Download i m. pharm. - Rajiv Gandhi University of Health Sciences

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