CN105886487A - 一种几丁质脱乙酰基酶突变体 - Google Patents
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Abstract
本发明公开了一种几丁质脱乙酰基酶的突变体，属于生物工程技术领域。本发明将专性海洋放线菌Salinispora arenicola几丁质脱乙酰基酶第52位缬氨酸V突变为甘氨酸G，第66位的脯氨酸P点突变为丝氨酸S。相比突变前，突变体酶的比活力是野生型酶的3.92倍。本发明提供的几丁质脱乙酰基酶更加适合工业化生产需求，满足社会生产的要求。
Description
技术领域
本发明公开了一种几丁质脱乙酰基酶的突变体，属于生物工程技术领域。
背景技术
几丁质(chitin)又称甲壳素、甲壳质等，是由N-乙酰-D葡萄糖胺(GlcNAc)单体通过β-1,4糖苷键链接而成的直链高分子化合物，是自然界中次于纤维素的天然高分子。几丁质具有几乎不溶于水和一般有机溶剂，限制了几丁质的应用(。几丁质脱乙酰后形成壳聚糖，化学名称为聚葡萄糖胺(1-4)-2-氨基-B-D葡萄糖。壳聚糖溶于酸性及中性水溶液，具有良好的生物相容性和生物可降解性，广泛应用于医药、食品、化工、化妆品、水处理、金属提取及回收、生化和生物医学工程等诸多领域(Pradip Kumar Dutta.Chitin andChitosan for Regenerative Medicine[M].Springer India,2016)。
目前，壳聚糖的工业生产是以几丁质为原料采用浓碱热化学法生产，此法不仅污染严重，而且程度不易控制，导致出现各种分子量范围的产品。采用酶法脱乙酰反应温和、环境友好，可以制备出高质量的壳聚糖。几丁质脱乙酰基酶(E.C.3.2.1.41)能将几丁质的乙酰基直接脱除生成壳聚糖，但是对几丁质分子的主链不发生降解，与特定酶结合使用还可以制备具有特定乙酰基位置的产物(Yong Zhao,Ro-Dong Park andRiccardo A.A.Muzzarelli.Chitin deacetylases:properties and applications[J].Mar.Drugs 2010,8:24-46)。
近十几年来，随着分子生物学与工业微生物研究的快速发展，研究者开始采用基因工程手段构建重组菌，以期实现几丁质脱乙酰基酶高效生产。目前，已相继出现了利用Escherichia coli、Bacillus subtilis和Pichia pastoris等作为宿主表达不同来源几丁质脱乙酰基酶基因的报道。尽管表达量较高，但是酶的催化效率等酶学特性还有待进一步改进。我国对几丁质脱乙酰基酶的研究主要集中在菌种选育、酶的纯化、表达和酶学性质、发酵工艺的改良方面，尚缺催化效率较高的几丁质脱乙酰基酶。
发明内容
发明要解决的技术问题：
本发明要解决的第一个技术问题是提供一种催化活性提高的几丁质脱乙酰基酶突变体。
所述突变体是以氨基酸序列如SEQ ID NO.2所示的几丁质脱乙酰基酶(野生型)为基础，将第52位缬氨酸V突变为甘氨酸G，第66位的脯氨酸P点突变为丝氨酸S。
本发明要解决的第二个技术问题是提供构确定突变氨基酸的方法，是通过模拟Salinispora arenicolaCAD三级结构，发现第52位缬氨酸和第66位的脯氨酸都位于链接催化活性中心的无规卷曲结构中，突变后有望改变催化结构域，并改变底物结合能力。
本发明要解决的第三个技术问题是提供构建所述几丁质脱乙酰基酶突变体的方法，是通过设计引物对编码几丁质脱乙酰基酶的基因进行定点突变后表达。
所述构建方法，以含有几丁质脱乙酰基酶质粒pEtac-His6-cad为模板，设计引物进行突变并将突变后的质粒转化进入E.coli DH5α进行扩增，然后以E.coli BL21(DE3)表达含突变基因的重组质粒，得到活性提高的突变体。
所述用于定点突变的引物是：
cadV52G primer1ATGGTGGCCACACCTGGCGGGTGTGG
cadV52G primer2CCACACCCGCCAGGTGTGGCCACCAT
cadP66S primer1ATCGCGTTGACCTTCGACGACGGACCCAA
cadP66S primer2TTGGGTCCGTCGTCGAAGGTCAACGCGAT
本发明要解决的第四个技术问题是提供应用基因工程菌发酵生产所述几丁质脱乙酰基酶突变体的方法，是将含有编码突变几丁质脱乙酰基酶的基因的重组菌E.coli BL21(DE3)(pEtac-His6-cad)活化培养后接种到含有100μg mL-1氨苄青霉素的发酵培养基中，装液量为30mL/250mL，于37℃，200r min-1培养；菌体生长到OD600＝0.6，加入终浓度0.5mM IPTG，30℃，诱导发酵24h。所用培养基组成：胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，pH7.0。
本发明提供的一种催化活性提高的几丁质脱乙酰基酶突变体，是将氨基酸序列如SEQ ID NO.2所示的几丁质脱乙酰基酶的第52位缬氨酸V突变为甘氨酸G，第66位的脯氨酸P点突变为丝氨酸S。
上述突变体的基因的编码也属于本发明的保护范围之内。
携带上述突变体的基因的载体或细胞也属于本发明的保护范围之内。
根据本发明的另一方面，提供了一种获得上述几丁质脱乙酰基酶突变体的方法，是通过设计引物对编码几丁质脱乙酰基酶的基因进行定点突变后表达。是以质粒pEtac-His6-cad为模板设计引物进行突变并将突变后的质粒转化进入E.coli DH5α进行扩增，然后以E.coli BL21(DE3)表达含突变基因的重组质粒，得到活性性增强的几丁质脱乙酰基酶突变体。
根据本发明的另一方面，提供了应用基因工程菌发酵生产上述几丁质脱乙酰基酶突变体的方法，是将含有编码突变几丁质脱乙酰基酶的基因的重组菌E.coli BL21(DE3)(pEtac-His6-cadm)活化培养后接种到含有100μg mL-1氨苄青霉素的发酵培养基中，所用培养基组成为胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，pH7.0。装液量为30mL/250mL，于37℃，200r min-1培养；菌体生长到OD600＝0.6，加入终浓度0.5mM IPTG，30℃，诱导发酵24h。取发酵液，8000r/min离心10min收集菌体，再适量蒸馏水悬浮细胞，于冰水中超声破碎4min，12000r/min离心15min去除细胞碎片，上清液即为粗酶液。
本发明的有益效果为：本发明通过定点突变改造几丁质脱乙酰基酶基因，使所编码的CAD催化活性增加；突变酶酶的比活力是野生型酶的5.33倍。酶的其它催化特性基本不变，本发明提供的几丁质脱乙酰基酶更加适合工业化生产需求，满足社会生产的要求。
具体实施方式
重组几丁质脱乙酰基酶纯化：按照Ni-NTA Purification System(美国Invitrogen公司nvitrogenTMK95001)说明书操作。细胞膜可溶性物直接上Ni-NTA柱，依次用含不同浓度咪唑(50mmol/l、100mmol/l、150mmol/l、200mmol/l)的洗脱液洗脱，收集洗脱峰，同步收集样品并进行酶活力与蛋白含量的测定。
重组蛋白质含量测定：改良型Lowry法蛋白浓度测定试剂盒(上海生工，C504041-1000)。
几丁质脱乙酰基酶活力检测：比色管中加入35℃预保温的浓度为0.05mol/L的pH 8.0的Tris-HCl缓冲溶液3mL、浓度为200mg/L的4-硝基乙酰苯胺溶液1mL、几丁质脱乙酰酶液1mL，在35℃下水浴反应15min后沸水浴终止酶促反应，用蒸馏水定容至10mL，混匀，3000r/min离心10min，测定上清液在400nm处的吸光值(A400)。无活性的酶液为空白对照。
几丁质脱乙酰基酶酶活单位定义：每小时几丁质脱乙酰酶催化底物产生lμg对硝基苯胺所需要的酶液的体积定义为一个酶活力单位。
以下通过实例进一步说明本发明
实施实例1、突变表达质粒的构建及重组大肠杆菌的获得
根据野生型酶基因设计引物，并引入酶切位点，所用引物如下：P1：5’-CGGAATTCATGGCGCGTGGTGTCCGCAT-3’(引入EcoR I酶切位点)；P2：5’-CCAAGCTTCTATGCCGTGGCTTCACCGGA-3’(引入HindIII酶切位点)。以Salinispora arenicola基因组DNA为模板进行PCR，PCR反应体系为：ddH2O(17.5μL)，buffer(2.5μL)，Mg2+(2.5μL)，dNTP(0.5μL)，P1(0.5μL)，P2(0.5μL)，模板(1μL)，Taq酶(0.2μL)。PCR程序：94℃预变性5min，然后94℃30s，54℃40s，72℃70s，循环35次；72℃延伸5min。扩增产物经1.0％琼脂糖凝胶电泳进行检测后测序。将上述PCR扩增产物经1.0％琼脂糖凝胶电泳、纯化，与pMD18-T连接后转化E.coli DH5α，从阳性菌落中提取重组质粒pMD18-T-cad。pMD18-T-cad和表达载体pEtac双酶切后，1％的琼脂糖凝胶电泳回收酶切片段，将酶切后的胶回收产物以一定比例(基因片段的摩尔量是载体的三倍)混合后16℃连接过夜。转化DH5α，将重组菌株涂布于含有50ug/mL的卡那霉素固体LB培养基内，37℃倒置培养过夜，随机挑选若干个菌落于含有50ug/mL卡那霉素的液体LB培养基内，37℃摇瓶培养过夜，提取质粒pEtac-his6-amy，测序验证后转化E.coli BL21(DE3)。
通过模拟Salinispora arenicola CAD三级结构，发现第52位缬氨酸和第66位的脯氨酸都位于链接催化活性中心的无规卷曲结构中，突变后有望改变催化结构域，并改变底物结合能力。以含有几丁质脱乙酰基酶质粒pEtac-His6-cad为模板，设计引物进行突变并将突变后的质粒转化进入E.coli DH5α进行扩增，然后以E.coli BL21(DE3)表达含突变基因的重组质粒，得到活性提高的突变体。。并将突变后的质粒转化进入E.coli DH5α进行扩增，然后以E.coli BL21(DE3)表达含突变基因的重组质粒，得到活性提高的突变体。
所述用于定点突变的引物是：
cadV52G primer1ATGGTGGCCACACCTGGCGGGTGTGG
cadV52G primer2CCACACCCGCCAGGTGTGGCCACCAT
cadP66S primer1ATCGCGTTGACCTTCGACGACGGACCCAA
cadP66S primer2TTGGGTCCGTCGTCGAAGGTCAACGCGAT
PCR管中加入10mmol/L MgCl2 2.5μL,10×PCR buffer 5μL,2.5μmol/L dNTP 4μL，Taq(5U/μl)0.25μL，上游引物和下游引物(10μmol/L)各1μL，模板DNA1μL，最后加双蒸水至50μL,混合后瞬间离心，置于PCR仪上95℃变性5min，然后94℃变性40s，55℃退火40s，72℃延伸1min,共35个循环，最后72℃延伸10min反应结束。
扩增产物经1.0％琼脂糖凝胶电泳进行检测后测序。将上述PCR扩增产物经1.0％琼脂糖凝胶电泳、纯化，并和表达载体pEtac双酶切后，1％的琼脂糖凝胶电泳回收酶切片段，将酶切后的胶回收产物以一定比例(基因片段的摩尔量是载体的三倍)混合后16℃连接过夜。转化DH5α，将重组菌株涂布于含有50ug/mL的卡那霉素固体LB培养基内，37℃倒置培养过夜，随机挑选若干个菌落于含有50ug/mL卡那霉素的液体LB培养基内，37℃摇瓶培养过夜，提取质粒pEtac-his6-amy，测序验证后转化E.coli BL21(DE3)。
实施实例2、突变表达载体的转化
所用培养基组成：胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，pH7.0。将含有编码突变几丁质脱乙酰基酶的基因的重组菌E.coli BL21(DE3)(pEtac-His6-cad)活化培养后接种到含有100μg mL-1氨苄青霉素的发酵培养基中，装液量为30mL/250mL，于37℃，200r min-1培养；菌体生长到OD600＝0.6，加入终浓度0.5mM IPTG，30℃，诱导发酵24h。180r/min诱导表达5h。4℃、10000r/min冷冻离心5min收集菌体。蒸馏水重悬菌体，以超声波细胞粉碎仪破碎菌体(200W，超3s，停7s，共超声5min)，4℃，10000r/min离心20min，收集上清液作为粗酶液。按照Ni-NTA Purification System(美国Invitrogen公司nvitrogenTMK95001)说明书一步纯化重组酶。和野生型重组酶相比，突变后的比活力剂催化活性提高3.92倍。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (6)
1.一种几丁质脱乙酰基酶的突变体，其特征在于，是将氨基酸序列如SEQ ID NO.2所示的几丁质脱乙酰基酶的第52位缬氨酸V突变为甘氨酸G，第66位的脯氨酸P点突变为丝氨酸S。
2.编码权利要求1所述突变体的基因。
3.携带权利要求2所述基因的载体或细胞。
4.一种获得权利要求1所述几丁质脱乙酰基酶突变体的方法，是通过设计引物对编码几丁质脱乙酰基酶的基因进行定点突变后表达。
5.根据权利要求4所述的方法，其特征在于，是以质粒pEtac-His6-cad为模板设计引物进行突变并将突变后的质粒转化进入E.coli DH5α进行扩增，然后以E.coli BL21(DE3)表达含突变基因的重组质粒，得到活性性增强的几丁质脱乙酰基酶突变体。
6.应用基因工程菌发酵生产权利要求1所述几丁质脱乙酰基酶突变体的方法，是将含有编码突变几丁质脱乙酰基酶的基因的重组菌E.coli BL21(DE3)(pEtac-His6-cadm)活化培养后接种到含有100μg mL-1氨苄青霉素的发酵培养基中，所用培养基组成为胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，pH7.0。装液量为30mL/250mL，于37℃，200r min-1培养；菌体生长到OD600＝0.6，加入终浓度0.5mM IPTG，30℃，诱导发酵24h。取发酵液，8000r/min离心10min收集菌体，再适量蒸馏水悬浮细胞，于冰水中超声破碎4min，12000r/min离心15min去除细胞碎片，上清液即为粗酶液。
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| CN111286498A (zh) * | 2020-03-26 | 2020-06-16 | 汪利平 | 用于生产乙酰氨基葡萄糖的高效几丁质酶 |
| CN111944793A (zh) * | 2020-07-28 | 2020-11-17 | 江苏海洋大学 | 一种Aspergillus nidulans几丁质脱乙酰基酶突变体 |
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| CN111286498A (zh) * | 2020-03-26 | 2020-06-16 | 汪利平 | 用于生产乙酰氨基葡萄糖的高效几丁质酶 |
| CN111286498B (zh) * | 2020-03-26 | 2024-03-29 | 汪利平 | 用于生产乙酰氨基葡萄糖的高效几丁质酶 |
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| CN111979218A (zh) * | 2020-07-28 | 2020-11-24 | 江苏海洋大学 | 一种Arthrobacter sp. AW19M34-1几丁质脱乙酰酶突变体 |
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