CN109355275A - 高热稳定性β-葡萄糖苷酶突变体及其应用 - Google Patents
高热稳定性β-葡萄糖苷酶突变体及其应用 Download PDFInfo
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- CN109355275A CN109355275A CN201811393399.9A CN201811393399A CN109355275A CN 109355275 A CN109355275 A CN 109355275A CN 201811393399 A CN201811393399 A CN 201811393399A CN 109355275 A CN109355275 A CN 109355275A
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- glucosidase
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Abstract
本发明公开了高热稳定性β‑葡萄糖苷酶突变体及其应用。本发明提供了一种热稳定性提高的β‑葡萄糖苷酶突变体,是在野生型β‑葡萄糖苷酶氨基酸序列的基础上突变得到,突变位点为:T167I/V181F/A298G或T167I/V181F/K186T/A187E/A298G,得到突变体2R1和4R1的氨基酸序列分别如SEQ ID NO.1和2所示。该β‑葡萄糖苷酶突变体2R1和4R1在50℃下的半衰期分别是野生酶Ks5A7的285倍和8640倍,对纤维二糖的水解活性分别是野生酶的1.75和1.5倍,且依然保持了对葡萄糖的高耐受性。因此,利用该β‑葡萄糖苷酶突变体可以大大提高β‑葡萄糖苷酶的热稳定性,为其在食品、生物能源、纺织、造纸、医药领域提供了应用基础。
Description
技术领域
本发明属于基因工程和酶工程技术领域,更具体地,涉及高热稳定性β-葡萄糖苷酶突变体及其应用。
背景技术
纤维素是地球上分布最广泛、含量最丰富的可再生资源,全球每年通过光合作用产生的纤维素多达1011-1012吨。纤维素的降解产物葡萄糖,是可替代能源生物乙醇的原料。因此,纤维素的高效降解对可替代能源的发展至关重要。纤维素酶是一类多组分酶系的总称,这些酶通过协同作用,分解纤维素产生纤维二糖和纤维寡糖,最终将其水解为葡萄糖。其中,β-葡萄糖苷酶能水解纤维二糖及纤维寡糖生成葡萄糖。
β-葡萄糖苷酶(EC 3.2.1.21),全称β-D-吡喃葡萄糖苷葡萄糖水解酶,是一类可以水解糖类和糖苷化合物的非还原性末端糖苷键,释放出相应糖苷配基和葡萄糖的酶。该类酶广泛分布于古菌、细菌和真核生物中,发挥着多种生物学功能,例如,微生物和动物体内生物质转化、糖脂和糖苷裂解、植物的木质化以及细胞壁的寡糖代谢等。β-葡萄糖苷酶广泛应用于食品、生物能源、纺织、造纸、医药等各个领域,在降解纤维素;水解果汁、果酒中的风味前体,释放风味活性物质,改善饮料的风味和品质;水解异黄酮苷元,增加食品和饲料中异黄酮的含量;合成寡糖和非离子表面活性剂烷基糖苷等方面发挥巨大作用。
为了进一步推动β-葡萄糖苷酶在工业中的应用,对其现有性能提出了更高的要求,如长时间内保持活性稳定,耐受更高浓度的葡萄糖,在极端环境中保持高的活性(极端温度或者pH值等)或者可以接受不同的底物(包括非天然底物)。其中,酶的热稳定性对于工业应用来说十分重要,高温条件下,酶的反应速度更快,能缩短反应周期,提高时空产率,节约成本,也有利于避免反应过程中被其他微生物污染。纤维素降解的工业反应温度在50℃,而野生β-葡萄糖苷酶的热稳定性较差,在50℃下的半衰期仅为1min,不利于β-葡萄糖苷酶在工业中的应用。因此,对提高β-葡萄糖苷酶稳定性的研究探索显得尤为重要。
发明内容
本发明要解决的技术问题是克服现有β-葡萄糖苷酶热稳定性差的问题,采用突变的方式,提供热稳定性显著提高、对纤维二糖的水解活性显著提高,且依然保持对葡萄糖的高耐受性的β-葡萄糖苷酶突变体。
本发明的第一个目的是提供热稳定性提高的β-葡萄糖苷酶突变体。
本发明的第二个目的是提供编码上述突变体的基因。
本发明的第三个目的是提供携带上述突变体基因的重组载体、重组细胞或重组工程菌。
本发明的第四个目的是提供一种提高β-葡萄糖苷酶热稳定性的方法。
本发明上述目的通过以下技术方案实现:
本发明提供了热稳定性提高的β-葡萄糖苷酶突变体,其氨基酸序列如SEQ IDNO.1所示。
本发明还提供了另一热稳定性提高的β-葡萄糖苷酶突变体,其氨基酸序列如SEQID NO.2所示。
本发明研究发现,对野生型β-葡萄糖苷酶的氨基酸序列进行T167I/V181F/A298G位点突变,可明显提高β-葡萄糖苷酶的热稳定性,进一步地,再增加两个突变位点K186T/A187E后,β-葡萄糖苷酶的热稳定性提高幅度更加显著。即本发明提供了两种热稳定性提高的β-葡萄糖苷酶突变体,其氨基酸序列分别如SEQ ID NO.1和2所示,均是在野生型β-葡萄糖苷酶氨基酸序列的基础上突变得到,SEQ ID NO.1包含三个突变位点:T167I/V181F/A298G,SEQ ID NO.2包含五个突变位点:T167I/V181F/K186T/A187E/A298G。
本发明还提供了热稳定性提高的β-葡萄糖苷酶突变体基因,编码SEQ ID NO.1所示突变体;具体如SEQ ID NO.3所示。
本发明还提供了另一热稳定性提高的β-葡萄糖苷酶突变体基因,编码SEQ IDNO.2所示突变体;具体如SEQ ID NO.4所示。
所述野生型β-葡萄糖苷酶Ks5A7编码基因如NCBI登录号:HV348683。
本发明还提供了携带所述突变体基因的重组载体、重组细胞或重组工程菌。
本发明还提供了一种提高β-葡萄糖苷酶热稳定性的方法,是将β-葡萄糖苷酶的氨基酸进行如下突变:T167I/V181F/A298G,即得到热稳定性显著提高的β-葡萄糖苷酶突变体2R1。
进一步地,将β-葡萄糖苷酶的氨基酸进行如下突变:T167I/V181F/K186T/A187E/A298G,即得到热稳定性更加显著提高的β-葡萄糖苷酶突变体4R1。
与现有技术相比,本发明具有以下有益效果:
本发明提供了热稳定性提高的β-葡萄糖苷酶突变体,所得的β-葡萄糖苷酶突变体2R1和4R1在50℃下的半衰期分别是野生酶Ks5A7的285倍和8640倍,对纤维二糖的水解活性分别是野生酶的1.75和1.5倍,且依然保持了对葡萄糖的高耐受性。因此,该热稳定性提高的β-葡萄糖苷酶突变体更适合工业应用,在食品、生物能源、纺织、造纸、医药领域具有广泛的应用前景。
附图说明
图1是在50℃条件下的β-葡萄糖苷酶突变体和野生酶的热稳定性折线图。
图2是以纤维二糖为底物时,底物浓度对β-葡萄糖苷酶突变体和野生酶活性影响的折线图。
图3是葡萄糖浓度对β-葡萄糖苷酶突变体和野生酶活性影响的折线图。
具体实施方式
以下结合具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
实施例1β-葡萄糖苷酶随机突变文库的构建
1、以携带野生型β-葡萄糖苷酶基因的pET 28a(+)-tac质粒(pET 28a(+)-tac-Ks5A7)为模板,采用低突变率(3mutations/kb)对该基因进行易错PCR(epPCR),参照II Random Mutagenesis kit的使用说明进行随机突变。
依次进行两轮突变:
第一轮突变的模板是pET 28a(+)-tac-Ks5A7,
第二轮突变是第一轮突变后所得质粒pET 28a(+)-tac-2R1。
2、具体地,上述epPCR的引物如表1所示,epPCR的反应体系如表2所示:
表1构建β-葡萄糖苷酶随机突变文库的引物(epPCR的引物)
表2构建β-葡萄糖苷酶随机突变文库的epPCR反应体系
epPCR反应的条件为:第一阶段:95℃预变性2min;第二阶段:95℃变性30s,58℃退火30s,72℃延伸1.5min,共30个循环;第三阶段:72℃延伸10min。
epPCR反应结束后,取5μL epPCR反应产物进行凝胶电泳分析,使用Gel Extraction Kit对epPCR产物进行胶回收、纯化。用NdeI和Xho I将纯化后的epPCR产物和载体pET 28a(+)-tac分别进行双酶切,酶切条件为:37℃下酶切1h,酶切体系如表3所示:
表3 epPCR产物和载体的双酶切体系
纯化上述得到的酶切产物,epPCR片段与pET 28a(+)-tac载体以摩尔比4:1混合,在22℃恒温水浴下连接1h,连接体系如表4所示:
表4酶切产物和载体的连接体系
上述得到的连接产物使用OMEGA公司MicroElute DNA Clean-Up Kit试剂盒进行回收。取5μL连接产物与100μL大肠杆菌DH5α电转化感受态细胞混合,放入无菌电击杯(0.2cm)中,使用2500V电压电击(时间约为5ms),之后加入900μL SOC培养基,转化物在37℃、180rpm条件下振荡培养1h。将培养物均匀涂布于LB培养基平板(添加50μg/mL卡那霉素和20μM IPTG),置于37℃恒温培养箱中培养过夜,即为随机突变文库。为了评估突变文库质量,随机挑选25个克隆子,在37℃、200rpm条件下培养过夜,提取质粒并测序,计算突变率。
实施例2β-葡萄糖苷酶随机突变文库的筛选
1、将实施例1得到的突变文库涂布在LB培养基平板(添加50μg/mL卡那霉素和20μMIPTG)上,在37℃恒温培养箱中培养过夜后,室温下再培养1d,以确保β-葡萄糖苷酶的足量表达。高温处理平板上的菌落以杀死细胞并使热稳定性没有提高的β-葡萄糖苷酶失活(第一轮突变:70℃处理40min;第二轮突变:85℃处理40min)。筛选指示剂是含有1g/L七叶苷,2.5g/L柠檬酸铁铵和5g/L琼脂的0.1M pH 6.0的磷酸钾溶液,将10mL筛选指示剂小心地倒在经热处理的菌落上,在室温下孵育10min后,基于菌落周围黑色晕圈的形成鉴定阳性菌落。用无菌牙签取出嵌在琼脂中的阳性菌落,使用OMEGA公司Plasmid Mini Kit提取质粒,转化到大肠杆菌DH5α中进行质粒扩增,纯化和DNA测序。
2、从野生酶Ks5A7出发,经过上述两轮筛选,得到了2个突变体:2R1(T167I/V181F/A298G)和4R1(T167I/V181F/K186T/A187E/A298G)。
实施例3β-葡萄糖苷酶突变体的表达、纯化
1、β-葡萄糖苷酶突变体的表达
将实施例2中测序确定突变的质粒转化到大肠杆菌BL21(DE3)中,挑取单菌落在LB液体培养基中以37℃、200rpm条件下培养过夜。按1:100的比例转接至LB液体培养基中,在37℃、200rpm条件下振荡培养至菌体密度OD600nm为0.85时,加入终浓度为0.8mM的IPTG。在25℃、200rpm条件下继续培养12h,10000×g离心5min,收集菌体。
2、β-葡萄糖苷酶突变体的纯化
β-葡萄糖苷酶突变体含有C端6×His标签,采用Novagen公司的Purification Kit进行纯化,具体步骤如下:
(1)取100mL诱导培养物,离心,去除上清,加入15mL预冷的1×Binding Buffer,混合均匀,使用超声波破碎菌体,10000×g离心8min,收集上清;
(2)取4mLHis·Bind resin,放于滤柱中形成纯化柱;
(3)依次用10mL去离子水、15mL 1×Charge Buffer和10mL 1×Binding Buffer洗涤纯化柱;
(4)将破碎后的菌体上清沿管壁缓慢加入纯化柱,在重力作用下自然通过纯化柱;
(5)依次使用20mL1×Binding Buffer和12mL 1×Wash Buffer洗涤纯化柱;
(6)用6mL 1×Elute Buffer洗脱,分批次收集洗脱液;
(7)洗脱液中的蛋白纯度用12%SDS-PAGE检测,选择纯化大于95%的批次合并;
(8)使用Millipore截留分子量为10kDa的超滤管,将目的蛋白置换于100mM、pH6.0的磷酸钾缓冲液中,于4℃保存备用。
实施例4β-葡萄糖苷酶突变体的热稳定性、纤维二糖水解活性和葡萄糖耐受性
1、β-葡萄糖苷酶突变体在50℃下的热稳定性的测定
将10U的野生酶Ks5A7及其突变体置于50℃下保温,隔一定时间取样,以5mM pNPG为底物测定剩余酶活,反应在pH 6.0(0.1M磷酸钾)和50℃条件下进行。
β-葡萄糖苷酶突变体在50℃下的热稳定性的测定结果如表5和图1所示,可以看出,β-葡萄糖苷酶突变体2R1和4R1的半衰期(t1/2)由突变前的1min分别提高到285min和8640min,分别是野生酶Ks5A7的285倍和8640倍。
表5β-葡萄糖苷酶突变体的酶学性质
2、β-葡萄糖苷酶突变体的纤维二糖水解活性的测定
以1%(w/v)纤维二糖为底物,在pH 6.0,50℃条件下,测定野生酶Ks5A7以及β-葡萄糖苷酶突变体2R1和4R1对纤维二糖水解活性,酶活测定方法如下:
使用蛋白质定量试剂盒(Bradford法)测定上述纯化得到的β-葡萄糖苷酶的浓度,以小牛血清白蛋白(BSA)作为对照。
酶活测定原理:对硝基苯酚-β-葡萄糖苷(pNPG)溶于水呈无色,其中的糖苷键水解后生成对硝基苯酚(pNP),在400~420nm有最大吸收峰,吸光度与pNP的浓度成正比。纤维二糖的糖苷键水解后,生成两分子葡萄糖,在葡萄糖氧化酶(GOD)的作用下生成葡萄糖酸和过氧化氢(H2O2),H2O2与4-氨基安替吡碄(4-AAP)、酚在过氧化物酶(POD)的催化下生成红色醌亚胺。醌亚胺在500nm附近有最大吸收峰,吸光度和葡萄糖浓度成正比。
酶活测定方法:以pNPG为底物时,反应体系为10μL酶液和490μL的5mM底物溶液(0.1M磷酸钾溶液,pH 6.0),混合均匀后,在50℃温育10min,加入500μL 10%(w/v)Na2CO3终止反应。取出300μL反应液加入酶标板中,在405nm测定吸光值,根据相应的标准曲线计算酶活单位。以灭活的酶液作为空白对照,每组做三个平行试验。以纤维二糖为底物时,反应体系为50μL酶液和450μL 1%(w/v)的纤维二糖溶液,混合均匀后于50℃温育10min,之后于90℃温育5min终止反应。冷却至室温后,取出10μL反应液加到750μL葡萄糖氧化试剂盒反应液中,在37℃温育15min。反应结束后,取出300μL加入酶标板中,在492nm测定吸光值,并根据葡萄糖标准曲线计算酶活性。以灭活的酶液作为空白对照,每组做三个平行试验。
酶活单位(1U)定义为:在标准反应条件下,1min催化生成1μmol pNP或葡萄糖所需的酶量。
对硝基苯酚(pNP)标准曲线的绘制:用磷酸钾缓冲液(0.1M,pH 6.0)配成1.0mMpNP母液。按表2所示,分别加入不同体积的pNP母液和磷酸钾缓冲液,混匀后加入300μL10%(w/v)Na2CO3。取300μL至酶标板上在405nm处测定吸光值(OD405),每次反应3次平行,并绘制曲线,其公式为:y=19.185x-1.6296,R2=0.9995。
表6pNP标准曲线的绘制
葡萄糖标准曲线的绘制:用磷酸钾缓冲液(0.1M,pH 6.0)配制浓度为2.5g/L的葡萄糖溶液母液,按表3所示,稀释成不同浓度的葡萄糖溶液。取10μL稀释后的葡萄糖溶液分别加入到750μL葡萄糖试剂盒测试液中,混匀后于37℃温育15min,之后取300μL测定其在492nm处的吸光值(OD492nm),并绘制曲线,其公式为:y=127.35x-0.9915,R2=0.9975。
表7葡萄糖标准曲线的绘制
测定结果如表5所示,可以看出,野生酶Ks5A7的比酶活是243.18U/mg,突变体2R1的比酶活是423.58U/mg,4R1的比酶活是374.26U/mg。因此,以1%(w/v)纤维二糖为底物时,其水解活性分别是野生酶的1.75和1.5倍。
3、纤维二糖浓度对β-葡萄糖苷酶突变体的影响
以不同浓度(0.1~5%,w/v)的纤维二糖为底物,测定野生酶Ks5A7以及β-葡萄糖苷酶突变体2R1和4R1的活性,计算比酶活。
结果如图2所示,可以看出,与野生酶Ks5A7一致,β-葡萄糖苷酶突变体2R1和4R1的活性随纤维二糖浓度升高而升高,高达5%(w/v)的纤维二糖,对β-葡萄糖苷酶突变体的活性没有抑制作用。
4、β-葡萄糖苷酶突变体的葡萄糖耐受性的测定
由于Ks5A7的活性被葡萄糖激活,所以选用半数抑制浓度(IC50)替代抑制常数(Ki)衡量其耐受能力,定义为抑制50%酶活性所需的葡萄糖浓度。以5mM pNPG为底物,测定不同浓度的葡萄糖对β-葡萄糖苷酶Ks5A7及其突变体活性的影响,反应在pH 6.0(0.1M磷酸钾)和50℃条件下进行。以不添加葡萄糖的酶活性为对照,设定为100%。
由表5和图3的结果可以看出,β-葡萄糖苷酶突变体2R1和4R1的葡萄糖耐受性与野生酶Ks5A7差别不大。
以上具体实施方式为便于理解本发明而说明的较佳实施例,但本发明并不局限于上述实施例,即不意味着本发明必须依赖上述实施例才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明所选用原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
序列表
<110> 中山大学
<120> 高热稳定性β-葡萄糖苷酶突变体及其应用
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tgcggcgttg ccttaccgat ctcagaaaat aaaaaagaca ttgaaattgc acgcaaacgc 720
tattttgata ttctggatct gaatgatgcg tatgtgtgga gcgtgagcct gtttttggac 780
ccaatcgtgt taggcgatta tccaaccaaa tattatgaac tgtataaaga acatttacct 840
aaaattacac aggaggacct gaaactgatc tcacagccgt tagattttct gggccagaat 900
atctataatg gctatcgtgt gagcgaagat gaaaatggca attatgtgta tcctaaacgc 960
aaagcaggtt atgatcatac ggatatgggt tggccaatta caccgtcagc cctgtattgg 1020
ggtcctcgct ttatctgcga acgctataat ctgccgtttt atattacgga aaatggctta 1080
gcctgtcatg atgttgtgag cttagataat aaagttcatg atcctaatcg catcgatttt 1140
ctgaataaat atctgctgga ttatagtcgc gcctcttgcg aaggttatga tattcgcggc 1200
tattttcagt ggtcactgat ggataatttt gaatggcgcg aaggctatag caaacgcttt 1260
ggtatggtgt atgtggattt tgaaacacag aaacgtacaa tcaaagatag cggttattgg 1320
tataaaaaag tgatcgaaga aaatggtgaa aatctg 1356
<210> 5
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
catatgaaat ttaatgaaaa ttttgtttgg gg 32
<210> 6
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ctcgagcaga ttttcaccat tttcttcgat cac 33
Claims (9)
1.热稳定性提高的β-葡萄糖苷酶突变体,其特征在于,其氨基酸序列如SEQ ID NO.1所示。
2.热稳定性提高的β-葡萄糖苷酶突变体,其特征在于,其氨基酸序列如SEQ ID NO.2所示。
3.热稳定性提高的β-葡萄糖苷酶突变体基因,其特征在于,编码权利要求1所述的突变体。
4.热稳定性提高的β-葡萄糖苷酶突变体基因,其特征在于,编码权利要求2所述的突变体。
5.根据权利要求3所述的突变体基因,其特征在于,其核苷酸序列如SEQ ID NO.3所示。
6.根据权利要求4所述的突变体基因,其特征在于,其核苷酸序列如SEQ ID NO.4所示。
7.携带权利要求3~6任一所述突变体基因的重组载体、重组细胞或重组工程菌。
8.一种提高β-葡萄糖苷酶热稳定性的方法,其特征在于,将β-葡萄糖苷酶的氨基酸进行如下突变:T167I/V181F/A298G。
9.根据权利要求8所述的方法,其特征在于,将β-葡萄糖苷酶的氨基酸进行如下突变:T167I/V181F/K186T/A187E/A298G。
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