蜡样芽胞杆菌磷脂酶C在乳酸克鲁维酵母中重组表达、纯化及酶学性质分析

【目的】构建蜡样芽胞杆菌(Bacillus cereus)磷脂酶C(Phospholipase C,PLC)的重组乳酸克鲁维酵母(Kluyveromyces lactis)菌株、纯化重组蛋白并对其进行酶学性质分析。【方法】以B.cereus基因组DNA为模板,PCR扩增得到磷脂酶C基因(bcplc),构建重组乳酸克鲁维酵母表达质粒并转化到乳酸克鲁维酵母中,实现bcplc基因的表达。利用镍柱亲和层析纯化和脱盐柱得到电泳纯的重组磷脂酶C(rbcPLC)。【结果】成功构建产磷脂酶C的重组乳酸克鲁维酵母并纯化了重组磷脂酶C,纯化后rbcPLC经SDS-PAGE分析在40 kDa附近出现显性条带。NPPC法测得rbcPLC酶活为19251 U/mg,最适反应温度为80°C,最适pH为9.0。在低于40°C时,pH 7.0-8.0时,rbcPLC重组酶较稳定。Cu~(2+)和Co~(2+)对其有明显的抑制作用;Zn~(2+)、Mn~(2+)、Ca~(2+)、Mg~(2+)对其有明显的促进作用。【结论】首次实现了对蜡样芽胞杆菌来源的磷脂酶C在乳酸克鲁维酵母中的重组表达、纯化及其酶学性质分析,为其它食品安全性微生物来源的磷脂酶C的研究提供了借鉴意义。     

芽孢杆菌P38中乳酸脱氢酶对其产L-乳酸光学纯度的影响

【目的】研究芽孢杆菌(Bacillus sp.) P38中乳酸脱氢酶对其产高光学纯L-乳酸(光学纯度>99%)的影响。【方法】全基因组测序显示在该菌中存在3个乳酸代谢关键酶,分别为L-乳酸脱氢酶(L-LDH)、D-乳酸脱氢酶(D-LDH)和苹果酸或L-乳酸脱氢酶(M/L-LDH)。通过将这3个酶进行异源表达、纯化与酶学特性分析,结合Native-PAGE、实时荧光定量PCR等方法,初步确定该菌高产光学纯L-乳酸的机理。【结果】Bacillus sp. P38中L-LDH对丙酮酸的催化活性(Kcat/Km值)最高,分别是D-LDH的2.9倍和M/L-LDH的4.3倍。其中M/L-LDH主要起L-LDH的功能。Native-PAGE实验中未检测到D-LDH活性。Bacillus sp. P38所有发酵阶段ldhL的转录水平均高于ldhD和ldhM/L。【结论】L-LDH是Bacillus sp. P38产高光学纯L-乳酸的主要关键酶。     

乳酸片球菌L-乳酸脱氢酶基因的克隆及在重组大肠杆菌JH12中的过量表达

以工程菌Escherichia coli SZ85基因组为模板,克隆得到乳酸片球菌(Pediococcus acidilactici)的L-乳酸脱氢酶基因(ldhL),连接到pUcm-T载体后,双酶切然后将其连接到表达载体pET-28a上,重组质粒经筛选后转入染色体上含有ldhL基因(来源P.acidilactici)的工程菌Escherichia coli JH12。P.acidilactici的ldhL基因过量表达体系E.coli JH12(pET-28a-ldhL)能利用浓度7%的木糖为碳源进行厌氧发酵,过量表达ldhL使L-乳酸产量提高了10 g/L,达64.86 g/L,糖酸转化率高达96%。     

米根霉菌ldhL基因的克隆及其在大肠杆菌中的表达

以米根霉菌基因组DNA为模板,根据GenBank上已公布的米根霉L-乳酸脱氢酶基因(ldhL)序列设计特异性引物,PCR扩增得到963 bp的DNA片段,经序列分析后将其亚克隆到原核表达载体pET30a上,构建成重组质粒pET30a-ldhL.将pET30a-ldhL转化到BL21感受态细菌中,经IPTG诱导表达后进行SDS-PAGE分析,可见约43 kD的与预期大小一致的目的蛋白条带,结果表明ldhL基因在大肠杆菌中进行了表达,经酶活分析产物的酶活力为98 U/mL,证明了表达产物具有预期的酶活性,这为进一步研究利用乳清为发酵原料高产L-乳酸的米根霉基因工程菌株奠定了基础.     

乙酸钙不动杆菌磷脂酶C在大肠杆菌中重组表达、纯化及酶学性质分析

【目的】构建乙酸钙不动杆菌(Acinetobacter calcoaceticus)ATCC17902磷脂酶C(Phospholipase C,PLC)的重组大肠杆菌菌株、纯化重组酶并进行酶学性质分析比较。【方法】以A.calcoaceticus ATCC17902基因组DNA为模板,PCR扩增得到两段磷脂酶C基因(PLC1、PLC2),构建重组大肠杆菌表达质粒并转化大肠杆菌BL21(DE3)中,实现PLC1、PLC2基因的表达。IPTG诱导表达后,经镍柱亲和层析纯化重组蛋白。【结果】成功构建两株产磷脂酶C的重组大肠杆菌并纯化,样品经SDS-PAGE分析在80 kDa附近均出现显著的特异性条带。NPPC法测得PLC1、PLC2酶活分别为31160±418 U/mg、13640±354 U/mg,最适反应温度分别为65、50℃,最适pH值分别为8、7.5。在低于30℃时,pH值7-8时,PLC1、PLC2重组酶较稳定,40℃处理30min,PLC1酶活稳定而PLC2残余酶活低于25%。Mg2+、Ca2+增强PLC1、PLC2的活性,Zn2+增强PLC1酶活性却抑制PLC2酶活。底物特异性分析表明PLC1、PLC2均水解磷脂酰肌醇(Phosphatidylinositol,PI),对其他种类磷脂不能水解或水解程度很低。【结论】本文首次实现了A.calcoaceticus ATCC17902来源的磷脂酶C的重组表达与功能验证,为其它食品安全性微生物来源的磷脂酶C的研究提供了一定的借鉴意义。     

丙酮丁醇梭菌ldhL基因的克隆及其在大肠杆菌中的表达

首次从丙酮丁醇梭菌(Clostridium acetobutylicum ATCC824)中克隆得到L-乳酸脱氢酶(L-lactate dehydrogenase,ldhL)基因,并将其连接到pSE380表达载体上,得到重组质粒pSE380ldhL,将重组质粒转化到乳酸脱氢酶和丙酮酸裂解酶缺陷的Escherichia coli FMJl44大肠杆菌中进行表达。SDS-PAGE分析表达产物的分子量约为34kD,摇瓶发酵后用HPLC检测分析L-乳酸产量为2.4g/L,纯度达到99.9%,不需要再进行手性分离,为以后在工业上生物法生产高纯度的L-乳酸打下基础。     

长梗木霉β-葡萄糖苷酶基因的克隆及表达

【目的】以实验室筛选获得的一株长梗木霉GM2(Trichoderma longibrachiatum)为材料,克隆出其β-葡萄糖苷酶(β-Glucosidase)基因bgl并在大肠杆菌和酵母中进行表达。【方法】利用同源克隆扩增出其β-葡萄糖苷酶基因bgl全长序列,分别亚克隆到质粒pET-32a(+)和pPICZα-B中,构建其原核表达载体pET32a(+)-bglI和真核表达载体pPICZα-B-bgl。【结果】bgl基因序列全长2 369 bp,含两个内含子,编码744个氨基酸。在大肠杆菌BL21(DE3)中表达bgl,重组蛋白以包涵体形式存在,上清液中没有β-葡萄糖苷酶的酶活。将载体pPICZα-B-bgl电转化入毕赤酵母GS115,得到78 kD左右重组蛋白,与预测大小相符。按9%接种量接入50 mL YP培养基(初始pH 5.5),30°C振荡培养96 h,添加终浓度1%的甲醇诱导后β-葡萄糖苷酶酶活达60 U/mL。重组酶bgl催化水杨苷水解反应的最适pH为5.0,最适温度为70°C;另外,此bgl在pH 3.0 10.0和40°C 60°C范围内具有比较好的稳定性。【结论】长梗木霉GM2的β-葡萄糖苷酶在P.pastoris中获得可溶性表达,并证明有一定的活性。     

几丁质酶基因的克隆表达及酶学性质

【背景】几丁质是自然界中储藏量仅次于纤维素的有机物,几丁质酶能降解几丁质生成几丁寡糖,实现废弃物的高值化利用,目前菌株产几丁质酶能力低限制了它的生产应用。【目的】克隆弧菌(Vibrio sp.)GR52的几丁质酶基因,实现其在大肠杆菌中的异源表达,对分离纯化的重组几丁质酶进行酶学性质研究。【方法】以弧菌GR52菌株基因组DNA为模板,克隆得到几丁质酶基因GR52-1,构建重组基因工程菌BL21(DE3)/p ET22b-chi GR52-1,诱导表达的产物通过Ni-NTA树脂纯化后进行酶学性质研究。【结果】重组酶的最适反应pH为6.0,在pH5.0-10.0范围内37°C保温1 h仍能保持85%以上的相对酶活力,具有较好的pH稳定性;最适反应温度为50°C,在45°C保温1 h其酶活力基本没有损失,在50°C保温1 h其残余酶活力仍达60%;在1 mmol/L浓度下,Cu~(2+)、Ca2+对该酶具有促进作用,Hg+对该酶具有明显的抑制作用;在5 mmol/L浓度下,Ni+对该酶具有一定的促进作用,Mn~(2+)、Co~(2+)、Li~+、Fe~(2+)、Hg~+、SDS(十二烷基硫酸钠)对该酶具有明显的抑制作用。以胶体几丁质为底物时,动力学参数Km、Vmax、kcat分别为0.85 mg/m L、0.19μmol/(m L·min)和7.02 s-1。底物特异性分析表明该重组酶能特异性降解几丁质。【结论】重组几丁质酶具有良好的酶学性质,为几丁质酶的开发应用奠定基础。     

麻类脱胶高效菌株果胶裂解酶基因克隆与表达

【目的】克隆麻类脱胶高效菌株Dickeya sp.DCE-01的果胶裂解酶基因并进行原核表达,对表达产物进行纯化和酶学性质研究。【方法】根据该菌株全基因组序列预测的果胶裂解酶基因Q59419设计引物,PCR扩增后将该基因连接到pEASY-E1和pACYCDuet-1载体上,导入E.coli BL21(DE3)进行表达。选择酶活力高的阳性克隆子进行大量诱导表达后,采用超滤和Sephadex G-100凝胶层析两步法纯化出果胶裂解酶,研究其酶学性质。【结果】克隆到果胶裂解酶基因pel(GenBank登录号:JX964997),其序列全长1 128 bp,编码375个氨基酸。pACYCDuet-1-pel-BL表达胞外果胶裂解酶活力最高,发酵液粗酶活达298.8 IU/mL。其最适反应温度为50°C,最适pH为9.0;保温1 h,酶活稳定温度≤45°C,稳定pH为9.0?10.0。酶催化作用依赖于Ca2+,其最适作用浓度为2 mmol/L;Zn2+、Ca2+和NH4+促进酶活力,Fe3+和Pb2+严重抑制酶活力;聚半乳糖醛酸钠为该酶的最适底物。【结论】从麻类脱胶高效菌株中发掘到碱性果胶裂解酶基因,其表达产物在生物质加工过程中具有重要工业化应用前景。     

低温几丁质酶基因在乳酸克鲁维酵母中的重组表达及酶学性质表征

【目的】通过构建假交替单胞菌(Pseudoalteromonassp.DL-6)低温几丁质酶(chitinaseA,chi A;chitinase C,chi C)的重组乳酸克鲁维酵母菌株、纯化重组蛋白并对其进行酶学性质表征,为低温几丁质酶潜在工业化生产几丁寡糖奠定理论基础。【方法】人工合成密码子优化的几丁质酶基因,构建重组乳酸克鲁维酵母表达质粒(p KLAC1-chi A、p KLAC1-chi C)并用电脉冲法转化到乳酸克鲁维酵母中,实现低温几丁质酶的可溶表达。利用镍柱亲和层析纯化得到高纯度的重组几丁质酶。【结果】成功构建产低温几丁质酶的重组乳酸克鲁维酵母并纯化获得高纯度的重组几丁质酶。经SDS-PAGE分析在110 k Da与90 k Da附近出现符合预期大小的蛋白条带。铁氰化钾法测得Chi A和Chi C的酶活分别为51.45 U/mg与108.56 U/mg。最适反应温度分别为20°C和30°C,最适p H分别为8.0和9.0。在低于40°C,p H 8.0–12.0时,Chi A和Chi C重组酶较稳定。Chi A和Chi C对胶体几丁质以及粉状底物α-几丁质与β-几丁质具有明显的降解活性,且具有一定协同降解能力。【结论】首次实现假交替单胞菌来源的低温几丁质酶在乳酸克鲁维酵母中的重组表达、纯化、酶学性质及其降解产物分析,为其他低温几丁质酶的研究提供借鉴意义。     

L—乳酸脱氢酶基因克隆及功能分析

构建了一株产D,L-乳酸的乳杆菌(Lactobaeillus sp．)MD—1的基因库。利用乳酸脱氢酶和丙酮酸裂解酶缺陷的Escherichia coli FMJ144作为宿主,通过互补筛选分离克隆到乳酸脱氢酶基因(ldhL)。核酸序列分析表明,该基因以ATG为起始密码子编码316个氨基酸残基组成的蛋白质,预测的分子量为33．84kD;5′端存在典型的启动子结构,3′端的终止子是不依赖于ρ因子的转录终止子。ldhL编码的蛋白质有3个保守区域,其中Gly13～Asp50保守区域是NADH的结合位点,Asp73～Ile100和Asn123～Arg154保守区是酶的活性部位。该ldhL和其他乳杆菌的ldhL基因和编码的氨基酸序列相似性较低,核苷酸序列相似性最高仅为64．1％,氨基酸序列相似性最高仅为68．9％,是新的L—乳酸脱氢酶基因。     

Unusual amino acid substitution in the anion-binding site of Lactobacillus plantarum non-allosteric L-lactate dehydrogenase.

In Lactobacillus plantarum non-allosteric L-lactate dehydrogenase (L-LDH), the highly conserved His188 residue, which is involved in the binding of an allosteric effector, fructose 1,6-bisphosphate [Fru(1,6)P2], in allosteric L-LDH is uniquely substituted by an Asp. The mutant L. plantarum L-LDH, in which Asp188 is replaced by a His, showed essentially the same Fru(1,6)P2-independent catalytic activity as the wild-type enzyme, except that the Km and Vmax values were slightly decreased. However, the addition of Fru(1,6)P2 induced significant thermostabilization of the mutant enzyme, as in the case of many allosteric L-LDHs, while Fru(1,6)P2 showed no significant effect on the stability of the wild-type enzyme, indicating that only the single-point mutation, G-->C, sufficiently induces the Fru(1,6)P2-binding ability of L. plantarum L-LDH. The mutant enzyme showed higher thermostability than the wild-type enzyme in the presence of Fru(1,6)P2. In the absence of Fru(1,6)P2, on the other hand, the mutant enzyme was more labile below 65 degrees C but more stable above 70 degrees C.     

Metabolic engineering of Lactobacillus helveticus CNRZ32 for production of pure L-(+)-lactic acid

Expression of D-(-)-lactate dehydrogenase (D-LDH) and L-(+)-LDH genes (ldhD and ldhL, respectively) and production of D-(-)- and L-(+)-lactic acid were studied in Lactobacillus helveticus CNRZ32. In order to develop a host for production of pure L-(+)-isomer of lactic acid, two ldhD-negative L. helveticus CNRZ32 strains were constructed using gene replacement. One of the strains was constructed by deleting the promoter region of the ldhD gene, and the other was constructed by replacing the structural gene of ldhD with an additional copy of the structural gene (ldhL) of L-LDH of the same species. The resulting strains were designated GRL86 and GRL89, respectively. In strain GRL89, the second copy of the ldhL structural gene was expressed under the ldhD promoter. The two D-LDH-negative strains produced only L-(+)-lactic acid in an amount equal to the total lactate produced by the wild type. The maximum L-LDH activity was found to be 53 and 93% higher in GRL86 and GRL89, respectively, than in the wild-type strain. Furthermore, process variables for L-(+)-lactic acid production by GRL89 were optimized using statistical experimental design and response surface methodology. The temperature and pH optima were 41 degrees C and pH 5.9. At low pH, when the growth and lactic acid production are uncoupled, strain GRL89 produced approximately 20% more lactic acid than GRL86.     

Lactobacillus plantarum ldhL gene: overexpression and deletion.

Lactobacillus plantarum is a lactic acid bacterium that converts pyruvate to L-(+)- and D-(-)-lactate with stereospecific enzymes designated L-(+)- and D-(-)-lactate dehydrogenase (LDH), respectively. A gene (designated ldhL) that encodes L-(+)-lactate dehydrogenase from L. plantarum DG301 was cloned by complementation in Escherichia coli. The nucleotide sequence of the ldhL gene predicted a protein of 320 amino acids closely related to that of Lactobacillus pentosus. A multicopy plasmid bearing the ldhL gene without modification of its expression signals was introduced in L. plantarum. L-LDH activity was increased up to 13-fold through this gene dosage effect. However, this change had hardly any effect on the production of L-(+)- and D-(-)-lactate. A stable chromosomal deletion in the ldhL gene was then constructed in L. plantarum by a two-step homologous recombination process. Inactivation of the gene resulted in the absence of L-LDH activity and in exclusive production of the D isomer of lactate. However, the global concentration of lactate in the culture supernatant remained unchanged.     

PURIFICATION AND CHARACTERIZATION OF A PROTEINASE FROM THE PROBIOTIC Lactobacillus rhamnosus OXY

A proteinase produced by the human gastrointestinal isolate Lactobacillus rhamnosus strain OXY was identified and characterized. The prtR2 gene coding for proteinase activity was detected in the examined strain. The PCR primers used were constructed on the basis of the sequence of the prtR2 proteinase gene from Lactobacillus rhamnosus GG. The enzyme was purified by fast protein liquid chromatography (FPLC) using CM-Sepharose Fast Flow and Sephacryl S-300 columns. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the enzyme had a relatively low molecular mass of 60 kD. Protease activity was observed at a pH range from 6.5 to 7.5 with optimum k (cat)/K (m) values at pH 7.0 and 40°C. Maximum proteolytic activity (59?U mL(-1)) was achieved after 48?hr of cultivation. The activity of the enzyme was inhibited only by irreversible inhibitors specific for serine proteinases (PMSF and 3,4-dichloro-isocumarine), suggesting that the enzyme was a serine proteinase. Proteinase activity was increased by Ca(2+) and Mg(2+), and inhibited by Cu(2+), Zn(2+), Cd(2+), and Fe(2+.).     

Cloning of a Neisseria meningitidis gene for L-lactate dehydrogenase (L-LDH): evidence for a second meningococcal L-LDH with different regulation.

We report the cloning of lldA, a Neisseria meningitidis gene for L-lactate dehydrogenase (L-LDH). Escherichia coli contains a single L-LDH gene (lldD) in the lld operon (previously lct). E. coli grown in complex media does not have L-LDH activity, but the activity is induced by growth in defined medium with L-lactate as the carbon source. In contrast, meningococci contain at least one L-LDH in addition to the lldA gene product. These enzymes are active in meningococci grown in complex media and are not dependent on growth in L-lactate. The predicted amino acid sequence of lldA is homologous to that of E. coli lldD and of other prokaryotic and eukaryotic flavin mononucleotide-containing enzymes that catalyze the oxidation of L-lactate and other small alpha-hydroxy acids. A mutant with a deletion in lldA was found to have reduced L-LDH activity. However, this mutant was able to grow on L-lactate, indicating that a second L-LDH must exist. Activity of the lldA enzyme was affected by growth conditions, being increased by growth on a defined medium with either L-lactate or pyruvate as the carbon source. For meningococci grown on a complex medium, activity of the lldA enzyme was increased by growth on plates or in well-aerated broth. A second L-lactate-oxidizing activity was seen in bacteria grown in poorly aerated broth. Neisseria gonorrhoeae contains a homolog of lldA. As for meningococci, mutation of the gonococcal lldA reduced L-LDH activity but did not affect growth on L-lactate.     

A molecular design that stabilizes active state in bacterial allosteric L-lactate dehydrogenases

l-Lactate dehydrogenase (l-LDH) of Lactobacillus casei (LCLDH) is a typical bacterial allosteric l-LDH that requires fructose 1,6-bisphosphate (FBP) for its enzyme activity. A mutant LCLDH was designed to introduce an inter-subunit salt bridge network at the Q-axis subunit interface, mimicking Lactobacillus pentosus non-allosteric l-LDH (LPLDH). The mutant LCLDH exhibited high catalytic activity with hyperbolic pyruvate saturation curves independently of FBP, and virtually the equivalent K(m) and V(m) values at pH 5.0 to those of the fully activated wild-type enzyme with FBP, although the K(m) value was slightly improved with FBP or Mn(2+) at pH 7.0. The mutant enzyme exhibited a markedly higher apparent denaturating temperature (T(1/2)) than the wild-type enzyme in the presence of FBP, but showed an even lower T(1/2) without FBP, where it exhibited higher activation enthalpy of inactivation (ΔH(?)). This result is consistent with the fact that the active state is more unstable than the inactive state in allosteric equilibrium of LCLDH. The LPLDH-like network appears to be conserved in many bacterial non-allosteric l-LDHs and dimeric l-malate dehydrogenases, and thus to be a key for the functional divergence of bacterial l-LDHs during evolution.     

Electron acquisition system constructed from an NAD-independent D-lactate dehydrogenase and cytochrome c2 in Rhodopseudomonas palustris No. 7

The activities of NAD-independent D- and L-lactate dehydrogenases (D-LDH, L-LDH) were detected in Rhodopseudomonas palustris No. 7 grown photoanaerobically on lactate. One of these enzymes, D-LDH, was purified as an electrophoretically homogeneous protein (M(r), about 235,000; subunit M(r) about 57,000). The pI was 5.0. The optimum pH and temperature of the enzyme were pH 8.5 and 50 degrees C, respectively. The Km of the enzyme for D-lactate was 0.8 mM. The enzyme had narrow substrate specificity (D-lactate and DL-2-hydroxybutyrate). The enzymatic activity was competitively inhibited by oxalate (Ki, 0.12 mM). The enzyme contained a FAD cofactor. Cytochrome c(2) was purified from strain No. 7 as an electrophoretically homogeneous protein. Its pI was 9.4. Cytochrome c(2) was reduced by incubating with D-LDH and D-lactate.