【作者】 帅玉英；

【导师】 江波；

【作者基本信息】 江南大学 ， 食品科学， 2011， 博士

【摘要】 L-茶氨酸是存在于茶叶内的一种主要氨基酸,是一种具有多种生理功能且不会产生任何副作用的一种功能因子,在食品、保健和医疗领域中具有重要的应用价值。因此,研究生物法合成L-茶氨酸具有重要的现实意义。目前生物法生产L-茶氨酸最有效的酶是γ-谷氨酰转肽酶（γ-Glutamyltranspeptidase, GGT, EC 2.3.2.2）。GGT是生物体内谷胱甘肽代谢途径中的关键酶,也是催化转移L-γ-谷氨酰基的特异性酶,可催化L-谷氨酰胺和乙胺合成L-茶氨酸（γ-谷氨酰基-乙胺）,在催化合成方面正成为研究者关注的热点。本研究首先从发酵食品虾酱中通过发酵法产GGT实验和酶法合成L-茶氨酸实验两步筛选法分离得到一株可用来高效合成L-茶氨酸的菌株SK11.004。通过形态观察和生理生化特性实验,结合16S rDNA序列分析,将该菌株鉴定为枯草芽孢杆菌,并将其命名为Bacillus subtilis SK11.004。其16S rDNA基因全长为1142 bp,已提交至GenBank数据库,数据库登记号为:FJ437210。GGT的合成与菌体生长同步,发酵16 h产酶活力最高,达到2.5 U/mL。在筛选获得Bacillus subtilis SK11.004的基础上,建立了以L-谷氨酰胺和乙胺盐酸盐为底物通过发酵液酶促反应合成L-茶氨酸的工艺,并完成了中试生产。首先对酶促反应条件进行了优化,在最适反应条件下,副反应得到了有效的抑制,L-茶氨酸的转化率约为94%,纯度达65.2%。此外,还建立了L-茶氨酸的脱色及分离纯化工艺,通过串联使用阴阳离子交换树脂去除反应液中的L-谷氨酸及其它杂质,再经洗脱、减压浓缩干燥,获得L-茶氨酸,纯度达到85%,收率为80.4%。最后建立了L-茶氨酸的结晶工艺,在优化后的结晶工艺条件下,晶体得率为48%,晶体纯度达97%,并利用扫描电镜对其晶体形态进行了扫描,晶体呈规则的致密的方形层状结构。对B. subtilis SK11.004的发酵液进行了分离纯化,采用超滤、硫酸铵沉淀、疏水作用层析和凝胶过滤等手段,得到两种具有GGT活力的酶（GGT-1与GGT-2）,比酶活分别达到684.9和193 U/mg。通过Native-PAGE、SDS-PAGE和凝胶过滤色谱对酶的纯度和分子量进行测定,结果表明GGT-1为二聚体,包含两个亚基（40 kDa和21 kDa）,分子量约为62 kDa;GGT-2为单体酶,分子量约为58 kDa。对比酶活高的GGT-1的酶学性质进行了系统研究。其转肽反应的最适pH为10;水解反应的最适pH为8.0;在pH6-12的缓冲液中酶活稳定性良好;等电点为7.8,说明该酶处于兼性离子及负离子时,才能更好地发挥活力;该酶的最适反应温度为37 oC,在50 oC以下具有良好的稳定性;Na⁺和Ca²⁺对酶活影响不明显,Al³⁺和Mg²⁺能促进酶活力,Cu²⁺和Zn²⁺对GGT的酶活有明显抑制作用,Zn²⁺的抑制作用最强,可抑制32.2%的活力,而酸根离子SO₄^2-、Cl^-、NO₃^-对酶活基本没有影响。另外,EDTA对酶活基本没有影响,说明GGT-1并非金属蛋白酶。对GGT-1的底物特异性进行研究,结果说明,空间位阻的大小、氨基酸的酸碱性是影响活力的最主要因素,侧链的增加以及酸性的增强会导致酶与底物亲和力的下降,且空间位阻的影响要大于酸碱性的影响。此外,底物的立体异构性对于酶的转肽活力也有一定影响。GGT-1对于谷氨酰供体具有相对低的K_m值,而对于谷氨酰受体则具有高的K_m值。对于γ-GpNA具有低的亲和力,而对于L-Gln亲和力高一些。L-Gln转肽反应和水解反应的特征常数K_m分别为0.83和3.16 mM,说明GGT-1易于催化L-Gln进行转肽反应。该酶催化反应速率较快,催化效率较高,可用于γ-谷氨酰类化合物的的生物转化。化学修饰剂PMSF和NBS对酶蛋白的活性影响显著,表明羟基和色氨酸残基均是GGT-1酶活性功能的必需基团。添加底物对NBS对酶的修饰不起保护作用,说明被修饰的色氨酸残基可能不处在酶与底物的契合部位,但它是维持酶空间结构保持其活性必需的基团,而底物的添加对PMSF对酶的修饰有一定保护作用,表明羟基处于底物结合区域。圆二色谱进一步考察了经PMSF和NBS修饰后GGT-1二级结构的变化。结果表明,GGT-1的二级结构由34.4%α-螺旋、11.7%β-折叠、22.2%转角和31.7%无规卷曲组成。经5 mmol/L PMSF处理1h后,其α-螺旋含量由34.4%减少至23.1%,β-折叠和无规卷曲的含量分别增加至17.6%和42.2%,而β-转角的含量则下降至17.1%,说明酶蛋白的二级结构受到了一定程度的破坏。而GGT被1mmol/L NBS修饰后,仅剩余6.8%的α-螺旋,其余结构均转变成了无规卷曲,基本失去了蛋白结构特征,进一步证实了色氨酸残基对于维持酶空间结构的重要性。对GGT-1进行了质谱鉴定,经数据库检索,获得一个Mascot score达到84的有效匹配,匹配蛋白为来自Bacillus amyloliquefaciens FZB42的GGT,氨基酸覆盖率达18%,初步判断GGT-1为γ-谷氨酰转肽酶。经492cLC型蛋白质测序仪分析,GGT-1大亚基的N端结构前八个氨基酸残基连接依次为:FSYDTYKQ。与通过肽指纹图谱匹配到的蛋白质比对,基本与其第35个氨基酸至第43个氨基酸吻合。证实肽指纹图谱鉴定的准确性。结合肽指纹图谱和N端测序结果,确证GGT-1是一种新的γ-谷氨酰转肽酶,且与B. amyloliquefaciens FZB42 GGT具有较近的同源性。更多还原

【Abstract】 Theanine, a unique amino acid found almost exclusively in tea plants, has various favorable physiological and pharmacological functions for human beings without any toxic or side effect. In addition, it has diverse applications in food and medicine areas. Therefore, a study on biological synthesis of theanine has important theoretical and practical significances. Gamma-glutamyltranspeptidase （GGT, EC 2.3.2.2） is considered to be the most effective enzyme for the production of theanine. GGT is the key enzyme in glutathione metabolism, and can catalyze the transfer of theγ-glutamyl group. It can catalyse L-glutamine and ethylamine to theanine （γ-glutamyl-ethylamine）. GGT’s application in catalysis has become a research focus.In this study, strain SK 11.004 isolated from fermented shrimp paste through two steps was demonstrated as an effective strain for theanine production. It was identified as Bacillus subtilis based on its physiological and biochemical properties, as well as its 16S rDNA sequence analysis, and named as Bacillus subtilis SK11.004. The 16S rDNA sequence data from B. subtilis SK11.004 was submitted to the GenBank databases under accession no. FJ437210. GGT production was synchronous with the growth of B. subtilis SK11.004, and the highest enzyme activity （2.5 U/mL） in the culture was achieved after 16h.The goal of developing an effective method for the production of theanine was achieved using the GGT from B. subtilis SK11.004. Firstly, the optimum conditions for the synthesis of theanine were determined. In the optimized conditions, the side reaction was inhibited effectively, and the conversion rate of L-Gln to theanine reached 94%. Secondly, the technology of decoloration and purification of theanine was established. The theanine with 85% purity was obtained through activated carbon decoloration, 001×7 strong acidic resin （H⁺ form）, 201×7 strong basic resin （Cl^- form）, vacuum concentration and freeze-drying, with the recovery rate of theanine as 80.4%. Finally, the crystallization process of theanine was also studied. After crystallization, the theanine with purity of 97% was obtained with a yield of 48%. The crystals appeared as compact layered-structure with tetra-rectangle under scanning electron microscopy.Two GGTs （GGT-1 and GGT-2） were purified to electrophoretical homogeneity by ultrafiltration, hydrophobic interaction and gel filtration chromatography, and exhibited the special activity of 684.9 and193 U/mg, respectively. Based on SDS-PAGE and gel filtration analysis, it was confirmed that GGT-1 was composed of one large subunit of 40 kDa and one small subunit of 21 kDa, with the molecular mass of 62 kDa, GGT-2 was showed as a monomer, with the molecular mass of 58 kDa.The properties of GGT-1 were investigated. The enzyme showed maximal activity in hydrolase reaction at pH 8, while in transpeptidase activity at pH 10. It exhibited strong stability in pH 6-12.The isoelectric point of GGT-1 was determined to be 7.8, suggesting that the enzyme had higher activity when it was zwitterion and anion. The enzyme showed maximal activity at 37 oC, and was highly stable below 50°C. Al³⁺ and Mg²⁺ stimulated the enzyme activity of GGT-1, whereas Cu²⁺, Zn²⁺, Fe²⁺and Fe³⁺ caused its inhibition. The acid radicals SO₄^2-, Cl^-, and NO₃^- did not influence enzyme activity.The enzyme exhibited the highest affinity to imino acids （L-Pro） and then decreasing affinities for aromatic amino acids, ethylamine and basic amino acids. GGT-1 has low K_m values forγ-glutamyl donors, but high ones for acceptors, and low affinity forγ-GpNA, but high for L-Gln. The K_m values of hydrolysis and of transpeptidation for L-Gln were 3.16 mM and 0.83 mM respectively, suggesting that the GGT-1 likely synthesizes valuableγ-glutamyl peptides using L-Gln asγ-glutamyl donor.Chemical modifiers PMSF and NBS showed strong inhibition of the GGT-1 enzyme activity, indicating the tryptophan residues and hydroxy groups of Ser or Thr are essential to enzyme activity. The addition of excess substrate did not reverse the inhibition process of NBS, it was suggested that the tryptophan residues were not located in the substrate binding site of GGT-1, but it was necessary group which maintain the structure of the enzyme. However, the addition of excess substrate reduced the inhibition effect of PMSF, revealing that the hydroxy groups were located in the substrate binding site of GGT-1.Circular dichroism spectra reflected the conformational changes of GGT-1. CD of the native enzyme showed that ratio ofα-Helix,β-sheet, turn and random coils structure were 34.4%, 11.7%, 22.2% and 31.7%. The modified GGT-1 by PMSF resulted in the decrease ofα-helix contente and increase of random coils content, showing that the secondary structure of the enzyme was destroyed to some extent. However, when GGT-1 was modified by NBS, there was only 6.8%α-Helix left, with 93.2% random coils. It was confirmed that the tryptophan residues were very important in maintaining the structure of the enzyme.The MALDI-TOF-TOF MS data were used to query the Mascot proteomics database, resulting in one significant match, GGT [Bacillus amyloliquefaciens FZB42]. After in-gel digestion, we obtained a Mascot score of 84 with a sequence coverage of 18%. The N-terminal amino acid sequence of the first 8 residues of heavy subunit was determined as FSYDTYKQ, which almost fully corresponded to residues 35-43 of B. amyloliquefaciens FZB42 GGT. The result confirmed the accuracy of identification by MALDI-TOF-TOF MS. A comparison with other existing GGT enzymes proved the observed N-terminal amino acid sequence to be different.更多还原