BIOCATALYSIS IN ORGANIC SOLVENT SYSTEM

About Authors:
Chhavi Bartaria^*, Javed Ali Khan
Institute of Biomedical Education and Research,
Mangalayatan University,
Aligarh.
*cbartaria@gmail.com

Abstract
Biocatalysis involving certain enzymes like NHase, Nitrilase, Tannase and Amidase produces certain commercially important compounds .NHase are used for the synthesis of certain compounds such as acrylamide and nicotinamide. Thermostable Nitrilase from the Rhodococcus strains is used for the modification of acrylic fiber surface. The activity of amidase enzymes is five times increased and inhibited with the use of Co²⁺and Hg^2+, 2-mercaptoethanol, dithiothreitol, and ethylene diaminetetraceticacid respectively. Furthermore the production of amidesis done as a by the help of a nitrilase-catalysed reaction of a heterolytic amino nitriles by a enzyme. For the hydrolysis of Gallic acid esters and hydrolysable tannis the tannase is use as catalyst and this gallic acid is produced by the hydrolysis of methyl gallate by fungal tannase, the tannase activity is also shown by lactic acid bacteria and the nitrilase is used for the modification of acrylic fibers by the help of a reaction media with some parameters like time, enzyme activity and its formulation.

Reference Id: PHARMATUTOR-ART-1294

Introduction
NHases such as Fe and Co containing Nitrile Hydratase can form commercially important in mild reaction parameters. It is used in the biotransformation and bioremediation (3, 20). Rhodococci can metabolize many toxic chemicals like aromatic and aliphatic nitriles and amides and hence are very useful for environmental pollution clearing (9).Nitrile Hydratase and nitrilase have potential to can convert nitriles to amides or acids where, nitrile hydratase produces acrylamide, and how other recent progress in nitrile biotransformation’s (12). Nitrilase convert the nitriles to chemo-, region-, orenantio-selective carboxylic acids in mild reaction conditions with high efficiency, selectivity (17, 18). Biocatalysis is used for the production of pharmaceutically important products by as nitrile hydratase, amidase and nitrilase converts acrylonitrile to commercially important products by Rhodococci like acrylamide and acrylic acid which are used as superabsorbents, dispersants and flocculants (5). Tannase is an enzyme which is used to produce Gallic acid which has commercial importance in beverage and food processing (31).

1. Use of nitrile hydratase to produce commercially important enzymes
NHase are usually metallenzymes is used for the production of industrial important compounds like acrylamide and nicotinamide (3, 20, 21). Nitrilehydratase is either iron type or and cobalt types involving the photo activation mechanism (20).Nitrile hydratases have interstetial filled by iron of hemE& Fe-S protein 3. The spin quantum resembles from nitrogen bond & they follow electron accepter & donor method (21).A hetero dimer cannot show a complex of anaerobically of aCys 112 --So2H & removed of ferric at low pH so the aCys112 & aCys114 are oxidized to Cys --So2H & CysSOH (22).Genus bacillus of strain DSM 2349 isolated. it grow optimally &thermostable intercellular nitrile hydratase. Benzonitrilase absent having a2b2 heterotetranular enzyme with homology to N terminal so the optimumcondition is 746 & 4580/ sec for acetonitrile &valeronitrile (23).Nitrile hydratase produced rhodococcusrhodochrous NCIMB 11216 they grow on propionitrile medium, both group hydrolyse PAN &acrylic fibersX-rays photo electron spectroscopy showed 16% of nitrile group so the dye were enhanced due to acrylic fibers are hydrophilic(25).The metal bound hydroxide involved in hydration of nitriles iron(III) or cobalt (III) center at active site 2 sulfers from a cysteine sulfenic ( Cys SO) & a cysteine sulfinic ( Cys SO2) acid moiety constitute the donor set around the metal center of this hydrolytic enzyme(26).E - pyridine -3- aldoxime bacteria responsible for nitrile hydratase of aldoxime metabolism. The strain YH3—3, in enzyme add cobalt ion so the activity is high .The enzyme is purified 108 fold with a 16% shield. mol. wt. 130000 & 2 units a & b subunit 27100 & 345000.the enzyme show high activity at 60 C & 40 C low pH 2.5 to 11 . the N-terminal of b-subunit show sequence(27).In mutant stain 19 gene is lost due to single enzyme but they congruent all the amidase activity of BREVIBACREIUM R312 . The Km has 12 different substrates. have high & low affinity of hydro carbon chain and specific activity is determined bynucleophilic and hydrophilic(28).In PCR identify nitrile hydratase (NHase) genomic & DNA screening, they have NHaseα- subunit and 10 β-subunits and 3 NHase activators encoding of natural genes are destroyed by low temperature and soluble in E.coli 6 were produced active of 9 NHaseinvestigated. So the homology based screening identified and at last the synthesis of new and distinct NHase enzyme(30).

2. Use of nitrilase to produce commercially important enzymes
Rhodococcus sp. NDB 1165 has aninducible nitrile-transformation for propionitrile, aromatic nitriles (benzonitrile, 3-cyanopyridine and 4-cyanopyridine) and unsaturated aliphatic nitrile (acrylonitrile) (9). The biocatalysts of nitrile from Rhodococcus strains show nitrile hydratase and activity to convert the β-hydroxynitriles where the activity is related to length of the aliphatic chain of the aryl aliphatic nitrile as well as the position of the substituted groups on side chains on the aromatic ring or aliphatic chain in these reactions the α-hydroxy carboxylic acids or amides are also formed from aldehydes in presence of cyanide (10). Fusariumsolani O1 and Aspergillusniger K10 have broad substrate specificity for carbocyclic and non-aromatic heterocyclic amino nitriles. These enzymes have a strong diastereopreference for cis- vs. trans-γ-amino nitriles where, the electronic and steric effects of N-protecting groups affected the reactivity of the nitriles (11). Pseudomonas marginales MA32 and Pseudomonas putida MA113 has nitrile hydratases which is resistant to cyanide andloses its activity above 50 mM under moderate cyanide concentrations. Substrate like acrylonitrile and also longer chain but also nitriles with longer side chains and even nitriles with quaternary alpha-carbon atoms show activity. They transformation of instable ketone cyanohydrins to release high amount of prussic acid than cyanohydrins formed from aldehydes. P.marginales MA32 which show a whole cell biocatalyst for the hydration of acetone cyanohydrin to α-Hydroxyisobutyramide, which is a precursor of methacrylamide, an important pre-polymer (13). Rhodococcusrhodochrous and Rhodococcusfascians has periplasmicnitrilase enzyme specific for benzonitrile where, the nitrilases of R. rhodochrous showed 50% more activity as compared to R. fascians and R. rhodochrous has better nitrile production and degradation potential having optimal conditions of 40?C and pH 8.0 and is thermo stable upto 45?C (14). Another important application is the modification of the acrylic fiber with nitrilase (EC 3.5.5.1) (15). The surface of an acrylic fiber was modified with a commercial nitrilase (EC 3.5.5.1). The effect of fiber solvents and polyols on nitrilase catalysis efficiency and stability was investigated. The nitrilase action on the acrylic fabric was improved by the combined addition of 1 M sorbitol and 4% N, N-dimethylacetamide. The color levels for samples treated with nitrilase increased 156% comparing to the control samples. When the additives were introduced in the treatment media, the color levels increased 199%. The enzymatic conversion of nitrile groups into the corresponding carboxylic groups, on the fiber surface, was followed by the release of ammonia and polyacrylic acid. A surface erosion phenomenon took place and determined the “oscillatory” behavior of the amount of dye uptake with time of treatment. These results showed that the outcome of the application of the nitrilase for the acrylic treatment is intimately dependent on reaction media parameters, such as time, enzyme activity and formulation (16).

3. Use of amidase to produce commercially important enzymes
Genetically engineered strains are used to obtain7-aminocephalosporanic acid when, an enzyme Cephalosporin acylases produces 7-aminocephalosporanic acid which further produces semisynthetic injectable cephalosporin’s (4). An enantio-specific amidase from Klebsiellaoxytoca produces (R, S)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide, giving (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid and (S)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide. Further, (S)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide (S)-amide could then be hydrolyzed chemically to (S)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid where, the starting material for the reaction is ethyl trifluoroacetoacetate, the productivity of the reaction (1). A homo-hexamerthermo stable -methionine amidaseof molecular wt.199 kDa from Brevibacillusborstelensis BCS-1. Shows specific activity of 207-times and 98% purity and having activity even at 65 °Cas well as broad pH range stability from 6.5 to 10.0, and maximum activity at pH 9.5 and 70 °C. Furthermore, the activity of this enzyme increases five times with the Co²⁺whereas; the enzyme is strongly inhibited by Hg²⁺, 2-mercaptoethanol, dithiothreitol, and ethylenediaminetetracetic acid. The thermo stable-methionine amidase has activity for amino acid amides and esters but not for -peptide hydrolysis (2). The peptide amidase from Citrus sinensis and Stenotrophomonasmaltophilia used N-protected amino acid amides when are substrates for the determination of V_max and K_M values for the deamidation reactions (6). A complex amidase (L-carnitineamidase) from DSM 6320 is competitively inhibited by the product L-carnitine as well as the unreactive enantiomer of the substrate, D-carnitine amide and also the second reaction product ammonia doesnot effect the rate of hydrolysis (7). The assay for nitrile hydratase and amidase activities can be performed by high-performance liquid chromatography,gas-liquid chromatographyand proton magnetic resonance spectrometry(8, 19).Amides are produced as by-products of the nitrilase-catalyzed reaction of heterocyclic amino nitriles by the A.niger enzyme but only from nitrile (±)-9a by the F. solani enzyme (11).

4. Use of tannase to produce commercially important enzymes
Tannase act as catalyst for the hydrolysis of Gallic acid esters and hydrolysable tannins. This enzyme is produced by plants and microorganisms and it is industrially used as catalysts in the manufacture of Gallic acid which could be determined by gas chromatography. Gallic acid could also be formed by the hydrolysis of methyl gallate by fungal tannase (31,33). Lactic acid bacteria species such as Lactobacillus, Leuconostoc, Oenococcus or Pediococcus can degrade tannins (32). An extracellular tannase of 45 kDa. (tannin acyl hydrolase) is isolated from Paecilomycesvariotii purified tannase was a monomeric enzyme with a molecular withstanding temperature range from 30 to 50°C and pH range 5.0 to 7.0 furthermore if, Tannase is immobilized on alginate beads it retains about 85% of the initial activity (34). Pomegranate juice is made tastier using tannase to degrade tannins of the juice which gives it bitter taste the Tannase treatment results in 25% tannin degradation whereas, tannase in combination with gelatin in ratio of (1:1) leads to 49% of tannin degradation (35).

Future Prospects:
NHaseis used to produce 30,000 tons/year of commodity chemical acrylamide from acrylonitrile (3). The purity of the product is more than 98%, when an enantio-specific amidase from Klebsiellaoxytoca produces (R,S)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide, giving (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid and (S)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide (1). The racemic N-acetyl amino acids amides form N-acetyl-L-amino acids of 99% optical purity on its complete conversion (6).The application of the nitrilase to modify acrylic fibers depends on reaction media parameters, such as time, enzyme activity and formulation (15).The inhibitor D-calamine is not normally present, a model which is used to manage the timings of batch conversions is created for describing the enzyme kinetics, so the activity and half-life of nitrilase is 2.39 ± 0.07 U/mg dry cell mass (dcm) and 150 min and 40 min at 45°C and 50°C respectively. Lactic acid bacteria have Tannase activity which has importance of selecting malolactic starter cultures which are used in winemaking process by reducing astringency and haze in wine (32). Enzymatic treatment of fruit juices to reduce the bitterness has got advantages such as the higher quality of juice due to the lower haze and non-deterioration of juice quality. The pomegranate has recently been acclaimed for its health benefits, in particular, for its disease-fighting antioxidant potential. Presence of high tannin content in the pomegranate is responsible for haze and sediment formation as well as for color, bitterness and astringency of the juice upon storage. Due to the inability of conventional fruit juice debittering processes for removing the bitterness effectively, enzymatic debittering should be preferred (35).

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Table 1: Showing the organic stability, temperature stability and pH range of various organisms which produce some enzymes:

Organism	Enzyme	Organic Solvent	Organic Solvent Stability	Temperature stability Max. Min	pH range Max. Min	Type whole Cell/Purified enzyme	Reference
Fusariumsolani	nitrilase	2-propanol	the enzyme shows more than 30% of the control activity with 5% 2-propanol.	55 35	10 5	Purified	Vejvoda, Vet al.(2008).
	nitrilase	Acetone	the enzyme shows more than 30% of the control activity with 5% acetone.	55 35	10 5	Purified	Vejvoda V et al.(2008).
	nitrilase	acetonitrile	the enzyme shows more than 30% of the control activity with 5% acetonitrile.	55 35	10 5	Purified	Vejvoda V et al.(2008).
	nitrilase	dichloromethane	the enzyme shows more than 30% of the control activity with 5% dichloromethane.	55 35	10 5	Purified	Vejvoda V et al.(2008).
	nitrilase	dimethylsulfoxide	the enzyme shows more than 30% of the control activity with 5-10% of dimethylsulfoxide.	55 35	10 5	Purified	Vejvoda V et al.(2008).
	nitrilase	Ethanol	more than half of activity is preserved at up to 15% of ethanol .	55 35	10 5	Purified	Vejvoda V et al.(2008).
	nitrilase	Ethyl acetate	the enzyme shows more than 30% of the control activity with 5% ethyl acetate.	55 35	10 5	Purified	Vejvoda V et al.(2008).
	nitrilase	Methanol	the enzyme shows more than 30% of the control activity with 5-10% of methanol.	55 35	10 5	Purified	Vejvoda V et al.(2008).
	nitrilase	n-heptane	more than half of activity is preserved at up to 50% of n-heptane.	55 35	10 5	Purified	Vejvoda V et al.(2008).
	nitrilase	n-hexane	more than half of activity is preserved at up to 50% of n-hexane.	55 35	10 5	Purified	Vejvoda V et al.(2008).
	nitrilase	toluene	the enzyme shows more than 30% of the control activity with 5-10% of toluene.	55 35	10 5	Purified	Vejvoda V et al.(2008).
Alcaligenesfaecalis	nitrilase	Acetone	extent of nitrile hydrolysis and enantiomeric purity of product are significantly enhanced in the range of 5-15%.	55 35	10 5	Whole cell	Kaul, P et al. (2008)
	nitrilase	dimethyl acetamide	extent of nitrile hydrolysis and enantiomeric purity of product are significantly enhanced at 5%.	55 35	10 5	Whole cell	Kaul, P et al. (2008)
	nitrilase	dimethyl formamide	extent of nitrile hydrolysis and enantiomeric purity of product are significantly enhanced in the range of 5-25%.	55 35	10 5	Whole cell	Kaul, P et al. (2008)
	nitrilase	dimethyl sulfoxide	signifantly enhances the synthetic potential of the enzyme as well as its enantioselectivity. Extent of nitrile hydrolysis and enantiomeric purity of product are significantly enhanced in the range of 5–25%.	55 35	10 5	Whole cell	Kaul P et al.(2008).
	nitrilase	dioxane	extent of nitrile hydrolysis and enantiomeric purity of product are significantly enhanced in the range of 5-10%.	55 35	10 5	Whole cell	Kaul P et al.(2008).
	nitrilase	Ethanol	xtent of nitrile hydrolysis and enantiomeric purity of product are significantly enhanced in the range of 5-20%.	55 35	10 5	Whole cell	Kaul P et al.(2008).
	nitrilase	isopropanol	extent of nitrile hydrolysis and enantiomeric purity of product are significantly enhanced in the range of 5-15%.	55 35	10 5	Whole cell	Kaul P et al.(2008).
	nitrilase	Methanol	extent of nitrile hydrolysis and enantiomeric purity of product are significantly enhanced in the range of 5-20%.	55 35	10 5	Whole cell	Kaul P et al.(2008).
	nitrilase	N-methyl pyrrolidone	addition results in decreased rate of hydrolysis.	55 35	10 5	Whole cell	Kaul P et al.(2008).
	nitrilase	n-propanol	extent of nitrile hydrolysis and enantiomeric purity of product are significantly enhanced in the range of 5-15%.	55 35	10 5	Whole cell	Kaul P et al.(2008).
	nitrilase	N-vinyl pyrrolidone	addition results in decreased rate of hydrolysis.	55 35	10 5	Whole cell	Kaul P et al.(2008).
	nitrilase	Pyridine	addition results in decreased rate of hydrolysis.	55 35	10 5	Whole cell	Kaul, P et al.(2008).
	nitrilase	tetrahydrofuran	addition results in decreased rate of hydrolysis.	55 35	10 5	Whole cell	Kaul, P et al.(2008).
Geobacilluspallidus-Nitrile Hydratase	Nitrile Hydratase	1,4-dioxane	the soluble enzyme shows about 80% relative activity in the presence of 1,4-dioxane.	60 50	7 -	Whole Cell.	Chiyanzu, I. et al. (2010).
	Nitrile Hydratase	Acetone	the soluble enzyme shows about 35% relative activity in the presence of acetone.	60 50	7 -	Whole Cell.	Chiyanzu, I. et al. (2010).
	Nitrile Hydratase	chloroform	the soluble enzyme shows about 70% relative activity in the presence of chloroform.	60 50	7 -	Whole Cell.	Chiyanzu, I. et al. (2010).
	Nitrile Hydratase	dichloromethane	the soluble enzyme shows about 80% relative activity in the presence of dichloromethane.	60 50	7 -	Whole Cell.	Chiyanzu, I. et al. (2010).
	Nitrile Hydratase	Ethanol	the soluble enzyme shows about 60% relative activity in the presence of ethanol.	60 50	7 -	Whole Cell.	Chiyanzu, I. et al. (2010).
	Nitrile Hydratase	formaldehyde dichloromethane	the soluble enzyme shows about 20% relative activity in the presence of formaldehyde	60 50	7 -	Whole Cell.	Chiyanzu, I. et al. (2010).
	Nitrile Hydratase	Methanol	the soluble enzyme shows about 70% relative activity in the presence of methanol.	60 50	7 -	Whole Cell.	Chiyanzu, I. et al. (2010).
	Nitrile Hydratase	tetrahydrofuran	the soluble enzyme shows about 40% relative activity in the presence of tetrahydrofuran.	60 50	7 -	Whole Cell.	Chiyanzu, I. et al. (2010).
Rhodoco- ccusrhodochrous	Nitrile Hydratase	dimethylformamide	30% v/v, no residual activity.	60 50	7 -	Whole Cell.	Kashiwagi, M. et al (2004)
Rhodoco- ccusrhodochrous	amidase	dimethylformamide	the enzyme is still active at 30% v/v.	- -	- 45	Whole cell	Kashiwagi, M. et al (2004)
Penicillium- variabile	Tannase	2-mercaptomethanol	39.6% residual activity at 1 mM.	- 50	- 5	Purified	Sharma, S et al. (2008)
	Tannase	Acetone	49% residual activity after 60 min at 20% (v/v).	- 50	- 5	Purified	Sharma, S et al. (2008).
	Tannase	carbon tetrachloride	57% residual activity after 60 min at 60% (v/v).	- 50	- 5	Purified	Sharma, S et al. (2008)
	Tannase	Ethanol	35.7% residual activity after 60 min at 60% (v/v).	- 50	- 5	Purified	Sharma, S et al. (2008)
	Tannase	formaldehyde	12% residual activity after 5 min at 20% (v/v).	- 50	- 5	Purified	Sharma, S et al. (2008)
	Tannase	heptane	62% residual activity after 60 min at 60% (v/v).	- 50	- 5	Purified	Sharma, S et al. (2008)
	Tannase	petroleum ether	33% residual activity after 60 min at 60% (v/v).	- 50	- 5	Purified	Sharma, S et al. (2008)
	Tannase	SDS	complete inactivation at 1% (w/v).	- 50	- 5	Purified	Sharma, S et al. (2008)
	Tannase	tetrahydrofuran	no activity after 5 min at 20% (v/v).	- 50	- 5	Purified	Sharma, S et al. (2008)
	Tannase	toluene	44% residual activity after 60 min at 60% (v/v).	- 50	- 5	Purified	Sharma, S et al. (2008)
	Tannase	tween 60	complete inactivation at 1% (v/v).	- 50	- 5	Purified	Sharma, S et al. (2008)
	Tannase	Tween 80	complete inactivation at 1% (v/v).	- 50	- 5	Purified	Sharma, S et al. (2008)
Aspergillusnige	Tannase	1-Propanol	activates at a concentration of 3.6-7.3% v/v, at higher concentration it inhibits the propyl gallate synthesis reaction causing disruption of essential membrane functions and denaturation of enzyme.	50 30	- 5	Whole cell.	Yu, X. et al. (2007)
	Tannase	acetone	-	50 30	- 5	Whole cell.	Yu, X. et al. (2007)
	Tannase	Benzene	optimum enzyme concentration should be 0.1 g mycelial dry weight per 10 ml benzene.	50 30	- 5	Whole cell.	Yu, X. et al. (2007)
	Tannase	Hexane	-	50 30	- 5	Whole cell.	Yu, X. et al. (2007)
	Tannase	N,N-Dimethylformamide	-	50 30	- 5	Whole cell.	Yu, X. et al. (2007)
	Tannase	Pyridine	-	50 30	- 5	Whole cell.	Yu, X. et al. (2007)
Aspergillusfoe- tidus	Tannase	SDS	the enzyme shows considerable stability in the presence of SDS.	40 15	7 5	Purified	Naidu, R.B. Saisubramanian et al. (2008)
	Tannase	Triton X-100	considerable loss in activity in the presence of Triton X-100.	40 15	7 5	Purified	Naidu, R.B. Saisubramanian et al. (2008)

The Cell-free nitrilases of the Rhodococcus strains show a short substrate range than resting whole cells of the same strains (10).In Rhodococcusstarins the nitrilase show the limited range of various substrates as compared to whole cells and the enzyme activity of Cephalosporin acylases is based on its structural subunits, post transitional modification, primary structure, and substrate specificity of the enzyme (2).

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