羰基还原酶不对称还原（R）-6-氰基-5-羟基-3-羰基己酸叔丁酯

选择R-羰基还原酶和葡萄糖脱氢酶双酶,协同催化（R）-6-氰基5-羟基-3-羰基己酸叔丁酯不对称还原制备阿托伐他汀关键手性合成子6-氰基-（3R,5R）-二羟基已酸叔丁酯。转化条件优化结果显示：在不添加外源性辅酶NADP（H）、菌体用量15．0g/L、147．0g/L（R）-6-氰基-5-羟基-3-羰基己酸叔丁酯、128．2g／L葡萄糖,30℃、pH6．5条件下反应6h后,底物转化率达到100％,产物d．e．值大于99．5％。     

具有差向选择性还原(R)-6-氰基-5-羟基-3-羰基己酸叔丁酯活性的微生物菌株筛选和鉴定

从土样中分离得到一株具有差向选择性还原(R)-6-氰基-5-羟基-3-羰基己酸叔丁酯活性的微生物菌株ZJB-09225,经生理生化特征鉴定和18S rDNA测序后鉴定为卡里比克毕赤酵母(Pichia caribbic ZJB-09225)。研究结果发现,在最适发酵条件下培养32 h,生物量为8.8 g/L,体积酶活达7.2 U/L;P.caribbic ZJB-09225最适作用温度、最适作用pH值分别为35℃和7.5。在最适的催化条件下,P.caribbic ZJB-09225细胞催化50.0 g/L(R)-6-氰基-5-羟基-3-羰基己酸叔丁酯3 h后,产物6-氰基-(3R,5R)-二羟基己酸叔丁酯得率3.4%,产物d.e.值99.5%以上。     

氧化葡萄糖酸杆菌生物催化1,3-丙二醇合成3-羟基丙酸

3-羟基丙酸是一种潜在的重要化工产品,可作为中间体合成多种有经济价值的工业用化合物。文中利用氧化葡萄糖酸杆菌生物催化1,3-丙二醇合成3-羟基丙酸。首先在50 mL摇瓶中(转化体系为10 mL)考察细胞加入量、底物和产物浓度等对催化反应的影响。在此基础上,在2 L鼓泡塔中(转化体系为1 L),采取适当的补料方式和生物转化与分离相耦合的手段解除抑制,以提高目标产物终浓度。结果表明:高底物和产物浓度通过降低反应初速度抑制转化的进行,并确定了最佳催化反应条件为6 g/L菌体量,pH 5.5。利用流加补料方式维持反应体系中底物浓度在15~20 g/L,经过60 h的反应,3-羟基丙酸的浓度达到60.8 g/L,生产强度为1.0g/(L.h),转化率为84.3%。采用生物转化与分离相耦合的方法,经过50 h的转化反应,3-羟基丙酸的总产量达76.3 g/L,生产强度为1.5 g/(L.h),转化率83.7%。研究结果对利用氧化葡萄糖酸杆菌的不完全氧化醇类化合物特性实现其在工业生物催化中的应用具有一定的指导意义。     

定点突变提高醇脱氢酶LkTADH催化制备他汀关键手性砌块的酶活

(S)-6-氯-3-羰基-5-羟基己酸叔丁酯[(S)-CHOH]是他汀类药物合成的关键手性中间体。利用醇脱氢酶催化6-氯-3,5-二羰基己酸叔丁酯不对称合成(S)-CHOH是很有潜力的制备路线,目前存在的主要问题是醇脱氢酶催化活性较低。首先对来源于Lactobacillus kefir DSM 20587的醇脱氢酶的四点突变体LkTADH(A94T/F147L/A202L/L199H)进行回复突变,确定了关键位点147和202,并获得比酶活提高1倍的突变体MF147L-A202L。对这两个位点进行饱和突变,获得比酶活比LkTADH提高1.47倍的突变体MF147I-A202L。其比酶活为10.17U/mg,为目前文献报道最高水平。通过动力学分析和分子对接,分析了突变位点对酶活影响的机制,为后续研究奠定了良好的基础。     

重组大肠杆菌合成聚（3-巯基丙酸）和聚（3-羟基丁酸-3-巯基丙酸）的研究

利用Clostridium acetobutylicum的丁酸激酶基因 (buk) 和磷酸转丁酰基酶基因(ptb),以及Thiocapsa pfennigii的PHA合成酶基因,设计了一条能够合成多种聚羟基烷酸的代谢途径,用构建的质粒转化大肠杆菌,获得了重组大肠杆菌菌株。前期的研究表明,在合适的前体物条件下,该重组大肠杆菌能够合成包括聚羟基丁酸、聚（羟基丁酸戊酸）等多种生物聚酯［Liu and Steinbüchel, Appl. Environ. Microbiol. 66:739743］。利用该重组大肠杆菌,通过生物催化作用合成了3巯基丙酸的同型共聚酯,同时利用该重组大肠杆菌还获得了含3-巯基丙酸单体的多种异型共聚物。实验首先研究了3巯基丙酸对大肠杆菌生长的影响,在此基础上优化了培养过程中添加3-巯基丙酸的时机和浓度,结果表明,在实验的条件下,细胞合成聚（3-巯基丙酸）可达6.7%(占细胞干重),合成聚（3-羟基丁酸—3-巯基丙酸）（分子中3-巯基丙酸:3-羟基丁酸=3:1）可达24.3%。实验进一步研究了同时或分别表达以上3个基因的重组大肠杆菌合成聚合物的能力,结果表明只有当3个基因同时表达时才能合成聚合物,说明3个基因对合成过程是必须的,从而表明了合成途径是按照设计的路线进行的。还通过GC/MS、GPC、IR等手段对合成的化合物进行了定性的研究。聚（3-巯基丙酸）或聚（3-羟基丁酸-3-巯基丙酸）等聚酯属于一类新型生物聚合物,它在分子骨架中含有硫酯键,不同于聚羟基烷酸酯的氧酯键,从而具有显著不同的物理、化学、光学等性质和具有重要的潜在应用价值。     

(S)-羰基还原酶Ⅱ与葡萄糖脱氢酶共催化高效合成(S)-苯乙二醇北大核心CSCD

【目的】通过优化获得最佳酶活配比,设计近平滑假丝酵母(Candida parapsilosis)CCTCC M203011的(S)-羰基还原酶Ⅱ与枯草芽孢杆菌(Bacillus sp.)YX-1葡萄糖脱氢酶在大肠杆菌中的共表达体系,实现重组菌高效催化2-羟基苯乙酮,合成(S)-苯乙二醇。【方法】分别从重组大肠杆菌中纯化了(S)-羰基还原酶Ⅱ和葡萄糖脱氢酶,研究了2种酶共催化2-羟基苯乙酮的最佳酶活比例,最适催化温度和pH,由此构建(S)-羰基还原酶Ⅱ和葡萄糖脱氢酶的共表达体系。【结果】(S)-羰基还原酶Ⅱ的比酶活力为1.3 U/mg,葡萄糖脱氢酶的比酶活力为13.5 U/mg。在总酶活力为1 U时,(S)-羰基还原酶Ⅱ和葡萄糖脱氢酶共催化体系中,确定了2种酶的最佳比例在1∶1到5∶1(U/U)之间,最适反应温度为30℃,pH为7.0。在此基础上构建了(S)-羰基还原酶Ⅱ和葡萄糖脱氢酶基因比为1∶1的共表达体系,共表达重组菌破碎上清液中(S)-羰基还原酶Ⅱ和葡萄糖脱氢酶酶活分别为0.76 U/mg和0.73 U/mg,两者的酶活比例为1∶1。在上述确定的最适催化条件下,其催化10 g/L 2-羟基苯乙酮,产物(S)-苯乙二醇的光学纯度和得率均高达99%以上。与仅含有(S)-羰基还原酶Ⅱ的重组大肠杆菌相比,共表达体系转化产物(S)-苯乙二醇的得率明显提高,且转化时间由原来的24 h缩短为13 h。【结论】通过确定(S)-羰基还原酶Ⅱ和葡萄糖脱氢酶最佳酶活配比,为构建手性催化的靶酶和辅酶再生酶共表达体系,为实现手性化合物的高效制备提供了研究基础。     

△^5—3β，7β-二羟基甾醇及其C-7差向异构体波谱特征及胆碱酯酶的抑制活性

本文对△^5-3β,7β-二羟基甾醇（1—3）和△^5-3β,7α-二羟基甾醇（4～6）的一些核磁共振波谱特征进行了比较。活性测试表明化合物1—6对乙酰胆碱酯酶（AChE）无明显的抑制活性,对丁酰胆碱酯酶（BuChE）则有较强的抑制活性,其中24-亚甲基胆甾-5-烯-3β,7α-二醇（6）的IC50值为9．5μM。通过活性数据比较我们发现7α-羟基甾醇对丁酰胆碱酯酶的抑制活性明显比相应的7β-羟基甾醇高。我们通过计算7位羟基和四环平面之间的二面角角度来尝试解释这些活性差别。     

Δ5-3β,7β-二羟基甾醇及其C-7差向异构体波谱特征及胆碱酯酶的抑制活性

本文对Δ5-3β,7β-二羟基甾醇(1~3)和Δ5-3β,7α-二羟基甾醇(4~6)的一些核磁共振波谱特征进行了比较。活性测试表明化合物1~6对乙酰胆碱酯酶(AChE)无明显的抑制活性,对丁酰胆碱酯酶(BuChE)则有较强的抑制活性,其中24-亚甲基胆甾-5-烯-3β,7α-二醇(6)的IC50值为9.5μM。通过活性数据比较我们发现7α-羟基甾醇对丁酰胆碱酯酶的抑制活性明显比相应的7β-羟基甾醇高。我们通过计算7位羟基和四环平面之间的二面角角度来尝试解释这些活性差别。     

重组固氮菌腈水合酶催化3-（4-氯苯基）戊二腈的选择性水解

通过基因数据挖掘方法（genome mining）获得了来源于固氮菌Herbaspirillum seropedicae SmR1中的腈水合酶基因hsn1。构建了hsn1／pETDuet-1／BL21的大肠杆菌共表达重组菌,经IPTG诱导获得了具有良好催化能力的Co^2＋依赖型腈水合酶HSN1。利用全细胞反应研究了HSN1的底物谱,发现HSN1对底物3-（4-氯苯基）戊二腈有良好的区域选择性及一定的对映选择性,它可以选择性地水解1个腈基得到3-（4-氯苯基）-4-氰基丁酰胺,该化合物可通过一步化学反应合成巴氯芬。     

Improving the catalytic efficiency of aldo-keto reductase KmAKR towards t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate via semi-rational design

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is an important chiral diol synthon of atorvastatin calcium. Previously, we constructed a variant KmAKR-W297H (M1) of Kluyveromyces marxianus aldo-keto reductase (KmAKR, designated as M0), possessing excellent diastereoselectivity but moderate activity towards t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1). In this work, KmAKR-W297H/Y296W/K29H (M3) was developed via semi-rational design. It exhibited much improved catalytic efficiency towards (5R)-1. The K_m values of M3 for NADPH and (5R)-1 were 0.15 mmol/L and 1.41 mmol/L, and the maximal reaction rate v_max was 55.56 μmol/min/mg. Compared with M1, the catalytic efficiency k_cat/K_m of M3 was increased 2.64-fold. Coupled with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration, M3 took 3.5 h to completely reduce (5R)-1 at up to 100.0 g/L, producing 237.4 mmol/L (3R,5R)-2 in d.e._P value above 99.5%. The space-time yield (STY) of M3-catalyzed (3R,5R)-2 synthesis was 372.8 g/L/d.     

Production of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate using carbonyl reductase coupled with glucose dehydrogenase with high space–time yield

tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is an important chiral intermediate for the synthesis of rosuvastatin. The biotechnological production of (3R,5S)-CDHH is catalyzed from tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH) by a carbonyl reductase, and this synthetic pathway is becoming a primary route for (3R,5S)-CDHH production due to its high enantioselectivity, mild reaction conditions, low cost, process safety, and environmental friendship. However, the requirement of the pyridine nucleotide cofactors, reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) limits its economic flexibility. In the present study, a recombinant Escherichia coli strain harboring carbonyl reductase R9M and glucose dehydrogenase (GDH) was constructed with high carbonyl reduction activity and cofactor regeneration efficiency. The recombinant E. coli cells were applied for the efficient production of (3R,5S)-CDHH with a substrate conversion of 98.8%, a yield of 95.6% and an enantiomeric excess (e.e.) of >99.0% under 350 g/L of (S)-CHOH after 12 hr reaction. A substrate fed-batch strategy was further employed to increase the substrate concentration to 400 g/L resulting in an enhanced product yield to 98.5% after 12 hr reaction in a 1 L bioreactor. Meanwhile, the space–time yield was 1,182.3 g L⁻¹ day⁻¹, which was the highest value ever reported by a coupled system of carbonyl reductase and glucose dehydrogenase.     

Synthesis and anti-glioma activity of 25(R)-spirostan-3β,5α,6β,19-tetrol

Malignant gliomas are common and aggressive brain tumours in adults. The rapid proliferation and diffuse brain migration are the main obstacles to successful treatment. Here, we show 25(R)-spirostan-3β,5α,6β,19-tetrol, a polyhydroxy steroid, is capable of suppressing proliferation and migration of C6 malignant glioma cells in a concentration-dependent manner. The compound 25(R)-spirostan-3β,5α,6β,19-tetrol was synthesised by seven steps starting from diosgenin in 8.55% overall yield. The structures of the synthetic compounds were characterised by infrared (IR), ¹H nuclear magnetic resonance (NMR), ¹³C NMR spectra and EA.     

Novel Potato Micro-Tuber-Inducing Compound, (3R,6S)-6-Hydroxylasiodiplodin,from a Strain of Lasiodiplodia theobromae

A novel potato micro-tuber-inducing compound was isolated from the culture broth of Lasiodiplodia theobromae Shimokita 2. The structure of the isolated compound was determined as (3R,6S)-6-hydroxylasiodiplodin by means of spectroscopic analyses, the modified Mosher method, and chemical conversion. The compound showed potato micro-tuber-inducing activity at a concentration of 10^?4 M, using the culture of single-node segments of potato stems in vitro.     

(3R)-2-羧乙基-3-氰基-5-甲基己酸乙酯水解酶产生菌的筛选与产酶条件优化

以2-羧乙基-3-氰基-5-甲基己酸乙酯为唯一碳源,采用手性气相法检测,筛选到一株能产对映选择性水解酶的假单胞菌Pseudomonas CGMCC No.4184.该菌产的水解酶能优先水解R型底物产生(3R)-2-羧乙基-3-氰基-5-甲基己酸,产物对映体过量值达到90%以上.对菌株Pseudomonas CGMCC ...     

Enhanced diastereoselective synthesis of t‐Butyl 6‐cyano‐(3R,5R)‐dihydroxyhexanoate by using aldo‐keto reductase and glucose dehydrogenase co‐producing engineered Escherichia coli

t‐Butyl 6‐cyano‐(3R,5R)‐dihydroxyhexanoate ((3R,5R)‐ 2 ) is a key chiral diol precursor of atorvastatin calcium (Lipitor®). We have constructed a Kluyveromyces lactis aldo‐keto reductase mutant KlAKR‐Y295W/W296L (KlAKRm) by rational design in previous research, which displayed high activity and excellent diastereoselectivity (de_p > 99.5%) toward t‐butyl 6‐cyano‐(5R)‐hydroxy‐3‐oxohexanoate ((5R)‐ 1 ). To realize in situ cofactor regeneration, a robust KlAKRm and Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) co‐producer E. coli BL 21(DE3) pETDuet‐esgdh (MCS1)/pET‐28b (+)‐klakrm was constructed in this work. Under the optimized conditions, AKR and GDH activities of E. coli BL 21(DE3) pETDuet‐esgdh (MCS1)/pET‐28b (+)‐klakrm peaked at 249.9 U/g DCW (dry cellular weight) and 29100 U/g DCW, respectively. It completely converted (5R)‐ 1 at substrate loading size of up to 60.0 g/L (5R)‐ 1 in the absence of exogenous NADH, which was one‐fifth higher than that of the separately prepared KlAKRm and EsGDH under the same conditions. In this manner, a biocatalytic process for (3R,5R)‐ 2 with productivity of 243.2 kg/m³ d was developed. Compared with the combination of separate expressed KlAKRm with EsGDH, co‐expression of KlAKRm and EsGDH has the advantages of alleviating cell cultivation burden and elevating substrate load. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1235–1242, 2017     

Large‐scale synthesis of tert‐butyl (3R,5S)‐6‐chloro‐3,5‐dihydroxyhexanoate by a stereoselective carbonyl reductase with high substrate concentration and product yield

To biosynthesize the (3R,5S)‐CDHH in an industrial scale, a newly synthesized stereoselective short chain carbonyl reductase (SCR) was successfully cloned and expressed in Escherichia coli. The fermentation of recombinant E. coli harboring SCR was carried out in 500 L and 5000 L fermenters, with biomass and specific activity of 9.7 g DCW/L, 15749.95 U/g DCW, and 10.97 g DCW/L, 19210.12 U/g DCW, respectively. The recombinant SCR was successfully applied for efficient production of (3R,5S)‐CDHH. The scale‐up synthesis of (3R,5S)‐CDHH was performed in 5000 L bioreactor with 400 g/L of (S)‐CHOH at 30°C, resulting in a space‐time yield of 13.7 mM/h/g DCW, which was the highest ever reported. After isolation and purification, the yield and d.e. of (3R,5S)‐CDHH reached 97.5% and 99.5%, respectively. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:612–620, 2017     

Production of (R)-3-quinuclidinol by a whole-cell biocatalyst with high efficiency

Optically pure (R)-3-quinuclidinol [(R)-3-Qui] is widely used as a chiral building block for producing various antimuscarinic agents. An asymmetric bioreduction approach using 3-quinuclidinone reductases is an effective way to produce (R)-3-Qui. In this study, a biocatalyst for producing (R)-3-Qui was developed by using Escherichia coli that coexpressed Kaistia granuli (KgQR) and mutant glucose dehydrogenase (GDH). KgQR catalyses the synthesis of (R)-3-Qui through the efficient reduction of 3-quinuclidinone. The specific activity of recombinant KgQR was 254?U/mg, and the Michaelis–Menten constant (K_m) for 3-quinuclidinone was 0.51?mM. The thermal stability of KgQR was relatively high compared with ArQR. Approximately 73% of the residual activity remained after incubation in 0.2 M potassium phosphate buffer (KPB) (pH 7.0) for 8?h at 30?°C. In addition, 80% residual activity remained for the double-mutant GDH (Q252L and E170K) after incubation in a buffer (pH 7.0) for 8?h at 30 and 40?°C. 3-Quinuclidinone (242?g/L) can be reduced to (R)-3-Qui in 3?h by coexpressing KgQR and mutant GDH in E. coli. The conversion rate reached 80.6?g/L/h, which is the highest reported to date. The results demonstrates that this whole-cell biocatalyst will have a great potential in industrial manufacturing.