4-氯乙酰乙酸乙酯羰基还原酶产生菌的筛选及产酶条件研究

从实验室保藏的菌株中,筛选到一株立体选择性较高的产4-氯乙酰乙酸乙酯(COBE)羰基还原酶的菌株———出芽短梗霉(Aureobasidiumpullulans)SW0202,菌体产酶条件研究表明,最佳的发酵培养基配方为:麦芽糖30.0g/L,酵母膏20.0g/L,蛋白胨3.0g/L,(NH4)2SO45.0g/L,KH2PO42.0g/L,MgSO4.7H2O0.7g/L,最适发酵温度及初始pH分别为:28°C和pH6.0。该菌在此条件下发酵培养24h,产菌丝体生物量16.78g干菌体/L,COBE羰基还原酶酶活力达到1007U/L。在COBE的转化反应中,产物S-CHBE的浓度达到10.12g/L,光学纯度>97%e.e.。     

表皮短杆菌ZJB-07021产R-酰胺酶培养条件的优化

采用响应面分析法(RSM)对R-酰胺酶产生菌Brevibacterium epidermidis ZJB-07021的发酵培养基进行了优化.首先运用了单因子试验筛选出了发酵培养的最佳pH与温度,在此基础上采用Plackett-Burman(PB)设计法,对 8 种影响产酶的因素进行评价,实验结果表明,葡萄糖、酵母粉与乙酰胺含量对菌株产酰胺酶的活力具有显著的影响.通过旋转中心组合实验考察了葡萄糖、酵母粉和乙酰胺这三个主要因素对菌株所产酰胺酶活力的影响.发酵培养基优化结果为葡萄糖 17.00 g/L,酵母粉 15.74 g/L,乙酰胺 7.05 g/L,采用优化后的发酵培养条件进行摇瓶发酵培养,酰胺酶的酶活达到 72.14 U/L,比优化前的初始发酵培养条件下的酶活提高了73.3%.     

2-氧代-4-苯基丁酸乙酯还原酶产生菌筛选及产酶条件

研究了利用生物催化不对称还原的方法制备(R)-2-羟基-4-苯基丁酸乙酯[(R)-HPBE]。以2-氧代-4-苯基丁酸乙酯(OPBE)为底物,通过对实验室保藏菌株进行筛选,得到一株产物立体选择性较高的菌株G2ndida krusei SW2026,并对其发酵产酶条件进行研究。其最适的发酵培养基组成为4.5%葡萄糖,3%蛋白胨,1.5%牛肉膏,0.05%Mn~(2+);适宜的产酶发酵条件为初始pH 6.0,温度28℃,摇床转速180 r/min,发酵周期48 h。将此条件下发酵培养的菌体用于OPBE的不对称还原反应,产物(R)-HPBE的对映体过量值(e.e.)可达97.33%,产率最高达到72.54%。     

重组大肠杆菌产NAD激酶发酵条件的优化研究

本研究将重组大肠杆菌E.coli BL21(DE3)/p ET30α(+)-NADK作为NAD激酶生产菌种,对其产酶发酵培养基及发酵条件进行优化。采用Placket-Burman(PB)设计先筛选出影响重组菌产NAD激酶的三个主要因素:葡萄糖浓度、Mg SO4浓度和诱导表达时间,试验结果表明,增加葡萄糖和Mg SO4的浓度及缩短诱导表达时间对产酶有利。根据中心组合实验设计(Central Composite Design,CCD)原理,利用PB设计确定的这三个显著影响因素,通过最陡爬坡实验逼近最大响应区域,挑选出实验范围内的最优点,以此作为响应面中心组合设计的中心点,用NAD激酶酶活作为响应值,使用Design Expert 8.0软件设计中心组合实验,通过对实验数据进行分析,得出最佳发酵培养基成分及发酵条件为:葡萄糖14.24 g/L、酵母粉8 g/L、胰蛋白胨8 g/L、Mg SO40.94 g/L、Na Cl 5g/L、NH4Cl 2 g/L、KH2PO42 g/L、K2HPO49 g/L,诱导表达时间8.34 h,接种量2%。在此最佳条件下,NAD激酶酶活实验验证值可达10.17 U/mg,与优化前相比提高了2.77倍。对诱导表达结束后的细胞上清液进行SDS-PAGE分析也证明优化取得了显著的效果。     

4,4,4-三氟乙酰乙酸乙酯羰基还原酶产生菌的筛选及产酶条件研究

对实验室保藏菌种进行筛选,得到一株葡萄汁酵母Saccharomyces uvarum SW-58,对其产酶条件进行优化,其发酵培养基组成:醋酸钠60 g/L,玉米浆30 g/L,KH2PO46 g/L,MgSO4.7H2O 1 g/L;培养条件为:发酵温度30℃,初始pH 6.0,发酵周期36 h。4,4,4-三氟乙酰乙酸乙酯羰基还原酶酶活最高可达388.1 U/L,产物的浓度由优化前的3.5 g/L提高到4.6 g/L,所得产物的光学纯度由优化前的60.8%e.e.提高到85.0%e.e。     

聚-ε-赖氨酸高产菌株的筛选及其发酵工艺的初步研究

利用亚甲基蓝平板初筛、摇瓶复筛,从土壤中筛选了一株聚-ε-赖氨酸(ε-PL)高产菌株,ε-PL摇瓶产量大于2g/L,经初步鉴定该菌株为白色链霉菌(Streptomyces albulus)。对该菌株发酵生产ε-PL的培养基成分进行初步研究表明:葡萄糖是最好的碳源,酵母粉和硫酸铵是最佳的复合氮源,在优化培养基下,ε-PL摇瓶产量达到3.9g/L。     

Optimization of fermentation medium for amidase production by response surface methodology

采用响应面分析方法,对阿萨希丝孢酵母(Trichosporon asahii)ZZB-1产酰胺酶的发酵培养基进行了优化.运用单因子试验筛选出麦芽糖和酵母浸膏为最适碳源、氮源,金属离子Ca2+、Mn2+可提高发酵酰胺酶产量;通过最陡爬坡实验逼近以上4个因子的最大响应区域后,采用Box-Behnken响应面分析法,确定产酰胺酶最佳发酵培养基为麦芽糖18.84 g/L、酵母浸膏9.55 g/L、NaCl 5g/L、KH2 PO41g/L、MgSO4·7H2O 0.2 g/L、FeSO40.001g/L、CaCO370.84 μmol/L、MnSO4 65.39 μmol/L(1％丙烯酸诱导),NH4·H2O调节pH至7.0.培养基优化后酰胺酶产量由初始2554U/L提高到4156 U/L,为原始发酵培养基配方酶活产量的1.63倍.     

重组Bacillus subtilis产B.stearothermophilus环糊精葡萄糖基转移酶的发酵优化

利用本研究室已构建的重组菌Bacillus subtilis/pBSMuL3-α/β-CGTase对产B.stearothermophilus环糊精葡萄糖基转移酶的发酵产酶进行了优化,考察了培养基中重要成分:碳源、有机氮源、无机氮源、有机与无机氮源质量比、碳源与氮质量比、金属离子种类等单因素对该重组菌产α/β-CGTase的影响,并采用正交实验对发酵培养基进行优化,对优化结果分析可知,重组菌B.subtilis/pBSMuL3-α/β-CGTase发酵产α/β-CGTase的最优培养基成本为:葡萄糖5 g/L,氮源(鱼骨蛋白胨∶NH4Cl=3∶1)25 g/L,1 mmol/L Mg^2+。在最优条件下发酵培养,α/β-CGTase的酶活由原来TB发酵培养基的9.20 U/mL提高至20.32 U/mL,是优化前酶活的2.2倍,为α/β-环糊精葡萄糖基转移酶的工业应用提供了理论支持。     

4-丁内酰胺水解酶法制备γ-氨基丁酸

以吡咯烷酮为唯一碳源,从采集的土样中分离、筛选得到具有4-丁内酰胺水解酶活性的菌株.采用响应面分析法对该菌株的产酶培养基组分进行了优化研究并对酶促转化反应条件也进行了研究.结果表明:编号为HHSW-16的菌株水解活性最高.优化后的培养基组成为:葡萄糖11.50 g·L-1,牛肉膏6.35 g·L-1,酵母粉5.58 g...     

谷氨酸棒状杆菌发酵生产L-瓜氨酸及发酵条件优化

以代谢控制发酵理论为指导,重点对C.glutamicum 366菌株进行摇瓶发酵条件的优化。应用响应面法优化发酵培养基的配比,优化后的发酵培养基:葡萄糖63.33 g/L、精氨酸196.96 mg/L、(NH4)2SO445.79 g/L、生物素35.72μg/L、K2HPO4·3H2O 1.0 g/L、KH2PO41.0 g/L、Mg SO4·7H2O、0.25 g/L、Mn SO4·H2O 0.02 g/L、Fe SO4·7H2O 0.02g/L、Zn Cl21 mg/L、Cu SO40.2 mg/L、VB1200μg/L、Ca CO330 g/L。摇瓶发酵培养条件:温度30℃、摇床转速200r/min、初始p H 7.0。在此发酵条件下,菌株进行摇瓶发酵72 h,产L-瓜氨酸14.96 g/L,相比优化之前提高了75.8%。     

Efficient biocatalytic synthesis of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol by a newly isolated Trichoderma asperellum ZJPH0810 using dual cosubstrate: ethanol and glycerol

(R)-[3,5-bis(trifluoromethyl)phenyl] ethanol is a crucial intermediate for the synthesis of Aprepitant. An efficient biocatalytic process for (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol was developed via the asymmetric reduction of 3,5-bis(trifluoromethyl) acetophenone, catalyzed by whole cells of newly isolated Trichoderma asperellum ZJPH0810 using ethanol and glycerol as dual cosubstrate for cofactor recycling. A fungal strain ZJPH0810, showing asymmetric biocatalytic activity of 3,5-bis(trifluoromethyl) acetophenone to its corresponding (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol, was isolated from a soil sample. Based on its morphological and physiological characteristics and internal transcribed spacer sequence, this isolate was identified as T. asperellum ZJPH0810, which afforded an NADH-dependent (R)-stereospecific carbonyl reductase and was a promising biocatalyst for the synthesis of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol. Some key reaction parameters involved in the bioreduction catalyzed by T. asperellum ZJPH0810 were subsequently optimized. The effectiveness of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol production was significantly enhanced by employing a novel dual cosubstrate-coupled system for cofactor recycling. The established efficient bioreduction system contained 50 mM of 3,5-bis(trifluoromethyl) acetophenone and 60 g l^?1 of resting cells, employing ethanol (6.0 %, v/v) and glycerol (0.5 %, v/v) as dual cosubstrate. The bioreduction was performed in distilled water medium, at 30 °C and 200 rpm. Under the above conditions, a best yield of 93.4 % was obtained, which is nearly a 3.5-fold increase in contrast to no addition of cosubstrate. The ee value of the product reached above 98 %. This biocatalytic process shows great potential in the production of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol, a valuable chiral building block in the pharmaceutical industry.     

Asymmetric biocatalytic reduction of 3,5-bis(trifluoromethyl) acetophenone to (<Emphasis Type="Italic">1R</Emphasis>)-[3,5-bis(trifluoromethyl)phenyl] ethanol using whole cells of newly isolated <Emphasis Type="Italic">Leifsonia xyli</Emphasis> HS0904

A novel bacterial strain HS0904 was isolated from a soil sample using 3,5-bis(trifluoromethyl) acetophenone as the sole carbon source. This bacterial isolate can asymmetrically reduce 3,5-bis(trifluoromethyl) acetophenone to (1R)-[3,5-bis(trifluoromethyl)phenyl] ethanol with high enantiometric excess (ee) value. Based on its morphological, physiological characteristics, Biolog, 16S rDNA sequence and phylogenetic analysis, strain HS0904 was identified as Leifsonia xyli HS0904. To our knowledge, this is the first reported case on the species L. xyli exhibited R-stereospecific carbonyl reductase and used for the preparation of chiral (1R)-[3,5-bis(trifluoromethyl)phenyl] ethanol. The optimization of parameters for microbial transformation of 3,5-bis(trifluoromethyl) acetophenone to (1R)-[3,5-bis(trifluoromethyl)phenyl] ethanol catalyzed by whole cells of L. xyli HS0904 was carried out by examining some key factors including buffer pH, reaction temperature, shaking speed, substrate concentration, and reaction time. The obtained optimized conditions for the bioreduction are as follows: buffer pH 8.0, 70 mM of 3,5-bis(trifluoromethyl) acetophenone, 100 g l⁻¹ of glucose as co-substrate, 200 g l⁻¹ of resting cells as biocatalyst, reaction for 30 h at 30 °C and 200 rpm. Under above conditions, 99.4% of product ee and best yield of 62% were obtained, respectively. The results indicated that isolate L. xyli HS0904 is a novel potential biocatalyst for the production of (1R)-[3,5-bis(trifluoromethyl)phenyl] ethanol.     

In vitro inhibition of translation initiation by N,N'-diarylureas--potential anti-cancer agents

Symmetrical N,N'-diarylureas: 1,3-bis(3,4-dichlorophenyl)-, 1,3-bis[4-chloro-3-(trifluoromethyl)phenyl]- and 1,3-bis[3,5-bis(trifluoromethyl)phenyl]urea, were identified as potent activators of the eIF2α kinase heme regulated inhibitor. They reduce the abundance of the eIF2·GTP·tRNA(i)(Met) ternary complex and inhibit cancer cell proliferation. An optimization process was undertaken to improve their solubility while preserving their biological activity. Non-symmetrical hybrid ureas were generated by combining one of the hydrophobic phenyl moieties present in the symmetrical ureas with the polar 3-hydroxy-tolyl moiety. O-alkylation of the later added potentially solubilizing charge bearing groups. The new non-symmetrical N,N'-diarylureas were characterized by ternary complex reporter gene and cell proliferation assays, demonstrating good bioactivities. A representative sample of these compounds potently induced phosphorylation of eIF2α and expression of CHOP at the protein and mRNA levels. These inhibitors of translation initiation may become leads for the development of potent, non-toxic, and target specific anti-cancer agents.     

Synthesis of substituted 4(Z)-(methoxyimino)pentyl-1-piperidines as dual NK1/NK2 inhibitors

The NK1 and NK2 receptor activity of a series of 5-[(3,5-bis(trifluoromethyl)phenyl)methoxy]-3-(3,4-dichlorophenyl)-4(Z)-(methoxyimino)pentyl-1-piperidines was evaluated. Compounds 11d, 11e, 11f, 12a, and 12k were found to be our most potent inhibitors.     

Synthesis and NK1/NK2 receptor activity of substituted-4(Z)-(methoxyimino)pentyl-1-piperazines

A series of 5-[(3,5-bis(trifluoromethyl)phenyl)methoxy]-3-(3,4-dichlorophenyl)-4(Z)- (methoxyimino)pentyl-1-piperazines was prepared and their affinity for the NK1 and NK2 receptors investigated. Compounds 7f, 10o, 10r, and 10s were found to be our most potent inhibitors.     

Identification of a newly isolated Rhodotorula mucilaginosa NQ1 and its development for the synthesis of bulky carbonyl compounds by whole-cell bioreduction

A strain NQ1, which showed efficient asymmetric reduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to enantiopure (S)-[3,5-bis(trifluoromethyl)phenyl]ethanol ((S)-BTPE), which is the key intermediate for the synthesis of a receptor antagonist and antidepressant, was isolated from a soil sample. Based on its morphological and internal transcribed spacer sequence, the strain NQ1 was identified to be Rhodotorula mucilaginosa NQ1. Some key reaction parameters involved in the bioreduction catalyzed by whole cells of R. mucilaginosa NQ1 were subsequently optimized, and the optimized conditions for the synthesis of (S)-BTPE were determined to be as follows: 5·0 ml phosphate buffer (200 mmol l⁻¹, pH 7·0), 80 mmol l⁻¹ of BTAP, 250 g (wet weight) l⁻¹ of resting cell, 35 g l⁻¹ of glucose and a reaction for 18 h at 30°C and 180 rev min⁻¹. The strain NQ1 exhibited a best yield of 99% and an excellent enantiomeric excess of 99% for the preparation of (S)-BTPE under the above optimal conditions, and could also asymmetrically reduce a variety of bulky prochiral carbonyl compounds to their corresponding optical hydroxyl compound with excellent enantioselectivity. These results indicated that R. mucilaginosa NQ1 had a good capacity to reduce BTAP to its corresponding (S)-BTPE, and might be a new potential biocatalyst for the production of valuable chiral hydroxyl compounds in industry.     

4-Amino-2-alkyl-butyramides as small molecule CCR2 antagonists with favorable pharmacokinetic properties

A systematic examination of the central aromatic portion of the lead (2S)-N-[3,5-bis(trifluoromethyl)benzyl]-2-(4-fluorophenyl)-4-(1'H-spiro[indene-1,4'-piperidin]-1'-yl)butanamide (9) led to the discovery of a novel class of CCR2 receptor antagonists, which carry small alicyclic groups such as cyclopropyl, cylobutyl, or cyclopropylmethyl attached at C2 of the carbon backbone. The most potent compound discovered, namely (2S)-N-[3,5-bis(trifluoromethyl)benzyl]-2-cyclopropyl-4-[(1R,3'R)-3'-methyl-1'H-spiro[indene-1,4'-piperidin]-1'-yl]butanamide (29), showed very high binding affinity (IC50 = 4 nM, human monocyte) and excellent selectivity toward other related chemokine receptors. The excellent pharmacokinetic profile of this new lead compound allows for extensive in vivo evaluation.     

Dual neurokinin NK(1)/NK(2) antagonists: N-[(R,R)-(E)-1-arylmethyl-3-(2-oxo-azepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamides and 3-[N'-3,5-bis(trifluoromethyl)benzoyl-N-arylmethyl-N'-methylhydrazino]-N-[(R)-2-oxo-azepan-3-yl]propionamides.

Based on the structure of N-[(R,R)-(E)-1-(4-chlorobenzyl)-3-(2-oxoazepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamide (1), attempts to improve the NK(2) affinity have resulted in the discovery of N-[(R,R)-(E)-1-(3,4-dichlorobenzyl)-3-(2-oxoazepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamide (9, DNK333) exhibiting a 5-fold improved affinity to the NK(2) receptor in comparison to 1. Simplification of the structure via elimination of a chiral centre led to 3-[N'-3,5-bis(trifluoromethyl)benzoyl-N-(3,4-dichlorobenzyl)-N'-methylhydrazino]-N-[(R)-2-oxo-azepan-3-yl]propionamide (22), a potent and fairly balanced NK(1)/NK(2) antagonist.     

An effective route to fluorine containing asymmetric alpha-aminophosphonates using chiral Bronsted acid catalyst

Asymmetric addition of dialkyl phosphites (--CH2CH3, --CH2CH2CH3, --CH(CH3)2, --CH2(CH2)3CH3, --CH2CH2OCH3 and --CH2CH2OC2H5) induced by chiral organocatalyst e.g. (R)- and (S)-3,3'-[3,5-bis(trifluoromethyl)phenyl]2-1,1'-binaphthyl phosphate on fluorinated aldimines derived from cinnamaldehyde has been found effective to give new bioactive alpha-aminophosphonates in good yields (58-73%) and high enantiomeric excess (64.6%-90.6%) under mild conditions.     

New chromogenic agar medium for the identification of Candida spp

A new chromogenic agar medium (Candida diagnostic agar [CDA]) for differentiation of Candida spp. is described. This medium is based on Sabouraud dextrose agar (Oxoid CM41) and contains (per liter) 40.0 g of glucose, 10.0 g of mycological peptone, and 15.0 g of agar along with a novel chromogenic glucosaminidase substrate, ammonium 4-(2-[4-(2-acetamido-2-deoxy-beta-D-glucopyranosyloxy)-3-methoxyphenyl]-vinyl)-1-(propan-3-yl-oate)-quinolium bromide (0.32 g liter(-1)). The glucosaminidase substrate in CDA was hydrolyzed by Candida albicans and Candida dubliniensis, yielding white colonies with deep-red spots on a yellow transparent background after 24 to 48 h of incubation at 37 degrees C. Colonies of Candida tropicalis and Candida kefyr were uniformly pink, and colonies of other Candida spp., including Candida glabrata and Candida parapsilosis, were white. CDA was evaluated by using 115 test strains of Candida spp. and other clinically important yeasts and was compared with two commercially available chromogenic agars (Candida ID agar [bioMerieux] and CHROMagar Candida [CHROMagar Company Ltd.]). On all three agars, colonies of C. albicans were not distinguished from colonies of C. dubliniensis. However, for the group containing C. albicans plus C. dubliniensis, both the sensitivity and the specificity of detection when CDA was used were 100%, compared with values of 97.6 and 100%, respectively, with CHROMagar Candida and 100 and 96.8%, respectively, with Candida ID agar. In addition, for the group containing C. tropicalis plus C. kefyr, the sensitivity and specificity of detection when CDA was used were also 100%, compared with 72.7 and 98.1%, respectively, with CHROMagar Candida. Candida ID agar did not differentiate C. tropicalis and C. kefyr strains but did differentiate members of a broader group (C. tropicalis, C. kefyr, Candida lusitaniae plus Candida guilliermondii); the sensitivity and specificity of detection for members of this group were 94.7 and 93.8%, respectively. In addition to the increased sensitivity and/or specificity of Candida detection when CDA was used, differentiation of colony types on CDA (red spotted, pink, or no color) was unambiguous and did not require precise assessment of colony color.