用P.pastoris表达L-阿拉伯糖异构酶及其酶活性分析

目的:用毕赤酵母表达L-阿拉伯糖异构酶。方法:用PCR法扩增大肠杆菌的L-阿拉伯糖异构酶基因,构建含L-阿拉伯糖异构酶基因的毕赤酵母分泌型表达载体pPIC9K-ai。通过电转法将pPIC9K-ai转化毕赤酵母GS115基因组。先筛选出高G418抗性的克隆,然后再从高拷贝的克隆中筛选出高表达重组L-阿拉伯糖异构酶的重组子作为工程菌GS115(pPIC9K-ai)。结果:在甲醇诱导下,摇瓶发酵GS115(pPIC9K-ai)3d,分泌表达L-阿拉伯糖异构酶32 mg/L。结论:毕赤酵母表达的L-阿拉伯糖异构酶具有转化D-半乳糖为D-塔格糖的生物活性。每升GS115(pPIC9K-ai)发酵液能转化D-半乳糖生成30 mgD-塔格糖。     

用毕赤酵母组成型分泌表达Canstatin-N

目的:用毕赤酵母的GAP启动子调控组成型表达Canstatin-N。方法:将canstatin-N基因重组于毕赤酵母表达载体pGAP9K的多克隆位点获得pGAP9K-can-N。用电转法将pGAP9K-can-N转化毕赤酵母GS115。筛选高G418抗性的克隆作为工程菌GS115(pGAP9K-can-N)。发酵GS115(pGAP9K-can-N)分泌表达Canstatin-N,用离子交换法纯化目标蛋白。结果:以葡萄糖为碳源,发酵48h分泌表达人血管能抑素蛋白56 mg/L。结论:用毕赤酵母的GAP启动子调控组成型表达的人血管能抑素蛋白具有诱发血管内皮细胞凋亡的生物活性。     

乳酸菌素Gassericin T基因的人工合成及其组成型表达载体的构建

目的:为了研究乳酸菌素Gassericin T的作用机制及应用价值,人工合成Gassericin T基因并构建能高效表达外源蛋白的毕赤酵母组成型表达载体。方法:应用PCR方法从毕赤酵母染色体中扩增GAP启动子,经测序正确后与已线性化的不含pAOX1启动子的毕赤酵母诱导型表达载体pPIC9K连接,转化大肠杆菌DH5α。根据Gassericin T的基因序列,把Gassericin T的结构基因gatA的密码子转换成毕赤酵母偏爱的形式,设计了6条59nt的寡聚核苷酸引物,通过3次连续PCR反应,人工合成gatA片段(简称gat基因),经测序正确后插入pGAP9K质粒的多克隆位点。结果:用GAP启动子(pGAP)取代了pPIC9K上的pAOX1,构建了毕赤酵母组成型表达载体pGAP9K;PCR拼接获得250bp的目的基因序列,将目的基因克隆于pGAP9K,获得组成型表达载体pGAP9K-gat。结论:为下一步在毕赤酵母中组成型表达外源蛋白,研究其作用机理和遗传机制奠定了基础。     

Enzymatic synthesis of D-tagatose from lactose with the combination of β-galactosidase and L-arabinose isomerase

将大肠杆菌K-12中的β-半乳糖苷酶基因lacZ和L-阿拉伯糖异构酶基因araA以串联方式克隆到载体pET-28a(+)上,并转入大肠杆菌BL21( DE3)中进行表达.通过SDS-PAGE分析发现,重组菌株能表达出大量可溶性β-半乳糖苷酶蛋白和L-阿拉伯糖异构酶蛋白.以重悬菌液为酶源,可将乳糖降解为D-半乳糖,并将D-半乳糖转化为D-塔格糖.在温度为50℃,pH 7.0的缓冲液中,经一段时间反应后,D-塔格糖的转化率可达21％以上.加入Mn2+、Co2+和Fe2+均能够使D-塔格糖的转化率提高.     

发酵乳杆菌来源l-阿拉伯糖异构酶理性设计及在d-塔格糖生产中的应用

L-阿拉伯糖异构酶(L-arabinose isomerase,L-AI)是D-半乳糖异构化生成D-塔格糖的关键酶。为提高L-阿拉伯糖异构酶对D-半乳糖的活性及在生物转化中的转化率,本研究对发酵乳杆菌(Lactobacillus fermentum)CGMCC2921来源的L-阿拉伯糖异构酶进行重组表达和生物转化应用,并对其底物结合口袋进行理性设计以提高酶对D-半乳糖亲和力和催化活性。结果显示,突变体F279I对D-半乳糖的转化率提高至野生型酶的1.4倍,进一步叠加获得的双突变体M185A/F279I的K_m和k_cat分别为530.8mmol/L与19.9s^-1,底物亲和力显著提高,催化效率提高至野生型酶的8.2倍。以400 g/L乳糖为底物时,突变酶M185A/F279I转化率高达22.8%。本研究在乳糖高值化生产塔格糖方面具有重要的应用价值。     

联合β-半乳糖苷酶和L-阿拉伯糖异构酶法合成D-塔格糖的研究

将大肠杆菌K-12中的B-半乳糖苷酶基因lacZ和L-阿拉伯糖异构酶基因araA以串联方式克隆到载体pET-28a（＋）上,并转入大肠杆菌BL21（DE3）中进行表达。通过SDS—PAGE分析发现,重组菌株能表达出大量可溶性B．半乳糖苷酶蛋白和L-阿拉伯糖异构酶蛋白。以重悬菌液为酶源,可将乳糖降解为D-半乳糖,并将D-半乳糖转化为D-塔格糖。在温度为50℃,pH7．0的缓冲液中,经一段时间反应后,D-塔格糖的转化率可达21％以上。加入Mn^2＋、Co^2＋和Fe^2＋均能够使D-塔格糖的转化率提高。     

利用游离型表达质粒强化毕赤酵母表达木聚糖酶

巴斯德毕赤酵母是用途广泛的蛋白表达系统。目前用于毕赤酵母的质粒主要以整合型质粒为主,很少见到游离的质粒用于外源基因的表达。文中通过将来源于酵母自身的自主复制序列连接到酵母整合型表达载体pGAP中构成自主复制的游离型表达载体pGAPZαA-PARS,将该载体用于表达木聚糖酶基因。转化毕赤酵母后同传统的整合型表达菌株相比,以甘油为碳源时最高酶活达到343 U/mL,比整合型表达提高了45.9%。同时游离载体表达重组酶比活相对整合表达提高了81.2%。为了节约发酵成本,进一步研究了分别以甘油、葡萄糖、蔗糖、混合碳源(蔗糖︰甘油=1︰2)等不同碳源下游离型重组菌株的表达水平。发现甘油表达水平最高,蔗糖最低,但是以工业葡萄糖为碳源时产酶成本最低。由于pGAP载体不需要以甲醇为碳源,因而文中所构建的游离载体pGAPZαA-PARS极大促进了毕赤酵母在食品行业中的应用。同时,游离型载体可大幅度提高表达水平,为进一步研究提高GAP启动子的高效表达奠定了基础。     

香菇菌C91-3凋亡相关蛋白24414在毕赤酵母GS115中的表达与鉴定

目的通过构建毕赤酵母表达载体将香菇菌C91-3凋亡相关蛋白24414在毕赤酵母GS115中进行表达,同时对表达产物进行鉴定。方法从香菇菌C91-3菌丝体中提取总RNA,根据转录组测序结果,用3＇-Full RACE、5＇-Full RACE方法获得24414基因,并将其克隆到毕赤酵母的表达载体pPIC9K中,构建真核重组表达质粒pPIC9K-24414。用电转化的方法将此质粒转化到毕赤酵母GS115中并进行诱导表达,对表达产物用Westen-blot方法进行鉴定。结果通过菌落PCR和基因序列分析确定插入pPIC9K中的片段为24414基因片段,通过Westen-blot方法确定所表达蛋白为目的蛋白。结论重组质粒pPIC9K-24414成功构建,目的凋亡相关蛋白24414在毕赤酵母GS115中成功表达,为进一步研究香菇菌C91-3凋亡相关蛋白24414的生物学功能奠定了基础。     

Bacillus Stearothermophilus IAM 11001 L-阿拉伯糖异构酶在大肠杆菌中的表达、纯化及活性研究

L-阿拉伯糖异构酶是生物法生产新型功能性因子D-塔格糖最为有效的酶。本文获得了一种新型耐热L-阿拉伯糖异构酶的编码基因araA,来源于Bacillus stearothermophilis IAM 11001,经NCBI Blastn分析,与GenBank中Thermus sp. IM6501 araA序列的同源性为95%,并将该新基因提交到GenBank,获得登陆号：EU394214。以pET-22b(+)为载体质粒,E. coli BL21(DE3)为宿主细胞,构建了基因重组菌,IPTG可诱导目的蛋白的过量表达;经亲和层析纯化的重组蛋白样品进行SDS-PAGE电泳分析,约在59 kDa处出现显著的特征蛋白条带;同时对重组L-AI的活性进行了初步研究,全细胞反应24小时D-塔格糖的转化率为39.8%。     

Bacillus Stearothermophilus IAM 11001 L-阿拉伯糖异构酶在大肠杆菌中的表达、纯化及活性研究球

L-阿拉伯糖异构酶是生物法生产新型功能性因子D-塔格糖最为有效的酶。一种新型耐热L-阿拉伯糖异构酶的编码基因araA,来源于Bacillus stearothermophilis IAM 11001。经NCBI Blastn分析,与GenBank中Thermus sp．IM6501 araA序列的同源性为95％,并将该新基因提交到GenBank,获得登陆号：EU394214。以pET-22b（＋）为载体质粒,E．coli BL21（DE3）为宿主细胞,构建了基因重组菌,IPTG可诱导目的蛋白的过量表达;经亲和层析纯化的重组蛋白样品进行SDS-PAGE电泳分析,约在59kDa处出现显著的特征蛋白条带;同时对重组L-AI的活性进行了初步研究,全细胞反应24小时D-塔格糖的转化率为39．8％。     

A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting ß-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from

ABSTRACT: BACKGROUND: D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a beta-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose. RESULTS: In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52[DEGREE SIGN]C; however, it exhibited over 60% of maximum activity at 30[DEGREE SIGN]C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50[DEGREE SIGN]C. In this study, a recombinant Pichia pastoris yeast strain secreting beta-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting beta-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield of lactose hydrolysis, the complete utilization of D-glucose and a 30% conversion of D-galactose to D-tagatose. CONCLUSIONS: The method developed for the simultaneous hydrolysis of lactose, utilization of D-glucose and isomerization of D-galactose using a P. pastoris strain secreting beta-D-galactosidase and recombinant L-arabinose isomerase seems to offer an interesting alternative for the production of D-tagatose from lactose-containing feedstock.     

用毕赤酵母表达理氏木霉的β-葡萄糖苷酶基因

目的:为提高β-葡萄糖苷酶的产量,用毕赤酵母取代理氏木霉用于生产,以弥补理氏木霉在大规模生产中的缺陷。方法:用套叠PCR法从理氏木霉基因组中扩增β-葡萄糖苷酶基因(bglⅠ)。用T4DNA连接酶和限制性DNA内酶将bglⅠ重组于P.pastoris表达载体pPIC9K的多克隆位点,获得含bglⅠ的重组表达载体pPIC9K-bglⅠ。通过电转法将其pPIC9K-bglⅠ载体转化于P.pastoris基因组,筛选高G418抗性以及高表达bglⅠ酶的重组子作为工程菌。结果:用BMGY-BMMY培养基体系,在摇瓶中发酵48 h,表达BglⅠ30 mg/L,在P.pastoris中表达的BglⅠ能水解对硝基苯-β-D-葡萄糖苷具有β-葡萄糖苷酶活性。其酶活力为56 U/L发酵液。结论:通过这种方法,可以成功地用毕赤酵母表达理氏木霉的β-葡萄糖苷酶基因。     

用pGAP启动子在P.pastoris中组成型表达cbhⅡ基因

用套叠PCR法扩增木霉的cbh Ⅱ基因,以EcoR I和Not I双酶切将其克隆进P.pastoris表达载体pGAP9K,获得重组表达质粒pGAP-cbh Ⅱ。通过电转法将其cbhⅡ基因重组于P.pastoris基因组,筛选高G418抗性的克隆作为工程菌。用葡萄糖作为碳源摇瓶发酵3 d,分泌的重组蛋白CBHⅡ达到50 mg/L。用CMC酶活法测定发酵液中的CMC酶活力为2.05 U/mL。     

里氏木霉纤维二糖水解酶Ⅱ在毕赤酵母中的高效表达

本工作采用巴氏毕赤酵母Pichiapastoris表达系统进行了里氏木霉Trichodermareesei纤维二糖水解酶Ⅱ(CellobiohydrolaseII)的表达。用RT-PCR的方法从经稻草粉诱导的里氏木霉培养物中分离出纤维二糖水解酶Ⅱ的基因,将其插入到巴氏毕赤酵母的表达载体pPICZαA中,并使之处于α-因子信号肽序列的下游,得到重组质粒pPICZαA-cbh2。通过电穿孔的方法用线性化的pPICZαA-cbh2转化巴氏毕赤酵母GS115菌株,经过大量筛选后得到可以高效表达纤维二糖水解酶的毕赤酵母菌株P.pastorisCBHⅡ1。在甲醇诱导的条件下培养P.pastorisCBHⅡ1,培养液中的CMC活性可达到3.82U/mL,SDS-PAGE分析结果表明纤维二糖水解酶在P.pastorisCBHⅡ1中的表达量远远高于里氏木霉。对表达产物进行了LC-MS分析,结果表明所表达的蛋白为里氏木霉的纤维二糖水解酶。     

在甲醇酵母Pichia pastoris胞内表达有活性的辣根过氧化物酶

为了开辟在甲醇酵母 Pichia pastoris中表达 HRP的新途径 ,将编码成熟 HRP同功酶 C基因克隆到表达载体 p PIK3.5K中 .p PIK3.5KHRP转化 GS1 1 5后 ,用 PCR筛选阳性 P.pastoris重组株 ,并用甲醇进行诱导 . Western印迹杂交分析表明目标蛋白 (约为 38k D)能被天然 HRP的多克隆抗体所识别 ,因此活性辣根过氧化物酶已在 P.pastoris胞内表达 .筛选菌株中显示了明显的过氧化物酶活性 ,同时诱导过程中血红素和 Ca Cl2 的加入对过氧化物酶的活力影响不大     

单纯疱疹病毒Ⅰ型糖蛋白D在酵母中的表达

从提取的HSV-1基因组中扩增得到编码gD蛋白胞外区1～314aa的基因gDt,将其插入毕赤酵母表达质粒pPIC9K的醇氧化酶(AOX1)启动子下游,构建携带gDt的重组载体,经电转化GS115菌株和G418筛选,得到了高效分泌表达gD蛋白的毕赤酵母菌株,表达量达到250mg/L,该目的蛋白可被gD单抗(1-I-9)特异性识别。表达产物经离子交换、金属螯合、分子筛柱层析纯化后得到纯度较高的重组蛋白。重组gD蛋白免疫BALB/c小鼠可诱生一定水平的特异性抗体,表明该蛋白具有较好的免疫原性,能够诱导小鼠产生体液免疫应答。     

强化表达SAM合成酶促进SAM在毕赤酵母中累积

S 腺苷甲硫氨酸 (S adenosyl L methionine ,SAM)是生物体硫代谢的重要中间代谢物质 ,在体内起着转甲基、转硫基、转氨丙基的作用 ,具有重要的药用和保健价值。将酿酒酵母来源的SAM合成酶 2基因置于GAP启动子调控下 ,构建胞内组成型表达质粒 ,并电转化至毕赤酵母菌株GS115。经Zeocin抗性和培养筛选到一株高产SAM的重组菌。对重组菌表达工艺的研究表明 ,碳源、氮源、pH和溶解氧对SAM的累积有较大影响。在优化条件下 ,重组细胞培养 3天 ,SAM累积量可达 2 .49g/L。     

Metabolite gene regulation: imidazole and imidazole derivatives which circumvent cyclic adenosine 3',5'-monophosphate in induction of the Escherichia coli L-arabinose operon

Imidazole, histidine, histamine, histidinol phosphate, urocanic acid, or imidazolepropionic acid were shown to induce the L-arabinose operon in the absence of cyclic adenosine 3',5'-monophosphate. Induction was quantitated by measuring the increased differential rate of synthesis of L-arabinose isomerase in Escherichia coli strains which carried a deletion of the adenyl cyclase gene. The crp gene product (cyclic adenosine 3',5'-monophosphate receptor protein) and the araC gene product (P2) were essential for induction of the L-arabinose operon by imidazole and its derivatives. These compounds were unable to circumvent the cyclic adenosine 3',5'-monophosphate in the induction of the lactose or the maltose operons. The L-arabinose regulon was catabolite repressed upon the addition of glucose to a strain carrying an adenyl cyclase deletion growing in the presence of L-arabinose with imidazole. These results demonstrated that several imidazole derivatives may be involved in metabolite gene regulation (23).     

Expression,purification, and characterization of recombinant human neurturin secreted from the yeast Pichia pastoris

Neurturin (NTN), a potent neurotrophic factor acting specifically on dopaminergic neurons, is comprised of 102 amino acids as a mature protein. We artificially synthesized a gene for mature human NTN (hNTN) using codons preferred by the yeast Pichia pastoris. This synthesized gene, fused in frame with sequences encoding the alpha-factor signal peptide gene from Saccharomyces cerevisiae was cloned into P. pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K-alpha-hNTN was then transformed into the yeast and stable multicopy recombinant P. pastoris strains were selected by G418 resistance. SDS-PAGE and Western blot assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hNTN, a 16kDa glycosylated protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using CM-Sepharose ion exchange and Superdex 75 size-exclusion chromatography steps. Bioactivity of the recombinant hNTN was confirmed by the ability of the protein to stimulate growth of nerve fibers from the dorsal root ganglia of chick embryos in vitro.