CN117384894B - 脱乙酰酶及其编码基因、重组质粒、重组菌与应用 - Google Patents
脱乙酰酶及其编码基因、重组质粒、重组菌与应用 Download PDFInfo
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Abstract
本发明涉及一种脱乙酰酶及其编码基因、重组质粒、重组菌与应用,属于酶工程领域,所述几丁质脱乙酰酶的氨基酸序列如SEQ ID NO.1所示。编码所述的几丁质脱乙酰酶的编码基因如SEQ ID NO.2所示。本发明还提供一种含有SEQ ID NO.2所述基因的重组质粒pPIC9K‑CDA和重组菌,以及利用所述几丁质脱乙酰酶催化几丁质来制备壳聚糖,所述几丁质脱乙酰酶对α‑几丁质的脱乙酰度达到52%,可实现工业上酶法催化几丁质制备壳聚糖的需求。
Description
技术领域
本发明属于酶工程领域,具体地涉及一种脱乙酰酶及其编码基因、重组质粒、重组菌与应用。
背景技术
几丁质(化学名称为β-(1,4)-2-乙酰氨基-2-脱氧-d-葡萄糖)是仅次于纤维素的第二丰富的天然聚合物,也是地球上最丰富的可再生含氮物质。它广泛存在于甲壳类动物、昆虫外骨骼、软体动物和真菌细胞壁中。然而,天然的几丁质具有致密的晶体结构,高度有序的三维网络,在常规溶剂中具有高度的聚合度和不溶性,这严重限制了几丁质的开发和商业应用。因此,天然几丁质生物资源通常被认为是海洋和生物技术工业的一种繁重的废弃物。
壳聚糖是由几丁质去乙酰化衍生的最关键的几丁质衍生物,是由β-(1,4)连接的葡萄糖胺葡萄糖和N-乙酰-d-胺葡萄糖组成的阳离子线性多糖,具有良好的生物相容性、生物降解性和无毒性。目前,市面上的壳聚糖仍可以通过热化学和物理过程获得。这些方法需要大量的浓缩碱处理,反应时间长,生产成本高,去乙酰化的程度不容易控制,这可能导致产品质量不稳定,可能破坏几丁质分子链,和对环境造成严重污染。几丁质的酶修饰可能是一种很有前途的替代传统的热化学和物理过程。几丁质去乙酰化酶(EC 3.5.1.41,CDA)可以通过酶去乙酰化将几丁质转化为壳聚糖来实现,避免了传统方法中存在的的缺点。CDA可以催化几丁质中N-乙酰葡糖胺的乙酰基水解,将几丁质转化为壳聚糖,并产生乙酸。
目前,对于脱乙酰酶异源表达的报道还较少。因此,发掘具有较高活性的脱乙酰酶基因,通过基因重组的方法,构建基因工程菌,高效异源表达脱乙酰酶,探索脱乙酰酶制备壳聚糖的酶解工艺,具有重要的工业应用价值和潜力。毕赤酵母表达系统是近几十年来迅速发展起来的新型外源蛋白表达系统,可实现外源蛋白的高效分泌表达,并对表达的蛋白进行翻译后的加工与修饰,确保表达出的蛋白具有生物活性。
发明内容
本发明的目是提供一种脱乙酰酶及其编码基因与应用。本发明是在基因组分析的基础上挖掘了南极磷虾CDA基因(EsCDA),并在毕赤酵母GS115中进行异源表达,其具有更高的酶活,以满足工业生产需求。
本发明是通过如下技术方案来实现的:
一种几丁质脱乙酰酶,所述几丁质脱乙酰酶的氨基酸序列如SEQ ID NO. 1所示。
编码前述所述的几丁质脱乙酰酶的编码基因,所述编码基因序列如SEQ ID NO.2所示。
一种重组质粒pPIC9K-CDA,所述重组质粒中含有SEQ ID NO. 2所示的核苷酸序列。
一种重组毕赤酵母工程菌,所述重组毕赤酵母工程菌含有所述重组质粒pPIC9K-CDA。
本发明还提供所述几丁质脱乙酰酶的应用,所述应用方法利用所述几丁质脱乙酰酶催化几丁质来制备壳聚糖。
本发明与现有技术相比的有益效果:本发明的EsCDA处理的α-几丁质的脱乙酰度为52 %,因此,本发明的脱乙酰酶EsCDA可实现工业上酶法催化几丁质制备壳聚糖的需求,且可被广泛应用于食品科学、生物医药、化工材料等方面,有良好的应用前景。
附图说明
图1为温度对几丁质脱乙酰酶活的影响的折线图;
图2为pH对几丁质脱乙酰酶活的影响的折线图;
图3为温度对几丁质脱乙酰酶稳定性的影响的折线图;
图4为pH对几丁质脱乙酰酶稳定性的影响的折线图;
图5为重金属离子对几丁质脱乙酰酶活的影响柱状图;
图6为α-几丁质电镜照片;
图7为EsCDA水解后的α-几丁质电镜照片;
图8为壳聚糖的电镜照片;
图9为脱乙酰红外光谱图,a为壳聚糖,b为EsCDA 处理的α-几丁质,c为α-几丁质。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法,下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1:通过基因组数据库发掘的方法分析了一批基因组序列,候选了一批在生物信息学上被预测为假定脱乙酰酶或具有潜在脱乙酰酶活性的未验证功能基因序列。所述的挖掘方法具体为,以文献中公开的几丁质脱乙酰酶基因为模板探针,在NCBI数据库中进行Blast搜索,筛选出与已知序列同源性在30-90% 的潜在序列,并进一步进行结构域分析及蛋白家族分类分析,确定候选序列。依据上述方法,从基因数据库中筛选得到候选基因EsCDA。
经过大规模的筛选和深入的研究,从南极磷虾中鉴定分离出一种新的几丁质脱乙酰酶,其不仅能够催化几丁质的脱乙酰化,还能够对不同底物具有特异性,具有非常好的应用前景。本发明的几丁质脱乙酰酶对于低温的适应性好,能够在毕赤酵母细胞中被表达。同时,本发明还优化了该几丁质脱乙酰酶的重组表达方法,从而使之在宿主细胞中得到了高效的表达。
本发明人首先根据表达情况进行了密码子优化,以提高表达效率及DNA片段的稳定性。将优化后的序列插入至质粒pPIC9K中,得到重组质粒pPIC9K-CDA,随后将重组质粒pPIC9K-CDA转化至巴斯德毕赤酵母GS115感受态细胞中进行异源表达。
实施例2:本实施例提供了一种重组表达载体的重组毕赤酵母工程菌的构建过程,具体如下:
EsCDA基因由青科生物技术有限公司(中国青岛)合成。以合成的EsCDA基因为模板,用正向引物5′-GAATTCATGCCAAACGGTATGAACCCAAAGAACGTGC-3′(SEQ ID NO.3),反向引物5′-CGCCGGCGTTAGTGGTGGTGGTGGTGGTGA-3′(SEQ ID NO.4)扩增EsCDA。将扩增产物纯化并用EcoR I/Not I双酶切,然后连接到EcoR I/Not I双酶切后的pPIC9K载体中。
用质粒提取试剂盒用回收质粒。基因测序后,转化到巴斯德毕赤酵母GS115感受态细胞中,用Sal I酶切测序正确的质粒。将转化体在BMGY培养基中培养,温度为30°C,振荡速度为180 r/min,直到OD600达到5.0,之后5000 r/min离心10分钟收集菌体,将收集到的菌体转移到BMMY培养基中。每天在培养基中添加1%甲醇,诱导发酵5天。
胶体几丁质制备方法如下:150 mL烧杯中加入15 g的α-几丁质,在冰水浴中缓慢倒入100 mL浓HCL并用玻璃棒轻轻搅拌至糊状,用保鲜膜将烧杯口封好置于4℃层析柜过夜。将过夜混合物加入到500 mL 95%的冰乙醇中,充分搅拌后用保鲜膜封口,室温静置过夜后在4℃,5000 r/min条件下离心20 min后取沉淀。用蒸馏水反复洗涤沉淀,直至pH呈中性后加入300 mL蒸馏水即配成胶体几丁质。配置好的胶体几丁质用保鲜膜封口,放入4 ℃冰箱保存备用。
重组酶的镍柱纯化:重组表达的EsCDA基因在C端携带6×His标签,可以与HisTrapTM HP柱填料的Ni结合,而Ni又可与咪唑结合,因此用不同浓度的咪唑洗脱可以达到纯化的目的,获得单一蛋白。将发酵液8000 r/min离心20 min,得到上清液,4°C冰箱冷藏备用。首先用ddH2O冲洗AKTA蛋白快速纯化仪的A、B泵和系统,然后用平衡缓冲液分别冲洗A、B泵;其次将流速调节为1 mL/min,连接镍柱后将流速调节为2 mL/min,将粗酶液过0.22 μm滤膜后上样,上样流速为0.5 mL/min;待穿透峰跑平后,用洗涤缓冲液冲洗,以去除杂蛋白;最后待基线平稳后,用洗脱缓冲液洗脱目的蛋白,出峰接样,直至峰跑平,停止接样。
平衡缓冲液为:20mM磷酸盐缓冲液;500mM NaCl;pH7.4;
洗涤缓冲液为:20mM磷酸盐缓冲液;500mM NaCl;10mM咪唑;pH7.4;
洗脱缓冲液为:20mM磷酸盐缓冲液;500mM NaCl;200mM咪唑;pH7.4。
实施例3:使用Megazyme公司乙酸试剂盒K-ACETRM Kit的方法进行乙酸含量的测定以表征实施例2制备的几丁质脱乙酰酶的酶活,具体测定方法如下:
酶活力的测定:检测催化反应的产物乙酸来表征重组几丁质脱乙酰酶的酶活力:200 μL反应体系中包含50 μL酶液、50 μL的胶体几丁质和100 μL的0.05 mol/L pH 7.0磷酸缓冲液,混匀后40℃反应2 h,100 °C煮沸3 min终止反应,8000 r/min离心20 min收集上清,测定上清中乙酸量,计算酶活力,详细步骤见乙酸检测试剂盒说明书。
酶学性质:
(1)最适温度测定:分别在20℃、25℃、30°C、35°C、40°C、45°C、50°C、55°C和60°C的不同温度条件下测定EsCDA的酶活性。在上述条件下反应2 h,测定相对酶活,以40°C所得的最高酶活为100%。
(2)最适pH测定:从pH的变化分析pH对酶活性的影响,配置柠檬酸缓冲液(pH 3.0-5.0)、磷酸缓冲液(pH 5.0-7.0)、Tris-HCl缓冲液(pH 7.0-9.0)和甘氨酸氢氧化钠缓冲液(pH 9.0-10.0)。将反应体系置于40°C水浴中反应2 h,测定相对酶活,以pH为8的最高酶活为100%。
(3)金属离子对重组EsCDA酶活影响:配制各种金属离子K+、Na+、Ba2+、Ca2+、Cu2+、Mg2 +、Zn2+、Co2+和Fe3+,使其金属离子的终浓度分别达到1 mM和10 mM,pH为7,温度为40°C,水浴中反应2 h,测定相对酶活。
上述反应结果如图所示,从图1-4中可以看出,酶活的较佳条件为pH7.0~9.0,较佳温度为30°C~50°C。酶的最适反应温度为40°C,当温度在30°C~50°C范围内,酶的相对活性均大于60%,当酶在4°C和25°C反应10 h后剩余酶活均大于85%,且随着温度升高剩余酶活逐渐降低,说明其在低温条件下具有良好的稳定性;最适pH为8.0,当pH 7.0~9.0范围内,酶的相对活性均大于60%,当酶在pH 7.0~9.0范围内反应10 h后剩余酶活均大于80%,说明其在该pH范围内具有良好的稳定性。
如图5所示,Co2+对几丁质脱乙酰酶EsCDA具有激活作用,剩余酶活为110%;Zn2+、Ba2 +和Mg2+抑制EsCDA的活性,剩余酶活分别为62%、80%和82%;剩余金属离子Na+、Ca2+和Fe3+对EsCDA的活性影响不大,剩余酶活均大于为90%。
实施例4:扫描电镜分析:(1)将0.1g α-几丁质、10 mL酶液与10 mL 0.05 mol/L磷酸盐缓冲液(pH 7.0)配制成20 mL混合液,40℃,180 r/min反应24 h。反应结束后4 ℃、8000 r/min离心30 min,保留沉淀,用无菌水洗涤沉淀3-5次,使沉淀 pH 值达到中性。
(2)在100℃干燥箱中烘干酶解产物至恒重。将样品分别用导电胶粘在金属样品台上,利用真空蒸发器喷镀一层金属膜于样品表面,并通过3 kv的扫描电子显微镜对样品进行观察。
结果如图6-8所示,图6中是酶液处理前的α-几丁质电镜扫描图,图7为EsCDA酶液处理后的α-几丁质电镜扫描图,图8为壳聚糖的电镜扫描图。
从图3中可以看出,几丁质为大颗粒致密颗粒,层状结构,表面结构紧密的晶态微纤维;而EsCDA处理后的几丁质的整体结构受到了严重的破坏,其纤维表面出现致密裂纹,表面结构变得模糊不清,界面分离不明显的颗粒状态与壳聚糖相似,与几丁质有明显的区别。该结果表明经EsCDA处理后几丁质的显微结构发生变化的原因是乙酰基含量降低,导致分子内和分子结构被破坏。
实施例5红外光谱测定:在100℃干燥箱中烘干溴化钾、α-几丁质和EsCDA酶解产物至恒重。在研钵中分别研磨适量的溴化钾和样品,并以 50:1的体积比混匀,利用压片机压制混合物至薄片状。利用波长 400-4000 cm-1的红外光谱仪对样品进行分析,并用 OMNNIC软件处理数据。样品的脱乙酰度与波数 1655 cm-1和 3450 cm-1的比值(A1655/A3450)呈线性关系。
结果如图9所示,图9为脱乙酰红外光谱图,从上到下依次为壳聚糖、EsCDA 处理的α-几丁质和α-几丁质。
从图中可以看出,3450 cm-1是多重吸收峰,因-OH伸缩振动峰和-NH的伸缩振动峰重叠而增宽1655 cm-1处为有较强的酰胺I特征峰;1655 cm-1处吸收峰与3450 cm-1处吸收峰的比值(A1655/A3450)与几丁质的脱乙酰度呈线性关系,所以常用来计算样品的脱乙酰度。壳聚糖、EsCDA处理的α-几丁质和α-几丁质的脱乙酰度分别为68 %、52 %和27 %。
SEQ ID NO.1
MPNGMNPKNVPQMITITFDDAVNTGNIDLYEELFGNQTLLNPNSCAIKGTFFVSHKYSNYSAVQELHRLGHEIAVHSISHNSSTDFWTSATEEQWTQEMAGARVISERFANITDQSIIGMRAPFNRVGSNKQFKMMEDQAFLYDSSVNAPLGKVPHWPYTLYYRMPHPCHGHLQECPTRSFAVWEMVMNEMDRREDPEFEEPLPGCVMVDSCFSSKPSGDQFYKFLTNNFDHHYNTNRAPLGLFFQSAFLKNNHEVRENFLRWIKEILATHNDVYFVTMTQVIQWMQDTRSVNELPNFGPWKDKCIVQGQPMCNGGNNCELNTQDLPGETLFLPTCMTCPNNYPWLQDPTGEGYF;
SEQ ID NO.2
ATGCCAAACGGTATGAACCCAAAGAACGTGCCCCAAATGATTACCATTACCTTTGACGACGCCGTTAACACCGGTAACATTGACTTGTACGAAGAATTGTTTGGTAACCAAACCCTGTTGAACCCAAACTCCTGCGCTATCAAAGGTACCTTCTTTGTTTCTCATAAATACTCTAACTACTCCGCCGTTCAAGAATTGCATAGATTGGGTCATGAGATTGCTGTCCATTCTATTTCTCACAACTCCTCTACTGATTTTTGGACTTCCGCCACTGAAGAACAATGGACCCAAGAAATGGCTGGTGCTAGAGTGATTTCTGAAAGATTTGCTAACATTACTGACCAATCCATTATTGGTATGAGAGCTCCATTTAACAGAGTTGGTTCTAACAAGCAATTTAAGATGATGGAGGATCAAGCTTTTTTGTACGACTCTAGTGTTAACGCTCCATTAGGAAAGGTTCCACATTGGCCATACACCCTGTACTACAGAATGCCACATCCTTGCCACGGTCATTTGCAGGAATGTCCAACCAGATCTTTTGCTGTTTGGGAAATGGTCATGAACGAAATGGATAGAAGAGAAGATCCAGAATTTGAAGAACCATTGCCCGGTTGTGTTATGGTTGATAGTTGTTTTTCTTCTAAGCCATCCGGTGATCAATTTTACAAGTTTTTGACTAACAACTTCGACCATCATTATAACACTAATAGAGCACCACTTGGTTTGTTTTTTCAATCTGCTTTTTTGAAGAACAACCATGAGGTTAGAGAAAACTTTCTGAGATGGATTAAGGAAATTTTGGCTACTCATAACGACGTCTACTTTGTGACTATGACCCAAGTTATTCAGTGGATGCAAGATACTAGATCCGTCAACGAATTGCCAAACTTTGGTCCCTGGAAGGACAAGTGTATTGTTCAAGGACAACCCATGTGTAACGGTGGTAACAACTGCGAATTGAACACCCAAGACCTTCCAGGTGAAACCTTGTTTTTGCCTACCTGTATGACTTGTCCAAACAACTACCCTTGGTTGCAAGATCCCACCGGTGAAGGATACTTTTAA。
Claims (5)
1.一种几丁质脱乙酰酶,其特征在于,所述几丁质脱乙酰酶的氨基酸序列如SEQ IDNO. 1所示。
2.编码权利要求1所述的几丁质脱乙酰酶的编码基因,其特征在于,所述编码基因的核苷酸序列如SEQ ID NO.2所示。
3.一种重组质粒pPIC9K-CDA,其特征在于,所述重组质粒pPIC9K-CDA中含有权利要求2中如SEQ ID NO. 2所示的核苷酸序列。
4.一种重组毕赤酵母工程菌,其特征在于,所述重组毕赤酵母工程菌含有权利要求3所述重组质粒pPIC9K-CDA。
5.一种几丁质脱乙酰酶的应用,其特征在于,所述应用为利用权利要求1所述几丁质脱乙酰酶催化α-几丁质来制备壳聚糖。
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