Constitutive acetonitrile hydrolysing activity of

Indian Journal of Experimental Biology Vol. 44, March 2006, pp. 240-245

Constitutive acetonitrile hydrolysing activity of Nocardia globerula NHB-2: Optimization of production and reaction conditions H Kumar, S Prasad, J Raj & T C Bhalla* Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla 171 005, India Received 24 June 2005; revised 7 December 2005 Nocardia globerula NHB-2 exhibited an intracellular acetonitrile hydrolysing activity (AHA) when cultivated in nutrient broth supplemented with glucose (10.0 g/l) and yeast extract (1.0 g/l), at pH 8.0, 30°C for 21 hr. Maximum AHA was recorded in the culture containing 0.1 M of sodium phosphate buffer, (pH 8.8) at 45°C for 15 min with 600 μmol of acetonitrile and resting cells of N. globerula NHB-2 equivalent to 1.0 ml culture broth. This activity was stable up to 40°C and was completely inactivated at or above 60°C. About five-fold increase in AHA was observed after optimization of culture and reaction conditions. Under the optimized conditions, this organism hydrolyzed various nitriles and amides such as propionitrile, benzonitrile, acetamide, and acrylamide to corresponding acids. This nitrile/amide hydrolysing activity of N. globerula NHB-2 has potential applications in enzymatic synthesis of organic acids and bioremediation of nitriles and amides contaminated soil and water system. Keywords: Acetonitrilase, Amidase, Nitrile hydratase, Nocardia globerula NHB-2

The nitrile and amide degrading enzymes (nitrilase, EC 3.5.5.1; nitrile hydratase, EC 4.2.1.84 and amidase, EC 3.5.1.4) have emerged as important biocatalysts for conversion of nitriles and amides to corresponding amides and acids of industrial and pharmaceutical value. The sources of these enzymes are plants and microbes. However, the microbes are most common source of nitrile and amide converting enzymes. A number of microorganisms such as Rhodococcus rhodochrous J11, Pseudomonas chlororaphis B 232, Rhodococcus sp. Strain YH3-33, R. rhodochrous PA-344,5, R. rhodochrous NHB-26, Rhodococcus equi A47 and Brevibacterium R 3128 have been isolated and explored for their application in the transformation of nitriles/amides to corresponding acids. Acetonitrile hydrolysing activity (AHA) has been reported in Agobacterium sp.9, Arthrobacter sp. J-110, Candida famata11, Nocardia rhodococcus LL 100-2112, R. erythropolis A1013, R. erythropolis BL114, Rhodococcus sp.15 as these organisms efficiently utilised acetonitrile as carbon and nitrogen source. In the present communication, optimisation of culture conditions for production of acetonitrile hydrolysing activity (AHA) of Nocardia globerula NHB-2 has been reported. ________________ *Correspondent author: Phone & Fax: +91-177-2832154 E-mail: [email protected]

Materials and methods Chemicals

Nitriles, amides and acids used in the present studies were purchased from Lancaster synthesis (England). All other chemicals and media ingredients were of AR grade from Merck (Germany) and HiMedia (Mumbai), respectively. Bacteria and culture conditions

Nocardia globerula NHB-2 is a versatile nitriledegrading organism, was isolated from the soil of a forest near Manali, India and maintained on nutrient agar slopes16. A pre-culture of N. globerula NHB-2 was prepared by transferring a loopful of culture from agar slope to 50 ml of modified nutrient broth4. Two ml of pre-culture was transferred to 50 ml of production medium17 supplemented with 0.4% (v/v) of acetonitrile (filter sterile) for induction of AHA in N. globerula NHB-2. It was incubated at 30oC in an incubator shaker (160 rpm) for 24 hr. Cells of N. globerula NHB-2 were harvested by centrifugation at 5,000 g for 15 min at 4°C. The cell pellet was washed twice with 0.1 M potassium phosphate buffer (pH 7.2) containing 5 mM of 2-mercaptoethanol and finally suspended in the same buffer. These cells were termed as resting cells and used as biocatalyst for hydrolysis of acetonitrile to acetic acid.

KUMAR et al.: ACETONITRILE HYDROLYSING ACTIVITY OF NOCARDIA GLOBERULA

Enzyme assay

AHA was assayed in 2 ml reaction mixture containing resting cells of N. globerula NHB-2 (i.e. equivalent to 1 ml of culture broth), 400 μmol acetonitrile and 0.1 M potassium phosphate buffer, (pH 7.2). It was incubated at 30oC for 15 min and the reaction was ceased by addition of 2 ml of 0.1 N HCl. In the control, acetonitrile was omitted during incubation and added after the reaction was stopped. The reaction mixture was centrifuged at 5,000 g for 10 min and ammonia released during enzymatic hydrolysis of acetonitrile was estimated in the supernatant following the method of Fawcett and Scott (1960)18. One unit of acetonitrile hydrolyzing activity (AHA) was defined as that amount of enzyme, which released 1 μmol of NH3/min under assay conditions. In case of hydrolysis of acrylonitrile to acrylamide, the amount of acrylamide formed was estimated in the supernatant of reaction mixture by high performance liquid chromatography, HPLC19. All experiments were carried out in triplicate and mean value was taken under consideration. Optimisation of culture conditions for production of AHA in N. globerula NHB-2

Six different media (M1-M6 supplemented with 0.4% v/v acetonitrile) were tested for the production of AHA by N. globerula NHB-2 (Table 1). Effect of temperature (20o-45oC), incubation time (0-63 h), inoculum size (1-10%, v/v) and pH (5.5-10.0) were also studied on this activity. Effect of acetonitrile concentration (0-4 % v/v) was studied on AHA, and growth of N. globerula NHB-2 in the production medium M2. Optimization of reaction conditions using AHA of N. globerula NHB-2

Effect of reaction buffer (pH 6.0-9.0), various buffer systems (i.e. sodium phosphate, potassium phosphate, borate, bicarbonate and Tris-HCl buffers of 0.1 M and pH 8.8), incubation temperature (20°60°C), amount of resting cell (i.e. equivalent to 1-5ml culture broth), incubation time (7-77 min), substrate (acetonitrile) concentration (200-900 μmol) on AHA of N. globerula NHB-2 were studied. Thermostability of AHA was studied after 1h incubation at 30°-80°C. Substrate affinity of AHA of N. globerula NHB-2

N. globerula NHB-2 was grown at optimized production conditions. The substrate affinity of AHA of this organism was studied atoptimized reaction conditions against 600 μmol of various nitriles/amides

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(e.g. acetonitrile, acrylonitrile, benzonitrile, acetamide, acrylamide and benzamide) to produce corresponding acids and ammonia. Effect of metal ions, carbonyl reagent and chelators on AHA of N. globerula NHB-2

Effect of metals ions (e.g. Zn++, Fe++, Ca++, Pb++, Cu++, Co++, Mn++, Al+++, Cd++, Hg++ and Ag+), carbonyl reagent (phenyl hydrazine hydrochloride), chelators (EDTA and sodium azide) and urea on AHA of N. globerula NHB-2 was also studied. Results and Discussion Optimization of culture conditions for production of AHA in N. globerula NHB-2

Medium ⎯ Among the six medium [M1-M6 supplemented with acetonitrile (0.4 % v/v)] tested, the maximum AHA (0.06 U/ml) was observed in the modified nutrient broth medium (M2) supplemented with glucose (10 g/l) and yeast extract (1 g/l), whereas slightly low enzyme activity (0.05 U/ml) was also recorded in N. globerula NHB-2 cells grown in nutrient broth medium (M1; Table 1). Aliphatic nitrile/amide hydrolysing activity of Bacillus pallidus Dac 521 was also noted in nutrient broth20. In media M3 and M6, 50 and 66% activity, respectively was observed in comparison to medium M2. No activity could be detected in cells cultured in mineral salt Table 1 ⎯ Production of AHA by N. globerula NHB-2 in various media Medium* M1 M2 M3 M4 M5

M6

Compostion (g/l, pH 7.2)

AHA (U/ml)

Nutrient broth Peptone 5.0 g and beef extract 3.0 g Modified nutrient broth Glucose 10.0 g, yeast extracts 1.0 g, peptone 5.0 g and beef extract 3.0 g MY medium Glucose 15.0 g, peptone 5.0 g, yeast extract 3.0 g and malt extract 3.0 g Modified MY medium Glycerol 10.0 g, peptone 5.0 g, yeast extract 3.0 g and malt extract 3.0 g Mineral salt medium Na2HPO4.12H2O 2.5 g, KH2PO4 2.0 g, MgSO4.7H2O 0.5 g, FeSO4.7H2O 0.03 g, CaCl2.2H2O 0.06 g and yeast extract 0.1 g APY medium (NH4)2 HPO4 5.0 g, peptone 5.0 g, yeast extract 3.0 g, K2HPO4 5.0 g, MgSO4 7H2O 0.2 g and FeSO4.7 H2O 0.02 g

0.05

*0.4% Acetonitrile was added in each medium ND: not detected

0.06 0.03 0.03 ND

0.04

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INDIAN J EXP BIOL, MARCH 2006

medium (M5) due to poor growth of the organism, while the optimum production of nitrilase has been reported in R. rhodochrous PA-34 cells grown in M5 by Bhalla et al. (1992)4. A comparison of the composition of media used in the present investigation showed that glucose and beef extract together in the medium M2 might have enhanced AHA in N. globerula NHB-2. Temperature ⎯ Optimum temperature for the production of AHA (0.06 U/ml) in N. globerula NHB-2 was found to be 30oC. It showed that AHA of N. globerula NHB-2 was mesophilic in nature. Most of the nitrile degrading organisms grew and nitrilehydrolysing enzymes operated around 30°C4. There was 67 and 50% decline in activity at 20o and 45oC, respectively. Time course of enzyme production ⎯ AHA in N. globerula NHB-2 was low during lag phase of growth (0-7 h), it increased rapidly in early exponential phase (7-14 h) and maximum activity (0.08 U/ml) was observed in mid exponential phase at 21 h (Fig. 1). Thereafter, a rapid fall in activity was recorded in stationary phase (28 h onward) and it was almost constant, 0.01 U/ml beyond 42 h incubation. Reduction in activity on prolonged incubation may be due to production of intracellular proteases and AHA may be prone to action of proteolytic enzymes. A similar trend has been reported in Arthrobacter sp. strain C-38 which exhibits maximum growth at 60 h, but increase in nitrile hydratase activity has been recorded only up to 30 h and a loss of enzyme activity has been observed on further incubation21. Inoculum size ⎯ At 2 and 4% (v/v) of inoculum, optimum production of AHA was recorded i.e. 0.08 and 0.083 U/ml, respectively. Above 4% (v/v) inoculum, gradual decline in enzyme activity was

Fig. 1 ⎯ Time course of AHA production in N. globerula NHB-2 (–○– indicates the growth of N. globerula NHB-2 and –●– shows status of AHA production)

observed and about 54% of enzyme activity was recorded at 8-10% (v/v) of inocula in comparison to control (0.4 % inoculum). Decrease in enzyme production at higher level of inocula may be due to relatively more abundance of cells which had achieved late log phase or stationary phase of growth because of early depletion of nutrients. Earlier investigators have also used 1-4% (v/v) pre-culture for optimum production of nitrile/amide hydrolysing activity5,22. pH ⎯ N. globerula NHB-2 was grown in the medium (M2) at pH 5.5 to 11, but AHA was only detected at pH range 6 to 9.5. Maximum activity (0.10 U/ml) was found at pH 8. At pH range 8.59.5, about 40% enzyme activity was observed as compared to optimum. AHA was not produced when pH of the medium was below 5.5 and above 10. It indicated that optimum pH for production of AHA was near neutral/slightly alkaline pH. Acetonitrilase (nitrile hydratase-amidase system) production in Brevibacterium R312 has been reported to be maximum at pH 7 (Ref.8). However, N. globerula NHB-2 expressed two significant activities, one at acidic (pH 6) and other at alkaline (pH 8) condition. It seemed that this organism produced two types of AHA (one at pH 6 and other at pH 8) for catalyzing the hydrolysis of acetonitrile to acetic acid. Acetonitrile concentration ⎯ In most of the nitrile-degrading enzyme systems reported hitherto, nitriles have been added in the culture medium as inducer of nitrile hydrolysing enzymes10,12. However, in the present studies, it was observed that maximum production of AHA in N. globerula NHB-2 (0.11 U/ml) was in the absence of acetonitrile in the medium M2. It seemed that this activity was constitutive. With increasing the concentration of acetonitrile (0-4% v/v) in medium M2, gradual decrease in AHA was recorded. It appeared that acetonitrile partially suppressed the genes involved in the expression of AHA in this organism. At 4% of acetonitrile concentration in the medium, the organism could exhibit only 17.6% activity. However, N. globerula NHB-2 could tolerate 4.5% (v/v) of acetonitrile concentration in the medium M2. Such a high tolerance of acetonitrile by N. globerula NHB-2 may be of potential application for removal of acetonitrile from industrial wastes, bioremediation of nitrile contaminated soil or water systems.

KUMAR et al.: ACETONITRILE HYDROLYSING ACTIVITY OF NOCARDIA GLOBERULA

Optimization of reaction conditions for AHA of N. globerula NHB-2

Buffer pH ⎯ The resting cells of N. globerula NHB-2 exhibited AHA in the pH range of 6-9. However, maximum activity (0.12 U/ml) was recorded at pH 8.8. As observed during production of AHA, two significant enzyme activities (0.09 and 0.12 U/ml) were also observed at pH 6.5 and 8.8, respectively. It further supported the apparent role of two different enzymes in the hydrolysis of acetonitrile. However, the optimum pH for hydrolysis of acetonitrile in Brevibacterium R 31223 was 7. Buffer system ⎯ Four buffers [sodium phosphate, potassium phosphate, borate, bicarbonate and tris-HCl (0.1M) and pH 8.8] were tested, and recorded maximum AHA (0.13 U/ml) in sodium phosphate buffer. However, AHA, 0.12, 0.05 and 0.09 U/ml, were reported in potassium phosphate, bicarbonate and borate buffer, respectively, while no AHA was detected in Tris-HCl buffer possibly due to inhibition of enzyme system. It seemed that the potentially reactive primary amine of Tris (hydroxymethyl)aminomethane (Tris) might be interfering with the enzyme protein leading to its inhibition24. Incubation temperature and thermal stability ⎯ AHA increased gradually from 0.05 U/ml at 20oC to 0.15 U/ml at 45oC and optimum temperature was found to be 45oC. However, insignificant decrease in activity (0.14 U/ml) was observed at 40° and 50oC. Above 50°C, a rapid decline in activity was recorded. It might be due to thermal inactivation of enzyme. Majority of the nitrile degrading enzymes optimally catalyse nitrile hydrolysis in the mesophilic range (23°-37°C), but the nitrile hydrolysing activity of Bacillus strain is thermophilic in nature20. Thermostability of AHA was also investigated by preincubating the resting cells of N. globerula NHB-2 at various temperatures (30°-80oC) for 1 hr. This activity was stable upto 40oC and thereafter, enzyme activity decreased with the rise in temperature. The residual AHA after 1 h preincubation at 50oC was 67% and above 60°C it was completely inactivated. Biocatalyst and substrate concentration ⎯ There was linear increase in total ammonia production with the increase in resting cell (biocatalyst containing AHA) in reaction mixture from 10-50% v/v (i.e. equivalent to 1-5 ml culture broth), however, AHA (0.15 U/ml) remained almost unchanged.

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AHA gradually increased from 0.04 U/ml at 200 μmol to 0.16 U/ml at 600 μmol of acetonitrile as substrate, then it declined with increasing concentration of acetonitrile. It might be due to substrate inhibition of enzyme. About 50 % AHA was observed at substrate concentration of 900 μmol. Substrate affinity for AHA of N. globerula NHB-2 Among six substrate, maximum AHA was recorded with acetamide as substrate followed by acetonitrile (Table 2). The higher relative activity against acetamide proved that this reaction was catalysed by two enzymes (i.e. nitrile hydratase and amidase) and amidase, which catalyse acetamides hydrolysis, had more activity in comparison to nitrile hydratase of acetonitrile hydrolysis pathway. Therefore, nitrile hydratase was a step regulating enzyme. Similar result has been observed in Kluyveromyces thermotolerans MGBY 3722. AHA of N. globerula NHB-2 could not hydrolyse acrylonitrile, while acrylamide was hydrolysed into acrylic acid. However, acrylamide peak was detected (Fig. 2), when reaction mixture of acrylonitrile hydrolysis was analysed using high performance liquid chromatography (HPLC), this indicated that acrylonitrile somehow inhibited amidase activity of the strain, NHB-2. In case of benzonitrile and benzamide hydrolysis, AHA activity could not be detected using benzamide as substrate, while benzonitrile was hydrolysed to small extent. This could be explained on the basis of presence of a third enzyme i.e. nitrilase, which directly converted benzonitrile to benzoic acid. Effect of metal ions on AHA of N. globerula NHB2 ⎯ Among the metal ions studied, Fe++, Ca++, Cu++, Co++, Cd++, Al+++ and Mn++ enhanced AHA ranging from 4 to 71%. These might have played an important role in the assembly of subunits of the enzyme and subsequently stabilization of the native enzyme in active form. However, Hg++, Pb++, Zn++ and Ag++ inhibited the activity up to 60, 35, 24 and 7%, respectively (Table 3). Table 2 ⎯ Substrate affinity of AHA of N. globerula NHB-2 Nitrile/Amide Acetonitrile Acrylonitrile Benzonitrile Acetamide Acrylamide Benzamide

Relative nitriles/ amides hydrolysing activity (%) 100.0 0.0 19.0 107.0 45.0 0.0

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INDIAN J EXP BIOL, MARCH 2006 Table 3 ⎯ Effect of metal ions and inhibitors on AHA of N. globerula NHB-2 Metal ions/Compound Control Metal ions Zn(NO3)2 CaCl2.2H2O FeSO4.7H2O Pb(NO3)2 CuCl2.2H2O CoCl2.6H2O MnCl2 4H2O AlCl3 CdCl2 HgCl2 AgNO3

Final concentration (mM)

Relative AHA (%)

0

100

1 1 1 1 1 1 1 1 1 1 1

76 108 145 66 122 171 166 107 104 40 93

Acknowledgement The author (HK) gratefully acknowledges CSIR, New Delhi, for financial support. References

Fig. 2 ⎯ (A)-HPLC analysis of acrylonitrile standard, (B) acrylamide standard; and (C) a sample from reaction mixture containing acrylamide and acrylonitrile [Retention time of acrylonitrile and acrylamide was 1.65 and 2.55 min, respectively]

AHA of N. globerula NHB-2 had some interesting features, e.g. its production was constitutive, it optimally catalysed the hydrolysis of nitrile/amides at 45°C, and the organism could tolerate up to 4.5% acetonitrile during growth that made it a potential biocatalyst for application in synthesis of organic amides and acids or bioremediation.

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