CN114369582B - 双向伯克霍尔德氏菌来源酯合成酶jg536_25355、编码基因及应用 - Google Patents
双向伯克霍尔德氏菌来源酯合成酶jg536_25355、编码基因及应用 Download PDFInfo
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- CN114369582B CN114369582B CN202210110054.8A CN202210110054A CN114369582B CN 114369582 B CN114369582 B CN 114369582B CN 202210110054 A CN202210110054 A CN 202210110054A CN 114369582 B CN114369582 B CN 114369582B
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Abstract
本发明属于基因工程技术领域，具体涉及双向伯克霍尔德氏菌来源酯合成酶JG536_25355、编码基因及应用。该酯合成酶氨基酸序列如SEQ ID NO.1所示。本发明通过克隆并诱导表达获得双向伯克霍尔德氏菌BJQ0010来源酯合成酶JG536_25355及其编码基因，并构建含有酯合成酶编码基因的大肠杆菌表达质粒，将该质粒转入大肠杆菌中，经过诱导表达获得该酶。本发明的酯合成酶经催化体系验证不仅具有在水相体系能催化合成己酸乙酯、辛酸乙酯的能力，还能催化合成丁酸乙酯、戊酸乙酯和癸酸乙酯，因此能用于白酒酿造领域中催化合成重要的风味酯。
Description
技术领域
本发明属于基因工程技术领域，具体涉及双向伯克霍尔德氏菌来源酯合成酶JG536_25355、编码基因及应用。
背景技术
风味分析表明，浓香型白酒中乙醇与水的含量约占酒体总量的98％，剩余2％为风味物质，是白酒中的微量成分。风味物质含量虽低，却对白酒的品质起到决定性的作用。目前已报道的微量成分约有2400多种，其中酯类是最重要的一类风味物质，共报道510种。因为原材料地理分布的差异、发酵剂以及酿造工艺的不同形成了12大香型的白酒。其中，浓香型白酒占白酒市场份额的70％以上，窖香浓郁、香味协调，是传统发酵酒精饮品的典型代表。而小分子脂肪酸乙酯如丁酸乙酯、戊酸乙酯、己酸乙酯、辛酸乙酯、癸酸乙酯等是浓香型白酒中的重要风味物质，尤其是己酸乙酯，作为浓香型白酒的代表性酯类物质，其生成机制的科学基础尚未明晰，造成浓香型白酒酿造过程中酯类物质合成效率低，降低了高品质白酒的生产效率，制约了产业发展。
已有的研究表明，微生物的代谢是浓香型白酒固态发酵的主要驱动力，其中微生物所产酯合成酶对己酸乙酯等小分子脂肪酸乙酯的合成具有显著贡献。具有酯类物质合成能力的微生物主要包括细菌、酵母菌和霉菌三大类，细菌属的伯克霍尔德氏菌、血红鞘氨醇单胞菌，以及霉菌属的根霉、曲霉、毛霉、多枝横梗霉和红曲霉均具有产酯能力。
虽然酯合成的微生物已有文献报道，然而聚焦到酯合成的关键酶，相关研究依然匮乏。已有研究表明有机相体系如溶剂为正己烷、正庚烷的反应体系有助于酶催化酯合成的反应，然而白酒为固态发酵体系，酒醅的含水量在53～58％之间，普遍被认为是一个水相体系，并不利于经典的酯合成反应的进行。所以白酒源酯合成的微生物应携带具有不同于已知的经典有机相催化酯合成的酶资源，这仍有待发现和研究。
而挖掘伯克霍尔德氏菌来源具有催化合成丁酸乙酯、戊酸乙酯、己酸乙酯、辛酸乙酯和癸酸乙酯等小分子脂肪酸乙酯能力的酯合成酶，有助于丰富白酒微生物源酯合成功能酶资源，相关酶资源的深入研究对于保障白酒中关键风味酯类的稳定合成具有重要意义。
发明内容
针对现有技术的不足，本发明的目的在于提供一种分离自白酒大曲的细菌属双向伯克霍尔德氏菌(Burkholderia ambifaria)BJQ0010来源酯合成α/β水解酶JG536_25355及其编码基因在水相体系催化合成丁酸乙酯、戊酸乙酯、己酸乙酯、辛酸乙酯和癸酸乙酯的应用。
本发明首先提供了双向伯克霍尔德氏菌(Burkholderia ambifaria)BJQ0010来源酯合成酶 JG536_25355，其氨基酸序列如SEQ ID NO.1所示。
本发明还提供所述双向伯克霍尔德氏菌来源酯合成酶JG536_25355的编码基因。
其中，所述双向伯克霍尔德氏菌来源酯合成酶JG536_25355的编码基因的核苷酸序列如 SEQ ID NO.2所示。
所述酯合成酶JG536_25355获取方式包括：大肠杆菌异源表达获取。
本发明还提供了含有上述双向伯克霍尔德氏菌来源酯合成酶JG536_25355编码基因的表达载体。
所述表达载体的宿主细胞为大肠杆菌细胞。
其中，所述载体克隆区域的核苷酸序列如SEQ ID NO.2所示。
本发明还提供上述双向伯克霍尔德氏菌来源酯合成酶JG536_25355在制备酒用酯香液或浓香型白酒中的应用。
进一步地，上述双向伯克霍尔德氏菌来源酯合成酶JG536_25355在水相体系中催化合成丁酸乙酯、戊酸乙酯、己酸乙酯、辛酸乙酯和癸酸乙酯中的应用。
所述应用中，双向伯克霍尔德氏菌来源酯合成酶JG536_25355是通过大肠杆菌工程菌诱导表达获得。
有益效果：本发明利用生物基因工程技术，克隆并诱导表达获得双向伯克霍尔德氏菌 (Burkholderia ambifaria)BJQ0010来源酯合成酶JG536_25355及其编码基因，并构建含有酯合成酶JG536_25355编码基因的大肠杆菌表达质粒，将该质粒转入大肠杆菌中，经过诱导表达获得该酶，与现有技术相比，本发明的酯合成酶JG536_25355经催化体系验证不仅具有在水相体系能催化合成己酸乙酯、辛酸乙酯的能力，还能催化合成丁酸乙酯、戊酸乙酯和癸酸乙酯，且丁酸乙酯、戊酸乙酯、己酸乙酯、辛酸乙酯和癸酸乙酯产量分别为4.23±0.85、 16.97±2.69、54.82±5.55、112.28±8.94、66.61±6.21mg/L。本发明的酯合成酶JG536_25355，能用于在白酒酿造的水相体系催化合成重要风味酯丁酸乙酯、戊酸乙酯、己酸乙酯、辛酸乙酯和癸酸乙酯。
附图说明
图1酯合成酶JG536_25355编码基因的PCR扩增结果；
图2酯合成酶JG536_25355编码基因诱导表达结果；
图3酯合成酶JG536_25355催化合成丁酸乙酯、戊酸乙酯、己酸乙酯、辛酸乙酯和癸酸乙酯气相色谱原始图谱；
图4酯合成酶JG536_25355催化合成丁酸乙酯、戊酸乙酯、己酸乙酯、辛酸乙酯和癸酸乙酯定量计算结果。
具体实施方式
本发明首先提供了一种双向伯克霍尔德氏菌(Burkholderia ambifaria)BJQ0010来源酯合成酶JG536_25355，该酶具有在贴合白酒发酵的水相体系催化合成丁酸乙酯、戊酸乙酯、己酸乙酯、辛酸乙酯和癸酸乙酯的能力，所述酯合成酶JG536_25355的氨基酸序列如SEQ ID NO.1所示。
所述酯合成酶JG536_25355的氨基酸序列SEQ ID NO.1：
METNVTAAAPSDHPVFVLVHGAWHGAWCYAHVAAALAERGYLSIARDLPAHGINARF PASYLERPLDKDAFGAEPSPVANTTLDDYATQVMEAVDDAYALGHGKVVLVGHSMGGLAITAAAERAPEKIAKIVYLAAFMPASGVPGLDYVRAPENKGEMLAPLMLASPRVAGALRIDPR SGDAAYRALAKRALYDDAAQADFEAMANLMTCDVPAAPFATAIPTTAARWGAIDRHYIKC LADRVILPALQQRFIDEADAFVPGNPTHVHQLDSSHSPFVSQPGVLAGVLVDIAKS。
本发明还提供所述双向伯克霍尔德氏菌来源酯合成酶JG536_25355的编码基因。
其中，所述双向伯克霍尔德氏菌来源酯合成酶JG536_25355的编码基因的核苷酸序列如 SEQ ID NO.2所示。
SEQ ID NO.2：
ATGGAGACGAACGTAACCGCCGCCGCACCATCCGACCATCCCGTTTTCGTGCTGGT GCACGGCGCGTGGCACGGTGCGTGGTGCTATGCGCACGTCGCGGCCGCGCTGGCCGAGCGCGGCTACCTGTCGATCGCGCGCGATCTGCCCGCACACGGCATCAACGCCCGCTTTCC CGCATCGTATCTCGAACGGCCGCTCGACAAGGACGCTTTCGGCGCCGAGCCGTCGCCGGTCGCGAATACCACGCTCGACGATTACGCGACGCAGGTGATGGAGGCCGTCGACGACGC GTACGCGCTCGGCCATGGCAAGGTCGTGCTGGTCGGCCACAGCATGGGCGGCCTCGCGATCACGGCCGCGGCGGAACGCGCGCCGGAGAAGATCGCGAAAATCGTCTATCTCGCCGC GTTCATGCCCGCGTCGGGCGTGCCGGGCCTCGACTACGTGCGCGCGCCCGAGAACAAGGGCGAAATGCTGGCGCCGCTGATGCTCGCGAGCCCGCGCGTTGCGGGCGCGCTGCGGA TCGATCCGCGCAGCGGCGATGCCGCGTATCGCGCGCTGGCCAAGCGCGCGCTGTACGACGACGCGGCGCAGGCCGACTTCGAGGCGATGGCGAACCTGATGACCTGCGACGTGCCGG CCGCGCCGTTCGCGACCGCGATCCCGACGACCGCCGCGCGCTGGGGGGCGATCGACCGTCACTACATCAAGTGCCTGGCGGATCGCGTGATCCTGCCAGCGCTGCAGCAGCGCTTCA TCGACGAAGCCGACGCGTTCGTGCCCGGCAACCCGACGCACGTGCACCAGCTCGACAG CAGCCATTCGCCGTTCGTGTCGCAGCCGGGCGTGCTGGCGGGCGTGCTCGTGGATATCGCGAAAAGCTGA。
本发明还提供上述双向伯克霍尔德氏菌来源酯合成酶JG536_25355在制备酒用酯香液或浓香型白酒中的应用。
进一步地，上述双向伯克霍尔德氏菌来源酯合成酶JG536_25355在水相体系中催化合成丁酸乙酯、戊酸乙酯、己酸乙酯、辛酸乙酯和癸酸乙酯中的应用。
本发明所述酯合成酶JG536_25355能用但不限于大肠杆菌异源表达获取。
其中，在水相体系反应条件下，催化合成丁酸乙酯、戊酸乙酯、己酸乙酯、辛酸乙酯和癸酸乙酯酯合成酶JG536_25355表达工程菌株的构建方法，其主要步骤如下：
A、以双向伯克霍尔德氏菌基因组DNA为模板，利用引物通过PCR技术扩增基因，然后利用整合酶
II把基因连接到但不限于pET-28a(+)表达载体上；
B、通过常规化学转化方法将连接基因的载体转化到大肠杆菌中，经诱导表达获得含有酯合成酶JG536_25355的大肠杆菌工程菌细胞；
C、利用超声波破碎仪破碎细胞，经离心后获得上清液(粗酶液)，水相体系条件下，该粗酶液可以催化底物丁酸、戊酸、己酸、辛酸或癸酸分别和乙醇发生酯化反应产生对应的乙酯。相应酯的浓度经气相色谱定量检测获知。
实验证实：在贴合白酒实际发酵过程的水相体系反应条件下，大肠杆菌工程细胞异源表达的酯合成酶JG536_25355粗酶制剂可以在含有1M乙醇和0.01M丁酸、戊酸、己酸、辛酸或者癸酸的底物分别转化合成丁酸乙酯、戊酸乙酯、己酸乙酯、辛酸乙酯和癸酸乙酯，产量分别为4.23±0.85、16.97±2.69、54.82±5.55、112.28±8.94、66.61±6.21mg/L。
酯合成酶功能差异的关键在于微生物的不同，虽然现有文献中已经有报道不同的伯克霍尔德氏菌，比如伯克霍尔德氏菌BJQ0011，是因为都是在研究过程中从白酒中分离获得的微生物，进行的依次编号，但不同编号的伯克霍尔德氏菌微生物在种水平上具有明显区别，属于不同的微生物。由于微生物不同，所以微生物所携带的功能基因及其所编码的酶在序列上有明显的差异。序列决定酶的结构，结构决定酶的功能，正是由于序列与现有专利相关的序列不同，使酶在催化特性上存在明显的差别。
本发明的创新点在于首次发现双向伯克霍尔德氏菌(Burkholderia ambifaria)BJQ0010 来源酯合成酶JG536_25355，且首次发现了该序列的基因所编码的酶具有在水相体系条件下高效催化合成白酒风味酯，不仅具有在水相体系能催化合成己酸乙酯、辛酸乙酯的能力，还能催化合成丁酸乙酯、戊酸乙酯、和癸酸乙酯的能力，且丁酸乙酯、戊酸乙酯、己酸乙酯、辛酸乙酯和癸酸乙酯产量分别为4.23±0.85、16.97±2.69、54.82±5.55、112.28±8.94、66.61±6.21 mg/L。
下面将结合具体实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
以下实施例中的伯克霍尔德氏菌BJQ0010为白酒源大曲样品中分离获得的一个具有在水相体系催化合成白酒风味酯的微生物菌种资源，经鉴定，命名为双向伯克霍尔德氏菌 (Burkholderia ambifaria)BJQ0010，于2021年12月24日保藏于中国微生物菌种保藏管理委员会普通微生物保藏中心(CGMCC)，地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，邮编：100101，保藏编号为CGMCC 24186。且该伯克霍尔德氏菌BJQ0010 的总DNA现已在NCBI数据库中能检索到。
实施例1酯合成酶JG536_25355编码基因的克隆
1.1伯克霍尔德氏菌的培养和基因组DNA提取
伯克霍尔德氏菌BJQ0010(CGMCC 24186)接种发酵培养基，摇床200±10r/min，30± 1℃培养24-72h。所述发酵培养基，其组成为可溶性淀粉10g/L、蛋白胨10g/L、NH4HSO41g/L、K2HPO4 1g/L、MgSO4 0.8g/L、橄榄油10mL/L，自然pH，121℃灭菌15min。
离心收集培养的双向伯克霍尔德氏菌，通过BIOMIGA细菌DNA提取试剂盒(BIOMIGA【Biomiga上海技术服务中心】，型号GD2411)提取总DNA。具体操作步骤按照试剂盒所属常规操作方法进行。
1.2酯合成酶JG536_25355编码基因的特异性扩增
通过PCR扩增伯克霍尔德氏菌来源的酯合成酶JG536_25355的编码基因。引物设计如下：正向引物(SEQ ID NO.3):5'-TAAGAAGGAGATATACCATGATGGAGACGAACGTAACC-3' 反向引物(SEQ ID NO.4):5'-GGTGGTGCTCGAGTGCGATGCTTTTCGCGATATCCAC-3'
PCR反应体系见下表。
表1实施例1 PCR反应体系扩增JG536_25355的编码基因
表2实施例1PCR扩增循环
通过凝胶电泳检测基因扩增结果，如图1所示。
实施例2构建表达酯合成酶JG536_25355的大肠杆菌工程菌株
2.1pET-28a(+)载体的线性化
利用PCR进行环状pET-28a(+)载体的线性化。引物设计如下：
正向引物(SEQ ID NO.5):5'-ATCGCACTCGAGCACCACC-3'
反向引物(SEQ ID NO.6):5'-CATGGTATATCTCCTTCTTA-3'
PCR反应体系和扩增循环分别见表3和表4。
表3实施例2环状pET-28a(+)的PCR线性化反应体系
| 试剂 | 体积(μL) |
| dd H2O | 72.0 |
| dNTP Mixture(2.5mM each) | 8.0 |
| 10×Ex Taq Buffer | 10.0 |
| 正向引物(10μM) | 4.0 |
| 反向引物(10μM) | 4.0 |
| pET-28a(+)载体 | 1.0 |
| Q5 DNA Polymerase(5U/μl) | 1.0 |
| Total | 100.0 |
表4实施例2PCR扩增循环
2.2质粒构建
通过
II进行质粒构建。反应体系组成见表5。
表5质粒连接反应体系
| 组成成分 | 使用量 |
| 5*CE II Buffer | 4μL |
| 线性化克隆载体 | 50–200ng |
| 基因扩增产物 | 20–200ng |
| Exnase II | 2μL |
| dd H2O | 补足到20μL |
配制完成的反应体系置于37±1℃反应20-50min，之后将反应管置于冰水浴中冷却5-10 min。
2.3质粒的转化
向200μL感受态细胞中加入20μL反应液，混匀后置于冰上20-30min，然后42℃热激60s，之后冰浴2-5min。向反应体系中加入600μL的SOC回复培养基，摇床37±2℃震荡培养45-90min。取回复培养后的转化菌液涂布含有硫酸卡那霉素的SOC培养基平板上，至于培养箱37±1℃过夜培养。所述SOC培养基配方：酵母粉10.0g/L、蛋白胨10.0g/L、NaCl 5.0g/L，固体添加2-4％的琼脂粉，调节pH为7.0，115℃灭菌30min。
实施例3酯合成酶JG536_25355粗酶液水相体系催化合成丁酸乙酯、戊酸乙酯、己酸乙酯、辛酸乙酯和癸酸乙酯
3.1酯合成酶JG536_25355的制备
利用菌落PCR验证培养菌落，选取正确转化子转接SOC液体试管(含有终浓度硫酸卡那霉素40-80mg/L)，摇床150-250r/min，37±2℃震荡培养10-15h，然后以1-5％(v/v) 接种含有SOC培养基的三角瓶，摇床150-250r/min，37±2℃震荡培养至细胞密度0.6-0.8(OD600nm)，之后加入浓度0.01-0.10mM的IPTG作为诱导剂，摇床150-250r/min，25±2℃震荡培养15-24h。8000rpm离心5min收集培养后的菌体，用0.05M Tris-HCl缓冲液(pH 7.5) 洗涤菌体2次并悬浮菌泥。悬浮菌体通过超声波细胞破碎仪破碎细胞，13000rpm离心5-10min，所得上清液即为含有JG536_25355的粗酶液。
3.2JG536_25355粗酶液的酯合成催化
粗酶液经SDS-PAGE电泳确认目标蛋白正确表达(结果如图2所示)，然后配制粗酶液反应体系，进行酯合成性能分析。
所述粗酶液反应体系如下。
粗酶液，2-5mL；丁酸、戊酸、己酸、辛酸和癸酸，终浓度均为10mM，加入乙醇0.92 g，添加pH 4.0-5.0的柠檬酸缓冲液至终体积20mL。反应体系至于摇床，恒温37±1℃，转速150±10rpm反应8-12h。利用正己烷进行反应液的震荡萃取，经滤膜过滤后利用气相色谱定量检测对应的风味乙酯浓度。
所属气相色谱仪定量检测的条件，包括但不限于如下。
仪器：Agilent 7890B，色谱柱型号Agilent 19091N-213I
检测条件：50℃，保持5-10min；以10℃/min的速度升至200℃，保持5-8min；以8℃/min的速度升至250℃，保持5-10min。进样量0.5-5.0μL。载气为氮气，流速为0.5-1.5mL/min，检测器为火焰离子检测器。
实验结果表明，在水相体系条件下，通过大肠杆菌工程菌表达所得酯合成酶JG536_25355 可以催化丁酸、戊酸、己酸、辛酸和癸酸分别与乙醇合成对应乙酯(如图3所示)，产量分别为4.23±0.85、16.97±2.69、54.82±5.55、112.28±8.94、66.61±6.21mg/L。(如图4所示)。
序列表
<110> 宜宾五粮液股份有限公司
<120> 双向伯克霍尔德氏菌来源酯合成酶JG536_25355、编码基因及应用
<130> A220051K
<141> 2022-01-29
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<212> PRT
<213> 双向伯克霍尔德氏菌(Burkholderia ambifariaBJQ0010来源酯合成酶JG536_25355)
<400> 1
Met Glu Thr Asn Val Thr Ala Ala Ala Pro Ser Asp His Pro Val Phe
1 5 10 15
Val Leu Val His Gly Ala Trp His Gly Ala Trp Cys Tyr Ala His Val
20 25 30
Ala Ala Ala Leu Ala Glu Arg Gly Tyr Leu Ser Ile Ala Arg Asp Leu
35 40 45
Pro Ala His Gly Ile Asn Ala Arg Phe Pro Ala Ser Tyr Leu Glu Arg
50 55 60
Pro Leu Asp Lys Asp Ala Phe Gly Ala Glu Pro Ser Pro Val Ala Asn
65 70 75 80
Thr Thr Leu Asp Asp Tyr Ala Thr Gln Val Met Glu Ala Val Asp Asp
85 90 95
Ala Tyr Ala Leu Gly His Gly Lys Val Val Leu Val Gly His Ser Met
100 105 110
Gly Gly Leu Ala Ile Thr Ala Ala Ala Glu Arg Ala Pro Glu Lys Ile
115 120 125
Ala Lys Ile Val Tyr Leu Ala Ala Phe Met Pro Ala Ser Gly Val Pro
130 135 140
Gly Leu Asp Tyr Val Arg Ala Pro Glu Asn Lys Gly Glu Met Leu Ala
145 150 155 160
Pro Leu Met Leu Ala Ser Pro Arg Val Ala Gly Ala Leu Arg Ile Asp
165 170 175
Pro Arg Ser Gly Asp Ala Ala Tyr Arg Ala Leu Ala Lys Arg Ala Leu
180 185 190
Tyr Asp Asp Ala Ala Gln Ala Asp Phe Glu Ala Met Ala Asn Leu Met
195 200 205
Thr Cys Asp Val Pro Ala Ala Pro Phe Ala Thr Ala Ile Pro Thr Thr
210 215 220
Ala Ala Arg Trp Gly Ala Ile Asp Arg His Tyr Ile Lys Cys Leu Ala
225 230 235 240
Asp Arg Val Ile Leu Pro Ala Leu Gln Gln Arg Phe Ile Asp Glu Ala
245 250 255
Asp Ala Phe Val Pro Gly Asn Pro Thr His Val His Gln Leu Asp Ser
260 265 270
Ser His Ser Pro Phe Val Ser Gln Pro Gly Val Leu Ala Gly Val Leu
275 280 285
Val Asp Ile Ala Lys Ser
290
<210> 2
<211> 885
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggagacga acgtaaccgc cgccgcacca tccgaccatc ccgttttcgt gctggtgcac 60
ggcgcgtggc acggtgcgtg gtgctatgcg cacgtcgcgg ccgcgctggc cgagcgcggc 120
tacctgtcga tcgcgcgcga tctgcccgca cacggcatca acgcccgctt tcccgcatcg 180
tatctcgaac ggccgctcga caaggacgct ttcggcgccg agccgtcgcc ggtcgcgaat 240
accacgctcg acgattacgc gacgcaggtg atggaggccg tcgacgacgc gtacgcgctc 300
ggccatggca aggtcgtgct ggtcggccac agcatgggcg gcctcgcgat cacggccgcg 360
gcggaacgcg cgccggagaa gatcgcgaaa atcgtctatc tcgccgcgtt catgcccgcg 420
tcgggcgtgc cgggcctcga ctacgtgcgc gcgcccgaga acaagggcga aatgctggcg 480
ccgctgatgc tcgcgagccc gcgcgttgcg ggcgcgctgc ggatcgatcc gcgcagcggc 540
gatgccgcgt atcgcgcgct ggccaagcgc gcgctgtacg acgacgcggc gcaggccgac 600
ttcgaggcga tggcgaacct gatgacctgc gacgtgccgg ccgcgccgtt cgcgaccgcg 660
atcccgacga ccgccgcgcg ctggggggcg atcgaccgtc actacatcaa gtgcctggcg 720
gatcgcgtga tcctgccagc gctgcagcag cgcttcatcg acgaagccga cgcgttcgtg 780
cccggcaacc cgacgcacgt gcaccagctc gacagcagcc attcgccgtt cgtgtcgcag 840
ccgggcgtgc tggcgggcgt gctcgtggat atcgcgaaaa gctga 885
<210> 3
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
taagaaggag atataccatg atggagacga acgtaacc 38
<210> 4
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ggtggtgctc gagtgcgatg cttttcgcga tatccac 37
<210> 5
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atcgcactcg agcaccacc 19
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
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catggtatat ctccttctta 20
Claims (3)
1.双向伯克霍尔德氏菌（Burkholderia ambifaria）BJQ0010来源酯合成酶JG536_25355在制备酒用酯香液或浓香型白酒中的应用，其特征在于：所述双向伯克霍尔德氏菌BJQ0010来源酯合成酶JG536_25355的氨基酸序列如SEQ ID NO.1所示。
2.双向伯克霍尔德氏菌（Burkholderia ambifaria）BJQ0010来源酯合成酶JG536_25355在水相体系中催化合成丁酸乙酯、戊酸乙酯、己酸乙酯、辛酸乙酯和癸酸乙酯中的应用，其特征在于：所述双向伯克霍尔德氏菌BJQ0010来源酯合成酶JG536_25355的氨基酸序列如SEQ ID NO.1所示。
3.根据权利要求2所述应用，其特征在于：所述双向伯克霍尔德氏菌来源酯合成酶JG536_25355是通过大肠杆菌工程菌诱导表达获得。
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