微生物谷氨酰胺转胺酶生产菌株的育种研究

在简要介绍微生物谷氨酰胺转胺酶的主要功能、应用和国外研究成果与目前状况后,指出该酶生产微生物菌种选育的重要性以及实际难度和艰巨性,阐述了进行初筛、复筛和常规诱变育种试验的关键技术手段及其成果效益。利用这些有效方法获得性能优良的高产酶突变菌株,进行规模化发酵工艺条件和酶制剂产品分离提纯技术研究,并在此基础之上,进一步开展通过“神舟”四号飞船搭载的微生物空间诱变育种研究强化产酶菌株的各种优良性能,说明了空间多种复合因素引发的综合搭载效果显著。     

碳源对轮枝链霉菌SK-2合成谷氨酰胺转胺酶的影响

研究不同的碳源对轮枝链霉菌(StreptoverticilliumSK-2产谷氨酰胺转胺酶的影响.结果2%甘油是最有效的碳源,其最高酶活达7.89μmol/(min*ml);添加油脂有助于菌体产酶,在各种油脂中,以添加橄榄油效果最好.并进一步研究了甘油和橄榄油作为复合碳源对产酶的影响.     

植物转谷氨酰胺酶

植物转谷氨酰胺酶廖祥儒,朱新产,赵军（西北农业大学,陕西杨陵７１２１００）（中国科学院西北水土保持研究所）关键词转谷氨酰胺酶,蛋白质交联蛋白质交联,作为转译后蛋白质的一种修饰方式,与细胞结构形成有关,对稳定组织结构和细胞骨架有重要意义。现已发现,和Ｎ...     

微生物转谷氨酰胺酶(MTGase)的AOT反胶束纯化研究

研究微生物转谷氨酰胺酶(MTGase)反胶束纯化的工艺和条件,调节MTGase离心上清液等电点,除去部分杂蛋白,MTGase活力升高7.5倍;用截留分子量为10000的超滤膜除去小分子杂蛋白,MTGase活力升高1.33倍;用0.05mol/L的AOT/异辛烷反胶束进一步纯化MTGase,其最适萃取条件是粗MTGase蛋白质浓度20mg/mL,[Na ]0.12mol/L,水相pH4.80～5.20,相比1:1(v/v);荷载MTGase的AOT反胶束用2.0mol/LKCl进行反萃取,MTGase活力为14.2U/g,纯化8.875倍;冷冻干燥脱盐反萃取液,获得MTGase冻干粉,其活力为110.3U/g,与粗酶液相比较,纯化689.4倍。经过AOT/异辛烷反胶束萃取纯化的MTGase,其SDS-聚丙烯酰胺凝胶电泳为一条带。Ca2 与表面活性剂非极性尾上丁二酰羰基氧、极性头磺酸基硫氧基氧及MTGase分子表面具有孤电子对的基团的配位结合放大了AOT反胶束的另一种萃取作用——配位萃取,致使其对MTGase的萃取率高于K 而接近Na 。     

微生物谷氨酰胺转胺酶研究进展

微生物谷氨酰胺转胺酶是一种在食品、医药、纺织、化妆品等领域具有广泛应用前景的酶制剂。就其理化性质、作用机理、工业化生产、在食品工业上的应用及当前国内外研究热点进行了概述。并讨论了微生物谷氨酰胺转胺酶在我国生产及应用中存在的问题和困难,对未来的研究方向做出展望。     

微生物转谷氨酰胺酶的纯化方法和酶学性质研究

由Streptoverticillium mobaTaense发酵生产的转谷氨酰胺酶经过除茵体、超滤浓缩、乙醇沉淀、干燥后得到粗酶产品,其活力回收率约70％。又经Superdex-75凝胶过滤和Source 30S阳离子交换两步纯化后得到纯酶,最终酶活力收率约37％。酶最适温度为50℃,在40℃以下稳定性良好;最适pH为6．0,pH4．0～8．0时比较稳定。离子强度对酶影响很小。     

谷氨酰胺转胺酶发酵条件的优化研究

通过优化种子培养条件和发酵培养基组分使谷氨酰胺转胺酵产酶水平有了很大的提高,确定种龄为20－24h,接种量8％左右。发酵培养基含淀粉15g/L、葡萄糖15g/L、蛋白胨25g/L、酵母膏3g/L、无水硫酸镁2g/L、磷酸氢二2g/L,无水磷酸二氢钾2g/L,24－28h添加质量浓度为0．5％的硫酸铵,在10L发酵罐实验中,验证了溶解氧对MTG合成至关重要,确定较适宜通气量1：1．25vvm,搅拌转速300mr/min,最高产酶单位最终稳定在3．2u/mL,放罐时间在44－46h左右较适宜。     

转谷氨酰胺酶的分子生物学与基因工程

来源于微生物特别是轮枝链霉菌的转谷氨酰胺酶是一种重要的酶制剂,在食品工业中有着广泛的应用前景。本综述了近年来对转谷氨酰胺酶的分子生物学研究成果,以及对其进行基因工程改造的最新进展,讨论了其进一步的研究发展方向。笔认为采用基因工程生产重组转谷氨酰胺酶是解决目前酶价高昂和来源困难问题的一个大有希望的办法。     

葡聚糖酶高产菌株的诱变筛选及其摇瓶发酵条件的初步研究

从土壤中分离出的一株产葡聚糖酶酶活31 U／ml的野生菌株,经UV、^60Co、LiCl诱变筛选后得到了产葡聚糖酶高产菌株SB126,经发酵条件优化试验后检测其酶活达到85U／ml,较野生菌株提高了近两倍。葡聚糖酶摇瓶较适发酵条件为：装量30ml(250m1)、转速180r／min、pH7．0、温度30℃、接种量8％、发酵周期5d。     

Production of transglutaminase by Streptomyces isolates in solid-state fermentation

Aims:  To screen Streptomyces isolates for transglutaminase (TGase) production in solid-state fermentation (SSF) on various substrates.
Methods and Results:  Streptomyces mobaraensis NRRL B-3729, Streptomyces paucisporogenes ATCC 12596 and Streptomyces platensis NRRL 2364 strains were screened for extracellular TGase production in SSF on different substrates. High-protein-content beans, peas and lentils proved to be the best substrates. Good TGase production was obtained on liver kidney beans and green mung beans in a 4- to 6-day SSF. Temperature optima of the enzymes varied between 45 to 50°C. Molecular weight determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS PAGE) indicated similar size (∼37 kDa) for all three enzymes. TGase was the dominating protein band on SDS PAGE for two Streptomyces strains in SSF extracts. Other enzymes were present in smaller quantities.
Conclusions:  Streptomyces mobaraensis NRRL B-3729, S. paucisporogenes ATCC 12596 and S. platensis NRRL 2364 strains were successfully propagated under SSF conditions on crushed/milled liver kidney bean and green mung bean to obtain good level of TGase.
Significance and Impact of the Study:  Owing to much reduced production cost and direct applicability, SSF TGase without downstream processing (cheap in situ enzyme, crude enzyme) may be an excellent candidate for some nonfood applications.     

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.     

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.     

Streptomyces hygroscopicus谷氨酰胺转胺酶酶原基因的鉴定及在大肠杆菌中的表达

[目的]鉴定来源于吸水链霉菌的谷氨酰胺转胺酶基因;研究其在大肠杆菌系统的克隆与表达;分析该酶与其同源酶的活性中心氨基酸序列.[方法]从本实验室筛选的吸水链霉菌(Streptomyces hygroscopicus;CCTCC M203062)发酵液中,分离纯化得到谷氨酰胺转胺酶酶原(pro-MTGase),测得N-端前十个氨基酸序列并与其它链霉菌来源的相应基因序列比较设计引物,扩增得到pro-MTGase 基因,将该基因插入到表达载体pET-20b( )信号肽pelB下游,构建分泌型表达载体pET/pro-MTG,并转化不同的大肠杆菌宿主BL21(DE3)和Rosetta(DE3)pLysS.[结果]获得了pro-MTGase的完整基因序列,多重碱基序列比对表明其与S.platensis和S.caniferus的pro-MTGase基因同源性高达92%.利用Rosetta(DE3)pLysS通过降温至24℃诱导策略,获得部分胞外表达的酶原.SDS-PAGE显示,胞外表达重组蛋白的分子量约为44kDa,与吸水链霉菌表达的天然酶原相符.诱导4 h后发酵液中的重组酶原经胰蛋白酶活化为成熟酶后测得最高酶活为0.24U/mL.[结论]该研究是对吸水链霉菌的谷氨酰胺转胺酶基因的首次报道,也是国内首次利用大肠杆菌实现pro-MTGase的胞外可溶性表达.     

吸水链霉菌发酵生产谷氨酰胺转胺酶的补料研究

在吸水链霉菌(Streptomyces hygroscopicus)分批发酵研究的基础上,通过在菌体生长阶段指数流加葡萄糖,进行高细胞密度培养,获得了较高的菌体量;待菌体生长进入产酶期后,通过补加氮源,为产酶提供充足的氮源,其中通过流加蛋白质氮源,可以减少蛋白酶对成熟MTG的分解,促进产酶。结果表明,8~16 h采用较高的的比生长速率(0.15 h-1),后期降低比生长速率(0.10 h-1),此时得到的菌体量较高,可达到36 g/L,比分批发酵下的菌体量提高了80%。同时在培养基中添加50g/L的豆饼粉,最终酶活可达到5.79U/ml,提高了83%。     

链霉菌发酵麦草产木聚糖酶的试验研究

通过正交设计试验 ,找出利用链霉菌和麦草基质发酵生产木聚糖酶的试验条件。培养基 (g/L) :麦草粉 ,4 5 ;(NH4 ) 2 SO4 ,7.5 ;酵母膏 ,8;K2 HPO4 ·3H2 O ,1;MgSO4 ·7H2 O ,0 .5 ;NaCl,0 .3。接种量为 5 .0× 10 8个孢子 / 5 0mL培养基 ,振荡培养 (12 0r/min) 5d     

根际益生菌链霉菌S506固体发酵条件优化

链霉菌S506是一株具有广谱防病、促生和降解根系毒素功能的根际益生菌.采用液固两相发酵工艺,以活菌总数和分生孢子产生量为检测指标,研究了S506活菌制剂固体发酵物料的配方和工艺参数.获得固体发酵物料的最佳参数为:麸皮100 g、M-115g、磷酸氢二钾0.3 g、氯化钠0.25 g、硝酸钾0.1 g、碳酸钙0.8 g,自然pH值,液体菌种种龄84 h,接种量10%,发酵温度30℃,料水比10:6,发酵时间60 h.在最佳发酵条件下,S506固体菌剂的活菌总量达到8.27×109CFU/g,分生孢子数达到6.23×109CFU/g.     

Genetic analysis of intraspecies recombinant formation by protoplast fusion with three species of Streptomyces

Abstract Protoplast fusion was shown to produce high frequencies of recombinant progeny in intraspecies crosses with auxotrophic mutants of Streptomyces canescens, Streptomyces griseus and Streptomyces limosus . The fused protoplasts were regenerated on non-selective media and the progeny spores subsequently analysed on selective media to allow detection of all possible genotypes. Prototrophic recombinants arose with frequencies of between 1% and 8%. All 4 possible genotypes were recovered in a series of 2-factor crosses and 6 of the 8 possible genotypes were detected in a 3-factor cross. In spite of attempts to equalise the ratios of parental protoplasts in the fusion mixture, there were noticeable deviations from unity in the ratios of parental genotypes in the progeny.     

Synthesis of vinyl arabinose ester catalyzed by protease from Streptomyces sp.

The transesterification of 0.5 M divinyladipate with 0.25 M arabinose in dimethylformamide for 7 days was catalyzed by Streptomyces sp. alkaline protease to give 5-O-vinyladipoyl-d-arabinofuranose at ca. 50% yield. Only enzymatic transesterification of primary hydroxyl group of arabinofuranose proceeded without esterification of arabinopyranose.