Biocatalytic synthesis of (R)-(-)-mandelic acid from racemic mandelonitrile by a newly isolated nitrilase-producer Alcaligenes sp. ECU0401

By using acetonitrile as the sole nitrogen source, a microbial strain with high nitrilase activity, named as Alcaligenes sp. ECU0401, was newly isolated from soil, which could enantioselectively transform racemic mandelonitrile into (R)-(?)-mandelic acid, with an enantiomeric excess of >99.9%.

Kinetics of Asymmetric Reduction of Phenylglyoxylic Acid to R-(-)-Mandelic Acid by Saccharomyces Cerevisiae FD11b

The kinetics of asymmetric production of R-（-）-mandelic acid （R-MA） from phenylglyoxylic acid （PGA） catalyzed by Saccharomyces cerevisiae sp. strain FD11b was studied by fed-batch cultures. The concentrations of glucose and PGA were controlled respectively with a dual feeding system. When the electron donor glucose was supplied at the rate of 0.0833mmol·gdw^-1·h^-1, the specific production rate （qp） and the enantiomeric excess of R-MA reached the maximum 0.353mmol·gdw^-1·h^-1 and 97.1%, respectively. The apparent reduction activity of yeast FD 11 b was obviously affected by both substrate PGA and product MA. The qp value reached the maximum 0.36-0.38mmol·gdw^-1·h^-1 when the PGA concentration was controlled between 25 and 35mmol·L^-1. The obvious substrate inhibition of bioconversion was observed at the PGA concentrations higher than 40mmol·L^-1. The accumulation of product MA also caused a severe feed-back inhibition for its production when the product concentration was above 60mmol·L^-1. The kinetic model with the inhibition effect of both substrate and product was simulated by a computer-based least-square arithrnatic. The established kinetic model was in good agreement with the experimental data.

IMMOBILIZATION OF Saccharomyces Cerevisiae USING POLY(ACRYLAMIDE)GEL FOR ASYMMETRIC SYNTHESIS OF R(-)-MANDELIC ACID

In this paper,the poly(acrylamide)hydrogel used to immobilize saccharomyces cerevisiae for asymmetric synthesis of R(-)-mandelic acid was prepared with free radical ploymerization in deionized water at room temperature under nitrogen atmosphere.The influence of the composition of hydrogel,loading amount of cells and culture conditions on the asymmetric synthesis was investigated.Results show that PAAm hydrogel is a feasible carrier for immobilization of cells which is a potential alternative method to prepare enantiomerically pure R(-)-mandelic acid.

L-LACTIC ACID FERMENTATION IN A ROTATING-DISC CONTACTOR WITH SIMULTANEOUS PRODUCT SEPARATION BY ION-EXCHANGE

A rotating disk contactor（RDC）was designed to perform L-lactic acid fermentation with afilamentous fungi,Rhizopus oryzae,which was immobilized on the surfaces of the rotating discs.Thebioreactor was operated using repeated-batch method as well as continuous feeding method.Ananionic resin,D354,slightly basic in nature and of high selectivity and capacity was chosen for lacticacid separation.A coupled process of L-lactic acid fermentation and ion-exchange separation wasevaluated experimentally.The results indicated that the pH value of the fermentation broth could bemaintained at about 3-3.5 without any addition of alkali.The conversion ratio of glucose to L-lacticacid was about 0.7 g·g-1 and the fermentation rate reached as high as 62.5 g·h-1·m-2.

Repeated-Batch and Continuous Production of L-Lactic Acid by Rhizopus oryzae Immobilized in Calcium Alginat Beads:ReactorPerformance and Kinetic Model

Repeated-batch and continuous production of L-lactic acid by immobilized Rhizopusoryzae with calcium alginate entrapment method in a three-phase fluidized-bed bioreactor was stud-ied.The operation conditions were optimized.The productivity based on total reactor volume wasabout 3 times higher than that with free cells in a traditional stirred tank bioreactor.A mathemat-ical model was proposed and the model predictions were in good agreement with the experimentaldat.

Identification and Mutagenesis of a New Isolated Strain Bacillus sp.B26 for Producing （R）-α-Hydroxyphenylacetic Acid

A bacterium strain B26 capable of producing(R)-α-hydroxyphenylacetic acid [(R)-HPA](yield,47.5%;enantiomeric excess,99.1%) from phenylglyoxylic acid(PGA) with high optical purity was isolated and identified as Bacillus sp.B26 by 16S rDNA(ribosomal DNA) sequencing.Phylogenic analysis showed that the strain was most similar to Bacillus sp.enrichment culture clone SYW5(FJ601635.1) and Bacillus cereus strain FM-4(EU794727.1).Efforts were made to further improve HPA-production and PGA-tolerance by UV irradiation and UV-LiCl cooperative mutagenesis.Among viable mutants,B.sp.UV-38 and B.sp.ULi-11 exhibited better productivities than the wild type.Comparisons of HPA production and time course among wild strain and two mutants showed that B.sp.ULi-11 was more competent than B.sp.UV-38.HPA production was increased by 39.1% with B.sp.ULi-11(yield,65.4%) compared to that with B.sp.B26(yield,47.0%) when cultured in fermentation broth(pH 7.2) at 32℃ with an agitation speed of 180 r·min-1 and PGA final concentration of 15 mmol·L-1 for 25 h.

Kinetic and Thermodynamic Studies of Acid Scarlet 3R Adsorption onto Low-cost Adsorbent Developed from Sludge and Straw

A low-cost adsorbent was prepared from sludge and straw by pyrolysis in a dried state with the surface area of the adsorbent of 829.49 ma. g-l, micropore volume of 0.176 cm2·g-1 and average pore radius of 5.0 nm. The kinetic, equilibrium isotherm and thermodynamic characteristics of trisodium 1-（1-naphthylazo）-2-hydroxynaphthalene- 4＇,6,8-trisulphonate （acid scarlet 3R） onto the adsorbent from sludge and straw were investigated. The results indicated that the pseudo second order adsorption was the predominant adsorption mechanism of acid scarlet 3R. Thus, the adsorption phenomenon was suggested as a chemical process. The adsorption data were fitted better with Langmuir model than Freundlich model, indicating that the adsorption of acid scarlet 3R belonged to the monolayer adsorption and mainly occurred in micropores.

A Novel Method for Production of 3-Hydroxydecanoic Acid by Recombinant Escherichia coli and Pseudomonas putida

3-hydroxydecanoic acid (3HD) is an interesting intermediate for chemical synthesis of many valuable compounds. A novel method to produce 3HD by recombinant bacteria was constructed in Escherichia coli HB101 and Pseudomonas putida GPpl04, respectively. Simultaneous expression of both phaG encoding (R)-3-hydroxydecanoyl-ACP:CoA transacylase and tesB encoding thioesteraseⅡin E. coli HB101 increased 3HD production approximate 1.7-folds compared with the expression of phaG gene alone under identical conditions. In addition, when the tesB gene was introduced into the strain, the polyhydroxyalkanoate synthase negative strain P. putida GPpl04 produced extracellular 3HD. Thus, a novel pathway to produce 3HD by recombinant Pseudomonas was constructed. It was also found that the ratio of carbon source to nitrogen source affected the production of 3HD by recombinant P. putida harboring tesB gene. Nitrogen limitation seemed to promote the extracellular 3HD production.

Asymmetric bioreduction of γ- and δ-keto acids by native carbonyl reductases from Saccharomyces cerevisiae

Optically pure(R)-γ-and(R)-δ-lactones can be prepared by intramolecular cyclization of chiral hydroxy acids/esters reduced asymmetrically from γ-and δ-keto acids/esters using Saccharomyces cerevisiae(S.cerevisiae) as a whole-cell biocatalyst.However,some of the enzymes catalyzing these reactions in S.cerevisiae are still unknown up to date.In this report,two carbonyl reductases,OdCRl and OdCR2,were successfully discovered,and cloned from S.cerevisiae using a genome-mining approach,and overexpressed in Escherichia coli(E.coli).Compared with OdCR1,OdCR2 can reduce 4-oxodecanoic acid and 5-oxodecanoic acid asymmetrically with higher stereoselectivity,generating(R)-γ-decalactone(99% ee) and(R)-δ-decalactone(98% ee) in 85% and 92%yields,respectively.This is the first report of native enzymes from S.cerevisiae for the enzymatic synthesis of chiral γ-and δ-lactones which is of wide uses in food and cosmetic industries.

Enantioselective esterification of(R,S)-2-(4-methylphenyl)propionic acid via Novozym 435:Optimization and application

This paper reports on the resolution of(R,S)-2-(4-methylphenyl)propionic acid(MPPA)enantiomers by enzymatic esterification in organic solvent.Novozym 435(CALB)has the best catalytic performance compared with other lipases.Of the alcohols screened,n-hexanol is the best acyl acceptor and gives the highest enzyme activity and enantioselectivity in n-hexane.Response surface methodology(RSM)was used to evaluate the influence of the factors,such as temperature,enzyme amount,substrate concentration and reaction time on the substrate conversion(c)and enantiomeric excess(ee).The correlation coefficient R2 for enantiomeric excess and the conversion are 0.9827 and 0.9910,respectively,indicating that can accurately predict the experimental results.By simulation and optimization,the optimal conditions were obtained,involving 600 mmol·L^-1 MPPA concentration(0.60 mmol),850 mmol·L^-1 hexanol concentration(0.85 mmol),58 mg enzyme amount,75℃temperature and 4.5 h reaction time,respectively.Under the optimized conditions,the experimental values of conversion and enantiomeric excess were 89.34%and 97.84%,respectively,which are in good agreement with the model predictions.

Role of miR-124 in the regulation of retinoic acid-induced Neuro-2A cell differentiation

Retinoic acid can cause many types of cells,including mouse neuroblastoma Neuro-2 A cells,to differentiate into neurons.However,it is still unknown whether microRNAs(miRNAs)play a role in this neuronal differentiation.To address this issue,real-time polymerase chain reaction assays were used to detect the expression of several differentiation-related miRNAs during the differentiation of retinoic acid-treated Neuro-2 A cells.The results revealed that miR-124 and miR-9 were upregulated,while miR-125 b was downregulated in retinoic acid-treated Neuro-2 A cells.To identify the miRNA that may play a key role,miR-124 expression was regulated by transfection of miRNA mimics or inhibitors.Morphological analysis results showed that inhibition of miR-124 expression reversed the effects of retinoic acid on neurite outgrowth.Moreover,miR-124 overexpression alone caused Neuro-2 A cells to differentiate into neurons,and its inhibitor could block this effect.These results suggest that miR-124 plays an important role in retinoic acid-induced differentiation of Neuro-2 A cells.

Synthesis of (R)(E)-3,7-Dimethyl-2-octene-1,8-dioic Acid, a Copulation Released Pheromone Component of Azuki Bean Weevil

Started from 5-hydroxy-2-pentanone, (R)(E)-3,7-dimethyl-2-octene-1,8-dioic acid, callosobruchusic acid, was synthesized via five steps with D-(-)-camphor sultam as the chiral auxiliary. It was of good optical purity and yield.

Study on Bicoordination Chiral Inducement Catalysis of (R)-4-Thiazolidinecarboxylic Acid

The bicoordination chiral inducement catalysis of (R)-4-thiazolidinecarboxylic acid inthe enantioselective borane reduction of prochiral ketones has been studied. The fact that theabsolute configuration of the main product can be changed by simply using different operationmethods was firstly verified. And the reason of bicoordination chiral inducement was discussed.

Synthesis,Characterization and Structure of Chiral Amino Acids and Their Corresponding Amino Alcohols with Camphoric Backbone

Chiral amino acids and their corresponding amino alcohols bearing camphoric backbone were prepared from D-（＋）-camphoric imide and characterized by infrared, elemental analysis, ESI-MS, and NMR measurements. Among them, one intermediate （1S,3R）-3-amino-2,2,3- trimethyl cyclopentane-1-carboxylic acid hydrochloride 3 was structurally elucidated by X-ray diffraction techniques. Versatile intermolecular hydrogen bonding interactions observed in its packing structure result in a two-dimensional framework.

茯苓酸对人骨肉瘤细胞系MG-63的增殖凋亡、迁移侵袭影响及PERK/CCAAT信号通路调控作用

目的观察茯苓酸(PA)对人骨肉瘤细胞系MG-63增殖、迁移、凋亡和侵袭的影响,并探讨PA对蛋白激酶R样内质网激酶(PERK)/CCAAT增强子结合蛋白同源蛋白(CHOP)信号通路的调控作用。方法体外培养MG-63细胞,分为对照组、低浓度PA组(PA-L组)、中浓度PA组(PA-M组)、高浓度PA组(PA-H组)、高浓度PA+蛋白激酶R样内质网激酶(PERK)抑制剂GSK2606414组(PA-H+GSK2606414组),PA-L组、PA-M组及PA-H组分别用含20、40及80μmol/L PA的培养基培养,PA-H+GSK2606414组用含80μmol/L的PA和5μmol/L的GSK2606414培养基培养,对照组用空白培养基培养。分别于培养24、48、72 h时采用MTT法观察各组细胞增殖情况;培养24 h时采用细胞划痕实验观察各组细胞迁移情况;培养48 h时采用Transwell实验观察各组细胞侵袭情况;培养48 h时采用流式细胞术测算各组细胞凋亡率;培养48 h时,采用RT-qPCR法检测PERK及CHOP的mRNA表达,并采用WESTERN Blotting法检测PERK/CHOP信号通路相关蛋白及凋亡相关蛋白真核生物起始因子2α(eIF2α)、活化转录因子4(ATF4)、CHOP、BCL2相关X蛋白(Bax)及B淋巴细胞瘤-2(Bcl-2)。结果与对照组比较,PA-M组及PA-H组培养48与72 h时细胞OD_(570nm)值低、细胞迁移率低、细胞侵袭数少、细胞凋亡率高(P均<0.05);与PA-M组相比,PA-H组培养48及72 h时的细胞OD_(570nm)值低、细胞迁移率低、细胞侵袭数少、细胞凋亡率高(P均<0.05);与PA-H组相比,PA-H+GSK2606414组细胞培养48及72 h时的OD_(570nm)值高、细胞迁移率高、细胞侵袭数多、细胞凋亡率低(P均<0.05)。与对照组比较,PA-M组及PA-H组细胞PERK及CHOP mRNA相对表达量高,PERK及eIF2α磷酸化水平高,ATF4、CHOP、Bax蛋白相对表达量高,Bcl-2相对表达量低(P均<0.05);与PA-M组相比,PA-H组细胞PERK及CHOP mRNA相对表达量高,PERK及eIF2α磷酸化水平高,ATF4、CHOP、Bax蛋白相对表达量高,Bcl-2相对表达量低(P均<0.05);与PA-H组相比,PA-H+GSK2606414组细胞PERK及CHOP mRNA相对表达量低,PERK及eIF2α磷酸化水平低,ATF4、CHOP、Bax蛋白相对表达量低,Bcl-2相对表达量高(P均<0.05)。结论PA可抑制MG-63细胞的增殖、迁移及侵袭,促进细胞凋亡。PA可能通过激活PERK/CHOP信号通路,抑制MG-63细胞的增殖迁移及侵袭,促进MG-63细胞的凋亡。

天冬氨酸酶体系中R-3-氨基丁酸相关物质HPLC方法研究

为了建立一种高效被大众认可的满足酶促体系中R-3氨基丁酸及相关物质的分析方法。基于显色法、衍生法本文探究了高效液相色谱UV检测器210 nm下采用特定流动相及检测参数的HPLC外标法。结果表明在选定的色谱条件下,R-3-氨基丁酸与相关物质可以和酶促反应中其它物质得到很好的分离,反应过程中底物巴豆酸在浓度0.05~0.4 g/L范围线性良好,R2大于3个9,产物R-3-氨基丁酸在此检测条件下灵敏度好,检测限、定量限符合规定,浓度0.35~2 g/L范围线性良好,R2大于3个9。该方法同时满足原辅料、催化反应、纯化结晶、成品含量及杂质检测。

酸性红R在UV-类Fenton体系中的光降解实验

本论文选用了偶氮染料ARR为主要研究对象,研究它在UV-类Fenton体系中的光催化降解行为。采用紫外杀菌灯(λ≥254 nm)为光源,光化学反应实验在自制的光催化反应装置中进行。光照反应时间50 min,间隔相同时间取样,采用分光光度法检测ARR在光催化降解过程中的浓度变化,系统地研究了ARR的初始浓度、反应体系pH值、蒙脱土/H_(2)O_(2)配比、以及NO_(3)^(-)、EDTA、Cl^(-)、丙酮酸钠等对偶氮染料ARR光催化降解的影响。结果表明:在pH=3.0、66 mg/L的30%H_(2)O_(2)和浓度为1 g/L的蒙脱土条件下,ARR在UV-类Fenton体系可以获得较高的反应速率,NO_(3)-、丙酮酸钠对ARR的光催化降解有促进作用,而Cl^(-)、EDTA对ARR的光催化降解有抑制作用。

R(-)-扁桃酸脱氢酶产生菌的筛选

选用酵母、乳酸菌、粪链球菌、假丝酵母等 4个种属 18种菌株 ,研究其对苯乙酮酸和苯乙酮酸甲酯的不对称还原催化特性。筛选出一株酵母sp .strainby1,对两种底物皆具有较高的转化活性和光学选择性。底物浓度为 10mmol L ,微生物转化 72h后 ,扁桃酸的得率 80 .83% ,对映体过量值e .e .达 95 .5 % ;微生物转化 2 8h后 ,扁桃酸甲酯的得率 92 .11% ,e .e .值达 91.1%。

生物不对称合成R-(-)-扁桃酸的影响因素

研究了酵母细胞(Saccharomyces cerevisiae)生物不对称还原苯乙酮酸合成R-(-)-扁桃酸的过程中,环境因子对底物的转化效率和产物对映体过量值的影响. 结果表明,高浓度的底物苯乙酮酸对酵母细胞的催化还原活性具有较显著的抑制效应. pH 6.5、温度32oC、严格厌氧为较适宜条件,底物苯乙酮酸的转化率和产物扁桃酸的得率分别可达97.0%和96.1%,R-(-)-扁桃酸的对映体过量值(ee)为95.1%.

β-环糊精接枝壳聚糖对酸性红R的吸附研究

为改善壳聚糖的吸附性能,用苯甲醛保护壳聚糖氨基,通过与环氧氯丙烷和β-环糊精的反应合成了新的吸附剂β-环糊精接枝壳聚糖,研究了其对偶氮染料酸性红R的吸附行为,考察了酸性红R的初始浓度、pH对吸附效果的影响。结果发现当酸性红R的浓度≤100mg/g时,合成物的吸附量随酸性红R浓度的增大而增加,当浓度进一步增大时,吸附量趋于平衡。合成物对酸性红R的吸附效果在pH为2～4之间最好。相比于Freundlich吸附等温式(R2=0.8476),β-环糊精接枝壳聚糖对酸性红R的吸附规律明显更符合Langmuir吸附等温式(R2=0.9977)。吸附动力学采用了准一级速率方程(R2=0.8609)和准二级速率方程来描述,结果表明吸附动力学符合准二级速率方程(R2=0.9945)。β-环糊精接枝后的壳聚糖对酸性红R的平衡吸附容量可以提高20.2mg/g。