【作者】 张东旭；

【导师】 陈坚；

【作者基本信息】 江南大学 ， 发酵工程， 2008， 博士

【摘要】 谷氨酰胺转胺酶(Transglutaminase EC 2.3.2.13简称TGase)是一种能够催化蛋白质或肽分子间形成共价交联的酶,是食品领域极为重要的酶制剂之一,同时在医药和纺织等工业领域具有广阔的应用潜力。链霉菌谷氨酰胺转胺酶是目前唯一商业化的能够交联蛋白的酶制剂。已有研究表明,茂源链霉菌(Streptomyces mobaraensis)中TGase以酶原(Pro-TGase)形式分泌,并受蛋白酶活化。然而有关链霉菌TGase活化过程的机制还不清楚,链霉菌TGase的生理功能也未见报道。本研究室近来从土壤中筛选到一株高产谷氨酰胺转胺酶的菌株——吸水链霉菌(Streptomyces hygroscopicus)。为进一步推动S. hygroscopicus产TGase的发酵水平,同时也为揭示更多链霉菌TGase的微生物学机制,本论文对S. hygroscopicus产TGase的活化过程及TGase的生理功能进行了研究。主要研究结果如下:1. S. hygroscopicus液体培养过程中存在从Pro-TGase到TGase的转化过程。TGase和Pro-TGase存在于S. hygroscopicus液体培养中,可通过乙醇沉淀和阳离子交换色谱(Fractogel EMD SO3-柱)分离等步骤进行纯化。TGase和Pro-TGase的分子量分别为40 kDa和44 kDa。Pro-TGase的N-端氨基酸序列为:ASGDDEEREG。S. hygroscopicus液体培养中TGase以酶原形式分泌并存在从酶原到成熟酶的转化过程。S. hygroscopicus液体培养中Pro-TGase出现在24h,在36h达到最高,之后Pro-TGase蛋白浓度逐渐下降。随着Pro-TGase蛋白浓度的降低,TGase蛋白浓度逐渐增加。与TGase蛋白浓度的增加相对应,TGase酶活力也开始增加。TGase酶活力在60h达到最高(2.4 U/mL),此时对应Pro-TGase的蛋白已全部转化为成熟酶TGase的蛋白。2. S. hygroscopicus液体培养中,TGase活化蛋白酶分泌到胞外环境中并受抑制因子抑制。TGase活化过程在无细胞上清液中发生沉默。在无细胞上清液培养过程中,从0至24h,Pro-TGase和TGase蛋白的浓度保持恒定,没有发生从Pro-TGase蛋白向TGase蛋白的转化;同时TGase活力保持恒定。10 mg/mL CTAB能够唤醒无细胞上清液沉默的TGase活化过程。添加CTAB的无细胞上清液中Pro-TGase蛋白明显的向TGase蛋白发生转化,同时对应着TGase活性的显著增加(从1.2 U/mL增加到2.3 U/mL)。3. S. hygroscopicus液体培养中,TGase活化过程受多种蛋白酶催化调控。通过DEAE-Sepharose F. F.柱纯化获得到了TAP样品。纯化的TAP样品(0.03 mg/mL)10μL加入到100μL Pro-TGase (0.26 mg/mL)中,30°C培养30min,TAP能够切割Pro-TGase获得TGase蛋白,同时可以检测到酶活从0提高到0.92 U/ml。纯化的TAP为丝氨酸蛋白酶。无细胞上清液中TGase活化过程是多种蛋白酶参与的:包括丝氨酸蛋白酶和金属蛋白酶。10 mmol/L的EDTA抑制下,无细胞上清液中TGase的酶活没有增加,Pro-TGase和TGase的电泳条带浓度也保持稳定;1 mmol/L的PMSF抑制下,TGase的酶活的增加以及Pro-TGase蛋白条带向TGase蛋白条带的转化过程明显滞后。4. TAPI的纯化和鉴定及性质研究。通过Fractogel EMD SO3-柱和Phenyl Sepharose 6 F. F.柱对TAPI进行纯化,TAPI的蛋白收率为6.36 mg/L。TAPI(0.4μg)能够抑制TAP(0.2μg)对Pro-TGase(2μg)的活化。TAPI的N-端氨基酸序列为:GLYAATTALV,属于SSI蛋白家族。TAPI具有表面活性,在液体培养中主要分布在气液界面。5分钟内,TAPI (0.1 mg/mL)使水的界面张力从72降到60 mJ/m2。在此基础上,提出了基于TAPI界面分布的TGase活化过程调控模型。5.在S. hygroscopicus固体培养中,TGase在菌体发生分化、形成气生菌丝时开始分泌和活化,并在形成孢子后停止活化,它可能参与了分化过程。TGase在S. hygroscopicus营养菌丝的的生长阶段并不分泌,而是在开始生分化形成气生菌丝时才开分泌和活化,并在形成孢子后停止活化。48 h固体平板上开始出现白色的气生菌丝的同时,检测到胞外蛋白浸提液中TGase活性显著提高(0.51 U/plate);144 h固体平板上开始出现黑色孢子,此后已检测不到TGase活性。Pro-TGase当量活性在48 h开始出现(0.03 U/plate),之后逐渐升高并在菌体形成孢子后保持稳定(0.65 U/plate左右)。胱胺能够抑制S. hygroscopicus液体培养种纯化到TGase的活性,25 mmol/L的胱胺可以抑制活性为0.17 U/mL的TGase 75 %的活性。25 mmol/L的胱胺能够显著延迟S. hygroscopicus的分化过程。更多还原

【Abstract】 Transglutaminase (TGase; Protein-glutamine-glutamyltransferase, EC 2.3.2.13) is an enzyme capable of introducing covalent cross-links between proteins as well as peptides. TGase is a very important enzyme in food industry and has great potential application in pharmaceutical and textile industries. Streptomyces TGase is the only commercial enzyme that could covalent proteins. It has been reported that TGase from Streptomyces mobaraensis was secreted as zymogen and could be activated by protease. However, no evidence explain the physiological function of Streptomyces TGase; and the mechanism of TGase activation was still unclear in Streptomyces. Recently, in our lab, a new strain capable of producing a high activity of TGase, classified as Streptomyces hygroscopicus, was isolated from soil. To provide theory support to enhance the fermentation of this strain, the activation mechanism and physiological function of TGase from S. hygroscopicus were studied in this dissertation. The main results are listed as following:1. Translation from Pro-TGase to TGase exists in liquid culure of S. hygroscopicus.TGase and Pro-TGase were secreted to the liquid media and could be purified by ethanol precipitation and Fractogel EMD SO3-. TGase and Pro-TGase exhibited molecular mass as 40 kDa and 44 kDa, respectively. N-terminal amino acids sequences of Pro-TGase were: ASGDDEEREG.During the liquid culture of S. hygroscopicus, TGase was secreted as a zymogen and was subsequently activated. Pro-TGase appeared at 24h and reached a maximum concentration at 36h. Subsequently, with the level of Pro-TGase decreasing, the level of mature TGase increased gradually accompanied by a significant increase in TGase activity. TGase activity reached the highest value of 2.4 U/mL at about 60 h, when Pro-TGase was entirely converted into mature TGase.2. TGase-activating protease is located in the cell-free fraction and is inhibited by a TGase-activating protease inhibitor.TGase activation was silenced in cell-removed system. Both Pro-TGase level and TGase activity in the cell-free fraction remained steady from 0 to 24 h. 10 mg/mL cetyltrimethyl ammonium bromide (CTAB) could waken TGase activation in cell-removed system. CTAB-treated supernatant showed that the TGase activation process resumed and the rate of activation (from 1.2 to 2.3 U/mL).3. Transglutaminase activaton is catalyzed by multi-proteases in S. hygroscopicus liquid culture.TAP preparation (0.03 mg/mL) was purified by DEAE-Sepharose F. F. TAP (10μL) was added to 100μL Pro-TGase (0.26 mg/mL). This mixture was incubated at 30°C for 30 min. Pro-TGase then was cuted into TGase, and Pro-TGase was readily converted to TGase by the TAP-rich preparation, and the TGase activity was clearly detected (0.92 U/mL). TAP was detected as serine protease. Serine protease and metalloprotease involve in the TGase activation in cell-removed system. Under the inhibition of EDTA (10 mmol/L), TGase activity in cell-removed system did not increased and concentration of TGase and Pro-TGase kept stady; while under the inhibition of PMSF (1 mmol/L), TGase activity and the convertion from Pro-TGase to TGase were absolutely delayed.4. Purification, indentification and characterization of TAPI.TAPI was purified by Fractogel EMD SO3- column and Phenyl Sepharose 6 F. F. column. Yeild of TAPI was about 6.36mg/mL. TAPI (0.4μg) could inhibite Pro-TGase (2μg) activation mediated by TAP (0.2μg).N-terminal acid sequences of TAPI were: GLYAATTALV. TAPI belongs to Streptomyces subtilisin inhibitor (SSI) family. TAPI has surface activity. TAPI (0.1 mg/mL) decreased the surface tension of water from 72 to 60 mJ/m2 within 5 min. Based on these findings, a model for TAPI-regulated TGase activation process was proposed.5. In S. hygroscopicus solid culture, TGase was secreted and activated during differentiation. TGase may be involved in differentiation of Streptomyces.TGase was sereted and activated during the emergence of aerial hyphae rather then during the vegetative growth. And the seretion and activation process stopped when spores were formed. When white aerial hyphae first appeared at 48 h, TGase activaty was detected from the extract liquid (0.51 U/plate); after spores appeared at 144 h, TGase activity could not be detected. Pro-TGase activity from bovine trypsin activation first appeared at 48 h (0.03 U/plate). Subsequently, this activity increased gradually till reach 0.65 U/plate.Cystamine could inhibite TGase activity. Cystamine at the concentration of 25 mmol/L inhibited 75 % activity of TGase (0.17 U/mL). Cystamine (25 mmol/L) remarkablely delayed differentiation of S. hygroscopicus.更多还原