极耐热性β-葡萄糖醛酸酶的高效表达和酶学性质及其应用

从海栖热袍菌克隆出编码热稳定性β-葡萄糖醛酸酶基因,以热激载体pHsh为表达质粒,在大肠杆菌中得到高效表达。基因表达产物通过一步热处理后,酶纯度达电泳均一。纯化重组酶酶学性质研究表明,β-葡萄糖醛酸酶的最适反应温度为80℃,最适反应pH为5．0,pH5．8～8．2之间酶的稳定性较好,80℃的半衰期为2h,SDS—PAGE结果显示分子量为65.9kD,与理论推算值相吻合。以对硝基苯-β-葡萄糖醛酸苷（pN/PG）为底物时,其动力学参数Km值0.18mmol／L,Vmax值为312u／mg。初步的应用分析表明,该重组酶能催化甘草酸转化为甘草次酸。     

海栖热袍杆菌来源的极耐热碱性果胶裂解酶的表达、纯化及定性

将来源于极端嗜热菌属海栖热袍杆菌Thermotoga maritima MSB8的编码碱性果胶裂解酶的结构基因pelA与新型热激质粒pHsh连接, 得到重组质粒pHsh-pelA, 运用mRNA二级结构预测软件对pHsh-pelA的翻译起始区的二级结构进行优化, 得到了具有最佳mRNA二级结构及自由能的质粒pHsh-pelC。将重组质粒pHsh-pelC转入大肠杆菌JM109(DE3)进行表达, 得到了一种极耐热性碱性果胶裂解酶(PelC)。对重组酶的酶学性质研究发现, 该酶的最适反应温度为90oC, 最适反应pH为8.5, 在pH 8.2~9.8之间酶活力稳定, 95oC酶活半衰期为2 h, 并且该酶依赖Ca2+作为活性离子。在工业生产常用温度60oC下, 该酶能够长时间保持稳定, 并具有较高的酶活力。以多聚半乳糖醛酸(PGA)为底物时, 其动力学参数Km值为0.11 mmol/L, Vmax值为327 U/mg。SDS-PAGE结果显示该重组酶的分子量为43 kD, 与理论值相符。基于热激载体pHsh的重组表达系统具有诱导表达简便、诱导方式廉价的优点, 且重组酶热稳定性非常好, 这对该酶的大规模发酵应用具有重要意义。     

水牛瘤胃宏基因组的一个新的b-葡萄糖苷酶基因umcel3G的克隆、表达及其表达产物的酶学特性

以木质纤维素为原料、应用同步糖化共发酵工艺发酵生产酒精时需要酸性中低温高活力纤维素酶包括b-葡萄糖苷酶。本工作分6次构建了水牛瘤胃未培养微生物宏基因组文库, 获得1.26×105个克隆, 文库含外源DNA的总长度 约为4.8×106 kb。从文库中筛选到118个表达b-葡萄糖苷酶活性的独立克隆。发现其中8个克隆表达的b-葡萄糖苷酶在pH5.0、37oC条件下活性较强。对其中一个克隆进行了亚克隆, 序列分析发现一个2223 bp的潜在的编码b-葡萄糖苷酶基因(umcel3G)的开放阅读框(ORF), 其编码产物的氨基酸序列与来自于 Bacillus sp.的一个b-葡萄糖苷酶同源性最高, 具有60%的一致性和73%的相似性。该ORF在E.coli中的表达产物Umcel3G的分子量与预测大小相似, 酶谱分析表明该表达产物具有b-葡萄糖苷酶活性, 证实该基因为一个b-葡萄糖苷酶基因。测定了用Ni-NTA纯化的Umcel3G的酶学特性, 其最适pH和最适温度分别为6.0~6.5和45oC。一些金属离子如Ca2+、Zn2+能显著提高该酶的酶活, 而另外一些金属离子如Fe3+、Cu2+能抑制Umcel3G的活性。在pH4.5、35oC和5 mmol/L的 Ca2+存在的条件下, 用Ni-NTA纯化的重组酶的比活为22.8 IU/mg, 说明该酶在用SSCF工艺发酵生产酒精中有潜在的应用价值。     

水牛瘤胃宏基因组的一个新的b-葡萄糖苷酶基因umcel3G的克隆、表达及其表达产物的酶学特性

以木质纤维素为原料、应用同步糖化共发酵工艺发酵生产酒精时需要酸性中低温高活力纤维素酶包括b-葡萄糖苷酶。本工作分6次构建了水牛瘤胃未培养微生物宏基因组文库, 获得1.26×105个克隆, 文库含外源DNA的总长度 约为4.8×106 kb。从文库中筛选到118个表达b-葡萄糖苷酶活性的独立克隆。发现其中8个克隆表达的b-葡萄糖苷酶在pH5.0、37oC条件下活性较强。对其中一个克隆进行了亚克隆, 序列分析发现一个2223 bp的潜在的编码b-葡萄糖苷酶基因(umcel3G)的开放阅读框(ORF), 其编码产物的氨基酸序列与来自于 Bacillus sp.的一个b-葡萄糖苷酶同源性最高, 具有60%的一致性和73%的相似性。该ORF在E.coli中的表达产物Umcel3G的分子量与预测大小相似, 酶谱分析表明该表达产物具有b-葡萄糖苷酶活性, 证实该基因为一个b-葡萄糖苷酶基因。测定了用Ni-NTA纯化的Umcel3G的酶学特性, 其最适pH和最适温度分别为6.0~6.5和45oC。一些金属离子如Ca2+、Zn2+能显著提高该酶的酶活, 而另外一些金属离子如Fe3+、Cu2+能抑制Umcel3G的活性。在pH4.5、35oC和5 mmol/L的 Ca2+存在的条件下, 用Ni-NTA纯化的重组酶的比活为22.8 IU/mg, 说明该酶在用SSCF工艺发酵生产酒精中有潜在的应用价值。     

腾冲嗜热杆菌热稳定海藻糖磷酸化酶的基因表达与酶的分子生物学性质研究

分离克隆了腾冲嗜热杆菌(Thermoanaerobacter tengcongensis)海藻糖磷酸化酶(TreP)的编码基因(treP), 该酶可催化以葡萄糖和α-1-磷酸葡萄糖为底物的海藻糖合成反应及其逆向的分解反应. 反向mRNA点杂交实验表明, 腾冲嗜热杆菌中treP基因在高盐胁迫条件下表达量增加, 而在海藻糖诱导条件下表达量降低. 将该基因导入不含TreP的大肠杆菌中进行诱导表达, SDS-PAGE表明, 异源表达的TreP分子量约为90 kD, 与预期值相同. 通过葡萄糖氧化酶法测定分解产物葡萄糖的产率表明: TreP催化海藻糖分解反应的最适温度是70℃, 最适pH值为7.0; 通过HPLC检测合成产物海藻糖的产率表明: TreP催化合成反应的最适温度为70℃, 最适pH值为6.0. 在最适反应条件下, 50 μg的TreP粗酶可催化25 mmol/L α-1-磷酸葡萄糖与葡萄糖在30 min合成11.6 mmol/L海藻糖; 而同量的酶在同样时间内仅能将250 mmol/L海藻糖分解生成1.5 mmol/L葡萄糖. 以上体内胁迫和诱导表达分析及体外酶学性质分析均证明该酶的主要功能是催化海藻糖的合成反应. 热稳定性实验表明, 该酶性质比较稳定, 在50℃下温育7 h还能保持77%以上的活性, 是一个有潜在工业用途的新的热稳定海藻糖合成酶.     

泰山土壤宏基因组DNA中耐热b-半乳糖苷酶基因的克隆、表达及性质

从泰山土壤宏基因组文库中发现可能的b-半乳糖苷酶基因pwtsA, 将其克隆到表达载体pET30a, 转化E. coli BL21(DE3)。工程菌在IPTG诱导下高效表达可溶性的重组蛋白PWTSA, 分子量为57 kD, 与预期大小一致。PWTSA能够水解ONPG产生o-硝基酚, 酶活力为13.6 U/mg, 确证了重组蛋白为b-半乳糖苷酶。PWTSA的最适反应温度在85°C ~95°C之间, 最适pH值为6.5, 对90°C左右的高温有很好的耐受力。在标准反应条件下, 酶作用于底物ONPG的米氏常数Km为0.83 mmol/L。     

巨大芽孢杆菌b-淀粉酶基因的克隆、表达和酶学性质分析

从巨大芽孢杆菌(Bacillus megaterium)的全基因组DNA文库中筛选出一个b-淀粉酶基因amyG, 分析测定了其核苷酸序列并进行了诱导表达; 其中amyG编码的蛋白有545个氨基酸、分子量为60.194 kD, 与已报道的巨大芽孢杆菌DSM319的b-淀粉酶序列有着94.5%的同源性。经氨基酸序列比较分析发现, AmyG从N末端到C末端依次由信号肽域、糖基水解酶催化功能域和淀粉结合域3个功能域组成。其中催化功能域里含有第14家族糖基水解酶常见的几个高度保守的酶催化活性区。经多步纯化, 重组酶的比活共提高了7.4倍, 获得凝胶电泳均一的蛋白样品; 经SDS-PAGE电泳测定, 酶AmyG的分子量为57 kD。该酶的最适反应温度为60oC, 最适反应pH为7.0; 在温度不超过60oC时, 酶活较稳定; AmyG能迅速降解淀粉生成麦芽糖, 属于外切b-糖苷酶。     

嗜热厌氧乙醇杆菌α-葡萄糖苷酶基因的表达及酶学性质的研究

用PCR方法从嗜热厌氧乙醇杆菌(Thermoanaerobacter ethanolicus)JW200中扩增出编码a-葡萄糖苷酶的基因,将其克隆到大肠杆菌(Escherichia coli)表达载体pTrc99A上并获得表达a-葡萄糖苷酶的大肠杆菌重组菌。重组菌经IPTG诱导表达,SDS-PAGE检测出蛋白相对分子量约89kDa,经阴离子交换层析和凝胶层析纯化后的a-葡萄糖苷酶最适反应温度为70℃,最适反应pH为5～5.5,且在pH 5.5～6.5之间有较高的稳定性。重组a-葡萄糖苷酶在70℃下105 min后酶活仍达到80%。     

海栖热袍菌葡萄糖异构酶基因xylA的克隆、表达及酶学性质

海栖热袍菌(Thermotoga maritima)是嗜极端高温的厌氧细菌,其产生的葡萄糖异构酶由于其出色的耐热性有着潜在的工业应用价值.由于海栖热袍菌苛刻的培养条件导致其葡萄糖异构酶产量较低.通过PCR方法克隆编码T. maritima MSB8葡萄糖异构酶基因xylA,构建重组质粒pHsh-xylA,转入Escherichia coli JM109,通过热激诱导表达.通过热处理和离子交换层析纯化两步得到电泳纯的酶制品,纯化倍数和回收率分别为8.02和49.02.对酶学性质研究表明,该重组酶为金属离子激活性酶,Mg2 ,Co2 对相对酶活有很强的激活作用,其最适pH为7.0,最适反应温度为95℃,且在pH 6～8之间有着较好的稳定性,在95℃下半衰期长达5 h以上.以葡萄糖为底物时的表观Km和Vmax分别为105 mmol/L和45.2 mol/min·mg.     

短小芽孢杆菌木聚糖酶基因在毕赤酶母中的分泌表达及酶学性质研究

将短小芽孢杆菌HB030的内切-1,4-木聚糖酶基因克隆到毕赤酵母表达载体pPIC9K,得到重组质粒pH-BM220,将pHBM220经酶切后分别转化三株毕赤酵母KM71、GS115、SMD1168,该木聚糖酶基因在三株毕赤酵母中均实现了分泌表达,将重组毕赤酵母KM71（pHBM220）,GS115（pHBM220),GS115(pHBM220),SMD1168（pHBM220）分别诱导产酶,对重组酶进行相关的酶学性质分析表明,三的最适反应pH值约为5.5,最适反应温度约为60℃,在其最适反应条件下测得三粗酶液酶活分别为10.80IU/mL,11.63IU/mL,9.68IU/mL,重组毕赤酵母KM71（pHBM220）所产酶的热稳定性较好,而在pH稳定性方面三没有太大的差异。     

Functional phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv: rapid purification with high yield and purity

Phosphoglucose isomerase (PGI) EC 5.3.1.9, is a housekeeping enzyme that catalyzes the reversible isomerization of d-glucopyranose-6-phosphate and d-fructofuranose-6-phosphate. We have previously reported expression and multistep purification of recombinant PGI from Mycobacterium tuberculosis using conventional methods. We now describe an improved and simplified single step approach for purification of functionally active mycobacterial rPGI. The gene encoding PGI from M. tuberculosis H37Rv was cloned in bacterial expression vector pET22b(+). Expression of recombinant PGI with six-histidine-tag protein was observed both in the soluble fraction and inclusion bodies. Approximately 116mg of recombinant enzyme was purified to near homogeneity with approximately 80% yield from the soluble fraction of 1L culture at shake flask level using one step Ni-NTA affinity chromatography. The specific activity of the purified six-histidine-tagged recombinant PGI (rPGI-His(6)) was approximately 800U/mg of protein. The apparent K(m) value of the active recombinant protein followed Michaelis-Menten kinetics and was 0.27+/-0.03mM. K(i) for the competitive inhibitor 6-phosphogluconate was 0.75mM. The enzyme had pH optima in the range of pH 7.6-9.0 and was stable up to 55 degrees C. rPGI-His(6) exhibited enzyme activity almost equal to that of enzyme without histidine tag.     

Purification and properties of a novel beta-glucosidase, hydrolyzing ginsenoside Rb1 to CK, from Paecilomyces Bainier

A novel ginsenoside-hydrolyzing beta-glucosidase was purified from Paecilomyces Bainier sp. 229 by a combination of QSepharose FF, phenyl-Sepharose CL-4B, and CHT ceramic hydroxyapatite column chromatographies. The purified enzyme was a monomeric protein with a molecular mass estimated to be 115 kDa. The optimal enzyme activity was observed at pH 3.5 and 60oC. It was highly stable within pH 3-9 and at temperatures lower than 55oC. The enzyme was specific to beta-glucoside. The order of enzyme activities against different types of beta-glucosidic linkages was beta-(1- 6)>beta-(1-2)>beta-(1-4). The enzyme converted ginsenoside Rb1 to CK specifically and efficiently. An 84.3% amount of ginsenoside Rb1, with an initial concentration of 2 mM, was converted into CK in 24 h by the enzyme at 45 degrees and pH 3.5. The hydrolysis pathway of ginsenoside Rb1 by the enzyme was Rb1-->Rd-->F2-->CK. Five tryptic peptide fragments of the enzyme were identified by a newly developed de novo sequencing method of post-source decay (PSD) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. By comparing the five identified peptide sequences with the NCBI database, this purified beta-glucosidase proves to be a new protein that has not been reported before.     

极端耐热纤维素酶Cel74的表达、纯化与特性

从海栖热袍菌中克隆出编码热稳定性的纤维素酶基因,以热激载体pHsh为表达质粒,构建重组质粒phsh—Ceff4,并转化至大肠杆菌中进行表达。基因表达产物通过热处理和离子交换层析,重组酶纯度达电泳纯。对纯化的重组酶酶学性质研究表明,最适反应温度85℃,最适反应pH4．6,pH4．5—6．0之间酶的相对酶活在80％以上。Co^2＋对酶活性有促进作用,Ca^2＋、Mg^2＋、Zn^2＋不影响酶活性,而Cu^2＋、Ni^2＋、Mn^2＋对酶活性有抑制作用。     

New effective sources of the Staphylococcus simulans lysostaphin

The gene encoding Staphylococcus simulans lysostaphin has been cloned into two Escherichia coli expression systems: pET23b+ (Novagen, UK) and pBAD/Thio-TOPO (Invitrogen, USA), which allow the overexpression of a target protein as a fusion protein. The enzyme produced in the pET system contains a cluster of six histidines at the C-terminus, and the protein produced in the pBAD system contains 133 additional amino acid residues at the N-terminus, including thioredoxin, a cluster of six histidines and a recognition site for endoprotease Factor Xa. The recombinant enzymes were purified by metal-affinity chromatography on a Co2+-Sepharose column. Approximately 20 mg of purified recombinant enzyme were obtained in the pET expression system and 39 mg in the pBAD system, from a 1-L culture. The obtained fusion protein from the pET system revealed specific activity that was approximately 10 times higher than that of the fusion protein from the pBAD system (970 U/mg versus 83 U/mg). The purified enzymes displayed maximum activity at close to 45 degrees C and pH 8.0 or 7.5 for the enzyme obtained from pET and pBAD system, respectively. The lysostaphin activity was strongly inhibited by Zn2+ or Cu2+ (2 mM) with a 70-80% decrease. The Ni2+ (2 mM) also inhibited the enzyme with a 60 and 20% activity decrease for enzyme from the pET and pBAD system, respectively. The Co2+ had no impact on enzymatic activity at the 2 mM concentration; however, 30 and 20% activity decreases were observed at the 10mM concentration for the enzyme obtained from the pET and pBAD expression systems, respectively. EDTA, known as a strong inhibitor of the native lysostaphin, had no impact on the antistaphylococcal activity of either recombinant enzyme.     

Molecular cloning and characterization of a bile salt hydrolase from Lactobacillus acidophilus PF01

Phenotypic screening for bile salt hydrolase (BSH) activity was performed on Lactobacillus acidophilus PF01 isolated from piglet feces. A gene encoding BSH was identified and cloned from the genomic library of L. acidophilus PF01. The bsh gene and surrounding regions were characterized by nucleotide sequence analysis and were found to contain a single open reading frame (ORF) of 951 nucleotides encoding a 316 amino acid protein. The potential bsh promoter region was located upstream of the start codon. The protein deduced from the complete ORF had high similarity with other BSHs, and four amino acid motifs located around the active site, FGRNXD, AGLNF, VLTNXP, and GXGXGXXGXPGD, were highly conserved. The bsh gene was cloned into the pET21b expression vector and expressed in Escherichia coli BLR(DE3) by induction with 0.1mM of isopropylthiogalactopyranoside. The BSH enzyme was purified with apparent homogeneity using a Ni2+-NTA agarose column and characterized. The overexpressed recombinant BSH enzyme of L. acidophilus PF01 exhibited hydrolase activity against tauroconjugated bile salts, but not glycoconjugated bile salts. It showed the highest activity against taurocholic acid. The maximum BSH activity occurred at approximately 40oC. The enzyme maintained approximately 70% of its maximum activity even at 60 degrees , whereas its activity rapidly decreased at below 37 degrees . The optimum pH was 6, and BSH activity was rapidly inactivated below pH 5 and above pH 7.     

重组黄杆菌肝素酶Ⅲ的纯化、表征及培养条件对酶生产的影响

肝素酶Ⅲ是一种特异性地裂解乙酰肝素的酶,在大肠杆菌中表达时容易形成包涵体.为实现肝素酶Ⅲ的可溶性表达,利用谷胱甘肽-S-转移酶(GST)与肝素酶Ⅲ融合性能,通过构建相应的表达质粒pGEX-heparinaseⅢ,在大肠杆菌中实现了肝素酶Ⅲ的可溶性表达.粗酶通过一步亲和纯化其纯度可达95%以上.通过对LB培养基摇瓶培养Escherichia coli BL21的诱导时机,诱导剂用量、诱导时间等培养条件的优化,确定了该可溶性肝素酶Ⅲ融合蛋白的最适生产条件.通过对纯酶的最适反应温度、pH、Ca~(2+)浓度等一系列性质研究,确定了该酶的最适反应条件.     

嗜热枯草芽孢杆菌普鲁兰酶基因的表达与重组酶的性质

克隆嗜热枯草芽孢杆菌WY-34普鲁兰酶基因并在大肠杆菌中进行表达,对重组酶进行纯化和酶学性质研究,根据枯草芽孢杆菌的普鲁兰酶蛋白序列,设计PCR引物对WY-34的普鲁兰酶基因进行克隆及异源表达.对表达蛋白的最适pH、pH稳定性及最适温度、温度稳定性等特性进行研究,并测定重组普鲁兰酶的底物特异性.将普鲁兰酶基因pluA克隆及分析序列后,发现基因长度为2.2 kb,编码718个氨基酸,在大肠杆菌中异源表达.通过Ni-IDA亲和层析一步纯化得到比活力为93.2 U/mg的纯酶,SDS-PAGE和凝胶层析测定的分子量分别为76.2 kD和74.3 kD.酶学性质研究表明,该酶的最适温度为40℃,在温度不高于45℃条件下稳定;最适pH为6.0,同一温度下pH 6.0-9.0范围内处理30 min可以保持80％以上的酶活力,此酶对普鲁兰糖有很强的底物特异性.此重组普鲁兰酶的酶学性质表明此酶具有一定的工业化应用价值.