Stereochemical Control in Microbial Reduction. Part 10. Asymmetric Reduction of Alkyl 3-Methyl-2-Oxobutanoate with Immobilized Bakers' Yeast in Hexane

Esters of 3-methyl-2-oxobutanoic acid are reduced with bakers' yeast by three methods: free bakers' yeast in water, immobilized bakers' yeast in water, and immobilized bakers' yeast in hexane. Although (R)-hydroxy esters are obtained in all cases, the enantiomeric excess varies from 3% (reduction of the methyl ester with free bakers' yeast in water) to 93% (reduction of the butyl ester with immobilized bakers' yeast in hexane) depending on the structure of substrate and on the reaction conditions. The mechanism of the present stereochemical control is discussed.     

Efficient kinetic resolution of 2-benzenesulfonylcyclopentanone derivatives

Efficient kinetic resolution of 2-benzenesulfonylcyclopentanones1, bearing 3-alkyl, 3-aryl, or 3-benzyl substituents, has been achieved by bakers' yeast mediated reduction. With the unsubstituted 2-benzenesulfonylcyclopentanone1a, efficient asymmetric reduction to form (1S,2R)-cis-2-benzenesulfonylcyclopentanol2a is observed under the same conditions. Excellent enantioselectivities (up to > 95% ee) are obtained.     

Effects of subzero temperatures on the kinetics of protease catalyzed dipeptide synthesis in organic media

A depeptide synthesis was drastically influenced by the reaction temperature, in the range from -30 degrees to 25 degrees C. This article shows the kinetic reasons of this effect. alpha-Chymotrypsin was immobilized on celite and used in four different water-miscible solvents containing small amounts of water-miscible solvents containing small amounts of water. The reaction studied was the aminolysis of N-acetyl-L-phenylalanine ethyl ester (Ac-PheOEt) with L-alaninamide (Ala-NH(2)) and water for the acylenzyme complex, the nucleophile was favoured by low reaction temperatures. This effect (quantified as p-values) was observed in all four solvents, and it was greatest in acetonitrile and tetrahydrofuran. The esterase and amidase activities of the enzyme were studies using AcPheOEt and N-acetyl-L-phenylalanyl-L-ananinamide (AcPheAla-NH(2)) as substrates. The Michaelis-Menten parameters, K(m,app) and V(max), were determined for ester hydrolysis and dipeptide hydrolysis. Both K(m,app) and V(max) tended to increase with increasing temperature. Secondary hydrolysis was reduced at subzero temperatures because ester hydrolysis was favoured in relation to depeptide hydrolysis. Depeptide synthesis was thus favored by low temperatures in two ways: first, in the competition between the nucleophile and water for the acyl enzyme; and, second, in the competition between the ester substrate and the peptide substrate for the free enzyme. As a result, in acetonitrile containing 10% water, the maximal yield was 99% at -20%C compared with 84% at 25 degrees C. (c) 1995 John Wiley & Sons, Inc.     

Effects of Acetonitrile-Water Mixtures on α-Chymotrypsin Catalyzed Dipeptide Synthesis

-Chymotrpysin (EC 3.4 21.1) was immobilized by deposition on celite and subsequent cross-linking with glutaraldehyde. The effects of different mixtures of aqueous buffer and acetonitrile on the immobilized preparation were evaluated using a dipeptide synthesis as model reaction. The initial reaction rate at 6-95% of water increased with increasing water content. The maximum yield of peptide had two maxima; the first one at 6% of water (92%) and the second one at 80% of water (39%). The presence of two maxima was due to severe enzyme inactivation at intermediate water contents (50-60%). The immobilisation procedure slowed the inactivation of -chymotrypsin. Cross-linked enzyme was inactivated to a lesser extent than both free enzyme and enzyme that had been deposited on celite. The increased resistance to inactivation was, however, not sufficient to make peptide synthesis attractive at intermediate water contents (50-60%). In order to obtain good peptide yields, low water contents (below 10%) should be used.     

Baker's yeast mediated reduction of β-keto esters and β-keto amides in an organic solvent system

The baker's yeast mediated reduction of four β-keto esters in petroleum ether indicated that the size of the group attached to the keto carbon affected their reactivity. Ethyl 3-phenyl-3-oxopropanoate (1), which has a phenyl group directly attached to the keto carbon, is incompletely reduced using 20 g yeast/mmol substrate, ethyl 4-phenyl-3-oxobutanoate (2), which has one methylene group between the phenyl and keto carbon, was also incompletely reduced using 20 g yeast/mmol, although the extent of reduction was about double that of (1), ethyl 5-phenyl-3-oxopentanoate (3), which has two methylene groups between the phenyl and keto carbon, is completely reduced using 10 g yeast/mmol and ethyl 3-oxobutanoate (4), which has a methyl group attached to the keto carbon shows complete reduction using only 1 g yeast/mmol. The corresponding β-keto amides are considerably less reactive than the corresponding β-keto esters with only the amides derived from ethyl 3-oxobutanoate indicating any significant reduction using 20 g yeast/mmol.     

α-Chymotrypsin Immobilized on Chitin. Relationships Between the Enzyme Kinetic Constant and Support Structure

-Chymotrypsin was immobilized on chitin from squills, lobsters and prawns by means of glutaraldehyde. Hydrolase and peptide synthetase activities were determined in aqueous and homogeneous aqueous-organic media, respectively.

The results show -chymotrypsin immobilized on chitin from prawn to be the most active immobilized derivative based on its synthetase activity (90% yield of Bz-Tyr-Leu-NH₂ in carbonate buffer, p^H 9 containing 70% 1,4- butanediol).

The relationship between the kinetic constant of hydrolysis and chitin structure was also studied. -Chymotrypsin immobilized on prawn chitin was found to be the best derivative in kinetic terms.

The stability of the three derivatives was studied at 37C.     

Aggregate Clearance of α-Synuclein in Saccharomyces cerevisiae Depends More on Autophagosome and Vacuole Function Than on the Proteasome

Parkinson disease is the second most common neurodegenerative disease. The molecular hallmark is the accumulation of proteinaceous inclusions termed Lewy bodies containing misfolded and aggregated α-synuclein. The molecular mechanism of clearance of α-synuclein aggregates was addressed using the bakers' yeast Saccharomyces cerevisiae as the model. Overexpression of wild type α-synuclein or the genetic variant A53T integrated into one genomic locus resulted in a gene copy-dependent manner in cytoplasmic proteinaceous inclusions reminiscent of the pathogenesis of the disease. In contrast, overexpression of the genetic variant A30P resulted only in transient aggregation, whereas the designer mutant A30P/A36P/A76P neither caused aggregation nor impaired yeast growth. The α-synuclein accumulation can be cleared after promoter shut-off by a combination of autophagy and vacuolar protein degradation. Whereas the proteasomal inhibitor MG-132 did not significantly inhibit aggregate clearance, treatment with phenylmethylsulfonyl fluoride, an inhibitor of vacuolar proteases, resulted in significant reduction in clearance. Consistently, a cim3-1 yeast mutant restricted in the 19 S proteasome regulatory subunit was unaffected in clearance, whereas an Δatg1 yeast mutant deficient in autophagy showed a delayed aggregate clearance response. A cim3-1Δatg1 double mutant was still able to clear aggregates, suggesting additional cellular mechanisms for α-synuclein clearance. Our data provide insight into the mechanisms yeast cells use for clearing different species of α-synuclein and demonstrate a higher contribution of the autophagy/vacuole than the proteasome system. This contributes to the understanding of how cells can cope with toxic and/or aggregated proteins and may ultimately enable the development of novel strategies for therapeutic intervention.     

Effect of gamma-ray on activity and stability of alcohol-dehydrogenase from Saccharomyces cerevisiae

The effect of γ-ray irradiation on alcohol-dehydrogenase activity of yeast was investigated. The results suggested that low doses of γ-ray (10 and 20 Gy) significantly increased the enzyme activity. This work also describes the impact of irradiation on immobilization efficiency of biocatalyst entrapped on to alginate gel beads. When yeast irradiated to a dose of 20 Gy was immobilized, ADH stability was improved up to 1.4 times at 45 °C compared to the immobilized non-irradiated cells. Also, the irradiated biocatalyst, when immobilized, can be reused more than eight times in oxidation reaction of ethanol. This preparation also permitted to reach high yields of immobilization (79%) and activity (88%).     

Enzymic Preparation of Benzyl β-D-Glucopyranoside

Benzyl β-D-glucopyranoside was prepared by an enzyme-catalysed direct reaction between D-glucose, or better cellobiose, and benzyl alcohol in the presence of a minimum amount of water. The enzyme β-glucosidase was used in the immobilized form (adsorbed onto macroporous polyethylene terephthalate or covalently bound on polyglycidyl methacrylate), enabling multiple application.     

α-Chymotrypsin shows higher activity in water as well as organic solvents after three phase partitioning

Alpha-Chymotrypsin was found to show a 119% increase in activity after three phase partitioning. The k_cat/K_m of the partitioned enzyme (TPP-C) for hydrolysis of Bz-Tyr-OEt in aqueous medium at 25°C was found to be 48.3×10⁴ mM^-1 min^-1 as compared to the corresponding value of 17.7×10⁴ mM^-1 min^-1 for the untreated control (C). The λ_max of the fluorescence emission spectrum of TPP-C showed 178% increase in the quantum yield when compared to C. TPP-C showed a 2.94 and 3.58 fold increase (as compared to C) in initial rates for formation of the ester Ac-Phe-OEt (from Ac-Phe and ethanol) in low water containing toluene and n-octane, respectively. It was found that TPP-C also showed the phenomenon of pH memory. At 5% (v v^-1) water (in t-amyl alcohol), while no esterification was observed with C, TPP-C still showed significant level of esterification activity.     

Regiospecificity and Enantiospecificity in Microbiological Reduction of Acyclic β-Diketones

A screening of microorganisms has been performed to study the regio- and stereospecificities of the reduction of carbonyl groups in 2,4 and 3,5 diketones to obtain all stereoisomers. (2S), (2R), (3S), (3R) and (5R) ketols are obtained along with (2S, 4S) and (3S, 5S) diols often in high optical yields. Chemo-enzymatic methodologies to obtain stereoisomers not reached by direct microbiological reduction are discussed.     

Substituent Effect on the Absolute Stereochemistry of the Asymmetric Reduction of Fluorine-Containing β-Diketones by Bakers' Yeast

Fluorine-containing β-diketones (la-d) were reduced by free bakers' yeast (FBY) and immobilized baker yeast (IMBY) in water, to give optically-active fluorinated β-hydroxyketones (2a-d). It was found that the reaction is highly regioselective, and that the stereochemistry of the reduction is controlled by the R substituent.     

Transesterification activity of lipases immobilized in a phyllosilicate sol-gel matrix**

Lipases from Pseudomonas cepacia (P.c.) and Thermomyces lanuginosa (T.l.) were immobilized in a phyllosilicate sol-gel matrix and studied for their ability to catalyze the alcoholysis of fats and oils to simple alkyl esters. At 50 degrees C and 48 h reaction immobilized T.l. lipase gave higher alkyl ester yields (70 to 100%) from fats and oils regardless of chain length or degree of unsaturation of the acyl groups in the triacylglycerols than did immobilized P.c. lipase (20-90%), which preferred unsaturated oils. Both immobilized lipases catalyzed ester formation (80-90%) from greases containing a range of free fatty acids (2.6 to 36%). Molecular sieves had no effect on ester yields in the immobilized T.l. lipase-catalyzed alcoholysis of greases but did improve yields (5-10%) in the immobilized P.c. lipase-catalyzed reactions.     

alpha-tocopherol increases the intracellular glutathione level in HaCaT keratinocytes

-Tocopherol is a lipophilic vitamin that exhibits an antioxidative activity. The purpose of this study was to clarify the roles of -tocopherol in the regulation of intracellular glutathione (GSH) levels in HaCaT keratinocytes. When HaCaT keratinocytes were cultivated with -tocopherol for 24 h, the intracellular GSH was increased at every concentration of -tocopherol tested. Furthermore, the HaCaT keratinocytes cultured with -tocopherol at 50 μM for 24 h exhibited resistance against H 2 O 2 . However, a short exposure of HaCaT keratinocytes to -tocopherol for 1 h did not influence either the GSH level or the resistance to H 2 O 2 . These findings suggest that GSH, which is inductively synthesized by -tocopherol, effectively reduces exogenous oxidative stress. To evaluate the effect of -tocopherol on the GSH level, BSO, which is a typical inhibitor of γ-glutamylcysteine synthetase ( γ-GCS), was used. When BSO was added to HaCaT keratinocytes, no action of -tocopherol on the GSH level was observed. On the other hand, -tocopherol resulted in the up-regulation of γ-GCS-HS (heavy subunit) mRNA. In addition, water soluble -tocopherol derivatives ( -tocopherol phosphate and trolox) caused no changes in GSH level. From these results, it was concluded that -tocopherol increases the intracellular GSH level of HaCaT keratinocytes through the up-regulation of γ-GCS-HS mRNA.     

Reduction of daunorubicin in the presence of sulfur-containing peptides

Daunorubicin, an anthracycline antitumor antibiotic, was reduced in the presence of reduced (GSH) or oxidized (GSSG) glutathione to evaluate the possibilities of detoxification or of potentiation of the drug by these compounds. The reductants were ^.COO⁻ free radicals produced by γ radiolysis. In both cases, the final product is 7-deoxydaunomycinone, i.e., the same as without glutathione. The reduction yield is also the same as without GSH or GSSG (0.23 μmol·J⁻¹). No glutathione depletion was observed. Limits for the rate constants of some possible nonenzymatic detoxification reactions are given. To evaluate the possible interactions of daunorubicin with sulfur-containing proteins, the reduction of this drug by ^.COO⁻ free radicals was also studied in the presence of a polypeptide containing two disulfide bridge are, respectively, 0.23 μmol·J⁻¹ 7-deoxydaunomycinone. The yields of reduction of the drug and of a protein disulfide bridge are, respectively, 0.23 μmol·J⁻¹ and ≤ 6 nmol·J⁻¹. These values indicate thet disulfide radical anions of the protein can reduce the drug, giving back the disulfide bridge, but that the drug transients niether oxidize nor reduce the protein.     

Integration of enzyme, strain and reaction engineering to overcome limitations of baker's yeast in the asymmetric reduction of alpha-keto esters

We report on the development of a whole-cell biocatalytic system based on the popular host Saccharomyces cerevisiae that shows programmable performance and good atom economy in the reduction of alpha-keto ester substrates. The NADPH-dependent yeast reductase background was suppressed through the combined effects of overexpression of a biosynthetic NADH-active reductase (xylose reductase from Candida tenuis) to the highest possible level and the use of anaerobic reaction conditions in the presence of an ethanol co-substrate where mainly NADH is recycled. The presented multi-level engineering approach leads to significant improvements in product optical purity along with increases in the efficiency of alpha-keto ester reduction and co-substrate yield (molar ratio of formed alpha-hydroxy ester to consumed ethanol). The corresponding alpha-hydroxy esters were obtained in useful yields (>50%) with purities of > or =99.4% enantiomeric excess. The obtained co-substrate yield reached values of greater than 1.0 with acetate as the only by-product formed.     

Stabilization of alpha-chymotrypsin by ionic liquids in transesterification reactions.

Five different ionic liquids, based on dialkylimidazolium and quaternary ammonium cations associated with perfluorinated and bis (trifluoromethyl) sulfonyl amide anions, were used as reaction media to synthesize N-acetyl-L-tyrosine propyl ester by transesterification with alpha-chymotrypsin at 2% (v/v) water content at 50 degrees C. The synthetic activity was reduced by the increase in alkyl chains length of cations and by increases in anion size, which was related to the decrease in polarity. Incubation of the enzyme (with and without substrate) in ionic liquids exhibited first-order deactivation kinetics at 50 degrees C, allowing determination of deactivation rate constants and half-life times (1-3 h). Ionic liquids showed a clear relative stabilization effect on the enzyme, which was improved by increased chain length of the alkyl substituents on the imidazolium ring cations and the anion size. This effect was 10-times enhanced by the presence of substrate. For example, 1-butyl-3-methylimidazolium hexafluorophosphate increased the alpha-chymotrypsin half-life by 200 times in the presence of substrate with respect to the 1-propanol medium. These results show that ionic liquids are excellent enzyme-stabilizing agents and reaction media for clean biocatalysis in non-conventional conditions.     

Influence of the microenvironment on the activity of enzymes immobilized on Teflon membranes grafted by γ-radiation

The effect of the microenvironment and immobilization method on the activity of immobilized β-galactosidase was investigated. Immobilization was done on Teflon membranes grafted with different acrylic monomers by γ-radiation and activated by two different coupling agents through the functional groups of the grafted monomers. 2-Hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAA) were grafted on the membrane, and 1,6-hexamethylenediamine (HMDA) was used as a spacer. Glutaraldehyde or cyanuric chloride were used as coupling agents to bind the enzyme to the membrane. Four different catalytic membranes were obtained using the same solid support. Direct comparison between the isothermal behaviour of the biocatalyst in its free and immobilized form was carried out. In particular the dependence of the isothermal activity on the temperature and pH was studied and the kinetic parameters determined. The influence of the microenvironment on the observed activity of the four membranes was evidenced and discussed. The way of improving the yield of these catalytic membranes is discussed also.     

固定化细胞有机相催化不对称还原β-羰基酯

将酵母细胞用海藻酸钙包埋后用于有机相催化不对称还原4-氯乙酰乙酸乙酯制备光学活性的4-氯-3-羟基丁酸乙酯,从中筛选得到具有较高立体选择性和还原能力的菌株假丝酵母SW0401,将此菌株的细胞固定化细胞作为研究对象,系统考察了固定化条件、固定化细胞大小、反应溶剂、初始底物浓度、辅助底物、固定化细胞热处理和抑制剂对还原反应的影响。结果表明,上述因素对反应的摩尔转化率和产物（S）-CHBE光学纯度有显著影响。固定化时所用缓冲液的pH值为7．0时和固定化细胞颗粒平均直径为2.5mm较合适,以正己烷为反应介质时反应的摩尔转化率和产物光学纯度最优,初始底物浓度以54.7mmol／L为宜,辅助底物以1-己醇为佳。对固定化细胞的热处理和添加抑制剂烯丙醇均能够明显改善产物的光学纯度,但对提高摩尔转化率有负面影响。     

Optical resolution of two isomeric naphthylalanines by immobilized enyzmes

Optical resolution of β-(1-naphthyl)alanine and β-(2-naphthyl)alanine have been efficiently carried out through enzymatic hydrolysis of their methyl ester and/or N-acetyl ester derivatives by immobilized enzymes. Difficulties related to the lipophilic character of these amino acids were overcome by using emulsions of n-butyl acetate–water as reaction medium. The use of an automatic recirculating apparatus allowed reproducible and repetitive use of the immobilized biocatalysts.