【作者】 张莉丽；

【导师】 张兰威；

【作者基本信息】 哈尔滨工业大学 ， 化学工程与技术， 2012， 博士

【摘要】 谷氨酰胺转氨酶（Transglutaminase，简称TGase，EC2.3.2.13，R-glutaminyl-peptide：amnie-γ-glutamyl-transferase）又名转谷氨酰胺酶，它通过催化蛋白质中谷氨酸残基上的甲酰胺与赖氨酸残基上的ε-氨基进行酰基转移反应，使蛋白质在分子内或分子间发生交联，从而改善蛋白质的乳化性、溶解性、凝胶性、热稳定性和流变性等性质，因此受到了广泛关注。但是目前食品级TGase来源单一、产量较低、价格昂贵，限制了其在食品工业上的应用。基于很多不利于菌体生长的条件可刺激链霉菌次级代谢产物合成的理论，研究了几种应激条件对Streptomyces mobaraensis菌体生长和TGase产量的影响，确定了其中最适宜提高TGase产量的条件；同时，对MgCl₂胁迫促进TGase产量的机制进行了初步探讨，并且研究了TGase的应用特性。通过研究热处理、向培养基中添加醇类、NaCl对TGase产量和菌体生长的影响，发现这些应激条件在不同程度上促进了TGase的合成。采用响应面设计的方法研究这三个因素对TGase产量的交互作用，结果发现它们的交互作用效果不显著，盐胁迫是提高TGase产量的最显著因素。为了更好地研究盐胁迫作用，分别将8种不同的中性盐（NaCl、Na₂SO₄、C₆H₅O₇Na₃、MgCl₂、CaCl₂、CH3COONa、KCl和Na₃PO₄）添加到培养基中，结果发现MgCl₂的促进作用最强。为了进一步研究MgCl₂胁迫对TGase产量的影响，对MgCl₂在培养基中的添加量进行了优化，同时研究了MgCl₂对菌体生长、产酶周期和蛋白酶产量的影响。结果发现当培养基中MgCl₂浓度为0.1mol/L时，茂原链霉菌的产酶高峰期比在初始培养基中提前24h，并且TGase活力达4.3U/mL，为初始发酵培养基的2.1倍。Western-blot结果表明，MgCl₂胁迫抑制了TGase酶原（Pro-TGase）的合成，促进了成熟酶的合成。MgCl₂胁迫使发酵液中的总蛋白酶、金属蛋白酶和丝氨酸蛋白的产量均提高了一倍。这些结果表明，MgCl₂胁迫促进了参与Pro-TGase激活的蛋白酶合成，从而促进了Pro-TGase向成熟酶的转化，最终提高了TGase产量。另外，为了确定Pro-TGase激活的最关键酶，本研究分别在发酵不同时期向初始培养基和MgCl₂培养基中添加金属蛋白酶抑制剂EDTA和丝氨酸蛋白酶抑制剂PMSF，检测发酵过程中酶活力变化情况，结果表明影响Pro-TGase激活的最关键酶为金属蛋白酶。本文系统地研究了茂原链霉菌在初始培养基和MgCl₂培养基发酵过程中菌体活力、形态分化和DNA断裂情况。通过激光共聚焦显微镜观察菌体活力变化，发现在发酵起始时MgCl₂胁迫抑制了菌体生长和菌球体形成；随着发酵时间延长，MgCl₂胁迫促进了菌体程序性死亡。通过扫描电镜观察菌体在两种培养基中形态变化，发现生长在MgCl₂培养基中的菌体更早地出现弯曲、分枝状和淀粉样化的现象，并且在发酵末期出现了初始培养基中不曾出现的菌毛和孢子，这表明MgCl₂胁迫促进了菌丝分裂。通过对比两种培养基中菌体DNA变化，发现MgCl₂胁迫促进了菌体DNA的裂解。这些结果表明，MgCl₂胁迫刺激了菌体的分化和程序性死亡，从而缩短了发酵周期。通过PDQuest软件分析了初始培养基和MgCl₂培养基中培养24h的菌体蛋白双向电泳图谱，得到53个差异蛋白点，将质谱鉴定成功的50个差异蛋白质与KEGG数据库中的信息进行比对，分析了这些蛋白质的生理功能。结果表明，菌体受到MgCl₂胁迫后，参与初级代谢，如糖代谢、氨基酸代谢、核苷酸代谢的酶均受抑制，而参与次级代谢的酶、压力应激蛋白以及能量代谢相关的酶合成量升高。这说明MgCl₂胁迫抑制了菌体的初级代谢，使菌体合成速率下降，同时促进了应激蛋白、参与次级代谢或合成次级代谢产物前体蛋白质/酶的合成，另外它还促进了合成TGase限制性氨基酸的生成，因此TGase提前合成，并且产量升高。通过超滤浓缩MgCl₂发酵液后利用凝胶层析和离子交换层析相结合的方法对MgCl₂发酵液中的TGase进行纯化，利用液相色谱串联质谱对纯化的蛋白质进行了鉴定，结果表明纯化得到的酶与S. mobaraensis合成的AAN01353是同一蛋白质。酶学性质研究发现纯酶比已知报道的酶具有更强的热稳定性和pH稳定性。将纯酶应用到脱脂和全脂酸奶中，发现脂肪能够影响TGase对蛋白质的交联。另外，它可以通过交联牦牛酸奶中的蛋白质，使其质地变得细腻、感观更易接受，为开发牦牛乳产品奠定基础。更多还原

【Abstract】 Transglutaminase （shorten as TGase, EC2.3.2.13, protein-glutamine: amineγ-glutamyltransferase） is a family of enzymes that can catalyze the cross-linking ofproteins to high molecular weight aggregates by transfer reaction betweenappropriate glutamine and lysine residues. It attacts a great interest, since it modifysthe functional properties of protein such as solubility, hydration ability, rheologicaland emulsifying properties as well as heat stability in model systems. However,scarcely sources, low yield and expensive price of TGase limit its application in thefood industry. Based on the theory that Streptomyces cell growth in disadvantageconditions are benefit to the synthesis of secondary metabolites, this paper aimed atimproving TGase production by stress methods. The most suitable condition wasfound. Furthermore, mechanism of enhancement of TGase production was studied.Meanwhile, TGase from MgCl₂media was purified and characterized, and appliedinto yogurt.Effects of heat treatment, supplementation alcohol or NaCl on the TGaseproduction and cell growth of Streptomyces mobaraensis showed that stress methodsimproved the TGase production in varying extent. Response of surface design ofheat treatment, addition alcohol and/or NaCl to media was used to estimate theirinteraction effects on TGase production. Results showed that the interaction effectwas not significant and salt stress was the best way to improve TGase production. Inorder to find better condition for TGase production, eight kinds of different neutralsalts （NaCl, Na₂SO₄, C₆H₅O₇Na₃, MgCl₂, CaCl₂, CH3COONa, KCl and Na₃PO₄）were added into the media. Results showed that the optimal salt for TGaseproduction was MgCl₂. In order to study the enhancement of TGase production byMgCl₂stress, effects of MgCl₂concentration on cell growth, period of TGasebiosynthesis and proteases production were studied. The highest yield of TGase wasobserved at96h of incubation （4.3U/ml）, when a basic medium was supplementedof0.1mol/l MgCl₂, compared with the basic media （2.1U/ml） by120h.Western-blot analysis showed faster transformation of pro-TGase into the matureenzyme in MgCl₂medium vs. the control medium. Total protease, metalloproteaseand serine protease were also biosynthesized in a fast rate in the media containingMgCl₂. The results demonstrated that the MgCl₂stress improved the production of key proteases involved in the activation of Pro-TGase. Furthermore, in order todetermine the key enzyme for Pro-TGase activation, the inhibitors formetalloprotease （EDTA） and serine protease （PMSF） were added into two kinds ofmedia separately, and TGase activities were determined during fermentation. Resultsshowed that the key enzyme for TGase production was metalloprotease.Strain viability, morphological differentiation and DNA fragment of S.mobaraensis during fermentation in the control media and MgCl₂media werecompared. MgCl₂inhibited cell growth and the formation of strain sphere. With theextension of the fermentation time, MgCl₂promoted the bacterial programmed celldeath. According to bacterial morphological differentiation in both kinds of media, itwas found that S. mobaraensis in MgCl₂media appeared bent, branched and amyloidearlier than in the control media; as well as, fimbriae and spores appeared in MgCl₂media, not in the control. These results suggested that the MgCl₂promotedmycelium differentiation. Comparing S. mobaraensis DNA changes in two kinds ofmedia, it was found that MgCl₂promote bacterial DNA cleavage, which indicatedthat MgCl₂stimulated morphological differentiation and programmed cell death,thus shortened the fermentation period.Comparing two dimensional gel electrophoresis of the bacterial cell protein incontrol medium and MgCl₂medium cultured for24h by PDQuest, fifty-threedifferentially expressed proteins were found and50proteins was identifiedsuccessfully by mass spectrometry. Their physiological functions were analyzedaccording to KEGG data. Results showed that proteins involved in primarymetabolism, such as sugar metabolism, amino acid metabolism, nucleotidemetabolism were inhibited by MgCl₂stress, while those involved in secondarymetabolism, pressure stress protein and energy metabolism-related enzymes wereincreased. These results showed that MgCl₂stress inhibited the primary metabolismof Streptomyces, decreased bacterial cell growth rates. However, it promotedproteins involved in secondary metabolism or synthesis of secondary metabolitesprecursors. Thus, TGase was biosynthesized earlier with high yields and myceliaprogrammed cell death was promoted.In order to purify TGase from high-salt media, gel filtration and ion exchangechromatography method were combined after concentrating fermentation liquid byultrafiltration. Using liquid chromatography/tandem mass spectrometryidentification and the protein database comparison, the purified enzyme showed high homology with AAN01353from S. mobaraensis. Characterization of purifiedenzyme showed higher heat and pH stability. Application of the pure enzyme intoskim and whole milk yogurt showed that it could improve the functional propertiesof yogurt, but the excessive cross-linking caused the oversize of yogurt gel particlesand the course texture. In addition, milk fat affected the protein cross-linking and thetexture of the yogurt. Furthermore, TGase was used to cross-link yak yogurt protein.Results showed that the texture of cross-linking yak yogurt was more delicate andacceptable. It provided the foundation for the development of yak milk products.更多还原