CN113999862A - 一种谷氨酰胺转氨酶的异源表达及其应用 - Google Patents
一种谷氨酰胺转氨酶的异源表达及其应用 Download PDFInfo
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- CN113999862A CN113999862A CN202010740486.8A CN202010740486A CN113999862A CN 113999862 A CN113999862 A CN 113999862A CN 202010740486 A CN202010740486 A CN 202010740486A CN 113999862 A CN113999862 A CN 113999862A
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- glutamine transaminase
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Abstract
本发明公开了一种链霉菌谷氨酰胺转氨酶基因的异源表达与表征及其在明胶交联中的应用，属于生物技术领域。本发明所述的谷氨酰胺转氨酶基因全长是通过挖掘Streptomyces sp.TYQ1024全基因数据而获得，其基因全长1233bp，编码410个氨基酸，并通过PCR体外扩增了该基因。本发明为首次实现链霉菌谷氨酰胺转氨酶基因在枯草芽孢杆菌SCK6中的异源表达、纯化和表征，并将重组谷氨酰胺转氨酶应用于明胶交联。该酶具有良好的酶学特性，且交联特性优异，在蛋白及明胶交联中拥有广阔的应用前景。
Description
技术领域
本发明涉及一种谷氨酰胺转氨酶的重组表达、分子优化及其应用的技术领域。
背景技术
谷氨酰胺转氨酶(TGase)是一类催化蛋白质发生交联反应的酶，其底物适用范围非常广，如乳清蛋白、大豆蛋白、酪蛋白、牛肌球蛋白等，并在生物医药、纺织、食品、化妆品、皮革、材料等领域具有广阔的应用前景。
从豚鼠、肝脏中获取的TGase，其分离纯化方法复杂，使酶的价格非常昂贵，限制了大规模的生产和应用。从猪、牛的血液中提取凝血因子XIII来生产商用TGase。但血液TGase需要凝血酶来激活其体外活性，影响产品的外观，所以很少应用于食物中。微生物谷氨酰胺转氨酶成本低廉，产量高效，目前已广泛应用于食品等商业中。
基因工程改造技术实现工程菌高效表达是提高酶活的主要方向。目前，TGase已成功在酵母菌、大肠杆菌、链霉菌和谷氨酸棒杆菌等多种宿主中表达。但表达量低、包涵体无催化活性，后期需稀释和复性等问题，令TGase的高效表达遇到许多困难。枯草芽孢杆菌表达宿主因其安全性高和对人体无害等特征，是蛋白和多种酶类克隆表达的常用工程菌。芽孢杆菌的高效胞外分泌能力可为蛋白提供更好的折叠条件，不仅防止了的包涵体形成，还能有效减少后续的分离纯化工艺，是TGase异源高效表达的理想系统。
发明内容
本发明首先提供了一种微生物谷氨酰胺转氨酶的基因TGase，是以实验室保藏的谷氨酰胺转氨酶产生菌Streptomyces sp. TYQ1024为出发菌株，提取其总基因组DNA，设计引物并结合其全基因组序列分析，通过PCR扩增出TGase基因片段（Genebank ID:MN700931），该基因全长1233 bp，编码410个氨基酸，其中1-28位氨基酸为信号肽，29-79位氨基酸为前肽，80-410位氨基酸为成熟肽。
本发明将获得的基因与载体pMA LipA连接，构建重组表达载体pMA LipA-TG。将重组质粒转化至Bacillus subtilis SCK6实现了TGase酶的异源表达，并对重组菌发酵液进行硫酸铵分级沉淀和离子交换层析纯化，获得了重组TGase酶。
本发明所得到的谷氨酰胺转氨酶分子量约为38 kDa，该酶最适反应温度为50 oC，最适反应pH为8，且在pH 5.0~9.0内稳定性较好；PMSF和SDS完全抑制酶活；还原剂DTT和β-ME对酶活有一定的促进作用。将TGase应用于明胶的凝胶交联过程中，促进了明胶的交联，形成更多的异肽键，具有良好的应用前景。
附图说明
图1重组质粒pMA LipA-TG的构建图
图2为重组质粒pMA LipA-TG双酶切鉴定：M1：Supercoiled DNA Marker；1：原始质粒pMA LipA；2：重组质粒pMA LipA-TG；3：重组质粒单酶切；4：重组质粒双酶切；M2：10000bpDNA Marker
图3为谷氨酰胺转氨酶的电泳分析：M：蛋白质marker；1：发酵上清液；2-6：纯化的目的TGase
图4为pH对谷氨酰胺转氨酶酶活(A)和稳定性(B)的影响。
图5为温度对谷氨酰胺转氨酶酶活(A)和稳定性(B)的影响。
图6为凝胶的红外光谱（FI-IR）分析
具体实施方式
实施例一：TGase基因的获得
提取Streptomyces sp. TYQ1024的基因组DNA，进行全基因组序列测序。对得到基因组数据进行分析，获取谷氨酰胺转氨酶基因TGase。
实施例二：TGase基因的异源表达
设计特异性引物扩增TGase基因，上游引物(5'-CGCGGATCCTCGCCACCGGCAGTGGCAGTGGCA-3')，下游引物(5'- CTAGCTAGCTCACGGCCAGCCCTGTGTCA-3')，通过PCR法获得TGase基因。对TGase基因和质粒pMA LipA进行BamH 
和Nhe 
酶切，并进行连接。然后，将连接产物转化至E. coli DH5α，提取重组质粒pMA LipA-TG，转化至B. subtilis SCK6。
实施例三：重组枯草芽孢杆菌工程菌培养条件
将重组菌B. subtilis SCK6/pMA LipA-TG接种至含50µg/mL卡那霉素的100 mL TB基础培养基中，37°C摇床振荡培养16h，以3%的接种量接种于含50µg/mL Kan的发酵培养基中（培养基成分：葡萄糖15 g/L，大豆蛋白胨20 g/L，尿素20 g/L，磷酸二氢钾2.31 g/L，磷酸氢二钾0.54 g/L，pH 6.5），37°C，220rpm摇床培养64h；离心，收集发酵上清液，并测定上清液中TGase活性。
实施例四：谷氨酰胺转氨酶活性测试
取100 μL菌体培养上清液和1 mL底物溶液（37 oC水浴恒温）混合，在37 ± 1 oC恒温水浴中精确反应10 min。反应结束后立即加入1 mL显色溶液终止反应，4000 r/min离心5min去除沉淀。以水为对照于525 nm处测定OD值，即为待测酶液吸收值。通过标准曲线计算得到具体酶活力单位。
实施例五：重组谷氨酰胺转氨酶的纯化
添加50%饱和度的硫酸铵至发酵上清液，4oC 静置过夜，离心收集上清，弃沉淀去除部分杂蛋白。缓慢加入硫酸铵粉末至硫酸铵饱和度达到80%，4oC静置过夜，离心收集沉淀，弃上清，进一步去除杂蛋白。然后，用适量的缓冲液(20 mM PB；pH 5.8)对沉淀进行充分溶解，离心收集上清，用SP阳离子交换柱对TGase进一步纯化，(20 mM PB；pH 5.8)平衡柱子后，用含1M NaCl线性梯度洗脱目的蛋白，进行SDS-PAGE鉴定。
实施例六：谷氨酰胺转氨酶的酶学性质研究
在pH 3~10的pH值范围内检测谷氨酰胺转氨酶酶活；将谷氨酰胺转氨酶置于不同pH缓冲液中，室温下保温30min，测定其pH稳定性；在10~70oC的不同温度下测定谷氨酰胺转氨酶酶活；将重组酶置于4~60oC 条件下，保温30min，测定其热稳定性。
实施例七：TGase交联明胶性能研究
分别将TGase改性前后的明胶凝胶冷冻干燥，获得干燥的海绵。称取5-10 mg干凝胶，用溴化钾研磨压片方法制成圆形片，固定在红外光谱仪的样品架上。在4000〜500 cm-1的光谱范围内进行红外扫描，获得改性前后凝胶的红外光谱图。
序列表
<110> 四川大学
<120> 一种谷氨酰胺转氨酶的异源表达及其应用
<141> 2020-07-22
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Claims (3)
1.一种微生物谷氨酰胺转氨酶TGase的基因序列，其特征是由1233 个核苷酸组成，编码410个氨基酸，其中1-28位氨基酸为信号肽。
2.将TGase基因连接入pMA LipA载体，构建重组质粒pMA LipA-TG并在Bacillussubtilis SCK6中实现高效表达，其特征是在明胶交联实验中展现出优异的蛋白交联特性。
3.TGase的特征在于最适温度50oC，最适pH 8，在明胶等蛋白交联中具有很好的应用前景。
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