CN116064484A - 一种突变的几丁质酶SsChi18A-2及其应用 - Google Patents
一种突变的几丁质酶SsChi18A-2及其应用 Download PDFInfo
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Abstract
本发明公开了一种几丁质酶SsChi18A的突变基因SsChi18A‑2,所述基因的突变位点为E287A,其核苷酸序列如SEQ ID No.1所示,蛋白质序列如SEQ ID No.2所示。本发明还公开了所述基因在降解胶体几丁质和真菌菌渣中的应用。实验证明,这种基因所编码的突变几丁质酶活性比野生型酶提高了35%,并且在降解真菌细胞壁时酶活较野生型提高49%,这些结果表明突变体在降解几丁质类底物方面有着广泛的应用。
Description
技术领域
本发明属于基因工程领域,具体涉及一种突变的几丁质酶SsChi18A-2及其应用。
背景技术
几丁质是一种多聚-β-1,4-N-乙酰氨基葡萄糖(NAG),是自然界中除纤维素外含量第二丰富的多糖。几丁质主要来源于甲壳类动物,每年水体中产生的几丁质高达1011吨。生物质中的几丁质去矿化并除蛋白以获取纯甲壳素的过程常用浓酸或碱来进行的,但会造成腐蚀及产生环境问题。酶法降解几丁质能够产生多种药理活性成分,例如N-乙酰葡糖胺(NAG)和几丁质寡糖(CHOS),可以作为药物输送载体和抗氧化剂,在抗肿瘤、伤口愈合、控制血液胆固醇和食品保存方面发挥作用。几丁质酶的来源广泛,酶解过程环境友好,在工业生产中应用潜力巨大。目前,几丁质酶制剂广泛应用在医药、食品、生物技术、植物病虫害防治等众多领域。
几丁质是仅次于纤维素的自然界中最丰富的天然多糖,分别由β-N-1,4-葡萄糖和β-N-1,4-氨基葡萄糖聚合而成。这些多糖具有高度结晶性,通过分子内和分子间复杂的氢键网络形成抗降解屏障,使其降解成为难题。但是生物体进化出高效利用结晶多糖的复杂机制,利用环境导向的糖苷水解酶协同作用将顽固多糖水解为可溶性多糖,特别是一些具有益生元价值的可溶性寡糖,在工业、农业及医疗领域具有重要应用价值。
Streptomyces sp.F-3是发明人课题组在高温生境分离筛选到的一株嗜热放线菌,该菌最适生长温度为50℃。全基因组测序分析结果显示,该菌含有9个几丁质降解相关酶,包含一个外切持续性几丁质酶SsChi18A。丰富的几丁质降解酶及嗜热特征使其具有发展成为几丁质高效降解工业菌的潜力。
SsChi18A的实际应用中,仍存在着亟待解决的问题;缩短工艺流程,提高生产效益亟待具有更高活性几丁质酶的出现。为了扩展SsChi18A的工业应用,提高其酶活性是一个很关键的任务。
发明内容
针对现有技术的不足,本发明提供了一种突变的几丁质酶及其应用。
本发明中,突变的几丁质酶命名为SsChi18A-2,也可以称之为SsChi18A-E287A,或者以E287A来指代。
一方面,本发明提供了一种突变的几丁质酶,所述突变的几丁质酶的氨基酸序列如SEQ ID No.2所示。
另一方面,本发明还提供了所述突变的几丁质酶的编码基因;优选的,所述基因的序列如SEQ ID No.1所示。
另一方面,本发明还提供了包含所述突变的几丁质酶的编码基因的重组载体;优选的,所述重组载体为重组表达载体;优选为pET系列的载体,例如,pET-15b、pET-22b、pET-28a。
另一方面,本发明还提供了包含上述重组载体的重组菌株,优选的,所述重组菌株为大肠杆菌,如,大肠杆菌BL21。
另一方面,本发明还提供了所述突变的几丁质酶、所述编码基因、所述重组载体或重组菌株在降解含有几丁质的材料中的应用。应当理解,含有几丁质的材料包括天然含有几丁质的材料,或者经加工的含有几丁质的材料;此外,几丁质单体也属于含有几丁质的材料的概念。
在一个实施方式中,含有几丁质的材料为来自真菌的菌渣,例如,出芽短梗霉的菌渣。
在一个实施方式中,所述出芽短梗霉菌渣是利用出芽短梗霉发酵生产特定产物后剩余的菌渣。
另一方面,本发明还提供了所述突变的几丁质酶、所述编码基因、所述重组载体或重组菌株在制备几丁质寡糖中的应用。
另一方面,本发明还提供了一种降解含有几丁质或者含有几丁质寡糖的材料的方法,所述方法包括利用所述突变的几丁质酶、所述编码基因、所述重组载体或重组菌株对含有几丁质或者含有几丁质寡糖的材料进行处理的步骤。应当理解,含有几丁质的材料包括天然含有几丁质的材料,或者经加工的含有几丁质的材料;此外,几丁质单体也属于含有几丁质的材料的概念。含有几丁质寡糖的材料包括天然含有几丁质寡糖的材料,或者经加工的含有几丁质寡糖的材料;此外,单纯的几丁质寡糖也属于含有几丁质寡糖的材料的概念。
优选的,所述寡糖为二糖、三糖、四糖、五糖或六糖。
进一步的,所述处理的温度为30℃-80℃,优选,50℃-70℃;所述处理的pH为3-11,优选,4-7。
本发明具有的有益效果是:
本发明公开的几丁质酶SsChi18A的突变体SsChi18A-2的活性比野生型酶提高了35%,并且在降解真菌细胞壁时酶活较野生型提高49%,这些结果表明突变体在降解几丁质类底物方面有着广泛的应用。在作为医药、食品、生物技术、植物病虫害防治等工业用酶时,能在更广泛的环境中保持较高活性,具体应用包括但不仅限于:
在医药生产过程中,此突变几丁质酶既可以作为微生物抑制剂来制备眼科制剂,也可以作为抗真菌药物的添加剂应用于真菌疾病的治疗。在食品工业中,此突变几丁质酶可以用于几丁质寡糖的生产,既能作为抗微生物剂和免疫增强剂,激活宿主防御***;又可以用作骨关节炎、风湿性关节炎治疗剂和食品添加剂,调节人体健康。在生物技术产业中,此突变几丁质酶可以和其它细胞壁降解酶一起用于真菌原生质体的制备。在植物病害防治过程中,此突变几丁质酶可以作为生防制剂来预防植物病原体和害虫,能够快速水解植物病害真菌的细胞壁,从而导致细胞裂解,进而杀死病原真菌。
附图说明
图1为蛋白纯化SDS-PAGE图。
图2为以胶体几丁质为底物野生型WT和突变体E287A的相对酶活性。
图3为突变体E287A与野生型WT降解胶体几丁质的产物谱。
图4为突变体E287A与野生型WT降解出芽短梗霉菌渣相对酶活。
图5为突变体E287A与野生型WT降解出芽短梗霉菌渣产物谱。
具体实施方式
下面结合附图以及具体实施例对本发明做进一步的说明,以下所述,仅是对本发明的较佳实施例而已,并非对本发明做其他形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容,依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化,均落在本发明的保护范围内。
实施例1、几丁质酶SsChi18A的突变以及重组载体的构建
从NCBI数据库中获取几丁质酶SsChi18A基因,将SsChi18A基因和质粒pET28a连接,获得pET28a-SsChi18A质粒。通过生物信息学预测关键氨基酸位点进行定点突变,以上述重组质粒为模板,设计突变引物,进行定点突变得到突变的几丁质酶SsChi18A-2,引物序列如下:
E287A-sense:CCTACGCAGCGGGCGTCGAGGACTA
E287A-antisense:AGTCCTCGACGCCCGCTGCGTAGGT。
突变的几丁质酶SsChi18A-2的核酸序列如SEQ ID No.1所示,氨基酸序列如SEQID No.2所示,其相对于野生型几丁质酶,第287位的E突变为A。
最终获得的突变质粒pET28a-SsChi18A-E287A的核苷酸序列如SEQ ID No.3所示。
实施例2、含有上述突变基因SsChi18A-2的重组工程菌构建
构建含有上述突变基因SsChi18A-2的重组工程菌,具体步骤如下:将测序正确的上述突变质粒pET28a-SsChi18A-E287A加入50μL大肠杆菌BL21感受态细胞,冰浴30min;42℃热击90s;冰浴2min,加入1mL液体LB,37℃摇床中培养1-1.5h;8000rpm离心2min,弃上清(留少许底部液体)。将剩余溶液涂布到含50μg/mL卡那霉素的LB平板,涂布均匀至干燥,37℃过夜倒置培养;次日挑取单克隆,接种在5mL含抗生素的LB培养基中,37℃200rpm过夜培养,即得到含有突变基因SsChi18A-2的重组工程菌。
实施例3、突变基因SsChi18A-2的重组表达
取得到的含有上述突变基因SsChi18A-2的重组工程菌,于LB培养基中(含50μg/mL卡那霉素)37℃200rpm发酵培养,至OD600=0.6-0.8;加入终浓度为0.2mM的IPTG,20℃诱导培养20h;8000rpm,4℃离心10min,得到菌体沉淀,重悬后,超声破碎细胞;离心得到上清液(粗酶液),用0.22μm的滤头过滤上清液;把柱子的填料与滤液结合,准备进行亲和纯化。使用GE Healthcare公司HisCap Co 6FF resin(Smart-life Sciences,Changzhou,China)对目的蛋白进行亲和纯化。纯化后的蛋白加pH 5.0的NaH2PO4-柠檬酸缓冲液,4900rpm于4℃超滤,至滤出的缓冲液pH=5.0;使用考马斯亮蓝染色方法测量纯化后的蛋白浓度。
如图1所示,为蛋白纯化后的SDS-PAGE图,Marker分别为25.0、35.0、45.0、66.2、116.0kDa,各泳道分别为Marker、粗酶液、沉淀、流出液、10mM咪唑洗脱液、10mM咪唑洗脱液、20mM咪唑洗脱液、100mM咪唑洗脱液、100mM咪唑洗脱液。由图可以看出目的蛋白条带位于45kDa附近,与蛋白大小相符。
实施例4、突变几丁质酶SsChi18A-E287A(SsChi18A-2)与野生型酶SsChi18A的酶学性质比较。
(1)胶体几丁质酶活测定
将突变几丁质酶SsChi18A-E287A和野生型酶WT稀释到0.01mg/mL;在对照组管中加入100μL pH 5.0的NaH2PO4-柠檬酸缓冲液,实验组管中加入100μL稀释过的酶液,每管再分别加入100μL浓度为20mg/mL的胶体几丁质(其溶于pH 5.0的50mM NaH2PO4-柠檬酸缓冲液),60℃反应10min;每管各加入300μL DNS,沸水浴10min;迅速冷却,摇匀,离心,取上清,测定OD 540;每种酶做三次重复实验;根据标准曲线算出还原糖量,再根据公式算出比酶活;比较两种蛋白的酶活性。
突变几丁质酶E287A与野生型几丁质酶WT在最适条件下的相对酶活性如图2所示,突变木聚糖酶E287A的酶活性是野生型木聚糖酶WT的1.35倍,这证明突变几丁质酶对于几丁质聚糖具有较强的酶活性,具有更优秀的应用价值。
(2)HPLC分析胶体几丁质降解产物
使用Bio-Rad Aminex HPX-42C,300×7.8mm(目录#125-0096)和SPD检测器(日本京都Shimadzu)采用高性能液相色谱(HPLC)***测定单糖和双糖的浓度。纯化酶(0.01毫克/毫升;250μL)与胶体几丁质(20mg/mL,500μL)在60℃反应30min。结束后,将溶液离心除去不溶底物,取400μL与100μL乙腈混合终止反应。得到的样品保存在-20℃,直到高效液相色谱分析。流动相为70%乙腈的纳米纯化水溶液,流速为0.2mL/min。在210nm处用SPD检测器检测分离产物。所有样品均为三份。
突变几丁质酶E287A与野生型几丁质酶WT降解胶体几丁质的产物谱如图3所示。二者的产物均为单糖(7min开始出峰)和二糖(9.5min开始出峰),且以二糖为主。二者相比,SsChi18A-E287A的产物中单糖和二糖的含量相对较高。
(3)降解菌渣酶活测定
将突变体E287A和野生型酶WT稀释到0.1mg/mL;实验组管中加入1mL稀释过的酶液,再加入0.05mg出芽短梗霉菌渣,60℃反应1h取样200μL,加入300μL DNS,沸水浴10min;迅速冷却,摇匀,离心,取上清,测定OD540;每种酶做三次重复实验;根据标准曲线算出还原糖量,再根据公式算出比酶活;比较两种蛋白的酶活性。
突变体E287A与野生型WT降解出芽短梗霉菌渣的相对酶活如图4所示。突变体E287A的酶活性是野生型WT酶活性的1.49倍,这证明突变几丁质酶对于出芽短梗霉菌渣具有较强的酶活性,具有更优秀的应用价值。
(4)FACE分析菌渣降解产物谱
将突变体E287A和野生型酶WT稀释到0.1mg/mL;实验组管中加入1mL稀释过的酶液,再加入0.05mg出芽短梗霉菌渣,60℃反应1h取样;采用FACE电泳的方法检测其产物谱:首先,用溶于15%乙酸的过饱和7-氨基-1,3-萘二磺酸盐(ANDS)溶液与样品等体积混合(各取5μL),避光反应1小时;随后将等体积的NaCNBH3溶液加入混合物中,42℃温育过夜;在混合液中加入15μL的50%蔗糖溶液;并将标记后的产物以7μL/孔的比例用于电泳;使用ChemiDocTM MP成像***扫描图像,并将图像文件以TIFF格式存储。
突变体E287A与野生型WT在60℃下处理1h出芽短梗霉菌渣产物谱如图5所示。
上述实施例只为说明本发明的技术构思及特点,其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施,并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰,都应涵盖在本发明的保护范围之内。
Claims (7)
1.一种突变的几丁质酶,所述突变的几丁质酶的氨基酸序列如SEQ ID No.2所示。
2.编码权利要求1所述突变的几丁质酶的基因;优选的,所述基因的序列如SEQ IDNo.1所示。
3.含有权利要求2所述基因的重组载体;优选的,所述重组载体为重组表达载体;更优选,所述重组载体的骨架载体来源于pET-15b、pET-22b或pET-28a。
4.包含权利要求3所述重组载体的重组菌株;优选的,所述重组菌株为大肠杆菌;更优选,大肠杆菌BL21。
5.权利要求1所述的突变的几丁质酶、权利要求2所述的基因、权利要求3所述的重组载体或权利要求4所述的重组菌株在降解含有几丁质的材料中的应用。
6.权利要求1所述的突变的几丁质酶、权利要求2所述的基因、权利要求3所述的重组载体或权利要求4所述的重组菌株在制备几丁质寡糖中的应用。
7.一种降解含有几丁质或者含有几丁质寡糖的材料的方法,所述方法包括利用权利要求1所述的突变的几丁质酶、权利要求2所述的基因、权利要求3所述的重组载体或权利要求4所述的重组菌株对含有几丁质或者含有几丁质寡糖的材料进行处理的步骤。
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