CN114672522B - 一种双酶催化n-乙酰-d-葡萄糖酸内酯生产2-酮基-3-脱氧-d-葡萄糖酸的方法 - Google Patents
一种双酶催化n-乙酰-d-葡萄糖酸内酯生产2-酮基-3-脱氧-d-葡萄糖酸的方法 Download PDFInfo
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Abstract
本发明涉及一种双酶催化N‑乙酰‑D‑葡萄糖酸内酯生产2‑酮基‑3‑脱氧‑D‑葡萄糖酸的方法。所述方法是以N‑乙酰‑D‑葡萄糖酸内酯为底物，向N‑乙酰‑D‑葡萄糖酸内酯中加入脱乙酰酶OngB和脱氨酶OngC，反应1～24h，得到2‑酮基‑3‑脱氧‑D‑葡萄糖酸。本发明构建了分别含有脱乙酰酶基因ongB和脱氨酶基因ongC的工程菌，再通过这两株工程菌制备得到重组蛋白OngB和OngC，进而双酶催化转化N‑乙酰‑D‑葡萄糖酸内酯得到2‑酮基‑3‑脱氧‑D‑葡萄糖酸，一方面解决了N‑乙酰‑D‑葡萄糖酸内酯难降解利用的问题，另一方面所得的2‑酮基‑3‑脱氧‑D‑葡萄糖酸具有广泛的工业用途，是合成工业上重要的呋喃衍生物、除草剂和食品添加剂等化合物的重要中间代谢产物，存在很高的经济价值。
Description
技术领域
本发明涉及一种双酶催化N-乙酰-D-葡萄糖酸内酯生产2-酮基-3-脱氧-D-葡萄糖酸的方法，属于生物技术领域。
背景技术
为了缓解不断减少的化石能源资源以及日益加剧的能源危机，利用生物质转化生产高附加值的化工品和燃料是未来实现低碳可持续发展经济的关键。几丁质是自然界中仅次于纤维素的第二大生物质资源，其来源广泛，是构成甲壳类动物、昆虫和海绵的外骨骼、鱿鱼软骨以及真菌细胞壁的主要结构成分。开发和利用几丁质这种生物质资源，将对缓解能源危机具有重要意义。几丁质是由N-乙酰-D-葡萄糖胺(N-acetyl-D-glucosamine，GlcNAc)单体通过β-1,4糖苷键连接形成的难溶性直链多糖，是自然界中难降解高分子量有机质的重要组分。因此，实现几丁质的高效降解是保证这种生物质资源得到充分开发和利用的关键。几丁质的利用主要通过化学法和酶法。与化学法相比，酶法具有反应条件温和、副反应少和环境友好等优点，因而具有更广阔的应用前景。
近年来发现的单加氧酶(lytic polysaccharide monooxygenase，LPMO)能够作用于几丁质等结晶多糖的表面，通过氧化断裂结晶态的多糖链，从而使得结晶底物的结构趋于松散，利于之后的糖苷水解酶进一步水解底物。LPMO与糖苷水解酶间的协同作用，极大地提高了难降解生物有机质的降解效率，这使得LPMO成为商业化生物质糖化酶制剂的重要组分，被应用于生物质能源转化等工业应用中。目前所有报道过的有活性的LPMO只能作用于几丁质的C1位原子产生末端为N-乙酰-D-葡萄糖酸内酯(2-(acetylamino)-2-deoxy-D-gluconic acid，GlcNAc1A)的几丁寡糖。在某些水解酶的作用下，氧化性的几丁寡糖最终被水解成N-乙酰-D-葡萄糖胺和N-乙酰-D-葡萄糖酸内酯。包括大肠杆菌在内的绝大多数微生物都能利用N-乙酰-D-葡萄糖胺，将其转换为乙醇或者其他高附加值的化工品。但是，包括大肠杆菌在内的绝大多数微生物都不能利用几丁质的氧化降解产物——N-乙酰-D-葡萄糖酸内酯，进而导致以几丁质为原料经微生物发酵生产乙醇或者其他高附加值的化工品的效率低下。因此，挖掘代谢N-乙酰-D-葡萄糖酸内酯的关键酶，将N-乙酰-D-葡萄糖酸内酯转化为绝大多数微生物能利用的中间代谢产物是非常必要的。
发明内容
针对现有技术的不足，本发明提供了一种双酶催化N-乙酰-D-葡萄糖酸内酯生产2-酮基-3-脱氧-D-葡萄糖酸的方法。利用脱乙酰酶OngB和脱氨酶OngC这两种酶将几丁质及其寡糖的氧化降解产物—N-乙酰-D-葡萄糖酸内酯转换为2-酮基-3-脱氧-D-葡萄糖酸(2-keto-3-deoxygluconate，KDG)，KDG是多种微生物碳代谢过程中的一个重要中间代谢产物，从而实现以几丁质为原料高效生产乙醇或者其他高附加值的化工品。
本发明技术方案如下：
一种双酶催化N-乙酰-D-葡萄糖酸内酯生产2-酮基-3-脱氧-D-葡萄糖酸的方法，以N-乙酰-D-葡萄糖酸内酯为底物，向N-乙酰-D-葡萄糖酸内酯中加入脱乙酰酶OngB和脱氨酶OngC，反应1～24h，得到2-酮基-3-脱氧-D-葡萄糖酸。
根据本发明优选的，所述脱乙酰酶OngB和脱氨酶OngC为依次加入，具体为：先加入脱乙酰酶OngB，在25℃下反应12h，再加入脱氨酶OngC继续反应12h。
根据本发明优选的，所述脱乙酰酶OngB和N-乙酰-D-葡萄糖酸内酯的摩尔浓度比为1:900～1100。
根据本发明优选的，所述脱氨酶OngC和脱乙酰酶OngB摩尔浓度比为1:9～11。
根据本发明优选的，所述脱乙酰酶OngB是通过工程菌E.coli/pET22b-ongB制备得到的重组蛋白；所述脱氨酶OngC是通过工程菌E.coli/pET22b-ongC制备得到的重组蛋白。
根据本发明优选的，所述工程菌为重组的大肠杆菌E.coli BL21(DE3)。
根据本发明优选的，所述脱乙酰酶OngB的核苷酸序列如SED ID NO.1所示，氨基酸序列如SED ID NO.3所示。
根据本发明优选的，所脱氨酶OngC的核苷酸序列如SED ID NO.2所示，氨基酸序列如SED ID NO.4所示。
上述工程菌E.coli/pET22b-ongB和工程菌E.coli/pET22b-ongC的构建方法，具体包括步骤如下：
(1)以海洋假交替单胞菌(Pseudoalteromonas prydzensis)ACAM 620基因组为模板进行PCR扩增，扩增得到脱乙酰酶基因ongB序列，PCR引物序列如下：
ongB_F:5′-AAGAAGGAGATATACATATGATGCAGTACGATATCTCGCAACCAG-3′(含NdeⅠ酶切位点)，
ongB_R:5′-TGGTGGTGGTGGTGCTCGAGATCATGTTTACTTGCTCCTAAGGATGTTAAAA ATTG-3′(含XhoI酶切位点)；
PCR扩增程序：预变性，95℃5min；变性，95℃30sec；退火，55℃30sec；延伸，72℃1min(30个循环)；终止延伸，72℃10min；最后16℃保温；
将载体质粒pET22b用NdeⅠ和XhoI双酶切后，经一步克隆试剂盒连接得到pET22b-ongB质粒；
(2)以海洋假交替单胞菌(Pseudoalteromonas prydzensis)ACAM 620基因组为模板进行PCR扩增，扩增得到脱氨酶基因ongC序列，PCR引物序列如下：
ongC_F:5′-AAGAAGGAGATATACATATGATGGAAAAGTTAGCCACAACAAGTGCT-3′(含NdeⅠ酶切位点)，
ongC_R:5′-TGGTGGTGGTGGTGCTCGAGAAAATACGTATCAAAGATCTCAATAACGTTATAGTCATCATCT-3′(含XhoI酶切位点)；
PCR扩增程序：预变性，95℃5min；变性，95℃30sec；退火，55℃30sec；延伸，72℃1min(30个循环)；终止延伸，72℃10min；最后16℃保温；
将载体质粒pET22b用NdeⅠ和XhoI双酶切后，经一步克隆试剂盒连接得到pET22b-ongC质粒；
(3)将步骤(1)得到的pET22b-ongB质粒或步骤(2)得到的pET22b-ongC质粒转化大肠杆菌感受态细胞BL21(DE3)中，挑选阳性重组子，得工程菌E.coli/pET22b-ongB或E.coli/pET22b-ongC。
上述脱乙酰酶OngB和脱氨酶OngC的制备方法，包括步骤如下：
将上述工程菌分别接种至LB液体培养基中，置于35～40℃、150～200rpm条件下培养至菌液OD600值为0.6～0.8，加入诱导剂IPTG至其终浓度为0.4～0.6mM，置于15～20℃、100～120rpm诱导表达16h，将培养液于4℃条件下7000～10000rpm离心5～10min收集菌体，然后将菌体重悬于缓冲液中，经破碎和纯化后，得到脱乙酰酶OngB和脱氨酶OngC。
有益效果：
1、本发明使用来源于海洋假交替单胞菌的脱乙酰酶基因ongB和脱氨酶基因ongC构建了工程菌E.coli/pET22b-ongB或E.coli/pET22b-ongC，再通过这两株工程菌制备得到重组蛋白OngB和OngC，进而双酶催化转化N-乙酰-D-葡萄糖酸内酯得到2-酮基-3-脱氧-D-葡萄糖酸，一方面解决了N-乙酰-D-葡萄糖酸内酯难降解利用的问题，另一方面所得的2-酮基-3-脱氧-D-葡萄糖酸具有广泛的工业用途，是合成工业上重要的呋喃衍生物、除草剂和食品添加剂等化合物的重要中间代谢产物，存在很高的经济价值。
2、本发明提供的工程菌能高效地催化N-乙酰-D-葡萄糖酸内酯生成KDG、乙酸和NH3，且几乎不降解N-乙酰-D-葡萄糖胺、N-乙酰-D-葡萄糖胺-6-磷酸、N-乙酰-D-谷氨酸和N-乙酰-D-丝氨酸，对N-乙酰-D-葡萄糖酸内酯表现出很高的底物特异性，能将N-乙酰-D-葡萄糖酸内酯转化成能被多种微生物所代谢的KDG，提高了以几丁质为原料生产乙醇或者其他高附加值的化工品的效率，因而在生物质转化方面具有很大的应用潜力。
附图说明
图1为通过PCR扩增克隆的脱乙酰酶OngB和脱氨酶OngC编码基因的琼脂糖凝胶电泳图；
图中：M、核酸分子量标准品(marker)；1、扩增的ongC基因片段；2、扩增的ongB基因片段；
图2为纯化后的重组酶OngB和OngC的SDS-PAGE电泳图；
图中：A、为纯化后的重组酶OngB(以箭头标出)的SDS-PAGE电泳图(M条带为蛋白质分子量标准品(marker))；B、为纯化后的重组酶OngC(以箭头标出)的SDS-PAGE电泳图(M条带为蛋白质分子量标准品(marker))；
图3为脱乙酰酶OngB和脱氨酶OngC降解N-乙酰-D-葡萄糖酸内酯的产物分析；
图中：A、OngB作用于N-乙酰-D-葡萄糖酸内酯的酶解产物的Q-TOF质谱分析；B、OngB和OngC共同作用于N-乙酰-D-葡萄糖酸内酯的酶解产物的Q-TOF质谱分析；
图4为脱乙酰酶OngB和脱氨酶OngC的底物特异性分析；
图中：A、脱乙酰酶OngB的底物特异性分析；B、脱氨酶OngC的底物特异性分析；
图5为包括脱乙酰酶OngB在内的脱-N-乙酰酶的系统发育分析；
图6为包括脱氨酶OngC在内的磷酸吡哆醛依赖酶的系统发育分析。
具体实施方式
下面结合实施例对本发明的技术方案作进一步说明，但本发明所保护范围不限于此。
海洋假交替单胞菌(Pseudoalteromonas prydzensis)ACAM 620的基因组序列(序列号：AQHH00000000)已于2015年提交至NCBI GenBank中。
实施例1：脱乙酰酶OngB和脱氨酶OngC编码基因的获取及其重组表达质粒的构建
将海洋假交替单胞菌脱乙酰酶基因ongB和脱氨酶基因ongC分别克隆入pET22b载体中，构建得到表达载体pET22b-ongB和pET22b-ongC。
具体操作步骤如下：以海洋假交替单胞菌ACAM 620为模板进行PCR扩增，根据海洋假交替单胞菌脱乙酰酶基因ongB序列和脱氨酶基因ongC序列和载体pET22b上多克隆位点的特征，利用生物信息学软件设计扩增引物，分别扩增得到脱乙酰酶基因ongB序列，其核苷酸序列如SEQ ID NO.1，以及脱氨酶基因ongC序列，其核苷酸序列如SEQ ID NO.2，引物序列如下：
ongB_F:5′-AAGAAGGAGATATACATATGATGCAGTACGATATCTCGCAACCAG-3′(含NdeⅠ酶切位点)，
ongB_R:5′-TGGTGGTGGTGGTGCTCGAGATCATGTTTACTTGCTCCTAAGGATGTTAAAA ATTG-3′(含XhoI酶切位点)，
ongC_F:5′-AAGAAGGAGATATACATATGATGGAAAAGTTAGCCACAACAAGTGCT-3′(含NdeⅠ酶切位点)，
ongC_R:5′-TGGTGGTGGTGGTGCTCGAGAAAATACGTATCAAAGATCTCAATAACGTTATAGTCATCATCT-3′(含XhoI酶切位点)。
PCR扩增体系按照试剂盒说明书配制；
PCR扩增程序：预变性，95℃5min；变性，95℃30sec；退火，55℃30sec；延伸，72℃1min(30个循环)；终止延伸，72℃10min；最后16℃保温。
所得的PCR产物经1％的琼脂糖凝胶电泳分析检测，分别得到大小约为1400bp的电泳条带和大小约为1200bp的电泳条带，将目的片段通过胶回收试剂盒回收，将载体质粒pET22b和回收之后的目的片段分别用NdeⅠ和XhoI双酶切后，经连接酶连接分别得pET22b-ongB质粒和pET22b-ongC质粒，将pET22b-ongB质粒和pET22b-ongC质粒分别通过热激法转入大肠杆菌DH5α感受态细胞中，涂布于含100μg/mL氨苄青霉素的LB固体平板上37℃过夜培养，挑取阳性重组子加入终浓度为20％的甘油于-80℃保存；重组菌命名为DH5α/pET22b-ongB和DH5α/pET22b-ongC；采用质粒提取试剂盒从重组菌中提取pET22b-ongB质粒和pET22b-ongC质粒，并送华大基因测序验证，所得序列无误。
实施例2：重组大肠杆菌工程菌的构建
将测序验证正确的重组质粒pET22b-ongB和pET22b-ongC分别转化至菌株E.coliBL21(DE3)中构建工程菌。
具体操作步骤如下：
取实施例1制备的pET22b-ongB质粒和pET22b-ongC质粒，取大肠杆菌为E.coliBL21(DE3)感受态细胞100μL，按《分子克隆实验指南》上的热激转化方法将重组质粒载体转至大肠杆菌BL21(DE3)感受态。将转化的大肠杆菌BL21(DE3)涂布于含100μg/mL氨苄青霉素的LB固体培养基平板上，37℃培养过夜。
挑取单菌落进行菌落PCR鉴定，鉴定结果如图1所示，凝胶电泳图中均出现目的条带且条带单一，E.coli/pET22b-ongB单菌落的目的片段长度约为1.4kb，E.coli/pET22b-ongC单菌落的目的片段长度约为1.2kb，均显示菌落为阳性克隆，即得工程菌E.coli/pET22b-ongB或E.coli/pET22b-ongC。
实施例3：重组蛋白OngB和OngC的异源表达及分离纯化
挑取实施例2中的工程菌E.coli/pET22b-ongB和E.coli/pET22b-ongC的单菌落接种至含有100μg/mL氨苄青霉素的LB液体培养基中，置于37℃、180rpm条件下培养至菌液OD600值为0.6～0.8，加入诱导剂IPTG至其终浓度为0.5mM，置于18℃、110rpm诱导表达16h，然后将培养液于4℃条件下8000rpm离心收集菌体，重悬于缓冲液Binding Buffer(50mMTris-HCl，100mM NaCl，pH 8.0)中，经过压力破碎得到粗酶液，经镍离子亲和层析柱纯化及PD10脱盐柱脱盐后，经SDS-PAGE电泳检测后，得到纯度较高的重组酶蛋白OngB和OngC，结果如图2所示。
由图2可知，目的条带单一，其中工程菌E.coli/pET22b-ongB表达的脱乙酰酶OngB大小约为53kDa，工程菌E.coli/pET22b-ongC表达的脱氨酶OngC的蛋白大小约为45kDa。
实施例4：双酶催化N-乙酰-D-葡萄糖酸内酯生产2-酮基-3-脱氧-D-葡萄糖酸
一种双酶催化N-乙酰-D-葡萄糖酸内酯生产2-酮基-3-脱氧-D-葡萄糖酸的方法，包括步骤如下：
(1)将10μΜ重组蛋白OngB、10mM Bis-Tris-HCl缓冲液(pH 7.5)和10mM的底物N-乙酰-D-葡萄糖酸内酯(GlcNAc1A)混合均匀，置于25℃条件下反应12h后，13,000rpm离心10min，得到产物1；
(2)将1μΜ重组蛋白OngC、10mM Bis-Tris-HCl缓冲液(pH 7.5)和10mM的步骤(1)所得产物1混合均匀，置于25℃反应12h后，13,000rpm离心10min，得到产物2。
将所得的产物1和产物2分别通过高分辨率的Q-TOF质谱进行产物分析，结果如图3所示。酶解产物在负离子的模式下进行检测，质谱参数设置为氮气流速为4L/min，温度180℃，雾化器压力6psi，毛细管4500V，扫描范围50～1500m/z。
由图3A可知，产物1为脱乙酰化的N-乙酰-D-葡萄糖酸内酯(2-(amino)-2-deoxy-D-gluconic acid，GlcN1A)，即重组蛋白OngB将N-乙酰-D-葡萄糖酸内酯(GlcNAc1A)脱乙酰生成脱乙酰化的N-乙酰-D-葡萄糖酸内酯(GlcN1A)。
由图3B可知，产物2为2-酮基-3-脱氧-D-葡萄糖酸(KDG)，即在重组蛋白OngB和OngC的双酶作用下，N-乙酰-D-葡萄糖酸内酯(GlcNAc1A)被转换成2-酮基-3-脱氧-D-葡萄糖酸(KDG)。
因此，本发明使用来源于海洋假交替单胞菌的脱乙酰酶基因ongB和脱氨酶基因ongC构建了工程菌E.coli/pET22b-ongB或E.coli/pET22b-ongC，再通过这两株工程菌制备得到重组蛋白脱乙酰酶OngB和脱氨酶OngC，进而双酶催化转化N-乙酰-D-葡萄糖酸内酯得到2-酮基-3-脱氧-D-葡萄糖酸。
实施例5：脱乙酰酶OngB和脱氨酶OngC的底物特异性分析
(1)重组蛋白OngB的底物特异性分析
检测重组蛋白OngB对包括N-乙酰-D-葡萄糖酸内酯(GlcNAc1A)、N-乙酰葡萄糖胺(GlcNAc)、N-乙酰葡萄糖胺-6-磷酸(GlcNAc-6-P)、N-乙酰-D-谷氨酸(Acetyl-D-glutamate)和N-乙酰-D-丝氨酸(Acetyl-D-serine)在内的不同底物的酶活性。
标准反应为：5μΜ重组蛋白OngB、25mM底物和10mM Bis-Tris-HCl(pH 7.5)混合后置于25℃反应30min后利用乙酸检测试剂盒(Megazyme，Ireland)检测反应体系中乙酸产物的生成量。结果如图4A所示。
酶活力定义为，在一定温度下，每分钟催化底物生成1μmol乙酸所需的酶量为一个酶活力单位(U)。
由图4A可知，重组蛋白OngB对N-乙酰-D-葡萄糖酸内酯表现出很高的底物特异性，且OngB几乎不降解N-乙酰-D-葡萄糖胺、N-乙酰-D-葡萄糖胺-6-磷酸、N-乙酰-D-谷氨酸和N-乙酰-D-丝氨酸。
(2)重组蛋白OngC的底物特异性分析
检测重组蛋白OngC对包括脱乙酰化的GlcNAc1A(GlcN1A)、D-葡萄糖胺(D-glucosamine)、D-半乳糖胺(D-galactosamine)和D-甘露糖胺(D-mannosamine)在内的不同底物的酶活性。GlcN1A是由10μΜOngB、10mM GlcNAc1A和10mM Bis-Tris-HCl(pH7.5)混合后置于25℃孵育4h制备而成。
标准反应为：0.5μΜOngC、10mM底物和10mM Bis-Tris-HCl(pH 7.5)混合后置于25℃反应30min后利用氨检测试剂盒(Megazyme，Ireland)检测反应体系中氨产物的生成量。酶活力定义为，在一定温度下，每分钟催化底物生成1μmol氨所需的酶量为一个酶活力单位(U)。
由图4B可知，重组蛋白OngC对GlcN1A表现出很高的底物特异性，且重组蛋白OngC几乎不降解D-葡萄糖胺、D-半乳糖胺和D-甘露糖胺。
实施例6：脱乙酰酶OngB和脱氨酶OngC的序列分析
(1)脱乙酰酶OngB的序列分析
在已表征的酶蛋白中，OngB与来自Alcaligenes faecalis的N-乙酰-D-氨基酸脱乙酰酶(序列号：1RJP)最相似，序列一致性为46％。为了确定OngB的进化位置，发明人从NCBI nr库中下载已报道N-乙酰-D-氨基酸脱乙酰酶(包括N-乙酰-D-谷氨酸脱乙酰酶(Acetyl-D-Glu DA)和N-乙酰-瓜氨酸脱乙酰酶(Acetylcitrulline DA))的代表序列、糖类-脱-N-乙酰酶(包括N-乙酰葡萄糖胺脱乙酰酶(GlcNAc DA)、N-乙酰葡萄糖胺-6-磷酸脱乙酰酶(GlcNAc-6-PDA)、几丁质脱乙酰酶(Chitin DA)、作用于肽聚糖中N-乙酰葡萄糖胺的脱乙酰酶(PGN GlcNAc DA)、几丁寡糖脱乙酰酶(Chitooligosaccharide DA)、乙酰半乳糖胺半乳聚糖脱乙酰酶(Galactoaminogalactan DA)和UDP-N-乙酰葡萄糖胺脱乙酰酶(UDP-GlcNAc DA))的代表序列以及OngB的同源序列。通过MUSCLE软件对获得的同源序列进行多重比对分析。选择WAG模型，用MEGA7.0构建了脱-N-乙酰酶的进化树。
如图5所示，在进化树上，OngB及其同源蛋白形成一个独立的分支，这表明OngB所在的分支可能代表了一个脱-N-乙酰酶新家族，而OngB是该新家族中第一个被研究的蛋白。与糖类-脱-N-乙酰酶相比，OngB及其同源蛋白与N-乙酰-D-谷氨酸脱乙酰酶(Acetyl-D-GluDA)的亲缘关系更近。但是，底物特异性分析表明(如图4A)，OngB几乎不降解N-乙酰-D-谷氨酸，却对酸性单糖——N-乙酰-D-葡萄糖酸内酯表现出很高的底物特异性。
(2)脱氨酶OngC的序列分析
在已表征的酶蛋白中，OngC与来自Arthrobacter sp.strain DK-38的D-苏氨酸醛缩酶(序列号：O82872.1)最相似，序列一致性为24％。为了确定OngC的进化位置，发明人从NCBI nr库中下载已报道D-氨基酸脱氨酶(包括D-丝氨酸脱水酶家族(D-Serinedehydratase)和D-丝氨酸脱水酶DSD1家族(D-Serine dehydratase DSD1))的代表序列、D-苏氨酸醛缩酶(D-Threonine aldolase)的代表序列以及OngC的同源序列。通过MUSCLE软件对获得的同源序列进行多重比对分析。选择JTT模型，用MEGA7.0构建了进化树。
如图6所示，OngC及其同源蛋白、D-丝氨酸脱水酶DSD1家族(D-Serinedehydratase DSD1)以及D-苏氨酸醛缩酶家族(D-Threonine aldolase)均属于折叠类型III磷酸吡哆醛依赖酶超家族(Fold-type III PLP-dependent enzymes)。但是，在进化树上，OngC及其同源蛋白形成一个独立的分支，这表明OngC所在的分支可能代表了折叠类型III磷酸吡哆醛依赖酶超家族中的一个新家族，而OngC是该新家族中第一个被研究的蛋白。底物特异性分析表明(如图4B)，OngC对酸性单糖——脱乙酰化的N-乙酰-D-葡萄糖酸内酯表现出很高的底物特异性。
综上所述，在脱乙酰酶OngB和脱氨酶OngC这两种酶的共同作用下，将几丁质及其寡糖的氧化降解产物——N-乙酰-D-葡萄糖酸内酯转化成能被多种微生物所代谢的KDG，不仅提供了一种制备KDG的新方法，还提高了以几丁质为原料生产乙醇或者其他高附加值的化工品的效率，因而在制备KDG及其衍生物以及生物质转化方面具有很大的应用潜力。
SEQUENCE LISTING
<110> 山东大学
<120> 一种双酶催化N-乙酰-D-葡萄糖酸内酯生产2-酮基-3-脱氧-D-葡萄糖酸的方
法
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1470
<212> DNA
<213> 海洋假交替单胞菌（Pseudoalteromonas prydzensis）
<400> 1
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gagcaagcat tcgatattaa ggtgattatt tcgcacctta aatgcgcagg taaaaataac 780
tgggggcggg ctcctgaatt attggctaaa ttttctgagc aaggtgagca ttcaaaatgc 840
agttgtgacg cttacccgta tgccgcaagt tccagcacct tagatttaaa ccaagtgacc 900
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cccgcgggag cggtttatca tggccttaat gaagacgatg taaaaaccat tctcgcgttt 1080
gataaaacca tgattggctc cgacggctta ccgtgcgatc cccatccgca tccgcgttta 1140
tggggctcat ttcctcgggt tttaggtcat tatagccgtg agcaaggcat tttttccttg 1200
gctacagcaa ttcataaaat gaccggttta agcgcagcaa attaccggtt agcaaaccgt 1260
ggggtgatca aggtgggtca ttttgctgat ttagtattat ttgatgccga tgaaattatt 1320
gataacgcga cctttgttga atctgcctta ccggcatcgg gtattcatca ggtttggact 1380
aatggccaaa ccacatttaa agataaacga gttttacccg cgtattcggg tcaattttta 1440
acatccttag gagcaagtaa acatgattag 1470
<210> 2
<211> 1248
<212> DNA
<213> 海洋假交替单胞菌（Pseudoalteromonas prydzensis）
<400> 2
atggaaaagt tagccacaac aagtgctgaa aatatcgcaa gtgtgaataa aggcttaggt 60
aatgatgagg cactcataca tgaggattgc aatgttgctt tgcagcaagt cagtttgcct 120
tgtgcatgta tttaccaaac tcggttagat aataacattg cgtggatggc aaagtttgcg 180
cagcaaagta aggtggagtt ttcaccccat ggtaaaacca ctatggcacc gggtattttt 240
aaaaagcagt taaatgcagg tgcttacgcg atcactattg caacggtaca acaggctgtg 300
gtggctgctc gcgctggtgc aaagcgtatt attatggcga atcaattagt tggcaaagcc 360
aacatgcagc agcttagcta tttattaaaa cagtataaga ttgattttta ttgtttagtg 420
gatgatgtta gtaatgtaac aaggcttggt gagtttttta gcgaacaagg tttacagctt 480
aaattactga ttgagctagg agtgcctggc ggacgttgtg gtgttgttga taatcaaact 540
cttcagcaat tggctgcgca tattcaacag tttgcttcat tacagcttgc gggtattgaa 600
gtgtatgaag gcgtattgag tacacaaagt gaagtgactg catttttagc agatgcggta 660
gcaaaatgta aagttttgat tgaaaagcag gcttttgcta cagagcaagt gatcattacc 720
gctggtggct ctgcttggta tgacttagtg tgtgatgcat ttgcaccgca caacttgtta 780
gacaacatga tacctgtgat ccgccctgga tgttacgttg cccatgatca aggaatatat 840
gagcacgcac agcagcaagt attagcgcgc aatccgctgg catgcaatat tggtagtgac 900
ttacagtcgt gcctagagat ttgggcgtat gtgcaatcat tgccagagcc tggtcgtgcc 960
attattggta tgggtaaacg agatgtcgcc ttcgatgccg gtttacccat tgccaattta 1020
catgtggatc aatcaggcac cgtaaaacct gcgggggccg attggaaagt agaaaaaata 1080
atggatcagc acgcgatgct gacttatacc ggcacagaac aactaaaagt gggcgatatt 1140
atagcatttg caacttcgca cccatgcctt acattcgata aatggcgttt tattaacgtc 1200
atagatgatg actataacgt tattgagatc tttgatacgt atttttag 1248
<210> 3
<211> 489
<212> PRT
<213> 海洋假交替单胞菌（Pseudoalteromonas prydzensis）
<400> 3
Met Gln Tyr Asp Ile Ser Gln Pro Val Asp Thr Ile Ile Tyr Gln Ala
1 5 10 15
Lys Val Phe Asp Gly Leu Gly Asn Ala Pro Val His Met Asp Val Ala
20 25 30
Ile Lys Gly Gln Gln Ile Val Ala Leu Gly Glu Leu Ser Ala Tyr Gln
35 40 45
Ala Thr Glu Glu Val Asn Ala Asp Gly Leu Cys Leu Ala Pro Gly Phe
50 55 60
Ile Asp Val His Thr His Asp Asp Leu Glu Val Leu Arg Asn Pro Glu
65 70 75 80
Met Ala Ala Lys Ile Ser Gln Gly Val Thr Thr Val Ile Thr Gly Asn
85 90 95
Cys Gly Ile Ser Ala Ala Pro Ala Glu Leu Ala Asn Asp Ala Pro Asp
100 105 110
Pro Met Asn Leu Leu Gly Glu Lys Ala Glu Phe Lys Phe Ala Gln Leu
115 120 125
Arg Asp Tyr Ile Asp Ala Tyr Lys Val Gln Lys Ala Asn Val Asn Val
130 135 140
Ala Ala Leu Val Gly His Thr Thr Leu Arg Asn Asn Val Met Ala Asp
145 150 155 160
Leu Leu Arg Pro Ala Thr Ala Ala Glu Ile Thr Leu Met Gln Gln Gln
165 170 175
Leu Asp Leu Ala Leu Ser Gln Gly Ala Leu Gly Leu Ser Thr Gly Leu
180 185 190
Ala Tyr Lys Asn Ala Asn Gln Ala Pro Ser Ser Glu Val His Ala Phe
195 200 205
Gly Glu Val Leu Lys Lys His Asp Ala Leu Tyr Thr Thr His Leu Arg
210 215 220
Thr Glu Phe Asp Ala Val Leu Asp Ala Met Asp Glu Ala Phe Ala Met
225 230 235 240
Glu Gln Ala Phe Asp Ile Lys Val Ile Ile Ser His Leu Lys Cys Ala
245 250 255
Gly Lys Asn Asn Trp Gly Arg Ala Pro Glu Leu Leu Ala Lys Phe Ser
260 265 270
Glu Gln Gly Glu His Ser Lys Cys Ser Cys Asp Ala Tyr Pro Tyr Ala
275 280 285
Ala Ser Ser Ser Thr Leu Asp Leu Asn Gln Val Thr Asp Asp Phe Asp
290 295 300
Ile Phe Ile Thr Trp Ser Asp Ser His Pro Glu Met Ala Glu Gln Leu
305 310 315 320
Leu Ala Asp Ile Ala Lys Gln Trp Gly Ile Ser Leu Leu Asp Ala Ala
325 330 335
Lys Gln Leu Gln Pro Ala Gly Ala Val Tyr His Gly Leu Asn Glu Asp
340 345 350
Asp Val Lys Thr Ile Leu Ala Phe Asp Lys Thr Met Ile Gly Ser Asp
355 360 365
Gly Leu Pro Cys Asp Pro His Pro His Pro Arg Leu Trp Gly Ser Phe
370 375 380
Pro Arg Val Leu Gly His Tyr Ser Arg Glu Gln Gly Ile Phe Ser Leu
385 390 395 400
Ala Thr Ala Ile His Lys Met Thr Gly Leu Ser Ala Ala Asn Tyr Arg
405 410 415
Leu Ala Asn Arg Gly Val Ile Lys Val Gly His Phe Ala Asp Leu Val
420 425 430
Leu Phe Asp Ala Asp Glu Ile Ile Asp Asn Ala Thr Phe Val Glu Ser
435 440 445
Ala Leu Pro Ala Ser Gly Ile His Gln Val Trp Thr Asn Gly Gln Thr
450 455 460
Thr Phe Lys Asp Lys Arg Val Leu Pro Ala Tyr Ser Gly Gln Phe Leu
465 470 475 480
Thr Ser Leu Gly Ala Ser Lys His Asp
485
<210> 4
<211> 415
<212> PRT
<213> 海洋假交替单胞菌（Pseudoalteromonas prydzensis）
<400> 4
Met Glu Lys Leu Ala Thr Thr Ser Ala Glu Asn Ile Ala Ser Val Asn
1 5 10 15
Lys Gly Leu Gly Asn Asp Glu Ala Leu Ile His Glu Asp Cys Asn Val
20 25 30
Ala Leu Gln Gln Val Ser Leu Pro Cys Ala Cys Ile Tyr Gln Thr Arg
35 40 45
Leu Asp Asn Asn Ile Ala Trp Met Ala Lys Phe Ala Gln Gln Ser Lys
50 55 60
Val Glu Phe Ser Pro His Gly Lys Thr Thr Met Ala Pro Gly Ile Phe
65 70 75 80
Lys Lys Gln Leu Asn Ala Gly Ala Tyr Ala Ile Thr Ile Ala Thr Val
85 90 95
Gln Gln Ala Val Val Ala Ala Arg Ala Gly Ala Lys Arg Ile Ile Met
100 105 110
Ala Asn Gln Leu Val Gly Lys Ala Asn Met Gln Gln Leu Ser Tyr Leu
115 120 125
Leu Lys Gln Tyr Lys Ile Asp Phe Tyr Cys Leu Val Asp Asp Val Ser
130 135 140
Asn Val Thr Arg Leu Gly Glu Phe Phe Ser Glu Gln Gly Leu Gln Leu
145 150 155 160
Lys Leu Leu Ile Glu Leu Gly Val Pro Gly Gly Arg Cys Gly Val Val
165 170 175
Asp Asn Gln Thr Leu Gln Gln Leu Ala Ala His Ile Gln Gln Phe Ala
180 185 190
Ser Leu Gln Leu Ala Gly Ile Glu Val Tyr Glu Gly Val Leu Ser Thr
195 200 205
Gln Ser Glu Val Thr Ala Phe Leu Ala Asp Ala Val Ala Lys Cys Lys
210 215 220
Val Leu Ile Glu Lys Gln Ala Phe Ala Thr Glu Gln Val Ile Ile Thr
225 230 235 240
Ala Gly Gly Ser Ala Trp Tyr Asp Leu Val Cys Asp Ala Phe Ala Pro
245 250 255
His Asn Leu Leu Asp Asn Met Ile Pro Val Ile Arg Pro Gly Cys Tyr
260 265 270
Val Ala His Asp Gln Gly Ile Tyr Glu His Ala Gln Gln Gln Val Leu
275 280 285
Ala Arg Asn Pro Leu Ala Cys Asn Ile Gly Ser Asp Leu Gln Ser Cys
290 295 300
Leu Glu Ile Trp Ala Tyr Val Gln Ser Leu Pro Glu Pro Gly Arg Ala
305 310 315 320
Ile Ile Gly Met Gly Lys Arg Asp Val Ala Phe Asp Ala Gly Leu Pro
325 330 335
Ile Ala Asn Leu His Val Asp Gln Ser Gly Thr Val Lys Pro Ala Gly
340 345 350
Ala Asp Trp Lys Val Glu Lys Ile Met Asp Gln His Ala Met Leu Thr
355 360 365
Tyr Thr Gly Thr Glu Gln Leu Lys Val Gly Asp Ile Ile Ala Phe Ala
370 375 380
Thr Ser His Pro Cys Leu Thr Phe Asp Lys Trp Arg Phe Ile Asn Val
385 390 395 400
Ile Asp Asp Asp Tyr Asn Val Ile Glu Ile Phe Asp Thr Tyr Phe
405 410 415
Claims (7)
1.一种双酶催化N-乙酰-D-葡萄糖酸内酯生产2-酮基-3-脱氧-D-葡萄糖酸的方法，其特征在于，以N-乙酰-D-葡萄糖酸内酯为底物，向N-乙酰-D-葡萄糖酸内酯中加入脱乙酰酶OngB和脱氨酶OngC，反应1~24 h，得到2-酮基-3-脱氧-D-葡萄糖酸；
所述脱乙酰酶 OngB的氨基酸序列如 SED ID NO.3所示，其编码核酸的核苷酸序列如SED ID NO.1所示；
所述脱乙酰酶 OngC的氨基酸序列如 SED ID NO.4所示，其编码核酸的核苷酸序列如SED ID NO.2所示。
2.如权利要求1所述的双酶催化N-乙酰-D-葡萄糖酸内酯生产2-酮基-3-脱氧-D-葡萄糖酸的方法，其特征在于，所述脱乙酰酶OngB和脱氨酶OngC为依次加入，具体为：先加入脱乙酰酶OngB，在25℃下反应12 h，再加入脱氨酶OngC继续反应12 h。
3.如权利要求1所述的双酶催化N-乙酰-D-葡萄糖酸内酯生产2-酮基-3-脱氧-D-葡萄糖酸的方法，其特征在于，所述脱乙酰酶OngB和N-乙酰-D-葡萄糖酸内酯的摩尔浓度比为1:900~1100。
4.如权利要求1所述的双酶催化N-乙酰-D-葡萄糖酸内酯生产2-酮基-3-脱氧-D-葡萄糖酸的方法，其特征在于，所述脱氨酶OngC和脱乙酰酶OngB摩尔浓度比为1:9~11。
5.如权利要求1所述的双酶催化N-乙酰-D-葡萄糖酸内酯生产2-酮基-3-脱氧-D-葡萄糖酸的方法，其特征在于，所述脱乙酰酶OngB是通过工程菌E. coli/pET22b-ongB制备得到的重组蛋白；所述脱氨酶OngC是通过工程菌E. coli/pET22b-ongC制备得到的重组蛋白。
6.权利要求5所述的工程菌E. coli/pET22b-ongB和工程菌E. coli/pET22b-ongC的构建方法，其特征在于，具体包括步骤如下：
（1）以海洋假交替单胞菌（Pseudoalteromonas prydzensis）ACAM 620基因组为模板进行PCR扩增，扩增得到脱乙酰酶基因ongB序列，PCR引物序列如下：
ongB_F:5′-AAGAAGGAGATATACATATGATGCAGTACGATATCTCGCAACCAG-3′，
ongB_R:5′-TGGTGGTGGTGGTGCTCGAGATCATGTTTACTTGCTCCTAAGGATGTTAAAA ATTG-3′；
PCR扩增程序：预变性，95℃ 5 min；变性，95℃ 30 sec；退火，55℃ 30 sec；延伸，72℃1 min，30个循环；终止延伸，72℃ 10 min；最后16℃保温；
将载体质粒pET22b用NdeⅠ和XhoI双酶切后，经一步克隆试剂盒连接得到pET22b-ongB质粒；
（2）以海洋假交替单胞菌（Pseudoalteromonas prydzensis）ACAM 620基因组为模板进行PCR扩增，扩增得到脱氨酶基因ongC序列，PCR引物序列如下：
ongC_F:5′-AAGAAGGAGATATACATATGATGGAAAAGTTAGCCACAACAAGTGCT-3′，
ongC_R:5′-TGGTGGTGGTGGTGCTCGAGAAAATACGTATCAAAGATCTCAATAACGTTATAGTCATCATCT-3′；
PCR扩增程序：预变性，95℃ 5 min；变性，95℃ 30 sec；退火，55℃ 30 sec；延伸，72℃1 min，30个循环；终止延伸，72℃ 10 min；最后16℃保温；
将载体质粒pET22b用NdeⅠ和XhoI双酶切后，经一步克隆试剂盒连接得到pET22b-ongC质粒；
（3）将步骤（1）得到的pET22b-ongB质粒或步骤（2）得到的pET22b-ongC质粒转化大肠杆菌感受态细胞BL21(DE3)中，挑选阳性重组子，得工程菌E. coli/pET22b-ongB或E. coli/pET22b-ongC。
7.权利要求1所述的脱乙酰酶OngB和脱氨酶OngC的制备方法，其特征在于，包括步骤如下：
将权利要求6所述的工程菌分别接种至LB液体培养基中，置于35~40℃、150~200 rpm条件下培养至菌液OD600值为0.6~0.8，加入诱导剂IPTG至其终浓度为0.4~0.6 mM，置于15~20℃、100~120 rpm诱导表达16 h，将培养液于4℃条件下7000~10000 rpm离心5~10 min收集菌体，然后将菌体重悬于缓冲液中，经破碎和纯化后，得到脱乙酰酶OngB和脱氨酶OngC。
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| CN104789546A (zh) * | 2015-02-04 | 2015-07-22 | 安徽正方生物科技有限公司 | 一种脱乙酰基酶突变体及其应用 |
| CN108070628A (zh) * | 2016-11-14 | 2018-05-25 | 中国科学院微生物研究所 | 一种生产氨基葡萄糖的方法 |
| CN112154206A (zh) * | 2018-05-17 | 2020-12-29 | Bp北美公司 | 丝状真菌中2-酮基-3-脱氧-d-葡萄糖酸的产生 |
| CN113122490A (zh) * | 2021-03-25 | 2021-07-16 | 淮阴工学院 | 双基因缺陷型工程菌及其在提高n-乙酰氨基葡萄糖产量的应用 |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104789546A (zh) * | 2015-02-04 | 2015-07-22 | 安徽正方生物科技有限公司 | 一种脱乙酰基酶突变体及其应用 |
| CN108070628A (zh) * | 2016-11-14 | 2018-05-25 | 中国科学院微生物研究所 | 一种生产氨基葡萄糖的方法 |
| CN112154206A (zh) * | 2018-05-17 | 2020-12-29 | Bp北美公司 | 丝状真菌中2-酮基-3-脱氧-d-葡萄糖酸的产生 |
| CN113122490A (zh) * | 2021-03-25 | 2021-07-16 | 淮阴工学院 | 双基因缺陷型工程菌及其在提高n-乙酰氨基葡萄糖产量的应用 |
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