利用毕赤酵母系统直接分泌表达具有活性的谷氨酰胺转胺酶

使用异源表达系统直接分泌表达具有活性的微生物谷氨酰胺转氨酶(Microbial transglutaminase,MTG)是目前最具前景的MTG生产方法之一,但由于产量较低无法实现工业化生产.毕赤酵母是近年来发展出的高效蛋白表达系统.通过采用pro序列与成熟MTG基因共表达的策略,成功地实现了用重组毕赤酵母分泌表达具有活性的茂原链霉菌Streptomyces mobaraense MTG.进一步通过对pro序列和MTG基因拷贝数以及重组酵母培养条件的优化,最终使得MTG在1L发酵罐中高密度发酵的酶活达到7.3 U/mL,为MTG的工业化生产奠定了基础.     

产耐高温谷氨酰胺转胺酶菌株的快速筛选方法

目的:采用亚硝基胍(NTG)诱变结合96孔板高通量筛选方法筛选产耐高温谷氨酰胺转胺酶(MTG)的茂原链霉菌(Streptomyces mobaraensis)。方法:通过优化96孔板高通量测定MTG活性的方法、确定筛选温度和时间,建立了产耐高温MTG菌株的快速筛选方法;通过优化NTG诱变条件建立了筛选突变库;通过96孔板高通量初筛、摇瓶复筛获得了产耐高温MTG的突变株12-82,并通过摇瓶发酵对12-82所产MTG进行热稳定性分析。结果:采用2mg/ml NTG、p H8.0、60min的诱变条件获得突变株,将突变株的发酵上清液于70℃水浴7.5min,再在37℃空气浴、反应10min的条件下测定MTG活性,从5 200株突变株中筛选出5株产耐高温MTG的突变株,其中突变株12-82在50℃水浴60min以及70℃水浴1.5min的酶活残留率均比出发株高出近20%,且80℃保温2min仍有11.9%的酶活残留率。结论:利用NTG诱变结合96孔板高通量筛选的方法筛选到5株所产MTG热稳定性相对较高的突变株,其中突变株12-82在50℃、70℃和80℃的酶活残留率均有10%~20%的提高。这为高温食品加工领域所需耐高温MTG生产菌株的高效筛选提供了可行性方案。     

羊毛防毡缩用蛋白酶的化学修饰

为减少防毡缩整理中蛋白酶对羊毛纤维主体结构的破坏作用,分别研究了戊二醛、微生物谷氨酰胺转氨酶(MTG)和水溶性碳二亚胺(EDC)对蛋白酶Savinase 16L的化学修饰,以期达到增大蛋白酶分子量,从而将水解作用限制在纤维表面的目的。主要通过体积排阻色谱、SDS-PAGE谱图以及荧光光谱研究修饰酶的分子量和结构变化。结果表明,戊二醛不能对蛋白酶分子进行有效修饰;MTG会被蛋白酶水解,无法催化酶分子间发生共价交联;而碳二亚胺既可以使蛋白酶分子间发生交联,又能将含有伯胺基的大分子修饰剂偶联到酶分子上。     

转谷氨酰胺酶基因在大肠杆菌中的克隆表达

从轮枝链霉菌Streptoverticilliummobaraense细胞中获得其基因组DNA ,用一对特异性的引物通过PCR的方法扩增出转谷氨酰胺酶 (transglutaminase,TGase)全长基因 ,回收片段并将其连接到表达载体pET30a中 ,转化大肠杆菌DH5α。双向测序表明获得的转谷氨酰胺酶全长基因序列正确。纯化重组质粒转化大肠杆菌BL2 1 (DE3) ,以 1mmol/LIPTG诱导 5h收集菌体进行SDS-PAGE电泳分析 ,与阴性对照相比 ,明显多出了一条蛋白条带 ,紫外扫描显示此带约占总蛋白量的1 7% ,Westernblotting证实此带能够特异性地与兔抗MTG(味之素公司 )的抗体发生反应。测得纯化后得到的TGase蛋白的酶活可以达到 15.1U/mg蛋白。     

基因拷贝数对重组毕赤酵母产谷氨酰胺转胺酶的影响

基于毕赤酵母核糖体DNA序列 (rDNA),构建多拷贝谷氨酰胺转胺酶基因表达载体pPICZα-rDNA- mtg,并转化到表达前导肽 (Pro peptide或pro) 的宿主菌pGAP9-pro/GS115,得到共表达菌株pro/rDNA-mtg (GS115)。实时荧光定量PCR (qPCR) 分析了4株阳性表达菌株中mtg基因拷贝数,进一步研究了不同基因拷贝数对重组毕赤酵母产酶的影响及高产菌株在3 L发酵罐高密度发酵。结果表明,被检测的4株阳性表达菌株中mtg拷贝数分别为2.21、3.36、5.72和7.62 (mtg-2c、mtg-3c、mtg-6c和mtg-8c),其发酵产酶能力和蛋白质表达水平为mtg-3c>mtg-2c>mtg-6c>mtg-8c;高密度发酵较低和较高拷贝数的两株菌mtg-3c和mtg-6c,发酵上清的最高酶活和单位菌体酶活分别为3.12 U/mL、52.1 U/g湿重和2.07 U/mL、36.5 U/g湿重,其中单位菌体酶活mtg-3c是mtg-6c的1.4倍;mtg-3c纯化酶的最高酶活达到7.21 U/mL,蛋白浓度为437.2 μg /mL。通过分析拷贝数对重组毕赤酵母产酶的影响,发现mtg-3c适合pro/rDNA-mtg中pro和mtg共表达,MTG高酶活与菌株较高分泌蛋白有关。     

肠球菌肽脱甲酰基酶在大肠杆菌BL21(DE3)中高效表达及其活性检测

肽脱甲酰基酶 (peptidedeformylase ,PDF)存在于所有原核生物中是其生长、代谢、繁殖必不可少的关键酶 ,但不存在于人类与其他哺乳动物细胞内 ,因而被视为新一代广谱抗生素药物筛选的理想靶点。将肠球菌 (Enterococcusfaecium)肽脱甲酰基酶基因连接到高效蛋白表达载体pET 30 A( )中 ,并转入宿主大肠杆菌BL2 1 (DE3)中进行诱导表达。在该基因的诱导表达中 ,采用不同表达条件进行诱导表达 ,最终获得表达效率极高且可溶的肽脱甲酰基酶。从宿主细胞中提取分离该酶 ,并进行酶活性检测 ,诱导表达的肽脱甲酰基酶有很高的酶活性     

添加CTAB促进吸水链霉菌产谷氨酰胺转胺酶

研究了添加十六烷基三甲基溴化铵(CTAB)对吸水链霉菌(Streptomyces hygroscopicus)合成谷氨酰胺转胺酶的影响。结果表明,添加CTAB可以提高发酵过程中谷氨酰胺转胺酶的酶活,摇瓶培养中,CTAB的最佳添加时间和添加量分别为32h和1%,发酵终了时,谷氨酰胺转胺酶酶活最高达5.04u/mL,比对照提高了21.8%。初步研究表明,CTAB的主要作用是促使谷氨酰胺转胺酶的酶原转化为成熟酶,因此,在发酵过程中添加适当浓度的CTAB,可使酶原快速、完全地转化为成熟的MTG,解除酶原的产物抑制作用,促进了细胞产酶。     

两端融合表达几丁质结合结构域提高几丁质酶抗真菌活性

【目的】通过两端融合表达几丁质结合结构域来提高几丁质酶的活性和生物防治植物病原真菌能力。【方法】以苜蓿链霉菌(Streptomyces alfalae)ACCC40021中唯一的GH19家族几丁质酶为模板,构建两端融合表达几丁质结合结构域的几丁质酶,并进行原核表达;利用3,5-二硝基水杨酸法(DNS)测定几丁质酶活。【结果】成功构建了CatD_(ChiB) (催化结构域)、rChiB (含N-端几丁质结合结构域)、DChBD_(ChiB)(含两端几丁质结合结构域)三种形式的酶,并在大肠杆菌中得到了高效表达;与CatD_(ChiB)和rChiB相比,DChBD_(ChiB)显著地提高了对α-几丁质、胶体几丁质和黑曲霉几丁质的结合能力和活性;同时增强了其对病原真菌长枝木霉的抑制作用。【结论】两端融合表达几丁质结合结构域是简单有效的提高几丁质酶活性及抗真菌活性的策略。     

琼胶酶AgaD在大肠杆菌中的高效表达

摘要:【目的】构建琼胶酶AgaD的高效表达体系,优化发酵条件提高重组酶的表达量。【方法】首先根据大肠杆菌(E.coli)密码子偏好性,优化并合成AgaD的基因,使其适合E.coli表达系统;考察了不同的E.coli表达宿主;根据N端法则构建了突变体;评价了培养基中添加CaCl2和甘氨酸(Gly)对重组酶表达的影响。【结果】成功构建了琼胶酶AgaD 的高效表达体系,确定了E.coli AD494(DE3)为最适表达宿主;利用N端法则提高了重组酶的稳定性,缩短了发酵时间;通过在培养基中添加CaCl2和甘氨酸(Gly)进一步提高了胞外酶产量。最终,发酵上清中重组酶的活力由20 U/L提高至11300 U/L,比优化前提高了500余倍。【结论】构建了琼胶酶AgaD的高效表达体系,为GH96家族琼胶酶的深入研究奠定了基础。     

Overproduction of microbial transglutaminase in Escherichia coli, in vitro refolding, and characterization of the refolded form

(MTG) The Streptoverticillium transglutaminase gene, synthesized previously for yeast expression, was modified and resynthesized for overexpression in E. coli. A high-level expression plasmid, pUCTRPMTG-02(+), was constructed. Furthermore, to eliminate the N-terminal methionine, pUCTRPMTGD2 was constructed. Cultivation of E. coli transformed with pUCTRPMTG02(+) or pUCTRPMTGD2 yielded a large amount of MTG (200-300 mg/liter) as insoluble inclusion bodies. The N-terminal amino acid residue of the expressed protein was methionine or serine (the second amino acid residue of the mature MTG sequence), respectively. Transformed E. coli cells were disrupted, and collected pellets of inclusion bodies were solubilized with 8 M urea. Rapid dilution treatment of solubilized MTG restored the enzymatic activity. Refolded MTG, purified by ion-exchange chromatography, which had an N-terminal methionine or serine residue, showed activity equivalent to that of native MTG. These results indicated that recombinant MTG could be produced efficiently in E. coli.     

添加CTAB促进吸水链霉菌产谷氨酰胺转胺酶

研究了添加十六烷基三甲基溴化铵（CTAB）对吸水链霉菌（Streptomyces hygroscopicus）合成谷氨酰胺转胺酶的影响。结果表明,添加CTAB可以提高发酵过程中谷氨酰胺转胺酶的酶活,摇瓶培养中,CTAB的最佳添加时间和添加量分别为32h和1%,发酵终了时,谷氨酰胺转胺酶酶活最高达5.04u/mL,比对照提高了21.8%。初步研究表明,CTAB的主要作用是促使谷氨酰胺转胺酶的酶原转化为成熟酶,因此,在发酵过程中添加适当浓度的CTAB,可使酶原快速、完全地转化为成熟的MTG,解除酶原的产物抑制作用,促进了细胞产酶。     

Improvement of shrink-resistance and tensile strength of wool fabric treated with a novel microbial transglutaminase from Streptomyces hygroscopicus

In this study, a novel microbial transglutaminase (MTG) from Streptomyces hygroscopicus WSH03-13 was applied in the processing of wool fabrics. The results indicated that MTG treatment could improve felting properties and decrease tensile strength loss of wool fabrics. For the wool fabrics used in this study, MTG treatment following chemical and protease pretreatment led to a 2.32% of area shrinkage and about 16% recovery in tensile strength based on the samples without MTG treatment. Moreover, a traditional resin treatment was compared with the role of MTG. Although the tensile strength of wool fabrics treated by MTG was lower than that treated by resin treatment, the fabrics had similar anti-felting properties, and the chemical oxygen demand of wastewater was only half of the latter.     

Enzymatic labeling of a single chain variable fragment of an antibody with alkaline phosphatase by microbial transglutaminase

Functional cross-linking of a single chain Fv fragment of anti-hen egg-white lysozyme antibody (scFv) and alkaline phosphatase (AP) was explored using microbial transglutaminase (MTG) from Streptomyces mobaraensis. A specific peptidyl linker for MTG was genetically fused to the N-terminus of each protein and the resultant proteins were obtained separately by bacterial expression. The recombinant peptide-tagged scFv and AP were site-specifically cross-linked by MTG through the extra peptidyl linkers in vitro, which mainly yielded the heterodimer (i.e., scFv-AP conjugate). The enzymatic cross-linking reaction had little influence on either the antigen-binding ability of the scFv moiety or the enzymatic activity of the AP moiety of the conjugate, allowing use within an enzyme-linked immunosorbent assay. The results obtained suggest that the enzymatic approach with MTG facilitates the posttranslational construction of functional fusion proteins.     

Kinetics of inclusion body formation and its correlation with the characteristics of protein aggregates in Escherichia coli

The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH) and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm) and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2-3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low β-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies.     

重组N-乙酰鸟氨酸脱乙酰基酶的表达、纯化和复性研究

报道重组N-乙酰鸟氨酸脱乙酰基酶(NAOase)的研究进展。重组NAOase由大肠杆菌argE基因编码,在重组菌BL21(DE3)-pET22b-argE中的表达量为32.5%,大多以无活性的包涵体存在。低温诱导可增大有活性的可溶表达部分的比例。可溶性NAOase经Ni-NTA凝胶亲和纯化后得到SDS-PAGE电泳纯的酶,比酶活为1193.2u/mg蛋白。诱导条件影响整菌蛋白的成分及比例。37℃诱导生成的包涵体经尿素梯度洗涤后纯度较22℃高。低的蛋白浓度和合适的氧化还原体系是影响复性的关键因素。稀释法和透析法皆可使包涵体部分复性。在合适的条件下以稀释法复性时,约有17.78%包涵体可顺利复活。包涵体经尿素洗涤、溶解、Ni-NTA凝胶柱亲和纯化后,获得了高纯度的NAOase。     

HBc-based virus-like particle assembly from inclusion bodies using 2-methyl-2, 4-pentanediol

Hepatitis B virus core antigen (HBc) has recently been used as carriers to develop recombinant vaccines. However, not virus-like particles (VLPs) but inactive inclusion bodies are often formed for the chimeric proteins when expressed in Escherichia coli. A novel method for in vitro assembly of chimeric HBc-MAGE3 II from inclusion bodies to VLPs was established in this study. The method utilized 2-methyl-2, 4-pentanediol (MPD), an amphipathic di-alcohol, to dissociate sodium dodecyl sulfate (SDS) from the solubilized chimeric protein to initiate VLP assembly. The HBc-MAGE3 II could assemble into VLPs only when the molar ratio of SDS/protein subunit was less than 0.14. After removing SDS/MPD by desalting and further purification, VLPs with similar morphology to the natural virus were obtained. This method could be used for preparation of other VLPs expressed as inclusion bodies.     

An enzymatic strategy for site-specific immobilization of functional proteins using microbial transglutaminase

A novel strategy for site-specific immobilization of recombinant proteins was investigated using microbial transglutaminase (MTG). Alkaline phosphatase (AP) was selected as a model protein and tagged with a short peptide (MKHKGS) at the N-terminus to provide a reactive Lys residue for MTG. On the other hand, casein, a well-known substrate for MTG, was chemically attached onto a polyacrylic resin to provide reactive Gln residues for the enzymatic immobilization of the recombinant AP. As a result, we succeeded in MTG-mediated functional immobilization of the recombinant AP onto casein-coated polyacrylic resin. It was found that the immobilized AP prepared using MTG exhibited much higher specific activity than that prepared by chemical modification. Moreover, enzymatic immobilization gave an immobilized formulation with higher stability upon repeated use than that obtained by physical adsorption. Use of this ability of MTG in posttranslational protein modification will provide us with a benign, site-specific immobilization method for functional proteins.