DEVELOPMENT OF NITRILE METABOLIZING ENZYME FOR SURFACE MODIFICATION OF POLYACRYLONITRILE

Abstract

Polyacrylonitrile (PAN) is a synthetic polymer and widely used in the textile industries and biomedical field. The main disadvantage with PAN is its poor hydrophilicity which affects the processability of fibers, restricting the application of finishing compounds such as dyes and other coloring agents in the textile industry and reduces the biocompatibility for use as biomaterials. Low hydrophilicity of PAN is due to the presence of-CN groups on the surfaces. Nitrile metabolizing enzymes has opened a new route for the transformation -CN to -COOH functional groups with many advantages over chemical methods of surface modification of polyacrylonitrile. During isolation and screening of nitrile metabolizing enzyme producing isolate, total seven isolates were obtained and among those, one isolate (No.6/b) was releasing ammonia from polyacrylonitrile powder. Therefore, this strain was selected for this research work. Adiponitrile was found to be the most efficient inducer or nitrogen source for the production of nitrile metabolizing enzyme which showed maximum activity with PMA (co polymer of PAN). Isolated strain found to have rounded configuration, convex elevations, and rough surface with white and creamy yellow pigmentation. Gram staining of isolated strain showed gram positive reaction and single/Y shaped morphology on visualization at 100X with oil immersion in a compound microscope. Isolated strain was identified by 16S rDNA phylogenetic analysis. Due to the maximum similarity with other Amycolatopsis strains, isolated strain named as Amycolatopsis sp.IITR215. Whole cells and cell free extract of Amycolatopsis sp.IITR215 showed activity towards aliphatic and aromatic nitriles as well as with arylacetonitriles and acrylamide. Thus, it indicates the presence of amidase and probably nitrilase and nitrile hydratase. Similar specificity was found with the cell free extract but specific activity was less as compare to whole cells activity. In presence of DEPA (an amidase inhibitor), no ammonia was released with acrylonitrile, isobutyronitrile, valeronitrile, propionitrile, acetonitrile, butyronitrile, isovaleronitrile, 3- hydroxypropionitrile, 4-cyanopyridine, 3-hydroxyglutaronitrile, malononitrile. Henceforth, it was concluded that ammonia released from these nitriles was solely due to nitrile hydratase and amidase. This was further confirmed by checking the presence of acrylamide in the reaction mixture when cells were incubated with 10 mM acrylonitrile along with 10 mM DEPA. In case of hexanenitrile, the presence of the inhibitor did not affect ammonia production. It was therefore concluded that ammonia released from hexanenitrile was probably due to nitrilase or another set of nitrile hydratase and amidase that was not affected by DEPA. For nitrile hydratase and amidase activities, acrylonitrile and acrylamide, respectively, were used as substrates. Further study on the nature of enzyme responsible for hexanenitrile hydrolysis was attempted by studying the effects of various reported inhibitors on enzymes activities with acrylonitrile, acrylamide and hexanenitrile respectively. At 1 mM N-bromosuccimide concentration, no activity was detected with acrylonitrile and 5% and 36% activities were retained with acrylamide and hexanenitrile, respectively. This result indicates high inhibitory effects of N-bromosuccimide on nitrile hydratase, amidase and less inhibition on enzyme involved in hexanenitrile hydrolysis. With 5 mM N-ethylmaleimide, enzyme with acrylonitrile and acrylamide showed 15% and 31% activities respectively and 56% activity was observed with hexanenitrile, which confirms that N-ethylmaleimide had a greater inhibitory effect on amidase and nitrile hydratase as compared to the enzyme involved in hexanenitrile hydrolysis. Amidase of the isolated strain was highly active for isobutyramide, propionamide, benzamide and hexanamide while acyl-transferase activity was maximum for hexanamide. To study the optimum conditions of nitrile hydratase, 10 mM acrylonitrile with DEPA was used as a substrate. The amidase and nitrile hydratase found to have an optimum temperature of 45°C. The optimum temperature for hexanenitrile hydrolysis was found to be 55°C. Optimum pH for amidase and nitrile hydratase was found to be 7.0 in 50 mM phosphate buffer while it was 5.8 for hexanenitrile metabolizing enzyme in 50 mM acetate buffer. Ferric salts were found to have no effect on hexanenitrile hydrolysis but affected the nitrile hydratase/amidase pathway marginally. Salts of Barium and Nickel were found to affect hexanenitrile hydrolysis but not the nitrile hydratase/amidase pathway. Dithiobisnitrobenzoic was also observed to inhibit both hexanenitrile hydrolysis and the nitrile hydratase/amidase. In absence of nitriles and amides in the MB media, Amycolatopsis n sp.IITR215 strain also produced nitrile metabolizing enzymes in media containing 1 g/1 yeast extract and 1 g/1 NH4C1 as sole nitrogen source which confirms the constitutive nature of all nitrile-metabolizing enzymes ofAmycolatopsis sp. IITR215 The half life of hexanenitrile metabolizing enzyme was found to be 252 min at 40°C, pH 5.8. Different stabilizers were checked to increase the stability. Maximum stability of hexanenitrile metabolizing enzyme was found in 100 mM NaCl, 50% activity was retained after 420 min. in 100 mM NaCl salt concentration at 40°C, pH 5.8. The whole cell enzyme for hexanenitrile hydrolysis from Amycolatopsis sp.IITR215 was highly active in 50% (v/v) alcohols. Relative activities of 29%, 50% and 14% were detected in 50 % (v/v) of ethyl alcohol, methyl alcohol and isopropyl alcohol respectively. For the separation of nitrile metabolizing enzymes from Amycolatopsis sp.IITR215, cells were lysed by using 2 g/1 lysozyme. Proteins in cell free extract were precipitated by 30% sodium sulphate and loaded onto the anion exchange (Q-sepharose) column. From the elution profile, total eight peaks were obtained on the basis of protein O.D. Active fractions were clubbed and used for activity determination. First three factions were of amidase (named amidase 1, 2 and 3) and next three fractions were active on nitrile thus confirmed as nitrile hydratase (named nitrile hydratase 1, 2 and 3). Substrate specificity pattern of these amidases were similar whereas maximum activity for nitrile hydratase 1 was found with acetonitrile and this nitrile hydratase was highly active for butyronitrile, valeronitrile, propionitrile and adiponitrile while nitrile hydratase 2 was showing totally different profile from nitrile hydratase 1. Maximum activity was found with butyronitrile and it was highly active for hexanenitrile, acrylonitrile, isobutyronitrile, adiponitrile, propionitrile and methacrylonitrile. Moreover, maximum activity for nitrile hydratase 3 was found with hexanenitrile and it was highly active for long chain nitrile such as propionitrile, butyronitrile and dinitrile (adiponitrile). Onthe basisof protein bandposition in native-PAGE analysis and zymogram, presence of one amidase was confirmed. Substrate specificity and native-PAGE analysis of nitrile hydratases supports the presence of three probable nitrile hydratase fractions but this needs to be further confirmed after subsequent purification of these three active fractions.

Cell free extract of Amycolatopsis sp.IITR215 was used to treat PAN powder at 30, 37 and 45°C. No ammonia was detected at 30°C and maximum was at 37°C after 12 hours of polymer treatment. Protein adsorption was higher at pH 7.0 as compared to pH 5.8. Nearly 4283 and 7962 uM ammonia was detected at pH 7.0 and 5.8 respectively after 30 hour of polymer treatment. Enzyme treated polymer of different pH was subjected to FTIR analysis. In FTIR spectra, a new peak was detected at 1547 cm"1 which corresponds to the formation ofcarboxylate group whereas peak at 1644 cm"1 gets broadened which supports the formation of amide group on PAN. Carboxyl groups on the enzyme treated polymer was quantified by Rhodamine 6G by decrease in absorbance at 480 nm. Untreated PAN was taken as the control. The carboxyl group formation was maximum at pH 5.8. At pH 5.8, 106 umole/g polymer and 456 umole/g polymer of carboxyl group were found in control and enzyme treated PAN respectively. At pH 7.0, 117 umoles/g polymer and 227 umoles/g polymer of carboxyl group were found in control and enzyme treated PAN respectively. On the basis of protein adsorption, ammonia release and formation of new peak at 1541 cm"1, 15 g/1 of PAN found to be the optimum concentration for surface modification of PAN. Protein adsorption and ammonia release on other PAN co-polymers such as PMA, PAN-co-butadiene-co-styrene and PAN-co-methacrylate were also studied at pH 7.0. Protein adsorption was higher on PMA. Nearly 60% proteins were adsorbed on PMA after 36 hour of polymer treatment while protein adsorption process was slow on PAN-co-butadine-costyrene. In FTIR spectra, no distinct peak for carboxylate group was detected with enzyme treated PAN-co-butadiene-co-styrene, PMA and PAN-co-methacrylate. Similarly, no ammonia was detected with PAN-co-butadiene-co-styrene, PMA and PAN-co-methacrylate which shows that nitrile metabolizing enzyme from Amycolatopsis sp.IITR did not convert -CN groups present on these PAN co-polymers to carboxylic acid. In FTIR spectra, no distinct peak was observed but peak at 1638 cm"1 get broader with these polymers which supports the formation of amide groups on these polymers.