【作者】 黄欣；

【导师】 袁文杰；

【作者基本信息】 大连理工大学 ， 生物化工， 2022， 硕士

【摘要】 二肽因其特殊功能广泛应用于食品、医药、保健品和化妆品等领域。化学合成法合成二肽,反应步骤繁琐、环境不友好,因此近些年利用生物酶催化法生产二肽的研究逐渐兴起。L-氨基酸连接酶（L-amino acid ligase,EC 6.3.2）能以氨基酸直接作为底物合成二肽,但该反应是ATP依赖型,需要外源补充ATP。本研究以大肠杆菌作为宿主,通过构建L-氨基酸连接酶偶联聚磷酸激酶表达系统,无需体外补充ATP（ATP-free）即可合成功能性二肽,通过反应条件优化,提高了二肽的产率,具体研究内容如下:首先,利用枯草芽孢杆菌（Bacillus subtilis ATCC 15245）中L-氨基酸连接酶Ywf E和丁香假单胞菌（Pseudomonas syringae NBRC14081 ATCC 27881）中L-氨基酸连接酶Tab S构建重组菌株,诱导表达并纯化得到Ywf E和Tab S。Ywf E催化丙氨酸（Ala）和谷氨酰胺（Gln）合成丙谷二肽（Ala-Gln）的反应在22-37℃、p H值8-9、Mg²⁺浓度20-25mmol/L、ATP浓度为30 mmol/L,以及底物Ala和Gln浓度分别为30和60 mmol/L时产率最高:反应6 h时,Ala-Gln产率高达61.0%;Tab S催化2分子Ala合成丙氨酸二肽（Ala-Ala）的反应在37℃、p H值9-9.5,ATP浓度为15 mmol/L时产率最高:反应11 h时,Ala-Ala产率达到47.3%。说明Ywf E和Tab S具有较好的合成不同二肽能力,可以完成多种功能性二肽的合成。其次,以谷氨酸棒杆菌（Corynebacterium glutamicum 299）、泗阳鞘氨醇杆菌（Sphingobacterium siyangensis CGMCC 1.6855）基因组为模板扩增聚磷酸激酶基因,并构建重组菌株,诱导表达并分离纯化得到有ATP合成活性的PPK2（1.6855）进行研究。PPK2（1.6855）催化ATP合成的反应在22-37℃、p H值7-9、Mg²⁺浓度为30 mmol/L、以及底物ADP和六偏磷酸钠的浓度为5和20 mmol/L时产率最高:当反应3 h时,ATP产率高达71.0%。最后,偶联L-氨基酸连接酶Ywf E和聚磷酸激酶PPK2（1.6855）双酶实现Ala-Gln高效合成:Ala和Gln浓度分别为30和60 mmol/L,ADP和六偏磷酸钠浓度为10和15mmol/L,控制反应温度为27℃,p H值7-8,PPK2反应3 h后加入Ywf E,反应7 h后Ala-Gln产率可高达64.1%,与直接添加ATP的产率相当。为减少酶的纯化步骤,降低反应成本,使其更适用于工业放大,利用单质粒和双质粒两种方法搭建了氨基酸连接酶Ywf E和聚磷酸激酶PPK2（1.6855）共表达系统,为实现全细胞催化合成功能性二肽奠定了基础。更多还原

【Abstract】 Because of its special function,dipeptide is widely used in food,medicine,health care products and cosmetics.Chemical synthesis of dipeptides is cumbersome and environmentally unfriendly,so in recent years,the use of enzyme catalytic method to produce dipeptides gradually emerged.L-amino acid ligase（EC 6.3.2）can directly use amino acids as substrates to synthesize dipeptides,however,for its ATP dependence,exogenous ATP supplementation or ATP regeneration system is needed.In this study,using Escherichia coli as the host,co-expressing L-amino acid ligase,polyphosphokinase and dual enzymes were explored.Functional dipeptides were synthesized by L-amino acid ligase and polyphosphokinase without ATP supplementation（ATP-free）,and the yield of dipeptides was improved by optimizing the reaction conditions.The specific research contents are as follows:Firstly,the recombinant strains were constructed by using L-amino acid ligase Ywf E from Bacillus subtilis ATCC 15245 and L-amino acid ligase Tab S from Pseudomonas syringae NBRC 14081 ATCC 27881.Ywf E and Tab S were induced,expressed and purified.Ywf E catalyzed the synthesis of alanine（Ala）and glutamine（Gln）to L-alanyl-L-glutamine（Ala-Gln）with the highest yield at 22-37℃,p H 8-9,Mg²⁺concentration 20-25 mmol/L,ATP concentration 30 mmol/L,and substrate Ala and Gln concentration 30 and 60 mmol/L respectively:the Ala-Gln yield reached 61.0%at 6 h;The synthesis of alanine dipeptide（Ala-Ala）from 2 molecules of Ala catalyzed by Tab S had the highest yield at 37℃,p H 9-9.5,ATP concentration 15 mmol/L,and the Ala-Ala yield reached 47.3%at 11 h.It shows that Ywf E and Tab S have good ability to synthesize different dipeptides,and can complete the synthesis of a variety of functional dipeptides.Secondly,using the genomes of Corynebacterium glutamicum 299 and Sphingobacterium siyangensis CGMCC 1.6855 as templates,the polyphosphate kinase gene was amplified,and the recombinant strain was constructed.PPK2（1.6855）with ATP synthesis activity was induced,expressed,isolated and purified.The yield of ATP synthesis catalyzed by PPK2（1.6855）was the highest at 22-37℃,p H 7-9,Mg²⁺concentration of 30 mmol/L,substrate ADP and sodium hexametaphosphate concentration of 5 and 20 mmol/L:when the reaction lasted for3 hours,the yield of ATP was as high as 71.0%.Finally,coupling L-amino acid ligase Ywf E and polyphosphate kinase PPK2（1.6855）to achieve high-efficiency synthesis of Ala-Gln:the concentrations of Ala and Gln were 30 and 60mmol/L respectively,the concentrations of ADP and sodium hexametaphosphate were 10 and15 mmol/L respectively,the reaction temperature was controlled at 27℃,the p H was 7-8,and the yield of Ala-Gln could be as high as 64.1%after 3 h of PPK2 reaction,which was equivalent to the yield of direct addition of ATP.In order to reduce the purification steps of the enzyme,reduce the reaction cost,and make it more suitable for industrial amplification,a whole cell catalyst with co-expression system of amino acid ligase Ywf E and polyphosphokinase PPK2（1.6855）was established by using single-plasmid and double-plasmid methods.This study laid the foundation for the efficient synthesis of functional dipeptide.更多还原