<Emphasis Type="Italic">Burkholderia cenocepacia</Emphasis>: a new biocatalyst for efficient bioreduction of ezetimibe intermediate

Ezetimibe is a selective acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor used in hypercholesterolemia. Synthesis of ezetimibe requires enantiopure 3-[5-(4-fluorophenyl)-5(S)-hydroxypentanoyl]-4(S)-4-phenyl-1,3-oxazolidin-2-one (FOP alcohol) as a crucial intermediate which is produced by reduction of the corresponding prochiral ketone (FOP dione). A new biocatalyst from acclimatized soil was screened for bioreduction of the above prochiral ketone. The microorganism was identified by 16S mRNA sequencing, as Burkholderia cenocepacia. Various physicochemical conditions were optimized to increase cellmass and enzyme activity. The overall increase in cellmass concentration and enzyme activity was 2.06 and 1.85-fold, respectively. Various reaction conditions, for example pH, temperature, agitation, and cellmass concentration, were optimized for maximum product yield (chiral alcohol) with excellent enantioselectivity. Best reduction was achieved in phosphate buffer (50 mM, pH 8.0) at 40°C (200 rpm) and the yield of enantiopure alcohol from the corresponding prochiral ketone was 54%. This biocatalyst was also used for the reduction of various other prochiral ketones.     

Asymmetric reduction of prochiral ketones to chiral alcohols catalyzed by plants tissue

As an important organic compound, chiral alcohols are the key chiral building blocks to many single enantiomer pharmaceuticals. Asymmetric reduction of the corresponding prochiral ketones to produce the chiral alcohols by biocatalysis is one of the most promising routes. Asymmetric reduction of different kinds of non-natural prochiral ketones catalyzed by various plants tissue was studied in this work. Acetophenone, 4'-chloroacetophenone and ethyl 4-chloroacetoacetate were chosen as the model substrates for simple ketone, halogen-containing aromatic ketone and beta-ketoesters, respectively. Apple (Malus pumila), carrot (Daucus carota), cucumber (Cucumis sativus), onion (Allium cepa), potato (Soanum tuberosum), radish (Raphanus sativus) and sweet potato (Ipomoea batatas) were chosen as the biocatalysts. It was found that these kinds of prochiral ketoness could be reduced by these plants tissue with high enantioselectivity. Both R- and S-form configuration chiral alcohols could be obtained. The e.e. and chemical yield could reach about 98 and 80% respectively for acetophenone and 4'-chloroacetophenone reduction reaction with favorable plant tissue. And the e.e. and yield for ethyl 4-chloroacetoacetate reduction reaction was about 91 and 45% respectively.     

Design of an enzyme cocktail consisting of different fungal platforms for efficient hydrolysis of sugarcane bagasse: Optimization and synergism studies

Lignocellulosic materials represent a very important and promising source of renewable biomass. In order to turn them into fermentable sugars, synergism among the different enzymes that carry out bioconversion of these materials is one of the main factors that should be considered. Experimental mixture design was performed to optimize the proportion of enzymes produced by native strains of Trichoderma harzianum IOC 3844, Penicillium funiculosum ATCC 11797, and Aspergillus niger ATCC 1004, resulting in a proportion of 15, 50, and 35%, respectively. This mixture was able to hydrolyze 25 g/L of pretreated sugarcane bagasse with 91% of yield after 48 h of enzymatic reaction. Synergism along the hydrolysis process, besides the influence of lignin, hemicellulose, and solids loading, were also studied. Response surface methodology (RSM) based on Central Composite Rotatable Design was used to optimize solids and protein loadings to increase glucose release and enzymatic hydrolysis yield. The optimum solid and protein loadings established with RSM were 196 g/L and 24 mg/g cellulose, respectively, and under these conditions (94.1 ± 8) g/L of glucose were obtained, corresponding to a hydrolysis yield of 64%. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1222–1229, 2016     

The effect of electrochemical regeneration upon the enzymatic catalysis of a thermodynamically unfavorable reaction

The association between enzymatic and electrochemical reactions, enzymatic electrocatalysis, had proven to be a very powerful tooth in both analytical and synthetic fields. However, most of the combinations studied have involved enzymatic catalysis of irreversible or quasi-irreversible reaction. In the present work, we have investigated the possibility of applying enzymatic electrocatalysis to a case where the electrochemical reaction drives a thermodynamically unfavorable reversible reaction. Such thermodynamically unfavorable reactions include most of the oxidations catalyzed by dehydrogenases. Yeast alcohol dehydrogenase (E.C. 1.1.1.1) was chosen as a model enzyme because the oxidation of ethanol is thermodynamically very unfavorable and because its kinetics are well known. The electrochemical reaction was the oxidation of NADH which is particularly attractive as a method of cofactor regeneration. Both the electrochemical and enzymatic reactions occur in the same batch reactor in such a way that electrical energy is the only external driving force. Two cases were experimentally and theoretically developed with the enzyme either in solution or immobilized onto the electrode's surface. In both cases, the electrochemical reaction could drive the enzymatic reaction by NADH consumption in solution or directly in the enzyme's microenvironment. However even for a high efficiency of NADH consumption, the rate of enzymatic catalysis was limited by product (acetaldedehyde) inhibition. Extending this observation to the subject of organic synthesis catalyzed by dehydrogenases, we concluded that thermodynamically unfavorable reaction and can only be used in a process if efficient NAD regeneration and product elimination are simultaneously carried out within the reactor.     

Dynamic resolution and stereoinversion of secondary alcohols by chemo-enzymatic processes

To overcome the maximum 50% yield limitation of classical resolution methods, deracemization processes involving a racemization step (dynamic resolution) or a prochiral intermediate (stereoinversion) have been developed. The use of transition metal complexes as racemizing agents, in combination with an enzymatic reaction, has been successfully extended to the deracemization of a number of simple or functionalized sec-alcohols. A two-enzyme process has been also investigated for their sequential or simultaneous deracemization. Other prominent results arise from an (apparently general) oxidoreduction process catalyzed by a single whole-cell microorganism.     

Enzymatic asymmetrization of prochiral 2-benzyl-1,3-propanediol through esterification in solvent media

The enzymatic esterification of the prochiral substrate, 2-benzyl-1,3-propanediol, has been studied in solvent media. Among the five tested lipases, Lipozyme and Novozym 435 led to higher reaction rates. Novozym 435 catalyzed faster reactions at low water activity and in solvents having log P above 2. However, the two positions of the diol, pro-(R) and pro-(S), led to the same reaction rate trends and no prochiral selectivity was obtained. When using Lipozyme in toluene, the reaction rates for the formation of both (R) and (S) products presented an optimum at a water activity of 0.22. In this case, the prochiral selectivity increased with the water activity, from a value of 5 at a _w < 0.01, to a value of 8 at a _w = 0.22, at which point it remained constant.     

Enantioselective properties of Fusarium solani pisi cutinase on transesterification of acyclic diols: activity and stability evaluation

The regio and enantioselectivity of a recombinant cutinase from Fusarium solani pisi was tested on three racemic and one prochiral phenylalkanediols via irreversible transesterification with vinyl acetate. The optimization of the reaction conditions involved the screening of different organic solvents as well as the variation of the substrate concentrations. Thus, the enzymatic activity was checked by measuring initial reaction rates, overall yields, and enantiomeric excess of the reaction products. Only the smaller molecules were recognized by the enzyme, and a denaturing effect of the acyl donor was observed. Nevertheless, a stabilising effect on the enzyme caused by a pre-incubation with the diol was also noted.     

Stereochemical inversion at C-15 accompanies the enzymatic isomerization of all-trans- to 11-cis-retinoids

all-trans-Retinol (vitamin A) is processed by membranes from the pigment epithelium of the amphibian or bovine eye to form 11-cis-retinoids. When the isomerization reaction is performed with either [15(S)-3H,14C]-all-trans-retinol or [15(R)-3H,14C]-all-trans-retinol as substrate, the resultant 11-cis-retinals, formed by the in vitro enzymatic oxidation of the retinols, retain their 3H in the former case and lose it in the latter. The ocular all-trans- (pro-R specific) and 11-cis-retinol (pro-S specific) dehydrogenases operate with different stereochemistries with respect to the prochiral methylene hydroxyl centers of their substrates. Inversion of stereochemistry at the prochiral retinol centers was shown to accompany the isomerization process in both the amphibian and bovine systems. The 11-cis-retinol formed from [15(S)-3H,14C]-all-trans-retinol was chemically isomerized with I2 to produce [15(R)-3H,14C]-all-trans-retinol. The 11-cis-retinol formed from [15(R)-3H,14C]-all-trans-retinol was chemically isomerized with I2 to produce [15(S)-3H,14C]-all-trans-retinol. The stereochemistry at the prochiral center of retinol is not affected by the I2-catalyzed double-bond isomerization process and, hence, inversion of stereochemistry at C-15 must accompany isomerization. The same inverted stereochemistry was found with the associated retinyl palmitates. Possible mechanistic reasons for the observed inversion of stereochemistry during isomerization are discussed.     

蒸汽爆破玉米秸秆酶解动力学

为了掌握蒸汽爆破玉米秸秆的酶解特性,研究了不同底物浓度、酶浓度、温度对反应速率的影响。运用米氏方程对酶解动力学过程进行拟合,结果表明,纤维素酶对该玉米秸秆的水解反应在反应前3 h符合一级反应,可用米氏方程对其进行拟合。在转速为120 r/min、酶浓度为1.2 FPU/mL、pH 5.0、温度为45 ℃时米氏常数Km为11.71 g/L,最大反应速率Vm为1.5 g/(L·h)。确立了包括底物浓度、酶浓度、温度在内的酶解动力学模型,该模型适合温度为30 ℃~50 ℃。     

Synthesis of peptide aldehydes via enzymatic acylation of amino aldehyde derivatives

Two ways for semi-enzymatic preparation of the peptide aldehydes are proposed: (1) enzymatic acylation of amino alcohols with acyl peptide esters and subsequent chemical oxidation of the resulting peptide alcohols with DMSO/acetic anhydride mixture or (2) enzymatic acylation of the preliminarily obtained by a chemical route amino aldehyde semicarbazones. Subtilisin 72, serine proteinase with a broad specificity, distributed over macroporous silica, was used as a catalyst in both cases. Due to the practical absence of water in the reaction mixtures the yields of the products in both enzymatic reactions were nearly quantitative. The second way seems to be more attractive because all chemical stages were carried out with amino acid derivatives, far less valuable compounds than peptide ones. A series of peptide aldehydes of general formula Z-Ala-Ala-Xaa-al (where Xaa-al=leucinal, phenylalaninal, alaninal, valinal) was obtained. The inhibition parameters for these compounds, in the hydrolysis reactions of corresponding chromogenic substrates for subtilisin and -chymotrypsin, were determined.     

Recombinant whole-cell mediated baeyer-villiger oxidation of perhydropyran-type ketones

Recombinant Escherichia coli cells expressing eight Baeyer-Villiger monooxygenases of bacterial origin have been utilized to oxidize prochiral heterocyclic ketones containing a pyran ring system. Within the biotransformation, two stereogenic centers were introduced with high control of enantioselectivity. The chemoselectivity of the enzymatic reaction was found to be high in favor of the Baeyer-Villiger process when using substituted ketone precursors incorporating functional groups labile to oxidation. A significantly different behavior was observed for two groups of monooxygenases with respect to substrate acceptance, which is consistent with our previous classification into two enzyme clusters.     

Comparisons of SPORL and dilute acid pretreatments for sugar and ethanol productions from aspen

This study reports comparative evaluations of sugar and ethanol production from a native aspen (Populus tremuloides) between sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) and dilute acid (DA) pretreatments. All aqueous pretreatments were carried out in a laboratory wood pulping digester using wood chips at 170°C with a liquid to oven dry (od) wood ratio (L/W) of 3:1 at two levels of acid charge on wood of 0.56 and 1.11%. Sodium bisulfite charge on od wood was 0 for DA and 1.5 or 3.0% for SPORL. All substrates produced by both pretreatments (except DA with pretreatment duration of 0) had good enzymatic digestibility of over 80%. However, SPORL produced higher enzymatic digestibility than its corresponding DA pretreatment for all the experiments conducted. As a result, SPORL produced higher ethanol yield from simultaneous saccharification and fermentation of cellulosic substrate than its corresponding DA pretreatment. SPORL was more effective than its corresponding DA pretreatment in reducing energy consumption for postpretreatment wood chip size-reduction. SPORL, with lower energy input and higher sugar and ethanol yield, produced higher sugar and ethanol production energy efficiencies than the corresponding DA pretreatment.     

鞘磷脂合成酶的提取和检测

在20℃条件下,采用相对饱和度为80％的（NH4）2SO4一步盐析并经低温冷冻干燥12h得到鞘磷脂合成酶粗酶粉后,采用体外酶促反应并结合HPLC法检测鞘磷脂合成酶的含量。     

稀有人参皂苷IH901酶法转化与制备研究

本研究利用酶制剂蜗牛酶,酶法转化三七二醇组皂苷制备稀有人参皂苷IH901,正交实验优化酶解条件,建立酶法转化工艺.结果表明:超声法提取三七总皂苷正交实验优化条件为用75%乙醇溶液,15倍溶剂用量,超声波提取210 min作为最佳条件,三七总皂苷得率为12.21%;酶法转化二醇组人参皂苷制备稀有人参皂苷IH901,正交实验优化的条件为物料比为6/1、反应时间9 h、反应温度为45℃、pH值为3.0,酶解得率为54.24%;经硅胶柱分离获得IH901单体化合物,HPLC测定纯度达98%.酶法转化制备皂苷IH901的工艺方法简便,切实可行,可为中试生产提供参考.     

生物酶法制备D-对羟基苯甘氨酸的研究

生物酶法生产D-对羟基苯甘氨酸具有良好的应用前景。通过介绍生物酶法生产D-对羟基苯甘氨酸的研究现状,从酶的分离纯化、酶和细胞的固定化、反应介质研究和反应动力学研究以及基因工程进展几个方面作了总结。     

Carbon-fate maps for metabolic reactions

MOTIVATION: Stable isotope labeling of small-molecule metabolites (e.g. (13)C-labeling of glucose) is a powerful tool for characterizing pathways and reaction fluxes in a metabolic network. Analysis of isotope labeling patterns requires knowledge of the fates of individual atoms and moieties in reactions, which can be difficult to collect in a useful form when considering a large number of enzymatic reactions. RESULTS: We report carbon-fate maps for 4605 enzyme-catalyzed reactions documented in the KEGG database. Every fate map has been manually checked for consistency with known reaction mechanisms. A map includes a standardized structure-based identifier for each reactant (namely, an InChI string); indices for carbon atoms that are uniquely derived from the metabolite identifiers; structural data, including an identification of homotopic and prochiral carbon atoms; and a bijective map relating the corresponding carbon atoms in substrates and products. Fate maps are defined using the BioNetGen language (BNGL), a formal model-specification language, which allows a set of maps to be automatically translated into isotopomer mass-balance equations. AVAILABILITY: The carbon-fate maps and software for visualizing the maps are freely available (http://cellsignaling.lanl.gov/FateMaps/).     

Quantitative analysis of enzymatic fractionation of multiple substrate mixtures

The enzymatic conversion of mixtures of multiple substrates was studied quantitatively, based on established methodology used for the enzymatic kinetic resolution of racemic mixtures, involving the use of competitive factors: ratios of specificity constants (k_cat/K_M) of substrate pairs. The competitive factors of the substrates were defined in relation to a reference substrate. These competitive factors were used to predict the composition of the reaction mixture as a function of the degree of conversion of the reaction. The methodology was evaluated using three different lipases to hydrolyze a model mixture of four fatty acid methyl esters and for the esterification of a mixture of the same fatty acids in free form with ethanol. In most cases, the competitive factors determined from the initial phase of the reactions predicted the product composition during the rest of the reaction very well. The slowest reacting fatty acid was erucic acid (both in free form and as methyl ester), which was thus enriched in the remaining substrate fraction, while the other fatty acids: lauric acid, palmitic acid and oleic acid were converted faster. Simulations of the compositions of reaction mixtures with different values of the competitive factors were carried out to provide an overview of what could be achieved using enzymatic enrichment. Possible applications include reactions involving homologous substrates and mixtures of multiple isomers. The analysis presented provides guidelines that can be useful in the screening and development of enzymes for enzymatic enrichment applications. Biotechnol. Bioeng. 2013; 110: 78–86. © 2012 Wiley Periodicals, Inc.     

Purification and Characterization of Two Epoxide Hydrolases from Corynebacterium sp. Strain N-1074

Enzymes II_a and II_b, which catalyze the conversion of epichlorohydrin (ECH) to 3-chloro-1,2-propanediol (MCP), were purified from Corynebacterium sp. strain N-1074, which catalyzes the formation of (R)-MCP from prochiral 1,3-dichloro-2-propanol via ECH. The specific activity of enzyme II_a for the formation of MCP from ECH was about 6.4-fold higher than that of enzyme II_b. Both enzymes catalyzed the conversion of 1,2-epoxides to the corresponding diol, although they differed in several enzymatic properties.     

A deep learning approach to evaluate the feasibility of enzymatic reactions generated by retrobiosynthesis

Retrobiosynthesis allows the designing of novel biosynthetic pathways for the production of chemicals and materials through metabolic engineering, but generates a large number of reactions beyond the experimental feasibility. Thus, an effective method that can reduce a large number of the initially predicted enzymatic reactions has been needed. Here, we present Deep learning-based Reaction Feasibility Checker (DeepRFC) to classify the feasibility of a given enzymatic reaction with high performance and speed. DeepRFC is designed to receive Simplified Molecular-Input Line-Entry System (SMILES) strings of a reactant pair, which is defined as a substrate and a product of a reaction, as an input, and evaluates whether the input reaction is feasible. A deep neural network is selected for DeepRFC as it leads to better classification performance than five other representative machine learning methods examined. For validation, the performance of DeepRFC is compared with another in-house reaction feasibility checker that uses the concept of reaction similarity. Finally, the use of DeepRFC is demonstrated for the retrobiosynthesis-based design of novel one-carbon assimilation pathways. DeepRFC will allow retrobiosynthesis to be more practical for metabolic engineering applications by efficiently screening a large number of retrobiosynthesis-derived enzymatic reactions. DeepRFC is freely available at https://bitbucket.org/kaistsystemsbiology/deeprfc .     

链霉菌来源D-甘露糖异构酶的性质及其在制备D-甘露糖中的应用

【背景】D-甘露糖具有多种功能活性,在食品、医药、饲料等行业应用广泛。D-甘露糖异构酶可以催化D-果糖与D-甘露糖之间的相互转化,在D-甘露糖的酶法制备中具有应用潜力。【目的】克隆一个链霉菌(Streptomycessp.)来源的D-甘露糖异构酶基因(ssMIaseA)并在大肠杆菌中表达,研究其酶学性质,并用于制备D-甘露糖。【方法】从链霉菌(Streptomycessp.)中发掘一个D-甘露糖异构酶基因(ssMIaseA),构建重组表达质粒pET-28a-ssMIaseA并在大肠杆菌BL21(DE3)中表达,经Ni-NTA亲和层析纯化后测定酶学性质,利用高效液相色谱对SsMIaseA制备D-甘露糖进行研究。【结果】SsMIaseA与嗜热裂孢菌(Thermobifda fusca)来源的D-甘露糖异构酶ManI相似性最高,为60.2%。该酶比酶活为525 U/mg,分子量约为45 kD,最适pH和温度分别为7.5和45°C,在pH 6.5-10.0范围内和45°C以下保持稳定。该酶对甘露糖具有最高催化活性,其次是果糖、塔罗糖和塔格糖。利用SsMIaseA转化600 g/L D-果糖,反应8 h达到平衡,生成185 g/L D-甘露糖,转化率为31%。【结论】SsMIaseA作为新型D-甘露糖异构酶为D-甘露糖的酶法制备奠定了基础。