CN116536289A - 具有溶菌活性的几丁质酶及其突变体和应用 - Google Patents
具有溶菌活性的几丁质酶及其突变体和应用 Download PDFInfo
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Abstract
本发明公开了具有溶菌活性的几丁质酶及其突变体和应用。本发明首先提供了具有溶菌活性的几丁质酶,其氨基酸序列为SEQ ID No.1所示。本发明进一步对几丁质酶进行Q8K、C28K、V121R、Q124E、D136E、N209R或K218R中的任何一种氨基酸单位点突变获得单位点突变体以及进行C28K/D136E双位点突变获得双位点突变体。本发明所提供的几丁质酶或各突变体具有较高的溶菌酶活性,可用于溶解或破坏细菌的细菌壁,实现杀菌或抑菌的效果,在杀灭有害细菌、食品防腐等方面具有应用前景。
Description
技术领域
本发明涉及几丁质酶,尤其涉及具有溶菌活性的几丁质酶及其突变体和应用,属于几丁质酶及其应用领域。
背景技术
几丁质酶(EC3.2.1.14)是一类能特异作用于几丁质糖苷键,将天然大分子高聚几丁质水解成低聚糖的一类糖苷水解酶。几丁质酶是生物体内降解或利用几丁质的关键,一方面几丁质酶能通过水解病原真菌细胞壁,破坏害虫体壁及杀虫增效等机制发挥生物防治作用,另一方面几丁质酶还能通过催化几丁质水解,促进其自然界循环的作用,维持生态系统平衡(PATEL S, GOYAL A. Chitin and chitinase: Role in pathogenicity, aller-genicity andhealth[J]. Int J Biol Macromol, 2017, 1864(9): 1253-1259)。
基于氨基酸序列相似性的糖苷水解酶家族分类方法,几丁质酶主要来源于CAZy数据库中GH18、GH19和GH20家族,而GH19家族的几丁质酶的催化结构域通常是富含α螺旋的溶菌酶样结构域,因而也常常具有溶菌活性。
细菌细胞壁主要成分为肽聚糖,其由 N-乙酰胞壁酸(NAM)、N-乙酰氨基葡糖(NAG)和4个氨基酸所组成的“尾巴”相结合而成。肽 聚 糖 的 基 本 双 糖 单 位由 N-乙 酰 胞 壁 酸(NAM)和 N-乙酰氨基葡糖(NAG)通过β-1,4糖苷键结合而形成。肽“尾”通过 D-乳酰羧基与 N-乙酰胞壁酸第3位碳原子相结合,再通过肽“桥”(肽键或少数几个氨基酸)使肽“尾”与肽“尾”相连接。N-乙酰胞壁酸(NAM)、N-乙酰氨基葡糖(NAG)、肽“尾”与肽“桥”(革兰氏阳性菌)或肽键(革兰氏阴性菌)相互连接构成肽聚糖片层,作为细胞壁骨架。由此可知,上述细胞壁的结构中,任何化学键的断裂都能破坏细菌的细胞壁,从而达到溶菌的效果。溶菌酶通过切断 N-乙酰胞壁酸与 N-乙酰氨基葡糖间的β-1,4糖苷键,在内部渗透压的作用下使细胞壁破裂、内容物逸出而达到溶菌的目的。
现有的几丁质酶大多存在溶菌活性较低的缺陷,限制了其在产业上的应用,有待改进。
发明内容
本发明的目的之一是提供具有溶菌活性的几丁质酶;
本发明的目的之二是提供几丁质酶突变体;
本发明的目的之三是提供含有几丁质酶编码基因或几丁质酶突变体编码基因的重组表达载体以及含有该重组表达载体的重组宿主细胞。
本发明的目的之四是将所述的几丁质酶或几丁质酶突变体作为溶菌酶应用于溶解细菌。
本发明的上述目的是通过以下技术方案来实现的:
本发明的一方面是通过对牛瘤胃中原虫的基因组进行分析,挖掘得到具有溶菌活性的几丁质酶G732,其氨基酸序列为SEQ ID No.1所示,其信号肽的氨基酸序列为SEQ IDNo.2所示。
本发明中所述的几丁质酶G732编码基因也属于本发明的保护范畴之内。
本发明的另一方面是提供几丁质酶的G732单位点突变体,所述几丁质酶G732的单位点突变体是将几丁质酶G732的氨基酸序列进行Q8K、C28K、V121R、Q124E、D136E、N209R或K218R中的任何一种氨基酸单位点突变所获得的单位点突变体;优选的,所述突变体是将几丁质酶G732的氨基酸序列进行C28K或D136E中的任何一种氨基酸单位点突变所获得的单位点突变体;更优选的,所述突变体是将几丁质酶G732的氨基酸序列进行C28K单位点突变所获得的单位点突变体。
本发明所述氨基酸单位点突变“C28K”表示将SEQ ID No.1所示的氨基酸序列的第28位氨基酸由半胱氨酸(C)突变成赖氨酸(K);其余的单位点突变的表述依此类推。
本发明中所述的各单位点突变体的编码基因也属于本发明的保护范畴之内。
本发明的另一方面是提供几丁质酶的G732双位点突变体,所述几丁质酶G732的双位点突变体是将几丁质酶G732的氨基酸序列进行C28K/D136E双位点突变所获得的双位点突变体。
本发明所述氨基酸双位点突变“C28K/D136E”表示将SEQ ID No.1所示的氨基酸序列的第28位氨基酸由半胱氨酸(C)突变成赖氨酸(K)同时将第136位氨基酸由天冬氨酸(D)突变为谷氨酸(E)。
本发明的另一方面是提供了了含有所述几丁质酶G732编码基因或几丁质酶G732的各突变体编码基因的重组表达载体或重组宿主细胞;其中,所述重组表达载体可以为重组原核表达载体或重组真核载体。
本发明的另一方面是提供制备几丁质酶G732或几丁质酶G732的各突变体的方法,包括:
(1)将几丁质酶G732编码基因或几丁质酶G732的各突变体编码基因可操作的与表达调控元件连接构建得到重组表达载体;
(2)将重组表达载体转化宿主细胞,培养宿主细胞,诱导表达重组蛋白,纯化,即得。
本发明所提供的几丁质酶G732或几丁质酶G732的各突变体具有优良的溶菌酶活性,因此可作为溶菌酶应用于溶解或破坏细菌的细菌壁,实现杀菌或抑菌的效果,在杀灭有害细菌、食品防腐等方面具有广泛的应用前景。
本发明所涉及到的术语定义
除非另外定义,否则本文所用的所有技术及科学术语都具有与本发明所属领域的普通技术人员通常所了解相同的含义。
术语“突变”和“突变体”在此具有它们的常用含义,指的是在核酸或多肽序列中的遗传的、天然存在的或引入的变化,它们的意义与本领域人员通常所知的意义相同。
术语“宿主细胞”或“重组宿主细胞”意指包含本发明多核苷酸的细胞,而不管使用何种方法进行插入以产生重组宿主细胞,例如直接摄取、转导、f配对或所属领域中已知的其它方法。外源性多核苷酸可保持为例如质粒的非整合载体或者可整合入宿主基因组中。
术语“转化”指真核细胞由于外源DNA掺入而获得新的遗传标志的过程。
附图说明
图1 为几丁质酶G732的异源表达与抑菌活性初筛结果;A :几丁质酶G732的异源表达结果;B :几丁质酶G732的抑菌活性初筛结果。
图2 为几丁质酶 G732的温度稳定性与最适反应pH检测结果;A :几丁质酶G732的温度稳定性检测结果;B :几丁质酶G732的最适反应pH检测结果。
图3为几丁质酶G732中氨基酸频率计算。
图4为几丁质酶G732单点突变体筛选;其中,较大黑点标记选中的突变位点。
图5为几丁质酶 G732单点突变体对藤黄微球菌抑菌活性检测;A:Q8K、C28K、T97R、V121R和Q124E活性初筛结果;B:D136E、A155R、N209R、K218R和N239K活性初筛结果。
图6 为几丁质酶G732双点突变体C28K/D136E对藤黄微球菌的酶活测定;A:比浊法酶活测定结果;B:国标法酶活测定结果。
具体实施方式
以下结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
试验例1几丁质酶G732的表达及溶菌活性测定试验
首先将几丁质酶基因g732构建到大肠杆菌表达载体pET30a中,转化到大肠杆菌BL21(DE3)中进行异源表达。SDS-PAGE结果显示,几丁质酶G732主要以可溶性蛋白的形式存在于大肠杆菌细胞内,大小为37KDa左右(图1中的A)。随后对几丁质酶G732溶菌活性进行初筛,结果表明G732对大肠杆菌(G-)不具有溶菌活性,但对溶壁微球菌(G+)的杀伤力显著(图1中的B)。
试验例2 几丁质酶G732的温度稳定性、最适反应pH和溶菌活性试验
为了进一步研究几丁质酶G732的功能,本试验选用国标法对其溶菌活性和性质进行研究。该方法主要参考GB 1886.257—2016,食品安全国家标准,食品添加剂—溶菌酶,该方法通过溶菌酶能够裂解藤黄微球菌的细胞壁,造成藤黄微球菌溶解而引起的溶液吸光值的降低。
温度稳定性检测:将纯化的几丁质酶G732置于4℃至65℃温度梯度内分别孵育15min,冷却1 min后,测定几丁质酶G732的剩余酶活。结果如图2A所示,在4℃至45℃范围内,几丁质酶G732能保持较高的溶菌活性;而在55℃孵育15 min后,几丁质酶G732的溶菌活性急速下降,55-65℃时几乎失去活性。
最适反应pH检测:使用不同pH的缓冲液分别重悬并稀释藤黄微球菌,将几丁质酶G732稀释到一定浓度后,加入至不同pH的菌悬液体系中进行反应,测定几丁质酶G732溶菌活性的最适pH。结果显示,在pH4.5-6.5范围内,G732的溶菌活性逐渐升高,pH6.5时,达到最高,当pH>7.5时,G732溶菌活性丧失。因此,几丁质酶G732的最适反应pH为6.5(图2中的B)。
溶菌活性检测:在最适反应pH及室温条件下,使用国标法对几丁质酶G732的溶菌活性进行检测,结果显示几丁质酶G732的溶菌活性为30303.03 U/mg。
试验例3几丁质酶G732的突变体设计以及活性筛选试验
为了鉴定几丁质酶G732中与溶菌活性相关的关键氨基酸位点,将几丁质酶G732序列与Uniparc数据库进行序列比对,获得与几丁质酶G732相似性较高的序列,然后计算出在所有序列的相同位点出现某一氨基酸的频率(图3),并根据这些氨基酸频率计算差异值,差异值是相同位点出现频率最高的某一个氨基酸的频率数减去模板中同一位点的氨基酸频率数,并根据差异值进行排序,排名靠前的序列说明在这一位点序列的保守性越强,即可作为优选突变体(图4);研究发现酸性氨基酸能够增强酶对细菌细胞壁的水解能力,而碱性氨基酸能够增加酶对底物细菌的结合能力,因而选择排名靠前且突变为带电氨基酸的10个突变体进行构建,这10个突变位点具体为:Q8K、C28K、T97R、V121R、Q124E、D136E、A155R、N209R、K218R、N239K。
1.单点突变体的活性初筛
将单点突变体分别成功构建和异源表达后,分别检测其对藤黄微球菌的溶菌活性实验,分两组进行。结果显示,单点突变体Q8K、C28K、V121R、Q124E、D136E、N209R和K218R的溶菌活性较野生型均有不同程度的提高(图5)。分别选择两组中溶菌活性最好的单点突变体 C28K和D136E进行后续实验。
2.组合突变体的活性检测
为了进一步增强几丁质酶G732的溶菌效果,将溶菌效果较为显著的突变位点C28K与D136E进行叠加构建双点突变体,即: C28K/D136E。双点突变体异源表达成功后,对其溶菌活性进行检测。比浊法结果显示突变体C28K/D136E的溶菌效果较野生型提升明显(图6中的A);进一步用国标法测定酶活显示C28K/D136E相对酶活是野生型的约2.5倍(图6中的B)。分析该位点突变情况可知28位的C存在于该蛋白的LysM结构域处,即决定着该酶对肽聚糖的结合能力,而碱性氨基酸的存在能够增强酶对底物细菌细胞壁的结合能力,因而该位点C28K的突变能够显著增强该酶对细菌的结合和水解效果。
Claims (10)
1.具有溶菌活性的几丁质酶,其特征在于,其氨基酸序列为SEQ ID No.1所示。
2.权利要求1所述的几丁质酶的编码基因。
3.权利要求1所述的几丁质酶的单位点突变体,其特征在于,所述单位点突变体是将权利要求1所述的几丁质酶的氨基酸序列进行Q8K、C28K、V121R、Q124E、D136E、N209R或K218R中的任何一种氨基酸单位点突变所获得的单位点突变体。
4.权利要求3所述的单位点突变体的编码基因。
5.权利要求1所述的几丁质酶的双位点突变体,其特征在于,所述的双位点突变体是将权利要求1所述的几丁质酶的氨基酸序列进行C28K/D136E双位点突变所获得的双位点突变体。
6.权利要求5所述的双位点突变体的编码基因。
7.含有权利要求2、权利要求4或权利要求6所述的编码基因的重组表达载体。
8.一种制备权利要求1所述的几丁质酶的方法,包括:
(1)将几丁质酶的编码基因可操作的与表达调控元件连接构建得到重组表达载体;
(2)将重组表达载体转化宿主细胞,培养宿主细胞,诱导表达重组蛋白,纯化,即得。
9.一种制备权利要求3所述的几丁质酶的单位点突变体或权利要求5所述的几丁质酶的双位点突变体的方法,包括:
(1)将所述的几丁质酶的单位点突变体的编码基因或几丁质酶的双位点突变体的编码基因可操作的与表达调控元件连接构建得到重组表达载体;
(2)将重组表达载体转化宿主细胞,培养宿主细胞,诱导表达重组蛋白,纯化,即得。
10.权利要求1所述的几丁质酶、权利要求3所述的单位点突变体或权利要求5所述的双位点突变体在溶解细菌细胞壁中的应用。
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