圈卷产色链霉菌硝基烷类氧化酶的分离纯化及酶学性质

圈卷产色链霉菌硝基烷类氧化酶基因naoA在大肠杆菌中获得了成功表达,从含有重组质粒pNA101(pET23b∷naoA)的工程菌株BL21(DE3)中分离纯化了硝基烷类氧化酶,SDSPAGE检测为均一。对纯酶进行了酶学性质及动力学研究。底物为1硝基丙烷、2硝基丙烷和硝基乙烷时,在04mol/L的磷酸缓冲液中,酶的最适反应pH值为7～8,最适反应温度为48℃～56℃。室温保存6d后,酶的活性保持了43.3%,但对60℃以上的高温敏感。硫醇化合物如巯基乙醇、还原型谷胱甘肽不同程度地抑制酶活性,特别是NADH,其浓度为1mmol/L时,酶活性几乎全部丧失。以1硝基丙烷为底物时,NaoA的Km为357mmol/L,Vmax为0199μmol/(μg.min)。     

苯丙氨酸合成的关键酶基因aroG与pheA串联表达

aroG和pheA是与苯丙氨酸合成有关的两个重要基因。在大肠杆菌(Escherichia coli)中,aroG基因编码脱氧阿拉伯糖型庚酮糖磷酸合酶(DS),该酶催化由糖代谢中心途径分流出来的磷酸烯醇丙酮酸(PEP)和赤鲜糖4磷酸(E4P)缩合形成脱氧阿拉伯糖型庚酮糖磷酸(DAHP)的反应;pheA基因编码一个双功能酶蛋白,它同时催化两步关键反应,即具有分枝酸变位酶(CM)和预苯酸脱水(PD)的两种功能。采用PCR技术分别从两个不同品系的大肠杆菌染色体DNA中扩增到aroG和pheA。当这两个基因串联在一个质粒上导入大肠杆菌P2392中进行表达时,它们编码的酶DS、CM和PD活性分别提高43、44和22倍;导入短杆菌(Brevibacterium)2731中表达时,相应的酶活性分别提高123、23和56倍。两基因的串联表达能大幅度地提高工程菌株的苯丙氨酸发酵产量。     

生物合成谷胱甘肽种间耦合ATP再生系统的构建

利用重组大肠杆菌Ⅱ 1中的谷胱甘肽合成酶系和面包酵母WSH J7中的ATP生物合成酶系 ,构建了一个以葡萄糖为能源的种间耦合ATP再生系统。经过通透性处理的酵母细胞几乎不能消耗葡萄糖。在反应体系中添加 1mmol/LAMP和 0 0 5mmol/LNADH ,即可启动酵母的酵解途径。提高耦合系统中的葡萄糖浓度 ,可促进GSH的合成。当葡萄糖浓度为 40 0mmol/L时 ,系统内GSH浓度达到 1 0 4mmol/L(3 2 g/L)。Mg2 +缺乏时 ,耦合系统和外加ATP的非耦合系统均不能合成谷胱甘肽。耦合系统中Mg2 +与ATP形成螯合物 ,可能是导致耦合系统中GSH产量较低的原因。在耦合系统中补加Mg2 +,反应 6h时GSH浓度达到 1 4 3mmol/L(4 4g/L)。     

氨甲酰磷酸不同合成途径在大肠杆菌中的比较

【背景】氨甲酰磷酸是生物合成代谢中精氨酸与嘧啶的重要前体物质,在工业微生物生产精氨酸与嘧啶及其衍生物中发挥关键作用。【目的】在大肠杆菌Escherichia coli BW25113中比较氨甲酰磷酸不同合成途径的催化效率。【方法】在大肠杆菌Escherichia coli BW25113中过表达鸟氨酸氨甲酰基转移酶(OTC)的基础上,分别过表达大肠杆菌自身的氨基甲酸激酶(CK)和氨甲酰磷酸合酶(CPSⅡ)并表征其反应效果。通过优化底物供应(调整底物浓度与引入L-谷氨酰胺合成酶)对CK与CPSⅡ的催化反应进行优化。【结果】在大肠杆菌中过表达OTC,建立细胞水平氨甲酰磷酸检测体系。在此基础上比较不同来源的CK,发现大肠杆菌来源的CK效果最好,50mmol/LNH4HCO3条件下全细胞催化9h得到2.95±0.15mmol/LL-瓜氨酸;过表达CPSⅡ时,50mmol/LL-谷氨酰胺催化9h得到3.16±0.29 mmol/L L-瓜氨酸。通过改变底物NH4HCO3浓度和引入外源L-谷氨酰胺合成酶(GS)等方式对CK与CPSⅡ的催化反应分别进行优化后,100 mmol/L NH4HCO3条件下,L-瓜氨酸浓度分别提高至4.67±0.55mmol/L和6.12±0.38mmol/L,且过表达GS后CPSⅡ途径可以利用NH3,不需要额外添加L-谷氨酰胺。【结论】引入L-谷氨酰胺合成酶后的CPSⅡ途径合成氨甲酰磷酸的能力优于CK途径,为精氨酸、嘧啶及其衍生物的合成提供了一种更加高效的策略。     

庚型肝炎病毒E2区cDNA在毕赤酵母中的表达及抗原性鉴定

从含有庚型肝炎病毒(GBVC/HGV)包膜蛋白E2 cDNA(559bp)的质粒pGEX\|E2中,扩增得到能够编码日本血吸虫谷胱甘肽硫转移酶(GST)和GBVC/HGV包膜蛋白E2的融合基因片段。将此长度为1324bp的DNA片段插入到酵母表达载体pPIC9K中,使之位于α因子信号肽下游,且与之同框。通过电激转化将构建的重组表达质粒pPIC9K\|GST\|E2插入到Pichia pastoris GS115菌株染色体中。筛选His\++Mut\+s表型的转化子,震荡培养,用05%甲醇诱导表达5d后,在培养液中得到表达的GSTE2融合蛋白。经过表达条件的优化,GSTE2蛋白可占培养液中总蛋白的50%。通过谷胱甘肽亲和层析柱纯化,GSTE2融合蛋白的纯度可达95%左右。以庚型肝炎病人血清为探针,进行免疫印迹及ELISA实验,结果表明该融合蛋白具有能被庚型肝炎病人血清特异性识别的抗原性。     

无机磷影响叠氮钠对ATP合成酶合成活性的抑制

利用ADP和放射性磷直接合成ATP的方法,研究了无机磷(P_i)和叠氮钠对猪心线粒体ATP合成酶（F₁F_O-ATPase）ATP合成活性的影响.结果发现无机磷除作为合成ATP的底物参与F₁F_O-ATPase的合成反应外,还对F₁F_O-ATPase的合成活性呈现抑制作用,在1 mmol/L ADP存在时,随着P_i浓度由0.01～10 mmol/L增加,抑制合成作用越来越强.与叠氮钠在低浓度时(小于1 mmol/L)只抑制ATP水解,不影响ATP合成的观点不同.实验结果显示0.1 mmol/L叠氮钠表观激活F₁F_O-ATPase的ATP合成活性,且激活程度与反应体系中所加P_i的浓度呈负相关.当固定P_i浓度（0.1 mmol/L）后,随着叠氮钠浓度的增加表观激活程度也在变化,叠氮钠与磷浓度相等时表观激活程度最大,直至叠氮钠浓度接近0.5 mmol/L时,开始呈现表观抑制现象,叠氮钠浓度高于1 mmol/L之后,就出现解偶联现象.     

大肠杆菌-分枝杆菌穿梭表达质粒pBCG-2100的构建及应用

利用分子生物学方法,构建了大肠杆菌分枝杆菌(E.coliMycobacterium)穿梭表达质粒pBCG-2100,研究了编码日本血吸虫中国大陆株谷胱甘肽S转移酶(Glutathione Stransferase,GST)抗原基因在卡介苗(Bacillus Calmette Guerin,BCG)中的表达。以含人结核杆菌热休克蛋白(Heat shock protein,hsp)70基因全长序列的质粒pMT-70为模板,扩增出hsp70启动子,测序选出无错配的启动子,将其定向克隆入E.coliMycobacterium穿梭质粒pBCG-2000中,构建成E.coliMycobacterium穿梭表达质粒pBCG-2100。再将编码GST的cDNA按正确的阅读框顺序,克隆到pBCG-2100中hsp70启动子的下游,得到分枝杆菌表达质粒pBCG-GST。将pBCG-GST电转化入BCG中,筛选出重组BCG疫苗,经热诱导后所表达的重组GST(rGST)抗原,为可溶性蛋白,经纯化后,在SDS-PAGE上分子量为26kD处可见明显的表达蛋白带,其表达量占BCG菌体总蛋白的13%。Western blot提示rGST能与抗GST的抗体反应。     

S-腺苷甲硫氨酸合成酶的组成型表达、产物纯化及鉴定

将大肠杆菌(E.coli K12) S 腺苷甲硫氨酸合成酶(SAMS)基因克隆至质粒pBR322中,获得的重组质粒pBR322-SAMS转入大肠杆菌JM109菌株,构建了能高效组成型表达SAMS的重组菌E.coli JM109 (pBR322-SAMS)。将重组大肠杆菌破碎后上清液经20%~40%硫酸铵分级盐析、Phenyl-Sepharose Fast Flow疏水层析和Q Sepharose Fast Flow离子交换层析,即可得到纯度提高5倍,比活为48.7 μ/mg的SAMS,三步纯化的总回收率为62%,纯度达到92%。SAMS表达量为1 176μ/L,占到菌体可溶性总蛋白的20%。重组酶的最适反应pH为8.5,4℃下在pH 7.5的缓冲液中保温10h酶活性几乎不改变。重组酶反应的最适温度为55℃ ,酶活性稳定的温度范围为20~35℃。重组酶的KmL Met为0.22mmol/L,Vmax L-Met为1.07mmol/(L·h),Km ATP为0.52 mmol/L,Vmax ATP为1.05 mmol/( L·h)。     

短双歧杆菌α-D-半乳糖苷酶基因aga1在大肠杆菌中的高效表达

短双歧杆菌（Bifidobacterium breve 203）α_D_半乳糖苷酶基因(aga1)被克隆到大肠杆菌温度诱导表达质粒pBV220中,构建重组质粒pBVaga1,转入大肠杆菌进行温度诱导表达,得到的重组酶Aga1在大肠杆菌DH5α、DH10B和BL21中的比活分别为28.08、19.44和13.85U/mg, 均高于短双歧杆菌α_D_半乳糖苷酶的比活1.76U/mg。重组质粒pBVaga1在E. coli BL21中稳定性较好。重组酶Aga1蛋白亚基分子量约67kD,最适反应温度为45℃,酶在40℃以下稳定,60℃仅剩余约5%的酶活性,70℃时酶全部失活;最适反应pH为4.0～4.4,酶在pH 3.6～6.0范围内稳定;酶对p_硝基苯酚_α_半乳糖苷的Km＝1.43mmol/L,Vmax＝35.71μmol/(L·min),对蜜二糖的Km＝261mmol/L,Vmax＝63.69μmol/(L·min);酶在蜜二糖、棉子糖水解体系中不显示转糖基活性。结果说明Aga1与已经报道的一种短双歧杆菌的α_D_半乳糖苷酶不同,是新发现的一种短双歧杆菌的α_D_半乳糖苷酶。     

重组大肠杆菌发酵生产谷胱甘肽的氨基酸添加策略优化

对表达双功能谷胱甘肽合成酶的重组大肠杆菌发酵生产谷胱甘肽(Glutathione,GSH)进行氨基酸添加策略优化,结果表明:基本培养基中未添加氨基酸时GSH产量为0.81 g/L;诱导2 h后添加17 mmol/L半胱氨酸GSH产量为1.16 g/L,比不加氨基酸提高43%;添加17 mmol/L的3种前体氨基酸,GSH产量达到3.86 g/L,比只添加半胱氨酸提高2.33倍;进一步提高3种氨基酸添加量至25 mmol/L,GSH产量可达4.64 g/L,比不添加氨基酸提高4.73倍,总生产强度高达317.8 mg/(L·h),半胱氨酸转化为谷胱甘肽达到0.60 mol/mol;考察氨基酸添加模式发现一次性添加25 mmol/L氨基酸较恒速流加模式生产速率提高了29.8%。后续在50 L罐放大生产GSH,产量为4.31 g/L,总生产强度达到310.1 mg/(L·h),为工业化放大生产GSH奠定了基础。     

谷胱甘肽合成酶系的克隆、测序及表达

谷胱甘肽(Glutathione,GSH)是由γ-谷氨酰半胱氨酸合成酶(GSHI)及谷胱甘肽合成酶(GSHII)连续催化合成的一种巯基化合物,有维持细胞正常的还原状态、保护细胞免受重金属的侵害等重要的生理功能,在临床、食品、保健品等方面有广泛的用途,如:重金属解毒、癌症的辐射和化疗的保护、HIV的抑制、抗氧化等.     

大肠杆菌BL21(pTrc-gsh)与酵母耦联合成谷胱甘肽的研究

谷胱甘肽 (ＧＳＨ)广泛存在于动、植物和微生物细胞内 ,有参与氨基酸的跨膜运输、维持细胞的还原状态等重要生理功能 ,在临床、保健品、食品等行业有广泛用途 ,如 :重金属解毒、抗氧化延缓衰老等 ,我国基本靠进口。开发高效、低成本的ＧＳＨ生产工艺势在必行。谷胱甘肽的制备有化学合成法[1 ] 、提取法[2 ] 、微生物发酵法[3] 、酶法[4] 等。由于酶法合成ＧＳＨ的产率高、后续的分离提取较简单而倍受关注。它是以ＡＴＰ为能量供体、由γ 谷氨酰半胱氨酸合成酶 (ＧＳＨＩ)和谷胱甘肽合成酶 (ＧＳＨＩＩ)连续催化合成的 :谷氨酸 半胱氨酸 Ａ…     

Improved glutathione production by gene expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

To utilize Pichia pastoris to produce glutathione, an intracellular expression vector harboring two genes (gsh1 and gsh2) from Saccharomyces cerevisiae encoding enzymes involved in glutathione synthesis and regulated by the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter was transformed into P. pastoris GS115. Through Zeocin resistance and expression screening, a transformant that had higher glutathione yield (217 mg/L) in flask culture than the host strain was obtained. In fed-batch culture process, this recombinant strain displayed high activity for converting precursor amino acids into glutathione. The glutathione yield and biomass achieved 4.15 g/L and 98.15 g (dry cell weight, DCW)/L, respectively, after 50 h fermentation combined with addition of three amino acids (15 mmol/L glutamic acid, 15 mmol/L cysteine, and 15 mmol/L glycine).     

A perturbation in glutathione biosynthesis disrupts endoplasmic reticulum morphology and secretory membrane traffic in Arabidopsis thaliana

To identify potentially novel and essential components of plant membrane trafficking mechanisms we performed a GFP-based forward genetic screen for seedling-lethal biosynthetic membrane trafficking mutants in Arabidopsis thaliana. Amongst these mutants, four recessive alleles of GSH2, which encodes glutathione synthase (GSH2), were recovered. Each allele was characterized by loss of the typical polygonal endoplasmic reticulum (ER) network and the accumulation of swollen ER-derived bodies which accumulated a soluble secretory marker. Since GSH2 is responsible for converting γ-glutamylcysteine (γ-EC) to glutathione (GSH) in the glutathione biosynthesis pathway, gsh2 mutants exhibited γ-EC hyperaccumulation and GSH deficiency. Redox-sensitive GFP revealed that gsh2 seedlings maintained redox poise in the cytoplasm but were more sensitive to oxidative challenge. Genetic and pharmacological evidence indicated that γ-EC accumulation rather than GSH deficiency was responsible for the perturbation of ER morphology. Use of soluble and membrane-bound ER markers suggested that the swollen ER bodies were derived from ER fusiform bodies. Despite the gross perturbation of ER morphology, gsh2 seedlings did not suffer from constitutive oxidative ER stress or lack of an unfolded protein response, and homozygotes for the weakest allele could be propagated. The link between glutathione biosynthesis and ER morphology and function is discussed.     

人源溶菌酶在大肠杆菌中的表达与复性研究

人源溶菌酶(Human lysozyme,HLZ)是一种糖苷水解酶,具有抗菌消炎的作用,其作为抗生素的替代品,已经被广泛应用于食品业、畜牧业和医疗等领域。如何获得高产量、高活性、高纯度的人源溶菌酶一直是亟待解决的技术问题。优化人源溶菌酶编码基因密码子,提高其在大肠杆菌中的适应度和表达量;将优化的基因克隆至大肠杆菌表达质粒pET21a,并将其在大肠杆菌表达菌株BL21(DE3)中诱导表达;利用8 mol/L尿素溶液对包涵体进行溶解变性后,探究一步透析、梯度透析和梯度稀释3种复性方式以及复性液中谷胱甘肽氧化还原对(GSSG/GSH)、精氨酸、甘油等复性物的浓度对重组人源溶菌酶复性的效果,获得最佳的复性方案。研究结果表明:37℃诱导温度下,利用0.5 mmol/L IPTG成功诱导了分子量约为14.7 kD的重组人源溶菌酶的表达,包涵体表达量约为380 mg/L(湿重)。包涵体经一步透析、梯度透析和梯度稀释3种复性方式复性后,测得比活力值分别为147 U/mg、335 U/mg、176 U/mg,表明最佳复性方法为梯度透析复性法。进一步探索了复性液中GSSG/GSH比值、精氨酸浓度、甘油浓度对人源溶菌酶复性效果的影响,表明当复性液中同时添加浓度比为1∶2的GSSG/GSH、4 mmol/L精氨酸和6%甘油时,复性后人源溶菌酶的最佳比活力值为1170 U/mg,显著高于3种复性物均不加时溶菌酶335 U/mg的比活力值,但低于溶菌酶标准品1732 U/mg的比活力值。成功地将人源溶菌酶基因在大肠杆菌中表达,并通过包涵体复性体系成功获得高活性重组人源溶菌酶。     

响应面分析法优化重组大肠杆菌生物合成谷胱甘肽的条件

通过响应面分析法和典型性分析得出重组大肠杆菌酶法合成谷胱甘肽的最优条件:菌体量249 mg/mL,磷酸钾缓冲液145 mmol/L,MgCl243 mmol/L和ATP 34 mmol/L,预测谷胱甘肽最大量为16.50 mmol/L。验证性实验证明在优化条件下,重组大肠杆菌酶法合成谷胱甘肽达16.42 mmol/L。响应面分析还表明,在重组大肠杆菌酶法合成谷胱甘肽各因素中,MgCl2和ATP,以及菌体量与磷酸钾缓冲液之间的交互作用较显著。     

The essential and ancillary role of glutathione in Saccharomyces cerevisiae analysed using a grande gsh1 disruptant strain

A grande gsh1 disruptant mutant of Saccharomyces cerevisiae was generated by crossing a petite disruptant to a wild-type grande strain. This strain was relatively stable, but generated petites at an elevated frequency, illustrating the ancillary role of glutathione (GSH) in the maintenance of the genetic integrity of the mitochondrial genome. The availability of the grande gsh1 deletant enabled an evaluation of the role of GSH in the cellular response to hydrogen peroxide independent of the effects of a petite mutation. The mutant strain was more sensitive to hydrogen peroxide than the wild-type strain but was still capable of producing an adaptive stress response to this compound. GSH was found to be essential for growth and sporulation of the yeast, but the intracellular level needed to support growth was at least two orders of magnitude less than that normally present in wild-type cells. This surprising result indicates that there is an essential role for GSH but only very low amounts are needed for growth. This result was also found in anaerobic conditions, thus this essential function does not involve protection from oxidative stress. Suppressors of the gsh1 deletion mutation were isolated by ethylmethanesulfonate mutagenesis. These were the result of a single recessive mutation (sgr1, suppressor for glutathione requirement) that relieved the requirement for GSH for growth on minimal medium but did not affect the sensitivity to H(2)O(2) stress. Interestingly, the gsh1 sgr1 mutant generated petites at a lower rate than the gsh1 mutant. Thus, it is suggested that the essential role of GSH is involved in the maintenance of the mitochondrial genome.     

Cloning and functional analysis of the GSH1/MET1 gene complementing cysteine and glutathione auxotrophy of the methylotrophic yeast Hansenula polymorpha

The Hansenula polymorpha GSH1/MET1 gene was cloned by complementation of glutathione-dependent growth of H. polymorpha gsh1 mutant isolated previously as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resistant and cadmium ion sensitive clone. The H. polymorpha GSH1 gene was capable of restoring cadmium ion resistance, MNNG sensitivity, normal glutathione level and cell proliferation on minimal media without addition of cysteine or glutathione, when introduced into the gsh1 mutant cells. It was shown that the H. polymorpha GSH1 gene has homology to the Saccharomyces cerevisiae MET1 gene encoding S-adenosyl-L-methionine uroporphyrinogen III transmethylase, responsible for the biosynthesis of sulfite reductase cofactor, sirohaem. The H. polymorpha GSH1/MET1 gene deletion cassette (Hpgsh1/met1::ScLEU2) was constructed and corresponding null mutants were isolated. Crossing data of the point gsh1 and null gsh1/met1 mutants demonstrated that both alleles were located to the same gene. The null gsh1/met1 mutant showed total growth restoration on minimal media supplemented with cysteine or glutathione as a sole sulfur source, but not with inorganic (sulfate, sulfite) or organic (methionine, S-adenosylmethionine) sources of sulfur. Moreover, both the point gsh1 and null gsh1/met1 mutants displayed increased sensitivity to the toxic carbon substrate methanol, formaldehyde, organic peroxide and cadmium ions.     

Glutathione production by Escherichia coli cells with hybrid plasmid containing tandemly polymerized genes for glutathione synthetase

Summary A DNA fragment containing the structural and promoter regions of glutathione synthetase (GSH II) gene (gsh II) from Escherichia coli B were polymerized. The dimeric and trimeric DNA fragments obtained were inserted into Bam HI site of vector plasmid pBR325 and the resulting hybrid plasmids were designated pGS401-02 and pGS401-03, respectively. The GSH II activity of E. coli cells with these hybrid plasmids increased depending on the number of the genes (gsh II) contained. To construct hybrid plasmids useful for glutathione production, another DNA fragment with a gene (gsh I) for -glutamylcysteine synthetase (GSH I) from E. coli B was inserted into Pst I sites of pGS401-02 and pGS401-03 and the hybrid plasmids obtained (pGS501-12 and pGS501-13, respectively) were introduced into E. coli B cells. Although the glutathione-producing activities of the cells with these plasmids were little improved as compared with that of cells with the hybrid plasmid (pGS501-11) containing both gsh I and gsh II because of the low activity of GSH I, our method has brought to light a new type of gene amplification.