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Biocatalytic ketone reduction-A study on screening and optimization of culture condition on the reduction of a selected Ketone SYNOPSIS FOR M. PHARM. DISSERTATION SUBMITTED TO RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA BY CAUVERY DUTTA I M. PHARM. DEPARTMENT OF PHARMACEUTICAL CHEMISTRY DAYANANDA SAGAR COLLEGE OF PHARMACY 2011 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES BANGALORE, KARNATAKA ANNEXURE-II PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION 1. Name of the candidate and CAUVERY DUTTA address (in block letters) I M. PHARM., DEPARTMENT OF PHARMACEUTICAL CHEMISTRY DAYANANDA SAGAR COLLEGE OF PHARMACY, KUMARASWAMY LAYOUT, BANGALORE-560078. PERMANENT ADDRESS D/O SARAT DUTTA D.R.Z.H ROAD HENGRABARI POST OFFICE BYE LANE NO-9 GUWAHATI-36 ASSAM 2. Name of the institute Dayananda Sagar College of Pharmacy, Shavige Malleswara Hills, Kumaraswamy Layout, Bangalore-560078, Karnataka. 3. Course of study and subject Master of Pharmacy in Pharmaceutical Chemistry 4. Date of admission to course 7th July 2011 5. Title of the project: “Biocatalytic ketone reduction-A study on screening and optimization of culture Condition on the reduction of a selected Ketone” 6.0 Brief resume of the intended work: 6.1 Need of the study: The asymmetric reduction of ketones is one of the most important, Fundamental and practical reactions for producing non-racemic chiral alcohols, which can be transformed into various functionalities, without racemization, to synthesize industrially important chemicals such as pharmaceuticals, agrochemicals and natural products. The catalysts for the asymmetric reduction of ketones can be classified into two categories: chemical and biological methodologies1. Enantioselective reduction of ketones to optically active secondary alcohols is One of the most interesting areas of research for a number of research groups. Microbial enzymes have been widely used in converting ketones to the corresponding optically active secondary alcohols. This technique has shown good to excellent levels of enantiomeric excess. A variety of aliphatic ketones, aryl ketones, α- and β-ketoesters that contain phenyl substituents were reported to be reduced to the corresponding enantiomerically pure chiral alcohols, while the reduction of aliphatic ketones gave a moderate levels of enantioselectivity2. From the economic point of view, preparation of enantiomerically pure chiral alcohols through green biocatalytic routes has become a subject of great interest due to the high enantioselectivity, mild reaction conditions and low environmental pollution of biocatalytic processes. Among various biological approaches, there has been much interest in whole cell-catalyzed biocatalytic enantioselective reduction of prochiral ketones to enantiopure chiral alcohols because of high theoretical yield of this reaction3. The advantage of biocatalysis is that enzyme-catalyzed reactions are often highly enantioselective and regioselective. They can be carried out at ambient temperature and atmospheric pressure thus avoiding the use of more extreme conditions that can cause problems with isomerization, racemization, epimerization and rearrangement of the product . Microbial cells and enzymes derived from them can be immobilized and reused for many cycles and enzymes can be over-expressed to make biocatalytic processes economically efficient4. Any medium for microbial growth should contain a minimum and specific elements in appropriate proportions5. Presence of specific carbon source, nitrogen source, metal ions and inducers in growth media has profound effect on the enzyme expression and hence media optimization is logical to get maximum activity during bioconversion. Statistical analysis of the dependent variables will yield a valuable information on the media composition for maximum enzymatic activity. Medium optimisation can be carried out in several ways. Most popular is the onevariable-at-a-time approach. This approach is however extremely inefficient in locating the true optimum when interaction effects are present. To overcome the problems with interaction effects, efficient medium will be achieved when mathematical optimisation techniques in-terms of Response Surface Methodology (RSM) were applied. RSM as an efficient tool has a long history in the literature. The microorganisms when inoculated in appropriate, suitable and selective medium using. RSM will enhance the concentration of target product and can be used for commercial and industrial application.5 6.2 - Review of literature: 1. Min-Hua Zong et al3., reported the biocatalytic reduction of 4-(trimethylsilyl)-3butyn-2-one to enantiopure (R)- 4-(trimethylsilyl)-3- butyn-2-ol with high enantioselectivity using immobilized whole cells of a novel strain Acetobacter sp. CCTCC M209061, newly isolated from kefir. Compared with other microorganisms that were investigated, Acetobacter sp. CCTCC M209061 was shown to be more effective for the bioreduction reaction and afforded much higher yield and product enantiomeric excess. The optimal buffer pH, co-substrate concentration, reaction temperature, substrate concentration and shaking rate were 5.0, 130.6 mM, 300C, 6.0 mM and 180 r/min, respectively. Under the optimized conditions, the maximum yield and the enantiomeric excess of the product were 71% and >99%, respectively, which are much higher than those reported previously. 2. J. Augusto R. Rodrigues et al6., showed whole cells of living organisms, Yeasts as reliable Biocatalysts to perform redox reactions of various functional groups. This review focuses on the potential of these whole cells to reduce acetophenones and α,β- unsaturated carbonyl compounds (aldehydes and ketones) furnishing relevant chiral building blocks for fine chemicals and the pharmaceutical industries. 3. Hiromichi Ohta et al7., carried out ketone reduction using Yamadazyma farinosa IFO 10896 to reduce α-hydroxyketones bearing a phenyl ring to give optically active diols with anti-Prelog selectivity. The distance between the carbonyl group and the phenyl ring was shown to have an interesting effect on the reactivity and selectivity of the enzyme system. 4. Adi Wolfson et al 8., successfully performed the asymmetric hydrogenation of methyl acetoacetate with baker's yeast in pure glycerol and mixtures of glycerol and water. Though yeast viability was very low after exposure to glycerol, the enzymatic activity in pure glycerol was preserved for some days. In addition, a mixture of glycerol and water combined the advantageous of each individual solvent and resulted in high catalytic performance and efficient product extraction yield. 5. Zhong-Hua Yang et al 9., showed asymmetric reduction of the prochiral aromatic Ketone, catalysed by yeast cells as one of the most promising routes to produce its corresponding enantio- pure aromatic alcohol, the toxicity of aromatic ketone and aromatic alcohol to the yeast cell is investigated in this work. It has been found that the aromatic compounds are poisonous to the yeast cell. The activity of yeast cell decreases steeply when the concentration of acetophenone is higher than 30.0 mmol/L. Asymmetric reduction of acetophenone to chiral S- phenylethyl alcohol catalysed by the yeast cell was chosen as the model reaction to study in detail the promotion effect of the introduction of the resin adsorption on the asymmetric reduction reaction. The resin acts as the substrate reservoir and product extraction agent in situ. The enantioselectivity and yield are acceptable even though the initial ACP concentration reaches 72.2 mmol/L. 6. Gensheng Yang et al10., reported that (S)-3-Chloro-1-phenylpropanol is an Important chiral precursor for numerous antidepressants such as tomoxene. A high enantiomeric excess of (S)-3-chloro-1-phenylpropanol can be achieved by asymmetric reduction of 3-chloropropiophenone using Saccharomyces cerevisiae CGMC 2266 cells immobilized in calcium alginate. Thermal pretreatment of the immobilized cells at 500C for 30 min resulted in high enantioselectivity (99%) and good percent conversion (80%). The effects of various conditions on the reduction reaction were investigated. The optimal conditions were found to be as follows: sodium alginate concentration 2%; bead diameter 2mm; temperature 300C; re-culture time 24 h; and batch addition of the substrate. After reusing these three times, the immobilized cells retained approximately 60% of their original catalytic activity with their enantioselectivity intact. 7. Zhang jian et al 11., used response surface methodology (RSM) to optimize the fermentation medium for enhancing pyruvic acid production by Torulopsis glabrata TP19. In the first step of optimization, with Plackett-Burman design, ammonium sulfate, glucose and nicotinic acid were found to be the important factors affecting pyruvic acid production significantly. In the second step, a 23 full factorial central composite design and RSM were applied to determine the optimal concentration of each significant variable. A second-order polynomial was determined by the multiple regression analysis of the experimental data. The optimum values for the critical components were obtained as follows: ammonium sulfate 0.7498 (10.75 g/L), glucose 0.9383 (109.38 g/L) and nicotinic acid 0.3633 (7.86 mg/L) with a predicted value of maximum pyruvic acid production of 42.2 g/L. Under the optimal conditions, the practical pyruvic acid production was 42.4 g/L. The determination coefficient (R2) was 0.9483, which ensures adequate credibility of the model. 8. Chanakya Pallem et al 12., carried out solid state fermentation (SSF) for the production of L-glutaminase by the fungal strain Trichoderma koningii using sesamum oil cake as the solid substrate. L-glutaminase has received significant attention in recent years owing to its potential applications in medicine as an anticancer agent, as an efficient anti- retroviral agent and as a biosensor. The overall maximum yield of L-glutaminase ( 19.41 U/gds) was achieved with the optimized process parameters of initial moisture content 65%, initial pH of the medium 7.0, supplemented with D-maltose (1.0% w/v) and malt extract (1.0% w/v), inoculated with 2ml of 6 day old fungal culture and incubated at 33°C for 5 days. Both physicochemical and nutritional parameters had played a significant role in the production of the enzyme, L-glutaminase. The enzyme production was found to be associated with the growth of the fungal culture. 9. Rekha Kaushik et al 13., used a response surface approach to study the production of an extracellular lipase from Aspergillus carneus, which has immense industrial importance. Interactions were studied for five different variables (sunflower oil, glucose, peptone, agitation rate and incubation period), which were found influential for lipase production by one-at a time method. 1.8-fold increase in production was seen with the final yield of 12.7 IU/ml in comparison to 7.2 U/ml obtained by one-ata-time method. Using the statistical approach (response surface methodology (RSM)) the optimum values of these most influential parameters were as follows: sunflower oil (1%), glucose (0.8%), peptone (0.8%), agitation rate (200 rpm) and incubation period (96 h) at 37 °C. The subsequent verification experiment confirmed the validity of the model. 10. Vahap yonten et al 14., optimized the growth conditions for Kluyveromyces Lactis Drosophilarum on artificially prepared whey in batch experiments by applying Response Surface Methodology (RSM). The significant medium parameters (lactose, yeast extract, MgSO4, NaCl, micronutrients and medium temperature) and the Steepest–Ascent was utilized to locate the optimum region. The optimum operational conditions of maximum growth rate were obtained 0.1404 1/h as 16.478 g/L, 1.683 g/L, and 32.242 °C, lactose concentration, yeast extract concentration and medium temperature respectively. 11. Vijayalakshmi et al 15.,used response surface methodology of growth parameters for carotenoid synthesis by a mutant strain of Rhodotorula gracilis (CFR 0-1) by a fractional factorial design involving 5 variables: 4-8% glucose; 6-12 days incubation; inoculum vol. 6-12 ml/100 ml; pH 4.5-7.7; and temp. 24-320C. The response equation developed indicated a linear relationship between period of incubation, temp., sugar concentration. and vol. of inoculum for carotenoid production. Results indicated max. carotenogenesis (0.09%) at 8% glucose level, pH 7.5 and 6.0 ml/100 ml of inoculum for an incubation period of 12 days at 240C. These theoretical values were verified by experimental data. 12. Haq Nawaz Bhatti et al 16., employed response surface approach to study the production of extracellular lipase from Ganoderma lucidum which has diverse applications in various fields. Interactions were studied for five different variables (moisture, canola oil cake, olive oil, pH and time of incubation) which were found influential for lipase production. Using the statistical approach (response surface methodology), the maximum yield of lipase (4838 U/gds) by G. lucidum was observed under optimum conditions. The optimum values of these parameters were as follows: canolaoil cake (12.50 g), moisture level (60%), pH (4.5), olive oil as inducer (2.0%) and incubation period (96 h) at 30°C. 13. Ehud Keinan et al 17., achieved the asymmetric reduction of aliphatic acyclic ketones (C4-Cl0 substrates) is by using alcohol dehydrogenase from Thermoanaerobium brockii either as a homogeneous, heat-treated, cell-free extract or following immobilization on a solid support. Both methods are superior to the use of whole-cell fermentation. The experimental conditions for working with (TBADH) Thermoanaerobium brockii were studied and optimized in order to improve reaction rates and the optical purity of the product. An interesting substrate size-induced reversal of stereoselectivity was observed. The smaller substrates (methyl ethyl, methyl isopropyl, or methyl cyclopropyl ketones) are reduced to R alcohols, whereas the higher ketones form the S enantiomer. 6.3 – Objective of the Study:The present project focuses on the :I. Screening of some fungal species for bioreduction of a selected ketone. II. Effect of culture condition on bioreduction for the selected fungi. Carbon source Nitrogen source Metal ion Inducers Initial pH Incubation temperature III. Optimization of media components by using statistical techniques. Screening for significant variables RSM-to find optimum concentration of the screened variables. 7.0 Materials and Methods:- 7.1- Source Of Data: Review of chemical abstracts and journals like Tetrahedron Asymmetry, Journal of molecular catalysisB:Enzymatic, Journal of Bioresource Technology, Journal of Physical Science, Food Technol. Biotechnol, International Journal of Applied Biology and Pharmaceutical Technology, Internet browsing. 7.2 – Method of collection of data: Microbial culture will be obtained from culture collection centers like MTCC, Chandigarh and NCL, Pune. All the chemicals and other reagents will be collected from standard companies. The reactions will be monitored by Thin layer chromatography. Qualification of the product (alcohol) will be done by HPLC. Enzymatic activity will be estimated spectrophotometrically. 7.3 – Does the study require any investigations or interventions to be conducted on patients or other humans or animals? If so, Please describe briefly: -No7.4 – Has ethical clearance been obtained from your Institution in case of 7.3? Not applicable 8.0 List of References: 1. Kaoru N, Rio Y, Tomoko M and Tadao H.. Recent developments in asymmetric reduction of ketones with biocatalysts. Tetrahedron: Asymmetry, 2003; 14: 2659–81. 2. Bawa RA, Ajjabou F and Shalfooh E. Enzymatic Reduction of Ketones to optically active secondary alcohols. Journal of Physical Science, 2008; 19: 1–5. 3. Zi-Jun Xiao, Min-Hua Zong, Wen-Yong Lou. Highly enantioselective reduction of 4-(trimethylsilyl)-3-butyn-2-one to enantiopure (R)-4(trimethylsilyl)-3-butyn-2-ol using a novel strain Acetobacter sp. CCTCC M209061. Bioresource Technology, 2009; 100: 5560–65. 4. Ramesh N. Patel. Biocatalytic Synthesis of Chiral Pharmaceutical Intermediates. Food Technol. Biotechnol, 2004; 42: 305–25. 5. Hanan M, Ibrahim and Elrashied EE. Response Surface Method as an Efficient Tool for Medium Optimisation. Trends in Applied Sciences Research, 2011; 6: 121-29. 6. Augusto J, Moran S, Conceicao A. Recent Advances in the Biocatalytic Asymmetric Reduction of Acetophenones and α,β- Unsaturated Carbonyl Compounds, Food Technol. Biotechnol, 2004; 42: 295–303. 7. Toshikuni T, Takeshi S and Hiromichi O. Microbial asymmetric reduction of αhydroxyketones in the anti-Prelog selectivity. Tetrahedron: Asymmetry,2001; 12: 2543–25. 8. Adi W, Nisim H, Chrstina D, Dorith T and Yoram S, Baker's yeast catalyzed asymmetric reduction of methyl acetoacetate in glycerol containing systems, Org. Commun. 2008; 1: 9-16. 9. Z. Yang, R. Zeng, X. Chang, Xuan-Ke and G. Wang, Asymmetric reduction of prochiral ketones to chiral alcohols catalyzed by plants tissue. J Ind Microbiol Biotechnol, 2008; 35:1047–51 10. Yanga G, Shanjing Z, Jiangyan Xu. Asymmetric reduction of 3-chloropropiophenone to (S)-3-chloro-1-phenylpropanol using immobilized Saccharomyces cerevisiae CGMCC 2266 cells, Journal of Molecular Catalysis B: Enzymatic, 2009; 57: 83–88. 11. Zhang J, Gao N. Application of response surface methodology in Medium optimization for pyruvic acid production of Torulopsis glabrata TP19 in batch fermentation, J Zhejiang Univ Sci B, 2007; 8: 98-104. 12. Chanakya P, Srikanth M, Subba RS, process optimization of l-glutaminase production by trichoderma koningii under solid state fermentation, International Journal of Applied Biology and Pharmaceutical Technology, 2010; 1: 1168-74. 13. Kaushik R, Saran S, Jasmine I, Saxena SK, Statistical optimization of medium components and growth conditions by response surface methodology to enhance lipase production by Aspergillus carneus, Journal of Molecular Catalysis B: Enzymatic, 2006; 40: 121-26. . 14. Yonten V and Aktas N, Optimization of growth rate of Kluyveromyces Lactis Drosophilarum using response surface methodology , Journal of Molecular Catalysis B: Enzymatic, 2006; 43: 9–14. 15. Vijayalakshmi and Shobha, response surface methodology for optimized of growth parameter for the production of carotinoids by a mutant strain of rhodotorula gracilis, European food research and tech, 2011; 3: 234-39. 16. Amin F, Nawaz H and Rehman S. Optimization of growth parameters for lipase production by Ganoderma lucidum using response surface methodology, African Journal of Biotechnology, 2011; 10: 5514-23. 17. Keinan E, Hafeli K, Seth K, and Lamed R. Thermostable Enzymes in Organic Synthesis. Asymmetric Reduction of Ketones with Alcohol Dehydrogenase from Thermoanaerobium brockii, J. Am. Chem. SOC, 1986; 108: 162-69. Signature of the candidate CAUVERY DATTA 10. Remarks of the guide: 11. Name and Designation (in block letters) 11.1. Guide Smt. M.S. SANDHYAVALI. Associate professor Department of Pharmaceutical Chemistry, Dayananda Sagar College of Pharmacy, Kumaraswamy Layout, Bangalore-560078. 11.2. Signature 11.3. Co-guide if any 11.4. Signature Smt. Kalpana Divekar 11.5. Head of the department Dr. V. MURGAN, Professor and Principal Department of Pharmaceutical chemistry, Dayananda Sagar College of Pharmacy, Kumaraswamy Layout, Bangalore-560078. 11.6. Signature 12. 12.1. Remarks of the principal Dr. V. MURUGAN, PRINCIPAL, Dayananda Sagar College of Pharmacy, Kumaraswamy Layout, Bangalore-560078. 12.2 Signature