微生物谷氨酰胺转胺酶的表达及分子改造研究进展

微生物谷氨酰胺转胺酶具有催化蛋白质和某些非蛋白物质交联的功能,被广泛应用于食品、医药及纺织等领域.为提高该酶的产量及建立相应的分子改造平台,上世纪90年代日本味之素公司便开展了微生物谷氨酰胺转胺酶重组菌构建的研究.目前,该酶已在多个表达系统中实现活性表达,部分重组菌较野生菌的产酶能力有显著提高.近年来,谷氨酰胺转胺酶的分子改造研究也取得了初步进展,酶的催化活力、热稳定性及底物专一性得到提升.文中对上述研究中涉及的蛋白质表达及改造策略进行了简要的总结及分析,并指出相关研究的发展趋势.     

谷氨酰胺转胺酶—理化性质、生产方法及其在食品工业中的应用

谷氨酰胺转胺酶(蛋白质-谷氨酸-γ谷氨酰胺转移酶EC2.3.2.13)催化体外大多数食品蛋白质的交联反应,如:酪蛋白,大豆蛋白,肌球蛋白,肌动蛋白,谷蛋白,禽蛋蛋白等等。通过催化肽键谷酰胺基残基的酰基转移反应,在各种蛋白质分子之间或之内形成ε-(γ-谷胺酰)赖氨酸键,从而改善各种蛋白质的功能性质。如:营养价值、质地结构、口感、贮存期等等。目前,商业化谷氨酰胺转胺酶主要从动物组织中提取,但由于其分离和纯化过程较复杂,且来源稀少,因而价格昂贵,近年来,人们开始转向于研究利用微生物发酵来生产谷氨酰胺转胺酶,并使之应用于食品工业,经过微生物谷氨酰胺转胺酶处理后的食品,其功能性质明显改善。本文就谷氨酰胺转胺酶的国内外研究现状作一综述,主要包括理化性质、生产及其应用。     

谷氨酰胺转胺酶—理化性质，生产方法及其在食品工业中的应用

谷氨酰胺转胺酶（蛋白质－谷氨酸－γ谷氨酰胺转移酶EC2,3,2,13）催化体外大多数食品蛋白质的交联反应,如：酪蛋白,大豆蛋白,肌球蛋白,肌动蛋白,谷蛋白,禽蛋蛋白等等。通过催化肽键谷酰胺基残基的酰基转移反应,在各种蛋白质分子之间或之内形成ε-（γ-谷胺酰）赖氨酸键,从而改善各种蛋白质的功能性质,如：营养价值,质地结构,口感,贮存期等等。目前,商业化谷氨酰胺转胺酶主要从动物组织中提取,但由于其分离和纯化过程较复杂,且来源稀少,因而价格昂贵,近年来,人们开始转向于研究利用微生物发酵来生产谷氨酰胺转胺酶,并使之应用于食品工业,经过微生物谷氨酸酰胺转胺酶处理后的食品,其功能性质明显改善,本文就谷氨酰胺转胺酶的国内外研究现状作一综述,主要包括理化性质,生产及其应用。     

添加胰蛋白酶促进Streptomyces hygroscopicus CCTCC M203062产谷氨酰胺转胺酶

研究了添加胰蛋白酶对Streptomyces hygroscopicus CCTCC M203062合成谷氨酰胺转胺酶的影响。结果表明,添加胰蛋白酶可以提高发酵过程中谷氨酰胺转胺酶的酶活。摇瓶培养中,在发酵起始时添加200U/ml的胰蛋白酶,谷氨酰胺转胺酶的酶活最高达到了6.61U/ml,比对照提高了27.1%。初步研究表明,添加胰蛋白酶可以直接切割发酵过程中产生的酶原,使其被快速地转化为成熟酶,因此推测胰蛋白酶提高谷氨酰胺转胺酶酶活的原因是解除了酶原的产物抑制作用,产生更多的酶原,从而促进了产酶。     

“微生物发酵法生产谷氨酰胺转胺酶”通过鉴定

具有“2 1世纪超级粘合剂”美称的谷氨酰胺转胺酶 (蛋白质 谷氨酸 γ 谷氨酰胺转移酶 ,ＥＣ2 .3 .2 .1 3 ) ,是一种可生产出满足人们需求的新型蛋白食品的重要酶制剂。它能够催化蛋白质分子内的交联、分子间的交联、蛋白质和氨基酸之间的连接以及蛋白质分子内谷氨酰胺酰基的水解 ,因而在食品工业、化妆品工业和制药工业中具有广泛的用途。源于动物的谷氨酰胺转胺酶价格较高 ,采用发酵法生产可望大幅度降低其生产成本 ,故而是拓宽谷氨酰胺转胺酶应用范围的根本途径。在日本 ,谷氨酰胺转胺酶已成为食品工业的第二大酶种 ,其用量仅次于α 淀粉…     

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。     

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。     

吸水链霉菌谷氨酰胺转胺酶基因的阻断及其对细胞分化的影响

【目的】通过对吸水链霉菌(Streptomyces hygroscopicus)中谷氨酰胺转胺酶基因的阻断,以期深入了解谷氨酰胺转胺酶生理功能,并为谷氨酰胺转胺酶发酵优化提供新的研究思路。【方法】以温敏型质粒pKC1139为出发质粒,构建阻断吸水链霉菌谷氨酰胺转胺酶编码基因的重组质粒pKC1139-TG1,转化吸水链霉菌原生质体,通过抗性筛选和PCR验证,成功得到一株谷氨酰胺转胺酶阻断菌株,命名为S.h-△TG。【结果】以原始菌株为对照,重组子基内菌丝生长不受影响,但是由基内菌丝分化形成气生菌丝的过程受到影响,重组子基本不产气生菌丝。【结论】谷氨酰胺转胺酶对吸水链霉菌气生菌丝的形成有着重要的影响,参与链霉菌气生菌丝的形成。     

分批发酵生产谷氨酰胺转氨酶的温度控制策略

微生物谷氨酰胺转氨酶 (Ｍｉｃｒｏｂｉａｌｔｒａｎｓｇｌｕｔａｍｉｎａｓｅ ,简称ＭＴＧ ,ＥＣ2 3 2 13)由于能催化许多食品中蛋白质的交联反应 ,改善各种蛋白质的功能性质 ,在食品工业具有广泛的应用潜力[1] ,因而引起了人们的极大兴趣。谷氨酰胺转氨酶的生产通常采用从豚鼠肝脏或组织中提取 ,由于豚鼠肝脏或组织来源稀少 ,谷氨酰胺转胺酶的分离纯化过程复杂 ,因而价格昂贵。 2 0世纪 80年代末 ,Ａｎｄｏ和Ｍｏｔｏｋｉ等人[2 ,3 ] 首先报道了利用微生物发酵法生产谷氨酰胺转胺酶的结果 ;近年来 ,Ｇｅｒｂｅｒ等人[4 ] 对其下游技术进…     

NaCl对轮枝链霉菌产谷氨酰胺转胺酶的促进作用研究

为了提高轮枝链霉菌发酵生产谷氨酰胺转胺酶的产量,研究了3种无机盐(NaCl、MgCl₂、KCl)对发酵产酶的影响。研究表明0.5%MgCl₂、0.5%NaCl均可促进菌体产酶,其中添加NaCl后谷氨酰胺转胺酶酶活提高最为显著。SDS-PAGE图谱分析显示,在各取样点实验组谷氨酰胺转胺酶酶原和成熟酶的总量高于对照,而且实验组成熟酶的增加也比对照快,从而显示NaCl是通过促进酶原的分泌和谷氨酰胺转胺酶的成熟,从而提高酶活、提早产酶。对NaCl的添加量进行优化,表明NaCl的最适添加量为0.5%,在此条件下,与对照相比,酶活水平提高了12%以上,发酵终点提前了约15h。     

Mitotracker Green Is a P-Glycoprotein Substrate

P-glycoprotein has a widespread expression on normal tissues. The protein has also been strongly associated with the multidrug resistance phenotype (MDR) on tumor cells. The employment of flow cytometry and confocal microscopy has contributed to the discovery and application of new particular fluorescent dyes. Nevertheless, several studies are being performed in different cellular types neglecting the expression/activity of MDR proteins. Because many fluorochromes have been reported as P-glycoprotein substrates, an especial attention must be given to the properties of new dyes in the presence of MDR proteins. Flow cytometric analyzes of Mitotracker Green (MTG) fluorescence profile were performed in a human erythroleukemic cell line and its resistant counterpart. In this report we demonstrated that MTG, a probe used to evaluate the mitochondrial mass, is a P-glycoprotein substrate and its staining profile is dependent on the activity of this protein. In vitro studies on a human erythroleukemic cell line and its resistant counterpart revealed that MDR modulators (Cyclosporin A, Verapamil, and Trifluoperazine) alter the MTG fluorescence pattern on a resistant cell line. The findings suggest that attention should be given to the expression of P-glycoprotein when performing an evaluation of mitochondria properties with MTG.     

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.     

Illuminating structure and acyl donor sites of a physiological transglutaminase substrate from Streptomyces mobaraensis

Transglutaminase from Streptomyces mobaraensis (MTG) has become a powerful tool to covalently and highly specifically link functional amines to glutamine donor sites of therapeutic proteins. However, details regarding the mechanism of substrate recognition and interaction of the enzyme with proteinaceous substrates still remain mostly elusive. We have determined the crystal structure of the Streptomyces papain inhibitory protein (SPI_p), a substrate of MTG, to study the influence of various substrate amino acids on positioning glutamine to the active site of MTG. SPI_p exhibits a rigid, thermo‐resistant double‐psi‐beta‐barrel fold that is stabilized by two cysteine bridges. Incorporation of biotin cadaverine identified Gln‐6 as the only amine acceptor site on SPI_p accessible for MTG. Substitution of Lys‐7 demonstrated that small and hydrophobic residues in close proximity to Gln‐6 favor MTG‐mediated modification and are likely to facilitate introduction of the substrate into the front vestibule of MTG. Moreover, exchange of various surface residues of SPI_p for arginine and glutamate/aspartate outside the glutamine donor region influences the efficiency of modification by MTG. These results suggest the occurrence of charged contact areas between MTG and the acyl donor substrates beyond the front vestibule, and pave the way for protein engineering approaches to improve the properties of artificial MTG‐substrates used in biomedical applications.     

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.     

Morphology and chemical composition of metathoracic scent glands in Dolycoris baccarum (Linnaeus, 1758) (Heteroptera: Pentatomidae)

One of the general defining characters of the Heteroptera is the presence of metathoracic scent glands (MTG). Using scanning electron microscopy, the morphology of the MTG of Dolycoris baccarum (Linnaeus 1758) (Heteroptera: Pentatomidae) was studied. The MTG belong to the diastomian type. The two glandular pores located between the mesothoracic and metathoracic coxae are associated with 'mushroom-like' structures. The MTG are composed of a reservoir and a pair of lateral glands is connected to the reservoir by a duct. A groove-like structure extends downwards from the ostiole. While this structure is long and wide, its ostiole is oval. Extracts of the volatile fractions from male and female MTG secretions were analysed by capillary gas chromatography–mass spectrometry (GC-MS) and exhibited a typical pentatomid composition. Seventeen chemical compounds were detected in female secretions, whereas 13 chemical compounds were detected in the male secretions. Most chemical compounds were similar between the sexes but were different in their quantities. In this regard, the compounds identified were investigated and the biological functions of the glandular secretions were discussed. In the analyses of the MTG of females of D. baccarum , tridecane (50.97%) was a major odour component and (Z,Z)-4,16-octadecadien-1-ol acetate (0.02%) was a minor odour component. In males, tridecane (50.80%) was a major odour component and 1,2-benzenedicarboxylic acid (0.02%) was a minor odour component.     

Microbial transglutaminase: An overview of recent applications in food and packaging

Abstract

The glue of proteins, microbial transglutaminase (MTG) has been adopted in the food processing industries for its broad enzymatic action. Microorganisms such as Streptoverticillium and Streptomyces are the major sources, to decrease the cost of manufacturing animal origin transglutaminase. The net % increase of its demands in the food processing is estimated at 21.9% per year. In fact, MTG is consumed by most food industries, spanning the meat, dairy, seafood and fish, plant proteins, edible film preparation and more. It used to improve gelation and change foaming, emulsification, viscosity, consistency and water-holding capacity properties. This paper presents an overview of the literature that described and explored the recent microbial origins, production media and applications of microbial transglutaminase.     

In vitro refolding process of urea-denatured microbial transglutaminase without pro-peptide sequence

Efficient refolding process of denatured mature microbial transglutaminase (MTG) without pro-peptide sequence was studied in the model system using urea-denatured pure MTG. Recombinant MTG, produced and purified to homogeneity according to the protocol previously reported, was denatured with 8M urea at neutral pH and rapidly diluted using various buffers. Rapid dilution with neutral pH buffers yielded low protein recovery. Reduction of protein concentration in the refolding solution did not improve protein recovery. Rapid dilution with alkaline buffers also yielded low protein recovery. However, dilution with mildly acidic buffers showed quantitative protein recovery with partial enzymatic activity, indicating that recovered protein was still arrested in the partially refolded state. Therefore, we further investigated the efficient refolding procedures of partially refolded MTG formed in the acidic buffers at low temperature (5 degrees C). Although enzymatic activity remained constant at pH 4, its hydrodynamic properties changed drastically during the 2h after the dilution. Titration of partially refolded MTG to pH 6 after 2h of incubation at pH 4.0 improved the enzymatic activity to a level comparable with that of the native enzyme. The same pH titration with incubation shorter than 2h yielded less enzymatic activity. Refolding trials performed at room temperature led to aggregation, with almost half of the activity yield obtained at 5 degrees C. We conclude that rapid dilution of urea denatured MTG under acidic pH at low temperature results in specific conformations that can then be converted to the native state by titration to physiological pH.     

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.     

S-peptide as a potent peptidyl linker for protein cross-linking by microbial transglutaminase from Streptomyces mobaraensis

We have found that ribonuclease S-peptide can work as a novel peptidyl substrate in protein cross-linking reactions catalyzed by microbial transglutaminase (MTG) from Streptomyces mobaraensis. Enhanced green fluorescent protein tethered to S-peptide at its N-terminus (S-tag-EGFP) appeared to be efficiently cross-linked by MTG. As wild-type EGFP was not susceptible to cross-linking, the S-peptide moiety is likely to be responsible for the cross-linking. A site-directed mutation study assigned Gln15 in the S-peptide sequence as the sole acyl donor. Mass spectrometric analysis showed that two Lys residues (Lys5 and Lys11) in the S-peptide sequence functioned as acyl acceptors. We also succeeded in direct monitoring of the cross-linking process by virtue of fluorescence resonance energy transfer (FRET) between S-tag-EGFP and its blue fluorescent color variant (S-tag-EBFP). The protein cross-linking was tunable by either engineering S-peptide sequence or capping the S-peptide moiety with S-protein, the partner protein of S-peptide for the formation of ribonuclease A. The latter indicates that S-protein can be used as a specific inhibitor of S-peptide-directed protein cross-linking by MTG. The controllable protein cross-linking of S-peptide as a potent substrate of MTG will shed new light on biomolecule conjugation.