多酶融合表达体系以木聚糖为辅助底物高效合成(S)-苯乙二醇

[目的] 利用木聚糖为辅助底物加强手性催化反应中的辅酶循环,构建来源于近平滑假丝酵母（Candida parapsilosis）CCTCC M203011的（S）-羰基还原酶II（SCRII）、枯草芽孢杆菌（Bacillussp.）YX-1葡萄糖脱氢酶突变体Ala258Phe/GDH和里氏木霉（Trichoderma reesei）Rut C-30木聚糖酶（XYN2）在大肠杆菌（Escherichia coli）BL21（DE3）中的融合表达体系,高效合成（S）-苯乙二醇。[方法] 调节3种酶编码基因在pET-28a载体上的位置,运用重叠延伸PCR技术,构建了E.coli/pET-SCRII-A258F-XYN2和E.coli/pET-A258F-SCRII-XYN2两种重组菌,研究了其合成（S）-苯乙二醇的最适反应条件。[结果] 重组菌株E.coli/pET-SCRII-A258F-XYN2在底物2-羟基苯乙酮与辅助底物木聚糖的比例为1：1、35℃、pH为7.0条件下,（S）-苯乙二醇的产率达98.8%（W/W）;而重组菌株E.coli/pET-A258F-SCRII-XYN2在底物与辅助底物的比例为2：1、35℃、pH为7.0条件下,（S）-苯乙二醇的产率达95.6%（W/W）,两者合成产物的光学纯度均>99%。[结论] 通过构建3种酶的融合表达体系,成功将木聚糖酶和葡萄糖脱氢酶突变体介导的辅酶再生循环体系引入不对称生物合成反应,提高了手性转化效率,为将大自然中丰富的木聚糖用于手性催化奠定了较扎实的研究基础。     

酿酒酵母B5不对称还原制备手性药物中间体R-2′-氯-1-苯乙醇的研究

从 11株微生物中筛选出 4株具有不对称还原 2′ 氯 苯乙酮能力的酵母 ,其中酿酒酵母B5的还原产率与对映体选择性最佳。确定了酿酒酵母B5对 2′ 氯 苯乙酮还原的最佳反应时间为 2 4h ;最佳pH 8 0 ;最佳反应温度为2 5℃ ;最佳共底物为 5 % (体积比 )乙醇。同时研究了底物浓度、微生物的量、微生物的培养条件等对反应产率和立体选择性的影响。细胞浓度为 10 75mg mL(细胞干重 反应体积 )的酿酒酵母B5可将 6 47mmol L的 2′ 氯 苯乙酮10 0 %地转化为R 2′ 氯 1 苯乙醇 ,其对映体选择性为 10 0 %。酿酒酵母B5可重复利用的特点可提高产物的产量。     

酿酒酵母B5不对称还原制备手性药物中间体R-2′-氯-1-苯乙醇的研究

从11株微生物中筛选出4株具有不对称还原2′-氯-苯乙酮能力的酵母,其中酿酒酵母B5的还原产率与对映体选择性最佳。确定了酿酒酵母B5对2′-氯-苯乙酮还原的最佳反应时间为24h;最佳pH 8.0;最佳反应温度为25℃;最佳共底物为5%（体积比）乙醇。同时研究了底物浓度、微生物的量、微生物的培养条件等对反应产率和立体选择性的影响。细胞浓度为10.75mg/mL（细胞干重/反应体积）的酿酒酵母B5可将647mmol/L的2′-氯-苯乙酮100%地转化为R-2′-氯-1-苯乙醇,其对映体选择性为100%。酿酒酵母B5可重复利用的特点可提高产物的产量。     

羰基还原酶产生菌Candida ontarioensis制备(R)-2-氯-1-(3-氯苯基)乙醇

从实验室保藏的菌株中筛选获得Candida sp.PT2A,并通过18S rRNA鉴定为安大略假单胞菌Candida on-tarioensis。对C.ontarioensis不对称还原合成(R)-2-氯-1-(3-氯苯基)乙醇的发酵产酶条件和转化条件进行优化,确定了最适的发酵产酶条件和转化条件:温度30℃,初始pH 6.5,摇床转速180 r/min,菌体质量浓度200 g/L。采用2-氯-1-(3-氯苯基)乙酮质量浓度为10 g/L时,还原反应72 h,(R)-2-氯-1-(3-氯苯基)乙醇的e.e.值为99.9%,产率为99%;底物质量浓度提高至30 g/L时,产率下降为84.3%。采用十六烷基三甲基溴化铵(CTAB)对C.ontarioensis细胞进行通透性处理(CTAB g/L,4℃下处理20 min),在30 g/L底物下反应24 h,产物的e.e.和产率分别达到99.9%和97.5%。     

YPS1/YPS2失活改进酿酒酵母An-α菌株β-葡萄糖苷酶分泌表达

[背景] 工业酵母菌株的蛋白质表达通常存在表达量低、分泌效率低的问题。[目的] 考察失活Yapsin蛋白酶Yps1p和Yps2p对β-葡萄糖苷酶在酿酒酵母An-α菌株中表达的影响。[方法] 利用CRISPR/Cas9基因组编辑技术,首先构建得到未折叠蛋白响应（Unfolded Protein Response,UPR）指示菌株An-α（leu2：：UPRE-lacZ）即An-αL,然后分别失活其YPS1和YPS2基因,导入以YEplac195为载体的β-葡萄糖苷酶表达质粒（简称BG）,进行生长和酶活分析评价。[结果] 菌株An-αL的YPS1和YPS2基因失活对其在酵母浸出粉胨葡萄糖（Yeast Extract Peptone Dextrose,YPD）培养基中的生长未造成明显的不利影响;导入质粒BG后将在酵母浸出粉胨纤维二糖（Yeast Extract Peptone Cellobiose,YPC）培养基中生长的最大OD₆₀₀分别提高了21.9%和7.4%;最大总酶活值为0.087 5和0.068 6 U/（mL·OD₆₀₀）,是对照菌株相应值的2.268倍和1.778倍;分泌比例提高了19.4%和22.2%;β-葡萄糖苷酶表达水平与β-半乳糖苷酶酶活水平所代表的UPR信号响应值之间呈现良好的相关性。[结论] YPS1和YPS2基因失活有助于改进酿酒酵母An-α菌株中β-葡萄糖苷酶的分泌表达。     

(R)-专一性羰基还原酶与甲酸脱氢酶基因在大肠杆菌中的共表达

【目的】通过研究(R)-专一性羰基还原酶和甲酸脱氢酶基因在大肠杆菌中的共表达,解决较高底物浓度下不对称转化反应的辅酶限制性问题。【方法】分别以近平滑假丝酵母(Candida parapsilosis CCTCC M203011)和博伊丁假丝酵母(Candida boidinii)基因组为模板,采用PCR方法扩增得到(R)-专一性羰基还原酶基因（rcr）和甲酸脱氢酶基因（fdh）,克隆到共表达载体pETDuetTM-1中进行表达。共表达质粒pETDuet-rcr-fdh转化稀有密码子优化型菌株E. coli Rosetta,获得重组菌E. coli Rosetta/pETDuet-rcr-fdh。【结果】在30℃条件下,经1 mmol/L IPTG诱导表达8 h后,SDS-PAGE结果表明(R)-专一性羰基还原酶和甲酸脱氢酶均有明显的表达,其相对分子质量分别为37 kDa和 40 kDa。以高浓度(6 g/L)2-羟基苯乙酮为底物时,0.1 g重组菌细胞催化产生(R)-苯基乙二醇,产物光学纯度为100% e.e.,产率为85.9%。与无甲酸脱氢酶参与辅酶再生循环的重组菌E. coli Rosetta/pETDuet-rcr相比,产物光学纯度和产率分别提高了1.3和2.7倍。【讨论】该重组菌的构建为基因工程法生物合成(R)-苯基乙二醇的工业应用奠定了基础。     

南海深海微生物酯酶EST12-7在制备(R)-2-氯丙酸乙酯中的应用研究

利用来源南海深海的微生物酯酶EST12-7不对称水解反应拆分制备（R）-2-氯丙酸乙酯。并探寻了温度、pH、底物浓度、有机溶剂和反应时间等因素对酯酶EST12-7催化制备（R）-2-氯丙酸乙酯的影响。结果表明,深海微生物酯酶EST12-7催化制备（R）-2-氯丙酸乙酯的最佳反应条件为：13.8 μg/ml酯酶EST12-7,50 mmol/L（±）-2-氯丙酸乙酯,2%正癸醇,pH8.5,30℃,0.05mol/L Tris-HCl,反应60 min。在最佳反应条件下,（±）-2-氯丙酸乙酯的转化率可达49%,所制备的（R）-2-氯丙酸乙酯的光学纯度为98%。通过对酯酶EST12-7拆分制备（R）-2-氯丙酸甲酯和（R）-2-氯丙酸乙酯进行比较,2-氯丙酸酯中的链长对酯酶EST12-7拆分反应有极大的影响。     

Peg3, Cdkn1c和 Gtl2基因在克隆绵羊和自然分娩绵羊中的DNA甲基化水平检测

尽管研究证明很多克隆动物存在DNA甲基化异常的情况,却很少有研究比较克隆绵羊与自然分娩绵羊之间的甲基化情况,可能是由于克隆绵羊的获得、绵羊基因组、绵羊基因组印记等因素的限制．本研究中,为了证明克隆绵羊重编程的状况,克隆了Peg3基因的差异甲基化区域(differential methylated region,DMR),并且分析了Peg3、Cdkn1c、Gtl2在克隆绵羊和自然分娩绵羊不同组织中的甲基化水平．研究发现,在克隆绵羊和自然分娩绵羊中Peg3呈现为超甲基化水平,在克隆绵羊的肾脏和肺脏中DNA甲基化水平为95.45%、81.18%,相对于正常分娩的绵羊组织中的98.18%、87.27%无显著性差异,而Cdkn1c在两组实验动物中的肾脏和肺脏中表现为非甲基化水平,分别为0%、0.53%、0.53%和0.53%,Gtl2则是低甲基化水平,并且克隆绵羊与正常分娩绵羊之间的DNA甲基化水平无显著性差异(r² = 0.77)．这些结果表明,Peg3、Cdkn1c、Gtl2三个印记基因在克隆绵羊和自然分娩绵羊组织中呈现类似甲基化水平,无显著性差异．     

基于谷氨酸消旋酶基因(murI)构建杀香鱼假单胞菌减毒活疫苗株的染色体-质粒平衡致死表达系统

【目的】构建一种基于谷氨酸消旋酶(MurI)基因的染色体-质粒平衡致死系统,用于杀香鱼假单胞菌减毒活疫苗株(Pseudomonas plecoglossicida ΔtssD-1, Pp ΔtssD-1)中表达外源抗原,为开发多联活疫苗提供新的思路和方法。【方法】利用同源重组技术,将亲本株Pp ΔtssD-1中的murI基因敲除,构建murI基因缺失突变株;将广宿主穿梭质粒pBBR1MCS-2的卡那霉素抗性基因替换为murI基因,构建平衡致死质粒(即无抗性回补质粒);在平衡致死质粒的多克隆位点处插入绿色荧光蛋白以检测外源抗原是否稳定表达,对重组菌株进行生物学特性分析,包括生长曲线、质粒稳定性和外源抗原表达水平。【结果】murI基因缺失株在不含D-谷氨酸的LB培养基上无法生长;无抗性回补株在不含D-谷氨酸的LB培养基上恢复了生长能力,但生长速度低于亲本株;经鉴定外源抗原可在无抗性质粒中稳定表达,并可在荧光显微镜下观察到明显的绿色荧光信号;此外,平衡致死质粒在重组菌株中具有良好的遗传稳定性。【结论】本研究以murI为靶点构建了新型的染色体-质粒平衡致死系统,可在无抗性筛选条件下在Pp ΔtssD-1中表达外源抗原,为开发多联活疫苗提供了新的策略和方法。     

乙醇脱氢酶与葡萄糖脱氢酶偶联催化制备(S)-1-(2,6-二氯-3-氟苯基)乙醇

(S)-1-(2,6-二氯-3-氟苯基)乙醇是抗癌药物克唑替尼的手性合成前体,可由2,6-二氯-3-氟苯乙酮经乙醇脱氢酶催化还原制备,还原中所需的还原型辅酶Ⅱ再生是该反应的技术瓶颈.本研究构建重组大肠杆菌E.coli BL21-ADH和E.coli BL21-GDH,实现了葡萄糖脱氢酶和乙醇脱氢酶的共表达,并进行偶联转化.结果表明,当在反应温度为30℃,pH为7的条件下,(S)-l-(2,6-二氯-3-氟苯基)乙醇的产量达到最高,在投料量为6％时,该体系转化率为93.75％.     

Transformation of 2-aminoacetophenone to (S)-2-amino-1-phenylethanol by Arthrobacter sulfureus

A new isolate of Arthrobacter sulfureus , when incubated at 50 g resting cells (dry cell wt) l(-1) with 50 g glucose l(-1) and 1 g 2-aminoacetophenone l(-1) in 50 mm potassium buffer (pH 7, 4 ml) at 30 degrees C, produced ( S )-2-amino-1-phenylethanol (e.e. >99%) with 75% yield in 6 h.     

(2S)-2-Amino-5-chloro-4-hydroxy-5-hexenoic acid,a new chloroamino acid,and related compounds fromAmanita gymnopus

Combining different chromatography systems, unusual nonprotein amino acids were isolated and unequivocally identified from a small amount (less than 100 g fresh weight) ofAmanita gymnopus fruit body. Without obtaining crystals of these amino acids, on the basis of¹H-NMR determination, high resolution mass spectrometry, chlorine analysis and oxidation with L-amino acid oxidase, one of them proved to be a new chloroamino acid, (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid (G2). The other three were (2S)-2-amino-5-hexenoic acid (G1), (2S)-2-amino-4,5-hexadienoic acid (G3) and (2S)-2-amino-5-hexynoic acid (G4). Amino acid (G1) was also encountered for the first time in natural products. Amino acid (G3) has been reported from several kinds of fungi belonging toAmanita, subgenusLepidella. The occurrence of amino acid (G4) was already reported fromCortinarius claricolor.Part 23 in the series Biochemical studies of nitrogen compounds in fungi. Part 22, Hatanaka, S. I. et al. 1985. Trans. Mycol. Soc. Japan26: 61–68.     

Antibody-supported screening of alcohol dehydrogenases

Polyclonal antibodies raised against purified (R)-specific alcohol dehydrogenase of Lactobacillus kefir were used in Western blot analyses to search for structurally or immunologically related proteins. No immunochemical reactions were found with commercially available alcohol dehydrogenases (from yeast, horse liver and Thermoanaerobium brockii), but screening among the genus Lactobacillus revealed that each strain of a subgroup of Betabacterium gave positive results whereas strains of the other subgroups of Lactobacillus were found to be inactive. However, enzymatic assays with these antibody-positive strains showed, that besides L. kefir itself, only the strains of L. brevis possess alcohol dehydrogenase activity with acetophenone and NADPH as substrates.     

Synthesis of (E)-9-(1-pyrenyl)-4-hydroxynon-2-enal, a fluorescent probe of the (E)-4-hydroxynon-2-enal with retained biological properties

(E)-9-(1-pyrenyl)-4-hydroxynon-2-enal (FHNE), a fluorescent probe of (E)-4-hydroxynon-2-enal (HNE) is synthesised in seven steps and in 35% overall yield, starting from commercially available 1-pyrencarboxyaldehyde. When incubated with cultured HeLa cells this fluorescent probe penetrates cells and particularly concentrates in the region surrounding the nucleus. As the parent compound, HNE it is able to induce the activation of heat shock factor (HSF) and it is able to induce the binding of HSF to heat shock element (HSE).     

Purification and characterization of a (R)-2,3-butanediol dehydrogenase from Saccharomyces cerevisiae

A NAD-dependent (R)-2,3-butanediol dehydrogenase (EC 1.1.1.4), selectively catalyzing the oxidation at the (R)-center of 2,3-butanediol irrespective of the absolute configuration of the other carbinol center, was isolated from cell extracts of the yeast Saccharomyces cerevisiae. Purification was achieved by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, affinity chromatography on Matrex Gel Blue A and Superose 6 prep grade chromatography leading to a 70-fold enrichment of the specific activity with 44% yield. Analysis of chiral products was carried out by gas chromatographic methods via pre-chromatographic derivatization and resolution of corresponding diasteromeric derivatives. The enzyme was capable to reduce irreversibly diacetyl (2,3-butanediol) to (R)-acetoin (3-hydroxy-2-butanone) and in a subsequent reaction reversibly to (R,R)-2,3-butanediol using NADH as coenzyme. 1-Hydroxy-2-ketones and C₅-acyloins were also accepted as substrates, whereas the enzyme was inactive towards the reduction of acetone and dihydroxyacetone. The relative molecular mass (M _r) of the enzyme was estimated as 140 000 by means of gel filtration. On SDS-polyacrylamide gel the protein decomposed into 4 (identical) subunits of M _r 35 000. Optimum pH was 6.7 for the reduction of acetoin to 2,3-butanediol and 7.2 for the reverse reaction.Abbreviations GC-MS gas chromatography-mass spectrometry - i.d. internal diameter - M _r relative molecular mass - MTPA-Cl -methoxy--trifluoromethylphenyl acetic acid chloride - PEIC 1-phenylethylisocyanate     

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

The biocatalytic reduction of 4-(trimethylsilyl)-3-butyn-2-one to enantiopure (R)-4-(trimethylsilyl)-3-butyn-2-ol was successfully conducted 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 (e.e.). The optimal buffer pH, co-substrate concentration, reaction temperature, substrate concentration and shaking rate were 5.0, 130.6 mM, 30 °C, 6.0 mM and 180 r/min, respectively. Under the optimized conditions, the maximum yield and the product e.e. were 71% and >99%, respectively, which are much higher than those reported previously. Additionally, the established biocatalytic system proved to be efficient for the bioreduction of acetyltrimethylsilane to (R)-1-trimethylsilylethanol with excellent yield and product e.e. The immobilized cells manifested a good operational stability under the above reaction conditions since they retained 70% of their catalytic activity after ten cycles of use.     

Stereoselective synthesis of (R)-1-chloro-3(3,4-difluorophenoxy)-2-propanol using lipases from Pseudomonas aeruginosa in ionic liquid-containing system

Direct transesterification of (R,S)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol (rac-CDPP) (a key intermediate in the synthesis of the chiral drug (S)-lubeluzole) with vinyl butyrate by lipases from Pseudomonas aeruginosa (P. aeruginosa) MTCC 5113 was performed in hexane with ionic liquids (ILs) 1-butyl-3-methyl imidazolium hexafluorophosphate [BMIm][PF₆] and 1-butyl-3-methyl imidazolium tetrafluoroborate [BMIm][BF₄] as co-solvents. The maximum conversion (>49%) and enantiomeric excess (ee > 99.9%) was achieved in 6 h of incubation at 30 °C with [BMIm][PF₆] as co-solvent in a two-phase system. The enzyme was able to perform with the same specificity even at 60 °C in the presence of ILs. It was possible to use lipases repeatedly for more than 10 times while still maintaining absolute enantioselectivity and reactivity. Stability studies on lipases from P. aeruginosa in ILs revealed the fact that the enzyme constancy and the reactivity in catalyzing transesterification of rac-CDPP into (S)-1-chloro-3-(3,4-difluorophenoxy)-2-butanoate was of the order of [BMIm][PF₆] > [BMIm][BF₄] in two-phase system.     

Synthesis of (<Emphasis Type="Italic">R</Emphasis>)-2-trimethylsilyl-2-hydroxyl-ethylcyanide catalyzed with (<Emphasis Type="Italic">R</Emphasis>)-oxynitrilase from loquat seed meal

The synthesis of optically active (R)-2-trimethylsilyl-2-hydroxyl-ethylcyanide by asymmetric trans-cyanation of acetyltrimethylsilane with acetone cyanohydrin in a biphasic system was achieved using (R)-oxynitrilase from loquat seed meal. Diisopropyl ether was the most suitable organic phase among the organic solvents examined. The optimal concentration of acetyltrimethylsilane, concentration of crude enzyme, volume ratio of the aqueous to the organic phase, temperature and the buffer pH value were 14 mM, 61.4 U ml^-1, 13% (v/v), 30 °C and 4, respectively. The substrate conversion and the product enantiomeric excess were 95% and 98% under the optimized conditions. Acetyltrimethylsilane was a better substrate of the enzyme than its carbon counterpart. Revisions requested 24 August 2004; Revisions received 12 November 2004     

NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit I sequences compared for members of the genus Taenia (Cestoda)

Nine members of the genus Taenia (Taenia taeniaeformis, Taenia hydatigena, Taenia pisiformis, Taenia ovis, Taenia multiceps, Taenia serialis, Taenia saginata, Taenia solium and the Asian Taenia) were characterised by their mitochondrial NADH dehydrogenase subunit 1 gene sequences and their genetic relationships were compared with those derived from the cytochrome c oxidase subunit I sequence data. The extent of inter-taxon sequence difference in NADH dehydrogenase subunit 1 (5.9–30.8%) was usually greater than in cytochrome c oxidase subunit I (2.5–18%). Although topology of the phenograms derived from NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit I sequence data differed, there was concordance in that T. multiceps, T. serialis (of canids), T. saginata and the Asian Taenia (of humans) were genetically most similar, and those four members were genetically more similar to T. ovis and T. solium than they were to T. hydatigena and T. pisiformis (of canids) or T. taeniaeformis (of cats). The NADH dehydrogenase subunit 1 sequence data may prove useful in studies of the systematics and population genetic structure of the Taeniidae.     

蜡样芽孢杆菌聚乙烯醇脱氢酶的表达及酶学特性

【背景】聚乙烯醇脱氢酶(polyvinyl alcohol dehydrogenase,PVADH)能够使聚乙烯醇(polyvinyl alcohol,PVA)氧化脱氢,在PVA的生物降解过程中起到重要作用。【目的】从PVA降解菌株蜡样芽孢杆菌DG01中获取pvadh基因,实现PVADH在毕赤酵母中的异源表达并探究其对不同型号PVA的降解特异性,为PVADH在PVA实际降解中的应用提供指导。【方法】通过反转录扩增技术获得长度为1 965 bp的pvadh基因片段,构建pPIC9K-cpvadh重组表达质粒并在毕赤酵母GS115中实现异源表达,甲醇诱导表达蛋白,进行分离纯化后对其酶学性质及降解特异性进行研究。【结果】最佳发酵条件下PVADH粗酶液酶活达到54.55 U/mL。经分离纯化后表达蛋白PVADH的比酶活为173.42 U/mg,分子量为67.1 kDa,等电点为6.06,该酶最适作用温度为41℃,最适作用pH值为7.5,在27-32℃、pH 7.0-8.0条件下酶的半衰期超过4 h,1 mmol/L的Ca2+对酶活力有激活作用。PVADH分别作用于PVA1788、PVA1799...