing sugars were correlated with each other. Furthermore, the weight loss caused by amylase obtained through the economical medium in this study was 31%, which was as good as the weight loss caused by commercial amylase (34.45%) and much higher than the 13% observed for the enzyme from synthetic medium. Nevertheless, immobilized porcine pancreatic α-amylase has been reported to cause only 12% weight loss [20]. The hydrophobic impurities present on the fiber surface, dried film of starch, and other hydrophobic components in the sized yarns contribute to poor absorbency. Hence, removal of these hydrophobic contents and starch will result in quick absorption of water. The lowest absorbency value reported was 24 Sec for desizing by B. amyloliquefaciens α-amylase [10]. Only when the bleaching treatment using glucose oxidase was combined with desizing did the time get reduced to 2 Sec. In contrast, in the present study, treated fabrics showed the lowest absorbency time as ∼3 Sec by S8-18 amylase treatment alone and thus the fabrics showed better absorbency (less time to


absorb water droplet) than the untreated control fabric. Almost instantaneous absorption of the water droplet was observed in the enzymatic desized fabric. This is because the amylase enzyme is expected to break the continuity of the waxy layer of the cuticle present in the surface of fibers, thereby improving the absorbency. Thus, with regard to drop absorbency, S8-18 amylase, particularly the enzyme from the low-cost medium, was proved to be the better desizing agent. Our finding is in agreement with the result of Saravanan et al. [11], who also reported ∼ 2 Sec as absorbency time. Thus, S8-18 α-amylase is hereby shown to be a potential desizing agent of cotton fabrics. In addition, the SEM images served as visible and strong evidence substantiating the desizing application of S818 enzyme. Further, the FTIR spectrograph illustrates that absorbance values in enzyme-treated fabrics, especially the economical enzyme and commercial enzyme, were reduced when compared with control fabric sample. Thus, the FTIR analysis also substantiates the desizing potential of S8-18 enzyme. In conclusion, because the desizing activity of S8-18 amylase is comparable to that of commercial enzyme and it can be produced in an economically feasible manner, the enzyme could prove useful in textile industries.

5. Acknowledgements Financial assistance rendered by the Council of Scientific and Industrial Research (CSIR), India, to Prof. S. Karutha Pandian for carrying out this work is gratefully acknowledged. The authors also gratefully acknowledge the computational and bioinformatics facility provided by the Alagappa University Bioinformatics Infrastructure Facility (funded by DBT, GOI; Grant No. BT/BI/25/001/2006). Financial support provided to Balu Jancy Kalpana in the form of an Innovation in Scientific Pursuit for Inspired Research (INSPIRE) Fellowship by Department of Science & Technology, Government of India (DST/INSPIRE Fellowship/2010 [IF10448]), is thankfully acknowledged.

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