CN116445456A - 耐酸性提高的β-甘露聚糖酶突变体N186D及其制备和应用 - Google Patents
耐酸性提高的β-甘露聚糖酶突变体N186D及其制备和应用 Download PDFInfo
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Abstract
本发明公开了一种耐酸性提高的β‑甘露聚糖酶突变体N186D及其制备和应用,涉及基因工程技术领域。本发明提供的突变体氨基酸序列如SEQ ID NO.3所示,编码该突变体基因的核苷酸序列如SEQ ID NO.4所示,该突变体是由氨基酸序列如SEQ ID NO.1所示的野生型β‑甘露聚糖酶突变所得,该突变体N186D的最适反应pH为6.5,在pH为4的缓冲液中耐受1h后,N186D保留74.6%的活性,而野生型β‑甘露聚糖酶仅保留55.92%的活性。本发明提供的耐酸性提高的β‑甘露聚糖酶突变体N186D在动物养殖和工业生产中具有较好的应用前景。
Description
技术领域
本发明涉及基因工程技术领域,具体涉及一种耐酸性提高的β-甘露聚糖酶突变体N186D及其制备和应用。
背景技术
β-甘露聚糖酶(EC 3.2.1.78)是一类半纤维素酶,能特异性地催化β-1,4糖苷键断裂,其作为绿色、环保、经济的饲用酶添加剂之一被广泛应用到动物养殖行业以此提高生产性能,降低养殖成本。β-甘露聚糖酶可以酶解非常规饲料原料中的抗营养因子甘露聚糖,生成动物可吸收利用的低聚寡糖,起到促进肠道益生菌增殖,抑制有害菌定殖的作用。通常情况下,动物胃肠道呈现弱酸环境,pH在2.0~5.5之间,因此,β-甘露聚糖酶需具备能在酸性介质中稳定,高效地发挥催化活性的特征。但目前现有的β-甘露聚糖酶的酸耐受性低,仍难以完全满足动物养殖和工业生产的需要。
发明内容
本发明的目的是提供一种耐酸性提高的β-甘露聚糖酶突变体N186D及其制备和应用,为解决β-甘露聚糖酶酸耐受性低,难以满足动物养殖和工业应用的需要等问题,本发明提供的β-甘露聚糖酶突变体N186D具有较高的酸耐受性,可被应用于动物养殖和工业生产中,具备显著的应用前景。
为了达到上述目的,本发明提供了一种耐酸性提高的β-甘露聚糖酶突变体N186D,该突变体N186D的氨基酸序列如SEQ ID NO.3所示,该突变体是由氨基酸序列如SEQ IDNO.1所示的β-甘露聚糖酶的第186位的天冬酰胺突变为天冬氨酸所得,与氨基酸序列如SEQID NO.1所示的野生型β-甘露聚糖酶相比,该突变体在pH为4~6时的耐酸性提高,尤其在pH=4的缓冲液中耐受1h后,N186D还保留74.6%的活性。
本发明提供的β-甘露聚糖酶突变体N186D可被应用于养殖业中,尤其可用于制备动物饲料。
本发明提供的β-甘露聚糖酶突变体N186D可被应用于β-甘露聚糖的工业生产中。
本发明还提供了该β-甘露聚糖酶突变体N186D的编码基因,其核苷酸序列如SEQID NO.4所示。
本发明还提供了一种包含上述编码基因的重组质粒。
优选地,上述的重组质粒的空载体选自pET-28a(+)。
本发明还提供了一种包含上述重组质粒的重组表达菌。
优选地,上述的表达菌选自大肠杆菌BL21(DE3)。
本发明还提供了一种上述β-甘露聚糖酶突变体N186D的制备方法,包含如下步骤:
S1.以含有核苷酸序列如SEQ ID NO.2所示的野生型β-甘露聚糖酶编码基因的载体为模板,以核苷酸序列如SEQ ID NO.15和SEQ ID NO.16所示的位点突变引物进行突变扩增,获得包含核苷酸序列如SEQ ID NO.4所示突变体编码基因的重组质粒;
S2.将步骤S1所得的重组质粒转入表达菌中,表达并纯化,得到如上所述的β-甘露聚糖酶突变体N186D。
本发明的耐酸性提高的β-甘露聚糖酶突变体N186D及其制备和应用,解决了现有的β-甘露聚糖酶酸耐受性低,难以满足动物养殖和工业应用的需要等问题,具有以下优点:
本发明通过对氨基酸序列如SEQ ID NO.1所示的野生型β-甘露聚糖酶的第186位的天冬酰胺突变为天冬氨酸,所得的突变体N186D和野生型β-甘露聚糖酶的最适反应pH为6.5。本发明提供的β-甘露聚糖酶突变体N186D具有较高的酸耐受性,在pH为4的缓冲液中耐受1h后,突变体N186D还能保留74.6%的活性,而野生型β-甘露聚糖酶仅保留55.92%的活性,这种高酸耐受性的β-甘露聚糖酶可被应用于动物养殖和工业生产中,具备显著的应用前景。
附图说明
图1为本发明中野生型β-甘露聚糖酶突变位点展示。
图2为本发明突变体β-甘露聚糖酶阳性重组子验证。
图3为本发明中野生型β-甘露聚糖酶与突变体N186D最适pH测定结果。
图4为本发明中野生型β-甘露聚糖酶与突变体N186D的pH耐受测定结果β-甘露聚糖酶突变体。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明实验例中的实验材料和试剂如下:
1、菌株和载体:大肠杆菌(Escherichia coli)BL21(DE3)购自北京博迈德基因技术有限公司,E.coli DH5α购自北京擎科生物技术有限公司,pET-28a-manDL4载体由云南能源持续开发利用教育部工程中心提供,其中,pET-28a-manDL4载体中连接有核苷酸序列如SEQ ID NO.2所示的野生型β-甘露聚糖酶编码基因manDL4,该野生型β-甘露聚糖酶ManDL4的氨基酸序列如SEQ ID NO.1所示。
2、主要试剂:QuickMutationTM基因定点突变试剂盒购自碧云天生物技术公司、IPTG(异丙基硫代半乳糖苷)购自德国BioFroxx公司。
3、主要培养基:
LB培养基:0.5%酵母粉,1%蛋白胨,1%NaCl,pH自然(约为7),固体培养基在此基础上加2%(w/v)琼脂。
说明:以下实施例中未作具体说明的分子生物学实验方法,均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行,或者按照试剂盒和产品说明书进行。
实验例1β-甘露聚糖酶耐酸性能改造突变位点确定
通过PyMOL可视化野生型β-甘露聚糖酶ManDL4的三维结构,查阅相关文献,避开该酶的催化活性位点(W103、W203、W329、W333、E198、E297),利用DNAMAN软件,选择不同物种来源的β-甘露聚糖酶进行多序列比对获得保守氨基酸位点,选择暴露在蛋白质表面的中性和碱性非保守氨基酸,包括以下7个氨基酸位点(Q90、M122、H146、K157、K158、N186、K228),其中,字母表示该位点的氨基酸,字母后的数字表示该位点在氨基酸序列的物理位置,将上述位点的氨基酸突变为谷氨酸或者天冬氨酸两类酸性氨基酸,选择和野生型相比负电势变化范围明显的位点作为候选突变位点,PyMOL可视化呈现野生型酶突变位点如图1所示。
实验例2含有β-甘露聚糖酶突变体的重组质粒构建
分别将氨基酸序列第90位的Q突变为谷氨酸E,记为突变体Q90E;第122位的M突变为天冬氨酸D,记为突变体M122D;第146位的H突变为天冬氨酸D,记为突变体H146D;第157位的K突变为谷氨酸E,记为突变体K157E;第158位的K突变为谷氨酸E,记为突变体K158E;第186位的N突变为天冬氨酸D,记为突变体N186D;第228位的K突变为谷氨酸E,记为突变体K228E;分别设计构建突变体的位点突变引物,具体引物序列如下所示,以pET-28a-manDL4质粒为模板,对β-甘露聚糖酶进行定点突变,获得突变体重组质粒。
突变引物序列分别为(5’→3’):
Q90E-F(SEQ ID NO.5):
GGGAATCGCCTGCTATTTACGGCTGCGATTAT;
Q90E-R(SEQ ID NO.6):
AATAGCAGGCGATTCCCCGGTGGCGCTTCGGAT;
M122D-F(SEQ ID NO.7):
CAGCGATTTAGAGTCGTATTGGAAAAATGGTGGAAT;
M122D-R(SEQ ID NO.8):
ACGACTCTAAATCGCTGTTGCAGCTTACATCT;
H146D-F(SEQ ID NO.9):
CTTTTCAGTCAGGGGATTTTAAAACACCGATTACAAACGATC;
H146D-R(SEQ ID NO.10):
ATCCCCTGACTGAAAAGCAGGATTCGCCAGGT;
K157E-F(SEQ ID NO.11):
CAAACGATCAGTATGAAAAAATACTAGATTCTTCAACAGCAGAA GG;
K157E-R(SEQ ID NO.12):
TTCATACTGATCGTTTGTAATCGGTGTTTTAA;
K158E-F(SEQ ID NO.13):
CGATCAGTATAAAGAAATACTAGATTCTTCAACAGCAGAAGGG;
K158E-R(SEQ ID NO.14):
TTTCTTTATACTGATCGTTTGTAATCGGTGTT;
N186D-F(SEQ ID NO.15):
AAGAGCTGGAGGACCAAGGTGTGCCTGTTTTGTTCA;
N186D-R(SEQ ID NO.16):
TTGGTCCTCCAGCTCTTGAAGTCCGTCAGCAA;
K228E-F(SEQ ID NO.17):
GCTCTACAAGGAAATCTATCATTATATGACCGACACAAGAG;
K228E-R(SEQ ID NO.18):
AGATTTCCTTGTAGAGCTGTTTATATAAAGAGATTCTTT。
定点突变扩增反应体系如下:
总体系25μL:16.5μL无菌水、2.5μL 10×BeyoFusion Buffer、上下游(F和R)引物各1.0μL、2.5μL dNTPmix、1.0μL模板质粒、0.5μLBeyoFusion DNApolymerase。
定点突变反应扩增程序如下:
先95℃预变性3min;再95℃变性30s、60℃退火30s、68℃延伸7min,循环20次后;68℃延伸15min后4℃保存。
利用上述的7对突变引物、反应体系和程序分别突变上述7个位点,获得7个突变质粒的突变产物。将定点突变扩增后的突变质粒产物分别转化至E.coli DH5α感受态细胞,利用pET-28a载体通用引物T7和T7ter进行菌落PCR扩增β-甘露聚糖酶突变体,其中,突变位点为Q90的突变体重组质粒的菌落PCR电泳结果如图2所示,其中泳道M为DNAladder,泳道1~5分别为5个不同的单菌落的PCR样,同时对所得的另外6个突变体(M122、H146、K157、K158、N186、K228)重组质粒分别做菌落PCR鉴定,并将扩增产物送至生物公司测序对β-甘露聚糖酶突变体重组质粒进行验证。
实验例3野生型β-甘露聚糖酶及其突变体的表达和纯化
将实验例2构建成功的重组质粒转化至大肠杆菌BL21(DE3)中,同时将质粒pET-28a-manDL4转化至大肠杆菌BL21(DE3)中,分别涂布在含有卡那抗生素平板,37℃培养12h,所得的阳性转化子即分别为野生型β-甘露聚糖酶和β-甘露聚糖酶突变体的重组表达菌。分别挑取阳性转化子接种至LB培养基中,于37℃、200r/min下过夜培养,按照1%的接种量接种至大瓶LB培养基(400mL)中培养,当OD600达到0.6时,加入终浓度为0.07mM的IPTG(异丙基硫代半乳糖苷),置于20℃、160r/min恒温摇床下诱导发酵20h。
在4℃、5000r/min条件下,离心10min收集菌体。用50mM、pH为7的磷酸缓冲洗吹悬浮菌体,超声波破碎重悬的菌体,4℃、10000r/min离心10min后收集上清液。将上清液用Ni2 +-柱纯化,咪唑洗脱液的浓度为500mM,分别获得了野生型β-甘露聚糖酶和β-甘露聚糖酶突变体酶液。
实验例4野生型β-甘露聚糖酶及其突变体的酶学性质测定
最适反应pH以及pH耐受测定:
配置不同pH的缓冲液,柠檬酸-磷酸氢二钠缓冲液(pH=3.0、4.0、4.5、5.0、5.5、6.0、6.5、7.0),Tris-HCl缓冲液(pH=8.0、9.0),甘氨酸-NaOH缓冲液(pH=10.0、11.0、12.0)。
以1%角豆胶溶液作为底物,将底物和pH分别为3.0、4.0、4.5、5.0、5.5、6.0、6.5、7.0、8.0、9.0的缓冲液混合,在37℃下,测定野生型β-甘露聚糖酶及其突变体的最适反应pH。
根据最适反应pH测定结果可知,野生型β-甘露聚糖酶及7个突变体的最适反应pH均为6.5,但突变体N186D在pH=5.0的条件下也具有很高的活性。野生型β-甘露聚糖酶及突变体N186D的最适反应pH测定结果如图3所示,其中WT为野生型β-甘露聚糖酶,N186D为β-甘露聚糖酶突变体N186D。
将所有突变体用pH为3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0、12.0的缓冲液按照浓度梯度依次稀释,直至反应产物的吸光度为0.5~1.5之间,因吸光度数值在这个范围内时的准确较高,在稀释250倍后的酶液达到该范围条件。吸取稀释250倍的酶液500μL置于37℃下耐受1h后取出放冰上,测定相对剩余酶活,测定条件为37℃、pH=5.5。
根据pH耐受测定结果可知,在pH为4~6的缓冲液中耐受1h后,突变体N186D突变体在所有突变体中的相对剩余酶活最高,约为74~91%,而野生型β-甘露聚糖酶仅保留约55%-87%的活性,尤其在pH=4的缓冲液中耐受1h后,N186D还保留74.6%的活性,而野生型β-甘露聚糖酶仅保留55.92%的活性;而突变体N186D在pH为8~11时的相对剩余酶活显著低于野生型酶,可见突变体N186D的耐酸性能显著提高,其余突变体和野生型相比区别不显著。其中,野生型β-甘露聚糖酶及突变体N186D的pH耐受测定结果如图4所示。同时,对实验例2中构建成功的N186D突变体的重组质粒进行测序,可知该突变体N186D编码基因的核苷酸序列如SEQ ID NO.4所示,该突变体N186D的氨基酸序列如SEQ ID NO.3所示。
综上结果表明,本发明提供的β-甘露聚糖酶突变体N186D的酸耐受性与野生型相比显著提高,而这种酸耐受性提高的β-甘露聚糖酶可被应用于养殖业和工业生产中,尤其可用于制备动物饲料,大大提高了饲料的吸收率,有助于提高养殖过程中的瘦肉率,具有巨大的应用价值。
尽管本发明的内容已经通过上述优选实施例作了详细介绍,但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后,对于本发明的多种修改和替代都将是显而易见的。因此,本发明的保护范围应由所附的权利要求来限定。
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Claims (10)
1.一种耐酸性提高的β-甘露聚糖酶突变体N186D,其特征在于,该突变体N186D的氨基酸序列如SEQ ID NO.3所示,与氨基酸序列如SEQ ID NO.1所示的野生型β-甘露聚糖酶相比,该突变体在pH为4~6时的耐酸性提高。
2.如权利要求1所述的β-甘露聚糖酶突变体N186D在养殖业中的应用。
3.根据权利要求2所述的应用,其特征在于,所述的应用包括制备动物饲料。
4.如权利要求1所述的β-甘露聚糖酶突变体N186D在β-甘露聚糖工业生产中的应用。
5.如权利要求1所述的β-甘露聚糖酶突变体N186D的编码基因,其特征在于,该编码基因的核苷酸序列如SEQ ID NO.4所示。
6.一种包含如权利要求5所述编码基因的重组质粒。
7.根据权利要求6所述的重组质粒,其特征在于,所述重组质粒选用pET-28a(+)。
8.一种包含如权利要求6所述重组质粒的重组表达菌。
9.根据权利要求8所述的重组表达菌,其特征在于,所述重组表达菌选用大肠杆菌BL21(DE3)。
10.如权利要求1所述β-甘露聚糖酶突变体N186D的制备方法,其特征在于,包含如下步骤:
S1.以含有核苷酸序列如SEQ ID NO.2所示的野生型β-甘露聚糖酶编码基因的载体为模板,以核苷酸序列如SEQ ID NO.15和SEQ ID NO.16所示的位点突变引物进行突变扩增,获得包含核苷酸序列如SEQ ID NO.4所示突变体编码基因的重组质粒;
S2.将步骤S1所得的重组质粒转入表达菌中,表达并纯化,得到β-甘露聚糖酶突变体N186D。
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