产碱杆菌ECU0401催化拆分扁桃酸及其衍生物

从土壤中分离的1株产碱杆菌Alcaligenes sp.ECU0401具有扁桃酸脱氢酶活性,可以以扁桃酸、苯甲酰甲酸或苯甲酸为唯一C源生长,并且具有较高的脱氢酶活力。以外消旋扁桃酸为C源,采用分批补料策略培养（或反应）99h,扁桃酸累计投入量为30.4g/L,（S）-（＋）-扁桃酸被完全降解,（R）-（-）-扁桃酸回收产率为32.8%,对映体过量值（e.e.）〉99.9%。利用静息细胞作为催化剂不对称降解外消旋扁桃酸的氯代衍生物,制备获得光学活性的（R）-（-）-邻氯扁桃酸、（S）-（＋）-间氯扁桃酸和（S）-（＋）-对氯扁桃酸,光学纯度均超过99.9%e.e.。     

恶臭假单胞菌CGMCC 1388对映选择性生物降解制备(S)-扁桃酸及其衍生物的研究

从土壤中分离得到一株（R）-扁桃酸选择性降解菌,经鉴定为恶臭假单胞菌,保存于中国普通微生物保藏中心,编号为CGMCC1388。考察了扁桃酸及其降解产物对扁桃酸脱氢酶活力的影响。研究表明,在培养基中添加少量扁桃酸、苯甲酰甲酸或苯甲酸均可显著提高其产量,以扁桃酸的诱导效果最佳。3种诱导物的最适添加质量浓度分别为4、4和2 g/L。当以消旋扁桃酸为反应底物时,该菌可高选择性降解（R）-扁桃酸,回收得到的（S）-扁桃酸对映体过量值（e.e.）高于99%,反应的对映选择率（E）达130。用静息细胞催化扁桃酸降解的最适温度和pH分别为30 ℃和6.0,最适底物浓度为60 mmol/L,以双倒数法求得Km为47 mmol/L。考察了该菌对扁桃酸苯环取代衍生物的生物转化,并以高产率制备获得高对映纯度的（S）-对羟基扁桃酸和（S）-对氯扁桃酸。     

葡萄酒酵母不对称还原苯甲酰甲酸合成（R）-扁桃酸

研究了葡萄酒酵母不对称还原制备（R）-扁桃酸的转化,并将其放大至反应罐进行小试研究。通过转化条件的优化,在密闭条件下,当底物质量浓度为10g/L时,苯甲酰甲酸的产率达到72％,扁桃酸的对应过量值（e.e）值达到99％以上。实验发现,该微生物具有很好的催化稳定性,全细胞经过10批次反应,产率无明显降低,产物对映体过量值均高于98％。转化反应放大至7L反应罐体系后,S.ellipsoideus,仍然具有良好的催化性能,产率提高到81％,e.e值保持在99％。     

抗生素

九1若石J任酶膜反应器进行扭一)扁桃酸的连续生产嵘】/Vasi二Racki,D,…11 ApPI.MICrobiol.Biotechnol‘一1989,31(3)一215一222〔译自DBA,1989,8(19),89一1 126司 使用扁桃酸盐脱氢酶(MDH)和甲酸盐脱氢酶(F DH)进行辅酶同时再生作用,在酶膜反应器中进行了苯乙醇酸向(R一)扁挑酸的连续转化.列出了祸联酶反应的数学模型,并用之确定(R一)扁挑酸生产的最适条件.结果显示,如果使用PEG一NADH代替天然NADH,FDH的活性会微弱增高.苯乙醇酸和扁桃酸都可抑制FDH,但按离子则可激活 MDH.在前12天中的2个小时的滞留时间内获得了98%的苯…     

新型R-扁桃酸脱氢酶的基因挖掘及表达鉴定

R-扁桃酸脱氢酶在苯乙酮酸的生物合成中起着关键的作用,挖掘具有高催化活性及稳定性的新型R-扁桃酸脱氢酶具有重要的意义。为了获得理想的R-扁桃酸脱氢酶,采用了基因组挖矿技术从Lactobacillus harbinensis菌株中获得了一个新型的R-扁桃酸脱氢酶LhDMDH,重组LhDMDH的比酶活高达1264.3 U/mg,约为探针的4倍,在已报道的R-扁桃酸脱氢酶中处于领先水平。同时,考察了4个重组酶主要的酶学特性,它们的最适反应温度在25~30℃,最适反应pH在9.0~9.5。动力学参数的结果表明,LhDMDH对底物的K_(cat)值为30.28 S~(-1),明显高于其它重组酶。此外,底物谱分析的结果也表明LhDMDH在外消旋扁桃酸的手性拆分及苯乙酮酸的生物合成中更具优势。在R-扁桃酸脱氢酶基因挖掘方面取得了较为理想的结果,为进一步的改造及应用奠定了坚实的基础,也为其它酶的挖掘提供了可资借鉴的经验。     

酿酒酵母脂酰-?9脱氢酶亚细胞定位表达及其对烟草脂肪酸合成的影响

以烟草Nicotiana tabacum L.为宿主植物,分别在细胞质内质网和质体内定位表达酿酒酵母Saccharomyees cerevisiae脂酰-CoA-Δ9脱氢酶(ScΔ9D),以期提高植物组织中棕榈油酸(16∶1Δ9)的积累量和分析该酶不同亚细胞定位表达对油脂代谢的影响。与野生型和空载体(对照)植物相比,转基因烟草植株叶片中单不饱和的棕榈油酸及顺式十八碳烯酸(18∶1Δ11)含量明显提高,而饱和的棕榈酸(16∶0)含量相应减少,多不饱和的亚油酸(18∶2Δ9,12)和亚麻酸(18∶3Δ9,12,15)含量亦降低。ScΔ9D质体定位表达烟叶中棕榈油酸及顺式十八碳烯酸含量分别是ScΔ9D细胞质内质网定位表达烟叶的2.7和1.9倍。这表明酵母脂酰-Δ9脱氢酶能在高等植物细胞中正确催化棕榈酸(16∶0)转化为棕榈油酸(16∶1Δ9),而且在质体内表达的效应显著高于在细胞质内质网上的效应。新建立了一种应用脂酰-CoA-Δ9脱氢酶代谢工程培育植物组织高水平合成积累棕榈油酸等ω-7脂肪酸的策略,有助于在生物量大的烟叶等营养器官中组装ω-7脂肪酸合成途径以生产优质生物燃油。     

酿酒酵母脂酰-△9脱氢酶亚细胞定位表达及其对烟草脂肪酸合成的影响

以烟草Nicotiana tabacum L.为宿主植物,分别在细胞质内质网和质体内定位表达酿酒酵母Saccharom,ees cerevisiae脂酰-CoA-△9脱氢酶(Sc△9D),以期提高植物组织中棕榈油酸(16∶1△9)的积累量和分析该酶不同亚细胞定位表达对油脂代谢的影响.与野生型和空载体(对照)植物相比,转基因烟草植株叶片中单不饱和的棕榈油酸及顺式十八碳烯酸(18∶1△11)含量明显提高,而饱和的棕榈酸(16∶0)含量相应减少,多不饱和的亚油酸(18∶2△9,12)和亚麻酸(18∶3△9,12,15)含量亦降低.Sc△9D质体定位表达烟叶中棕榈油酸及顺式十八碳烯酸含量分别是Sc△9D细胞质内质网定位表达烟叶的2.7和1.9倍.这表明酵母脂酰-△9脱氢酶能在高等植物细胞中正确催化棕榈酸(16∶0)转化为棕榈油酸(16∶1△9),而且在质体内表达的效应显著高于在细胞质内质网上的效应.新建立了一种应用脂酰-CoA-△9脱氢酶代谢工程培育植物组织高水平合成积累棕榈油酸等ω-7脂肪酸的策略,有助于在生物量大的烟叶等营养器官中组装ω-7脂肪酸合成途径以生产优质生物燃油.     

(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。     

S-丙二酰转移酶基因失活对地中海拟无枝酸菌产利福霉素的影响

为提高利福霉素的产量,构建了S-丙二酰转移酶基因失活的地中海拟无枝酸菌。利用融合PCR构建S-丙二酰转移酶基因的同源重组载体,通过电击转化导入到地中海拟无枝酸菌中,使之发生同源重组,以安普霉素为标记,筛选了S-丙二酰转移酶基因失活菌株,并对比了突变菌株与原始菌株的利福霉素SV产量。成功构建了S-丙二酰转移酶基因的同源重组载体,获得了地中海拟无枝酸菌突变株A.mediterranei △fab D,失活菌株的利福霉素SV产量为168.08 mg/L,比原始菌提高了9.94%。S-丙二酰转移酶基因的失活,弱化了突变菌株脂肪酸的合成,强化了利福霉素的合成。     

过表达Dbtnbt基因提高中国红豆杉细胞的紫杉醇含量

本文通过克隆3′-N-去苯甲酰紫杉醇N-苯甲酰转移酶（3′-N-debenzoyltaxol-N-benzoyltransferase,DBTNBT）基因Dbtnbt,构建其表达载体p1303-SDbtnbtN,转化中国红豆杉细胞,经潮霉素抗性筛选获得转基因细胞系. 转基因细胞分析结果表明,T-DNA及其包含的基因与宿主细胞染色体成功整合;转基因细胞中报告基因GusA-mgfp5正常表达;转基因细胞的Dbtnbt mRNA表达量是未转化细胞的1.33 倍;转基因细胞的紫杉醇产量约为27.3 μg/g,是未转化细胞的1.37倍. 本研究结果表明,过表达Dbtnbt基因将中国红豆杉细胞的紫杉醇产量提高约37%.     

荧光假单胞菌天冬氨酸转氨酶的基因克隆及其在大肠杆菌中的表达

采用克隆基因测序技术,从荧光假单胞菌GcM5-1A基因组文库中筛选到了天冬氨酸转氨酶的编码基因aspC。通过聚合酶链式反应（PCR）扩增目的基因,插入pET-15b构建重组表达质粒pET-15bAAT,转化E.coli BL21(DE3),IPTG诱导天冬氨酸转氨酶在大肠杆菌中高效表达,利用亲和层析法初步分离纯化了重组蛋白。生物活性分析表明,纯化的重组天门冬氨酸转氨酶具有氨基转移活性。     

Studies on structure-function relationships of indolepyruvate decarboxylase from Enterobacter cloacae, a key enzyme of the indole acetic acid pathway.

Enterobacter cloacae, isolated from the rhizosphere of cucumbers, produces large amounts of indole-3-acetic acid. Indolepyruvate decarboxylase, the key enzyme in the biosynthetic pathway of indole-3-acetic acid, catalyses the formation of indole-3-acetaldehyde and carbon dioxide from indole-3-pyruvic acid. The enzyme requires the cofactors thiamine diphosphate and magnesium ions for catalytic activity. Recombinant indolepyruvate decarboxylase was purified from the host Escherichia coli strain JM109. Specificity of the enzyme for the substrates indole-3-pyruvic acid, pyruvic acid, benzoylformic acid, and seven benzoylformic acid analogues was investigated using a continuous optical assay. Stopped-flow kinetic data showed no indication for substrate activation in the decarboxylation reaction of indole-3-pyruvic acid, pyruvic acid or benzoylformic acid. Size exclusion chromatography and small angle X-ray solution scattering experiments suggested the tetramer as the catalytically active state and a pH-dependent subunit association equilibrium. Analysis of the kinetic constants of the benzoylformic acid analogues according to Hansch et al. [Hansch, C., Leo, A., Unger, S.H., Kim, K.H., Nikaitani, D & Lien, E.J. (1973) J. Med. Chem.16, 1207-1216] and comparison with indole-3-pyruvic acid conversion by pyruvate decarboxylases from Saccharomyces cerevisiae and Zymomonas mobilis provided some insight into the catalytic mechanism of indolepyruvate decarboxylase.     

编码Synechococcus sp. PCC 7002 FNR 中FNR区的基因克隆及其在大肠杆菌中的表达

用ＰＣＲ的方法克隆出了编码蓝细菌Ｓｙｎｅｃｈｏｃｏｃｃｕｓｓｐ．ＰＣＣ７００２ＦＮＲ中ＦＮＲ区的基因ｐｅｔＨＬ,克隆到达载体ｐＥＴ３ａ上,转化大肠杆菌ＢＬ２１（ＤＥ３）后实现了大量表达。重组ＦＮＲ区（ｒＦＮＲＤ）经ＤＥＡＥＳｅｐｈｄｅｘＡ５０离子交换层析及ＳｅｐｈａｄｅｘＧ１００凝胶层析得到大量的电泳均一的ｒＦＮＲＤ。Ｎ末端氨基酸序列分析表明,表达产物确为ｐｅｔＨＬ所编码。且起始Ｍｅｔ翻译后未被除去。ｒＦＮＲＤ与ｒＦＮＲ的吸收光谱相同,其黄递酶活性的最适ｐＨ和最适温度也相同。ｒＦＮＲＤ能在体外催化电子从Ｐ７００到ＮＡＤＰ＋的传递     

Cloning of the Gene Encoding FNR Domain of FNR of Cyanobacterium Synechococcus sp. PCC 7002 and its Overexpression in Escherichia coli

The petHL of Synechnococcus sp. PCC 7002 encoding FNR domain (FNRD) of FNR was amplified by PCR, and cloned into expressing vector pET-3a. Ovemxpression of petHL was achieved with E. coli BL21 (DE3). The recombinant FNRD was purified to homogeneity by DEAE-Sephadex A-50 and Sephadex G-100 chromatography. N-terminal amino acid sequencing showed that rFNRD was encoded by petHL and initial Met was not posttranslationally removed, rFNRD had the same absorption spectrum, optimal pH and optimal temperature as those of rFNR. rFNRD could catalyze photosynthetic electron transport from PT00 to NADP+ in vitro.     

大豆ω-3脂肪酸脱氢酶基因GmFAD3C在酿酒酵母中的表达

α亚麻酸(ALA)被称为必需脂肪酸,对人体有一系列的保健作用。ω-3脂肪酸脱氢酶(FAD)催化亚油酸(LA)生成ALA。大豆种子油中ALA含量较高,为了研究大豆ω3FAD的功能,用RTPCR方法从大豆未成熟种子中扩增出GmFAD3C的cDNA,克隆到酵母表达载体p416中,并用醋酸锂法转化酿酒酵母营养缺陷型K601,经筛选鉴定,得到阳性克隆。气相色谱分析脂肪酸成分,发现工程菌产生了新的脂肪成分ALA,含量占总脂肪酸的3.1%,LA含量与对照相比相应地下降,证明该基因编码的蛋白具有催化18碳多不饱和脂肪酸(PUFA)底物LA在Δ15位脱氢生成ALA的ω3FAD功能,首次实现大豆ω-3脂肪酸脱氢酶基因在酿酒酵母K601p416系统中的表达,建立了一种新的高效低成本的FAD酵母表达系统。     

Cloning and characterization of chironomidae ferrochelatase: Copper activation of the purified ferrochelatase

All organisms utilize ferrochelatase (EC 4.99.1.1) to catalyze the insertion of ferrous iron into protoposphyrin IX in the terminal step of the heme biosynthetic pathway. Different metal-binding affinity for the enzyme leads to changes in enzyme activity. In this work, we have cloned and over-expressed the enzyme from chironomidae in E. coli. The enzyme was purified and characterized. The recombinant enzyme showed higher enzymatic activity (four-fold increase) in the presence of copper ions and unaffected by calcium ions. Other divalent metal ions including magnesium, manganese, lead, reduced the enzyme activity by >60%. Over 90% of the enzyme activity was inhibited by Zn2+. The sequence alignment of amino acid residues reveals 83% homology with other ferrochelatases. The results of electron proton resonance (EPR) analysis showed that Fe2+ ion was present in the cluster of the recombinant enzyme complex. The recombinant enzyme also contained the [2Fe-2S] center with two-fold higher enzymatic activity than human ferrochelatase.     

野桑蚕GST-Omega1基因在草地贪夜蛾Sf9细胞中的表达

谷胱甘肽 S-转移酶（glutathione S-transferases,GSTs）是昆虫的重要解毒酶之一。为了研究野桑蚕Bombyx mandarina中谷胱甘肽S-转移酶在真核表达系统中的表达情况。本研究通过RT-PCR从野桑蚕中肠中获得GST-Omega1基因的cDNA序列,该基因的开放读码框为771 bp,编码256 个氨基酸。对推导的氨基酸序列用NCBI的蛋白质Conserved Domains工具进行在线分析,结果显示GST-Omega1的氨基酸序列中具有Cys38和8个GSH结合位点的Omega类基因保守序列。对所获得的基因克隆进表达载体pFastBacHT b中获得pFast-GST-Omega1,将其转化DH10Bac感受态细胞,获得Bac-GST-Omega1重组病毒DNA,用脂质体法转染草地贪夜蛾Sf9细胞,获得重组病毒。对表达产物经SDS-PAGE和Western blotting分析,能检测到一条分子量约为33 kD的特异性条带,与推导的融合蛋白大小相符,该目的蛋白的表达量占总蛋白的14.4%。目的蛋白经His·Bind树脂纯化,用Lineweaver Burk作图法测定其K_m和V_max,结果显示其K_m为2.81 µmol/L,V_max为2.70 µmol/(mg·min）。     

Cloning, expression and preliminary application of a alpha-hydroxynitrile lyase from cassave]

alpha-Hydroxynitrile lyase (ME-HNLs, E.C. 4.1.2.3.37) from the cyanogenic crop cassava(Manihot esculentz, Crantz) catalyze the condensation of hydrocyanic acid and aldehydes or ketone into (s)-cyanohydrins, which are valuable starting material for various optically active compounds, such as pharmaceuticals and agrochemicals. The cDNA of a ME-HNL were obtained by RT-PCR and cloned. The sequencing result for the cDNA showed that the sequence encoded for the ME-HNL was inconsistent with all those which are published, such as hnl10, hnl24, hnl4. The full sequence analysis demonstrated that the cloned cDNA was about 75.2%, 79.8%, 99.2% homologous to other three reported HNL genes from cassava, respectively, among which the last was the same to the cloned gene except the five base substitution at the site 142, 337, 476, 634 and 636, respectively. The two base substitutions lead to change the amino acid sequence, i.e., Ser113-->Gly113, Phe158-->Tyr158. To construct the recombinant plasmid pET30a-hnl, the cDNA was inserted into an expression vector pET30a. After transformation of pET30a-hnl and induction with IPTG, the ME-HNL was efficiently expressed in E. coli. BL21 (DE3) and reached over 2100 units/L of culture with the specific activity 8.5 u/mg protein. By one simple treatment, incubating 10 minutes at 70 degrees C, the recombinant ME-HNL may be used as an catalyst for production of (S)-mandelonitrile with enantiomeric excess of 95.2% and 98.2% yield.     

黑芝麻多酚氧化酶的重组表达及其酶学特性

为明确黑芝麻多酚氧化酶的酶学性质,利用大肠杆菌Escherichia coli原核表达了黑芝麻多酚氧化酶 (Black sesame polyphenol oxidase,BsPPO)。将合成的基因构建至pMAL-c5x载体,并在大肠杆菌中进行表达,对重组蛋白进行分离纯化及融合标签切除,获得的BsPPO蛋白用于酶学性质探究。结果表明,合成的Bsppo基因1 752 bp,编码585个氨基酸,理论蛋白分子量为65.3 kDa;构建的pMAL-c5x-Bsppo重组质粒在大肠杆菌Escherichia coli BL21(DE3) 中可溶表达了MBP-BsPPO蛋白;酶切去除MBP融合标签后对BsPPO进行了酶学性质研究,结果表明BsPPO的最适温度和pH分别为25 ℃和4.0,在低温和弱酸性环境中有较好的稳定性。短时间低强度的光照和Cu2+可激活BsPPO的活性,Zn2+和Ca2+能抑制其活性。BsPPO可催化单酚、二酚以及三酚类化合物,对l-酪氨酸以及香草酸表现出较高的催化活性,此外BsPPO还对黑芝麻中含有的2-甲氧基肉桂酸、吲哚3-羧酸和根皮素表现出良好的催化活性。研究结果为黑芝麻多酚氧化酶酶学特性的明确奠定了理论基础。     

Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori

Alcohol dehydrogenases (ADH) are a class of enzymes that catalyze the reversible oxidation of alcohols to corresponding aldehydes or ketones, by using either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), as coenzymes. In this study, a short-chain ADH gene was identified in Bombyx mori by 5'-RACE PCR. This is the first time the coding region of BmADH has been cloned, expressed, purified and then characterized. The cDNA fragment encoding the BmADH protein was amplified from a pool of silkworm cDNAs by PCR, and then cloned into E. coli expression vector pET-30a(+). The recombinant His-tagged BmADH protein was expressed in E. coli BL21 (DE3), and then purified by metal chelating affinity chromatography. The soluble recombinant BmADH, produced at low-growth temperature, was instrumental in catalyzing the ethanol-dependent reduction of NAD(+), thereby indicating ethanol as one of the substrates of BmADH.