DEVELOPMENT OF BIOCATALYTIC PROCESSES FOR HYDROXAMIC ACID AND ACID HYDRAZIDE SYNTHESIS

Abstract

Hydroxamic acids and acid hydrazides are important chemical compounds with considerable applications in pharmaceutical, medicinal, polymer and agrochemical industries. Nicotinic acid hydroxamate, a heterocyclic class of hydroxamic acid has roles as bioligand, urease inhibitor, antityrosinase, antioxidant, antimetastatic and vasodilating agents due to strong metal chelating ability and possibly their NO releasing property. Isoniazid and its derivatives are potent antituberculosis agents and isoniazid has been employed in the treatment of tuberculosis for over half a century. Chemical methods for syntheses of hydroxamic acids and acid hydrazides present several disadvantages and thus there is a need to explore efficient enzymatic routes for syntheses of these compounds. Amidases have been studied as biocatalyst for syntheses of hydroxamic acids and acid hydrazides, however amidase catalysed bioprocesses for productions of nicotinic acid hydroxamate and isoniazid are still not developed. The major objective of the present thesis was to develop biocatalytic processes for syntheses of nicotinic acid hydroxamate and isoniazid using acyltransferase activity of whole cell amidase. At the beginning of this thesis, Chapter 1 introduces briefly about roles of hydroxamic acids and acid hydrazides in various industrial applications with special emphasis on nicotinic acid hydroxamate and isoniazid. The present scenario of different existing chemical and enzymatic methods for syntheses of these compounds was discussed. Further, it also dealt with the limitations and disadvantages associated with the chemical methods and suitability of amidase catalyzed biocatalytic methods to overcome these drawbacks was highlighted. Finally the objectives to be attained in this study were specified in brief. Followed by this, Chapter 2 presents (1) the detail review on hydroxamic acids and acid hydrazides, their structures, functions and industrial applications of nicotinic acid hydroxamate and isoniazid; (2) a detailed description of various chemical methods of syntheses of these compounds; (3) information on enzymatic routes for syntheses of hydroxamic acids and acid hydrazides reported in various literatures, factors affecting enzyme catalyzed processes, advantages of enzymatic methods over chemical methods; (4) ii and finally classification, mechanism of action, occurrence and properties of reported amidases with their industrial applications in various fields. Further, Chapter 3 deals with materials and methods used in present work. Detailed descriptions of methods, different experimental techniques utilized in this study were mentioned. Detailed results, obtained during this work along with discussion are presented in Chapter 4. This chapter is divided into four sections. The work presented in Section 1 aimed at screening of suitable bacterial isolate producing amidase enzyme having acyltransferase activity. Among ten available nitrile metabolizing bacterial isolates, 6b2 was selected as biocatalyst as it possessed acyltransferase activity for a broad range of amides, higher acyltransferase to amide hydrolase activity ratio for heterocyclic amides and the highest mole ratio of product (nicotinic acid hydroxamate) to by-product (nicotinic acid). Isolate 6b2 was identified as Bacillus smithii strain IITR6b2, based on biochemical characteristics and 16S rDNA sequence. The influences of various physiochemical parameters on amidase production were studied using one variable at a time approach. Amidase was found to be inducible in nature. Under optimized conditions, maximum amidase production was obtained in mid exponential growth phase at 48 h with glycerol (10 g/l) as carbon source, phenylacetonitrile (10 mM) as sole source of nitrogen and inducer at pH 7.0 and temperature 45 °C. Whole cell amidase had pH and temperature optima of 7.0 and 55 °C respectively. Acyltransferase activity of amidase was thermally stable with half lives of 29, 14 and 10 h at 30, 45 and 55 °C respectively. Amidase showed acyltransferase activity for a broad range of amides like aliphatic, aromatic and heterocyclic amides with highest activity for nicotinamide. Whole cell amidase was compatible in presence of both water miscible and water immiscible solvents at 15% (v/v) concentration. This solvent compatible property is highly desirable for syntheses of hydroxamic acids and acid hydrazides from hydrophobic amides that are poorly soluble in aqueous medium. Although amidases from different microbial sources having acyltransferase activity for a broad range of amides have been studied, only a few have been reported to develop bioprocesses for syntheses of hydroxamic acids. Bioprocess development for synthesis of nicotinic acid hydroxamate using acyltransferase activity of B. smithii strain IITR6b2 is described in Section 2. Amidase with acyltransferase activity for nicotinamide is suitable iii for nicotinic acid hydroxamate production. However amidase can also simultaneously hydrolyzes nicotinamide to nicotinic acid. Nicotinic acid is an undesirable by-product and thus any biocatalytic process involving amidase for nicotinic acid hydroxamate production needs to have high ratio of acyltransferase to amide hydrolase activity. Isolate B. smithii strain IITR6b2 was found to have 28 fold higher acyltransferase to amide hydrolase activity. This higher ratio resulted in limited undesirable by-product, nicotinic acid synthesis. Further the effects of various parameters on bioconversion yield were investigated in detail. The optimal substrate/co-substrate ratio, pH, temperature, incubation time and resting cells concentration were 200/250 mM, 7.0, 30 °C, 40 min and 0.7 mgdcw/ml respectively. Under these optimized reaction conditions, 94.5% molar conversion of nicotinamide to nicotinic acid hydroxamate was achieved. To avoid substrate inhibition effect, a fed batch process based on the optimized parameters was developed and a molar conversion yield of 89.4% with the productivity of 52.9 g/h/gdcw was achieved in laboratory scale. Finally, 6.4 g of powder containing 58.5% (w/w) nicotinic acid hydroxamate was recovered after lyophilisation and further purification resulted in 95% pure product. In Section 3, a biocatalytic route for the synthesis of isoniazid, in aqueous system is presented. Acyltransferase activity of B. smithii strain IITR6b2 was utilized for its ability to transfer acyl group of isonicotinamide to hydrazine-2HCl in aqueous medium. Whole cell amidase possessed 3 folds higher acyltransferase activity as compared to amide hydrolase activity for isonicotinamide and this ratio was further improved to 4.5 by optimizing concentration of co-substrate hydrazine–2HCl. Various key parameters were optimized and under the optimum reaction conditions of pH (7.0, phosphate buffer 100 mM), temperature (30 °C), substrate/co-substrate concentration (100/1000 mM) and resting cells concentration (2.0 mgdcw/ml), 90.4% conversion of isonicotinamide to isoniazid was achieved in 60 min. Under these conditions, a fed batch process for production of isoniazid was developed and resulted in the accumulation of 439 mM of isoniazid from 500 mM isonicotinamide with 87.8% molar conversion yield and productivity of 6.0 g/h/gdcw. Finally, 9.1 g of powder containing 33.3% (w/w) isoniazid was recovered after lyophilisation and further purification resulted in 94% pure product. In addition, amidase from B. smithii strain IITR6b2 was purified and characterized as reported in Section 4. Purified amidase was characterized for temperature optima, pH optima, substrate specificity, organic solvent compatibility, effects of chemical reagents iv including metals, inhibitors and surfactants and was compared with amidases reported in literature from other microbial sources. Intracellular amidase was purified to 12.11 fold with yield of 37.5% by a procedure involving ammonium sulphate precipitation (30-60%), ultrafiltration by amicon (10 kD), column chromatographies with Q sepharose and phenyl sepharose resins. The purified amidase was a monomer with molecular mass of 63 kDa as determined by gel filtration chromatography and SDS-PAGE. Purified amidase had pH and temperature optima of 7.0 and 45 °C respectively. The substrate specificity of purified amidase was determined for both acyltransferase and amide hydrolase activities. It had highest acyltransferase activity with nicotinamide followed by benzamide and hexanamide. This amidase also hydrolyzed different amides but acyltransferase activity was considerably higher as compared to amide hydrolase activity. Purified amidase was not a metalloenzyme as metal chelating reagent EDTA did not affect the enzyme activity. The amidase activity was highly inhibited by heavy metal ions and –SH modifying reagents (DTNB and NEM), while reducing agent DTT improved enzyme activity to 2.2 fold suggesting thiol group involvement at amidase active site. Purified amidase was compatible with organic solvents and maintained more than its 90% activity in 10% (v/v) of methanol, ethanol, isopropanol, acetonitrile, 1-Decanol, DMF, Xylene, hexane, heptane and n-hexadecane. In presence of methanol, ethanol and isopropanol at 20% (v/v) concentration activity reduced to 76, 56 and 53% respectively. Amidase activity was enhanced to 285, 302, 365 and 157% in the presence 0.05% (v/v) of non ionic surfactants such as Tween 20, Tween 60, Tween 80 and Triton X-100 respectively. On the other hand SDS and CTAB caused complete inhibition of activity. The substrate specificity profiles of purified amidase and whole cell amidase were found considerably different and it led to predict the presence of one more amidase having affinity for short chain aliphatic amides. This second amidase was partially purified. The two amidases purified from B. smithii strain IITR6b2 totally differed in substrate specificity and temperature optima, while pH optima were nearly same. Finally, in Chapter 5, summary of the complete work and the conclusions drawn are presented. In conclusion, the present work demonstrates the development of efficient bioprocesses for nicotinic acid hydroxamate and isoniazid syntheses using acyltransferase activity of B. smithii strain IITR6b2. These results also presents that this bacterial isolate can be a potential biocatalyst for syntheses of other hydroxamic acids and acid hydrazides.