CN116497005A - 热耐受性降低的β-木糖苷酶突变体K130GK137G及其应用 - Google Patents
热耐受性降低的β-木糖苷酶突变体K130GK137G及其应用 Download PDFInfo
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Abstract
本发明公开了一种热耐受性降低的β‑木糖苷酶突变体K130GK137G及其应用,涉及基因工程技术领域,该突变体K130GK137G的氨基酸序列如SEQ IDNO.1所示。本发明提供的突变体K130GK137G最适pH为4.5,最适温度为50℃,与氨基酸序列如SEQ ID NO.7所示的重组野生β‑木糖苷酶JB13GH39P28相比,该突变体K130GK137G的热性质发生了改变,其在50℃的稳定性下降,在60℃的活性变低且失活更迅速,有利于酶的安全使用,且在低温生物技术领域有较高的应用价值。
Description
技术领域
本发明涉及基因工程技术领域,具体涉及一种热耐受性降低的β-木糖苷酶突变体K130GK137G及其应用。
背景技术
木聚糖是半纤维素的主要成分且分布广泛,在木聚糖的降解过程中,β-1,4-内切木聚糖酶以内切的方式作用于木聚糖主链上的β-1,4糖苷键,降低聚合度,并将其切割为大小不一的低聚木糖片段和少量木糖;β-木糖苷酶主要负责木聚糖降解过程中寡聚糖的降解,以外切方式作用于低聚木糖的非还原性末端,并且释放木糖单体(PatelHetal.Internationaljournalofbiological macromolecules,2018,109:1260-1269.)。木糖可作为生产木糖醇、生物乙醇的原料;作为不能被人体吸收的无热量甜味剂,木糖可满足糖尿病人的需要。除降解寡聚糖外,β-木糖苷酶可以水解去除木糖残基来制造生物活性物质。因此,β-木糖苷酶可应用于食品、酿造、医疗、洗涤等行业。
实际应用中,一些工业生产过程在低温或常温条件下开展,例如食品工业中,β-木糖苷酶可用于萃取和澄清果汁,苹果汁在20℃澄清效果较好,而且果汁中的维生素B和C族属于热敏感物质高温下容易变性,使用不耐热的β-木糖苷酶,在低温下反应,不仅可以澄清果汁,还能保证营养;酿造工业中,β-木糖苷酶可以降低葡萄汁的粘稠度和浑浊度,提高过滤的澄清度,提升酒的稳定性,而红葡萄酒的发酵温度一般在20℃-32℃,故不耐热的酶具有的热稳定性差,受热容易失活的特点使其在低温生物技术领域有较高的应用价值(ChenQetal.TrendsinFoodScience&Technology,2022,125:126-135),此外,该特性使酶可以通过简单的操作控制反应过程,降低能耗。因此,不耐热的β-木糖苷酶更有利于应用于食品、酿造、洗涤等行业。
发明内容
本发明的目的是提供一种热耐受性降低的β-木糖苷酶突变体K130GK137G及其应用,该突变体K130GK137G的氨基酸序列如SEQ ID NO.1所示,与氨基酸序列如SEQ ID NO.7所示的重组野生酶JB13GH39P28相比,该突变体K130GK137G在50℃的稳定性下降,在60℃的活性变低且失活更迅速,在低温生物技术领域有较高的应用价值。
为了达到上述目的,本发明提供了一种热耐受性降低的β-木糖苷酶突变体K130GK137G,该突变体的氨基酸序列如SEQ ID NO.1所示。
本发明还提供了上述热耐受性降低的β-木糖苷酶突变体K130GK137G的编码基因,该编码基因的核苷酸序列如SEQ ID NO.2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,上述重组质粒选自pET-28a(+)。
本发明还提供了一种包含上述编码基因的重组菌。
优选地,上述重组菌选自大肠杆菌BL21(DE3)。
本发明提供的热耐受性降低的β-木糖苷酶突变体K130GK137G可被用于食品、酿酒或洗涤行业中,尤其在低温生物技术领域中,当需要热稳定性较差,受热容易失活的情况时,该突变体K130GK137G具有更大的适用性。
本发明提供的一种热耐受性降低的β-木糖苷酶突变体K130GK137G,拓宽了β-木糖苷酶在不同温度需求下的适用性,具有以下优点:
本发明提供的突变体K130GK137G与重组野生β-木糖苷酶JB13GH39P28相比,热性质发生了改变,其在50℃的稳定性下降,在60℃的活性变低且失活更迅速。重组野生酶JB13GH39P28的最适温度为50℃,在10℃、20℃、30℃、40℃和60℃时分别具有10.67%、23.94%、43.61%、69.58%和77.97%的酶活;突变体K130GK137G的最适温度也为50℃,在10℃、20℃、30℃、40℃和60℃时分别具有8.43%、21.68%、39.49%、76.89%和39.20%的酶活;50℃处理60min后,野生酶JB13GH39P28的酶活下降至83.17%,而突变体K130GK137G的酶活下降至28%;野生酶JB13GH39P28在60℃的半衰期约为10min,而突变体K130GK137G在60℃下处理10min酶活下降至5%。本发明提供的热耐受性降低的β-木糖苷酶突变体K130GK137G可应用于食品、酿酒、洗涤等行业。
附图说明
图1为本发明中重组野生酶JB13GH39P28和突变体K130GK137G的SDS-PAGE分析结果。
图2为本发明中重组野生酶JB13GH39P28和突变体K130GK137G的pH活性对比结果。
图3为本发明中重组野生酶JB13GH39P28和突变体K130GK137G在不同pH中稳定性对比结果。
图4为本发明中重组野生酶JB13GH39P28和突变体K130GK137G的热活性对比结果。
图5为本发明中重组野生酶JB13GH39P28和突变体K130GK137G在50℃的热稳定性对比结果。
图6为本发明中重组野生酶JB13GH39P28和突变体K130GK137G在60℃的热稳定性对比结果。
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。
本发明中所用到的部分实验材料和试剂:
1)菌株及载体:大肠杆菌EscherchiacoliBL21(DE3)购于北京博迈德基因技术有限公司;pET-28a(+)表达载体来源于江苏苏州泓迅生物科技有限公司。
2)酶类及其它生化试剂:pNPX购自Sigma公司;Nickel-NTA蛋白纯化树脂购自QIAGEN公司;QuickMutationTM基因定点突变试剂盒购自碧云天生物技术公司;Dpn1消化酶购自Takara生物技术公司;其他都为国产试剂(均可从普通生化试剂公司购买得到)。
3)培养基:LB培养基:Peptone10g,Yeastextract5g。NaCll0g,加蒸馏水至1000mL,pH自然(约为7)。固体培养基在此基础上加2.0%(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1重组野生β-木糖苷酶JB13GH39P28表达载体的构建和转化
1)从GenBank中下载氨基酸序列如SEQ ID NO.3所示的β-木糖苷酶JB13GH39(编号为AZC12019.1)及其核苷酸序列如SEQ ID NO.4所示的编码基因jb13gh39(编号为MG838204.1),去除jb13gh39编码信号肽的序列(第1-57位核苷酸)后,委托苏州泓迅生物科技股份有限公司对jb13gh39编码成熟肽的序列(第58-1614位核苷酸)进行密码子优化,结果序列的GC含量由60%降低至48%,将密码子优化后所获得的目的片段命名为jb13gh39p28,其核苷酸序列如SEQ ID NO.5所示,其长度为1557bp,也可以通过基因合成得到jb13gh39p28。
2)通过PCR的方式在jb13gh39p28的5’和3’端分别引入限制性酶切位点NcoⅠ(5’CCATGG3’)和XhoⅠ(5’CTCGAG3’),得到序列nxjb13gh39p28,其核苷酸序列如SEQ ID NO.6所示,通过限制性酶酶切上述产物和表达载体pET-28a(+),将nxjb13gh39p28和pET-28a(+)的酶切产物通过连接酶连接,获得包含nxjb13gh39p28的重组质粒nxjb13gh39p28-pET-28a(+)。
3)以nxjb13gh39p28-pET-28a(+)为模板,设计2条突变引物(F1和R1),具体序列如下所示。通过PCR去除重组时在C端组氨酸标签前引入的1个亮氨酸和1个谷氨酸序列,PCR反应参数为:95℃变性30sec;然后95℃变性15sec,70℃退火15sec,72℃延伸3min30sec,30个循环;72℃保温5min。最终获得包含jb13gh39p28的重组质粒jb13gh39p28-pET-28a(+),重组后的jb13gh39p28形成的核苷酸序列如SEQ ID NO.8所示,编码重组野生酶JB13GH39P28的氨基酸序列如SEQ ID NO.7所示。
突变引物序列如下(5’→3’):
F1(SEQ ID NO.9):
GAACGTAAACACCACCACCACCACCACTGAGAT
R1(SEQ ID NO.10):
GTGGTGGTGTTTACGTTCTTTCGGTGCAATACT
4)将重组质粒jb13gh39p28-pET-28a(+)通过热激方式转化到大肠杆菌BL21(DE3)中,获得包含jb13gh39p28的重组菌株BL21(DE3)/jb13gh39p28。
实验例2突变体K130GK137G表达载体的构建和转化
1)将实验例1中所得包含重组质粒jb13gh39p28-pET-28a(+)的重组菌株以0.1%含量接种于LB培养基中(含50μg/mL卡那霉素),37℃下过夜培养,通过试剂盒提取质粒。
2)以重组质粒jb13gh39p28-pET-28a(+)为模板,设计2条突变引物(F2和R2),具体序列如下所示。利用QuickMutationTM基因定点突变试剂盒进行突变,PCR反应参数为:95℃变性30sec;然后95℃变性15sec,70℃退火15sec,72℃延伸3min30sec,30个循环;72℃保温5min。PCR扩增得到包含核苷酸序列如SEQ ID NO.2所示的编码基因k130gk137g的线性化重组质粒k130gk137g-pET-28a(+),该编码基因编码的突变体为K130GK137G,其氨基酸序列如SEQ ID NO.1所示。k130gk137g和k130gk137g-pET-28a(+)也可以通过基因合成得到。
突变引物序列如下(5’→3’):
F2(SEQ ID NO.11):
AGGCAATACCAGTCATCCGGGACCGGACGGTTGGCGTAAT
R2(SEQ ID NO.12):
GGATGACTGGTATTGCCTCCCCAATAAAAGATGGTTTGCGG
3)对PCR产物进行Dpn1酶消化,于37℃消化3h。
4)将3)中的消化产物通过热激方式转入大肠杆菌BL21(DE3)中,得到包含K130GK137G编码基因k130gk137g的重组菌株BL21(DE3)/k130gk137g。
实验例3重组野生β-木糖苷酶JB13GH39P28和突变体K130GK137G的制备
1)将实验例2中所得的重组菌株BL21(DE3)/jb13gh39p28和BL21(DE3)/k130gk137g以0.1%的接种量分别接种于LB(含50μg/mL卡那霉素)培养液中,37℃、180rpm/min摇床中振荡16h进行活化。
2)将1)中活化的菌液以1%接种量分别接种到新鲜的LB(含50μg/mL卡那霉素)培养液中,于37℃、180rpm/min摇床中振荡培养约2-3h(OD600达到0.6-1.0)后,加入终浓度0.7mM的IPTG进行诱导,于20℃、160rpm/min摇床中继续振荡培养约20h诱导重组蛋白产生。
3)于4℃、6000rpm/min离心8min,收集菌体。用适量的pH=7.0McIlvaine buffer悬浮菌体后,于低温水浴下超声波破碎菌体。以上胞内浓缩的粗酶液经12000rpm/min离心l0min后,吸取上清并用Nickel-NTAAgarose和0-500mM的咪唑分别亲和和纯化目的蛋白。两个纯化所得的蛋白SDS-PAGE结果如图1所示,其中M为蛋白质Marker,W为野生酶,K130GK137G为突变体。结果表明,重组野生酶JB13GH39P28和突变体K130GK137G得到了表达和纯化,产物为单一条带。
4)把样品体积量100倍的透析液(pH=7.0McIlvainebuffer)加入透析装置。将透析袋(mw:14000)剪成适当长度(10-20cm)的小段,在沸水中煮沸30min后,用蒸馏水彻底清洗透析袋,将3)中得到的纯化的重组野生酶JB13GH39P28和突变体K130GK137G分别装入透析袋,透析袋两端预留3-5cm的长度用透析夹夹住。将透析样品放在透析缓冲液中,置于4℃透析,每隔2小时更换透析液,更换3次。
实验例4重组野生β-木糖苷酶JB13GH39P28及突变体K130GK137G的性质测定
1)重组野生酶JB13GH39P28和突变体K130GK137G的活性分析
活性测定方法采用PNP法,以对硝基苯基-β-D-吡喃木糖苷(pNPX)为底物测定纯化的重组野生酶JB13GH39P28及突变体K130GK137G活性。将pNPX溶于缓冲液中,使其终浓度为2mM;反应体系含50μL酶液,450μL的含底物的缓冲液;底物在反应温度下预热5min后,加入酶液后再反应10min,然后加2mL1MNa2CO3终止反应,冷却至室温后在405nm波长下测定释放出的pNP的量;1个酶活单位(U)定义为每分钟分解底物产生1μmolpNP所需的酶量。
2)重组野生酶JB13GH39P28及突变体K130GK137G在不同pH中的活性测定
将实验例3中纯化的酶置于pH=3.0-7.0(3.0、4.0、4.5、5.0、5.5、6.0、7.0)McIlvainebuffer中,于37℃的条件下进行酶促反应,测定纯化的重组野生酶JB13GH39P28及突变体K130GK137G的酶活。
重组野生酶JB13GH39P28和突变体K130GK137G在不同pH中活性对比结果如图2所示,结果表明,纯化的重组野生酶JB13GH39P28及突变体K130GK137G的最适pH为4.5。
3)重组野生酶JB13GH39P28及突变体K130GK137G在不同pH中的稳定性测定
将实验例3中纯化的酶置于pH3.0-10.0缓冲液中(pH=3.0、4.0、5.0、6.0、7.0、8.0:McIlvainebuffer,pH=9.0、10.0:1mol/LGlycine-NaOHBuffer),在37℃下处理60min。按照酶活力测定方法,在pH4.5及37℃条件下进行酶促反应,以pNPX为底物,反应10min,测定纯化的重组野生酶JB13GH39P28及突变体K130GK137G的酶活。
重组野生酶JB13GH39P28和突变体K130GK137G在不同pH中稳定性对比结果如图3所示,结果表明,与重组野生酶JB13GH39P28相比,突变体K130GK137G在碱性条件下的稳定性下降,在pH=4.0-9.0条件下处理60min,野生酶JB13GH39P28仍能保持80%以上的活性,而突变体K130GK137G在pH=4.0-7.0中有80%以上的酶活,经pH=8.0条件下处理60min,酶活下降至76.96%,经pH9.0处理60min,仅剩余5.31%的酶活。
4)重组野生酶JB13GH39P28及突变体K130GK137G的热活性测定
在pH=4.5的缓冲液中,于0-70℃下进行酶促反应。以pNPX为底物,反应10min,测定纯化的重组野生酶JB13GH39P28及突变体K130GK137G的酶活。
重组野生酶JB13GH39P28和突变体K130GK137G的热活性对比结果如图4所示,结果表明,纯化的重组野生酶JB13GH39P28和突变体K130GK137G最适温度为50℃。在10℃、20℃、30℃、40℃、60℃和70℃下,纯化的重组野生酶JB13GH39P28分别具有10.67%、23.94%、43.61%、69.58%、77.97%和19.50%的酶活,突变体K130GK137G分别有8.43%、21.68%、39.49%、76.89%、39.20%和0.56%的酶活。与野生酶JB13GH39P28相比,突变体K130GK137G在60℃下活性显著降低,低于野生酶约38%,并在70℃时失活,提示与野生酶JB13GH39P28相比,该突变体对60℃及以上的热耐受性低。
5)重组野生酶JB13GH39P28及突变体K130GK137G的热稳定性测定
将同样酶量的酶液置于50℃、60℃处理60min,每10min测一次酶活,在pH=4.5及37℃条件下进行酶促反应,以未处理的酶液作为对照。以pNPX为底物,反应l0min,测定纯化的重组野生酶JB13GH39P28和突变体K130GK137G的酶活。
重组野生酶JB13GH39P28和突变体K130GK137G在50℃的热稳定性对比结果如图5所示,其在60℃的热稳定性对比结果如图6所示,结果表明,与重组野生酶JB13GH39P28相比,突变体K130GK137G在50℃和60℃中热稳定性均降低,在50℃下处理60min后,野生酶JB13GH39P28有83%的酶活,而突变体K130GK137G的酶活下降至28%;野生酶JB13GH39P28在60℃时的半衰期约为10min,60℃时处理10min后野生酶JB13GH39P28还保持约50%活性,而突变体K130GK137G在60℃下处理10min后酶活下降至5%。
综上可知,本发明提供的突变体K130GK137G最适pH为4.5,最适温度为50℃,与氨基酸序列如SEQ ID NO.7所示的重组野生酶JB13GH39P28相比,该突变体K130GK137G的热性质发生了改变,其在50℃的稳定性下降,在60℃的活性变低且快速失活。本发明提供的热耐受性降低的β-木糖苷酶突变体K130GK137G在低温生物技术领域有较高的应用价值,尤其在食品、酿造、洗涤等行业中,当需要热稳定性较差,受热容易失活的情况时,本发明提供的突变体K130GK137G具有更大的适用性。
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。
Claims (7)
1.一种热耐受性降低的β-木糖苷酶突变体K130GK137G,其特征在于,该突变体的氨基酸序列如SEQ ID NO.1所示。
2.如权利要求1所述热耐受性降低的β-木糖苷酶突变体K130GK137G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.包含如权利要求2所述编码基因的重组质粒。
4.根据权利要求3所述的重组质粒,其特征在于,所述的质粒选自pET-28a(+)。
5.包含如权利要求2所述编码基因的重组菌。
6.根据权利要求5所述的重组菌,其特征在于,所述的菌选自BL21(DE3)。
7.如权利要求1所述热耐受性降低的β-木糖苷酶突变体K130GK137G在食品、酿酒或洗涤行业中的应用。
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CN116555233B (zh) * | 2023-03-10 | 2024-06-04 | 云南师范大学 | 热不稳定的β-木糖苷酶突变体E179GD182G及其应用 |
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