CN114317495A - 一种热稳定性提高的葡聚糖酶突变体及其应用 - Google Patents
一种热稳定性提高的葡聚糖酶突变体及其应用 Download PDFInfo
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Abstract
一种热稳定性提高的葡聚糖酶突变体及其应用,所述突变体的氨基酸序列为SEQ ID NO:1所示的野生型葡聚糖酶的第43位的Ala突变为Pro,氨基酸序列如SEQ ID NO:2所示;第59位的Thr突变为Ile,氨基酸序列如SEQ ID NO:3所示;或第165位的Gly突变为Lys,氨基酸序列如SEQ ID NO:4所示。以地衣多糖为底物时,突变体较野生型的最适pH值无明显变化,比活无明显变化,突变体A43P和T59I的最适温度较野生型提高5℃,本发明中三个突变体A43P、T59I和G165K在60℃下的半衰期分别比野生型(16分钟)延长34分钟、49分钟和24分钟。
Description
技术领域
本发明涉及基因工程和酶工程技术领域,具体涉及一种热稳定性提高的葡聚糖酶突变体及其应用。
背景技术
β-葡聚糖是一种广泛存在于植物细胞壁以及一些真菌、藻类和细菌中的非淀粉多糖,其中大麦中含量最高,可达2-20g/100g干重(65%可溶)。自然界中的β-葡聚糖多将β-葡萄糖残基以(1,3)/(1,4)或(1,3)/(1,6)键连接而成。因此根据β-葡聚糖中糖苷键的类型,可分为:β-1,3-1,4-葡聚糖(地衣聚糖)、β-1,4-葡聚糖(纤维素)、β-1,3-葡聚糖(海带多糖)和β-1,3(4)-葡聚糖。
大麦等谷物中的β-葡聚糖对其工业应用具有一定阻碍。例如,在饲料中,β-葡聚糖作为一种抗营养因子会增加肠道中食糜粘度,抑制了肠道对营养物质的吸收,降低了饲料的利用率。其次,在啤酒酿造过程中,酿酒原料麦芽含有的β-葡聚糖,会导致提麦芽汁和啤酒粘度升高、麦芽汁分离和啤酒过滤速度缓慢最终导致酒体质量下降。
β-葡聚糖酶是水解葡聚糖的关键酶类,根据其水解糖苷键的类型可分为四类,即β-1,4-葡聚糖酶(纤维素酶);β-1,3-葡聚糖酶(昆布多糖酶),β-1,3-1,4-葡聚糖酶(地衣多糖酶)和β-1,3(4)-葡聚糖酶。其中β-1,3-1,4-葡聚糖酶能够特异性水解3-O-取代的吡喃糖中的β-1,4-糖苷键。由于其专一的水解特性,β-1,3-1,4-葡聚糖酶在一些以谷物,如燕麦或大麦为原料的工业中需求量较大。例如,作为饲料添加用酶,β-1,3-1,4-葡聚糖酶能够将β-葡聚糖水解成低分子寡糖,消除抗营养作用;在啤酒酿造中,β-1,3-1,4-葡聚糖酶能够降低酒浆粘度,提高麦芽提取率。另外,β-1,3-1,4-葡聚糖酶还用于产生具有益生作用的功能性寡糖,常用于食品加工。因此,开发具有优良酶学性质(高活性且耐高温)的β-葡聚糖酶对于工业生产具有重要意义。
发明内容
解决的技术问题:本发明提供一种热稳定性提高的葡聚糖酶突变体及其应用。
技术方案:一种热稳定性提高的葡聚糖酶突变体,所述突变体的氨基酸序列为SEQID NO:1所示的野生型葡聚糖酶的第43位的Ala突变为Pro,氨基酸序列如SEQ ID NO:2所示;第59位的Thr突变为Ile,氨基酸序列如SEQ ID NO:3所示;或第165位的Gly突变为Lys,氨基酸序列如SEQ ID NO:4所示。
编码所述热稳定性提高的葡聚糖酶突变体的基因。
上述基因的核酸序列如SEQ ID NO:5所示。
上述基因的核酸序列如SEQ ID NO:6所示。
上述基因的核酸序列如SEQ ID NO:7所示。
包含上述基因的重组表达载体。
包含上述重组表达载体的重组酵母菌株。
上述重组酵母菌株在制备热稳定性提高的葡聚糖酶中的应用。
本发明还是提供一种制备热稳定性提高的葡聚糖酶突变体的方法,包括以下步骤:
1)用上述的重组载体转化宿主细胞,得重组菌株;
2)培养重组菌株,诱导葡聚糖酶表达;
3)回收并纯化所表达的热稳定性提高的葡聚糖酶突变体。
有益效果:将含有突变体基因的重组表达载体线性化后转化毕赤酵母GS115感受态细胞,通过对小管水平的发酵液进行酶活筛选阳性转化子。对酶活最高的转化子进行大瓶诱导,并对粗酶液进行蛋白浓缩及纯化。利用SDS-PAGE电泳检测纯化后野生型和突变体的纯度。以纯化后的蛋白为对象,利用DNS法测定野生型与突变体的基本酶学性质。结果表明,以地衣多糖为底物时,三个突变体A43P、T59I和G165K的最适pH值在pH3.5-pH4.0之间,与野生型葡聚糖酶相比无明显变化,最适温度在55-60℃之间,最适条件下的比活无明显降低,三个突变体A43P、T59I和G165K在60℃下的半衰期分别比野生型(16分钟)延长34分钟、49分钟和24分钟。
附图说明
图1葡聚糖酶野生型及其突变体的SDS-PAGE分析,其中,M为低分子量蛋白质Marker;A、C、E、G分别为纯化的的野生酶BisGlu16B_ΔC和三个突变体A43P、T59I、G165K;B、D、F、H分别为脱糖基处理的野生酶BisGlu16B_ΔC和三个突变体A43P、T59I、G165K。
图2为3个葡聚糖酶突变体与野生型的最适pH;
图3为3个葡聚糖酶突变体与野生型的pH稳定性;
图4为3个葡聚糖酶突变体与野生型的最适温度;
图5为3个葡聚糖酶突变体与野生型的热稳定性(60℃下的半衰期)。
具体实施方式
试验材料和试剂:
1.菌株及载体:质粒扩增大肠杆菌DH5α感受态细胞购自上海生工生物,表达宿主Pichia pastoris GS115为本实验室保存。
2.试剂盒及其它生化试剂:点突变试剂盒购自北京全式金,地衣多糖购自Magzyme公司。其它均为国产分析纯试剂均可从普通生化试剂公司购买得到。
3.培养基:
1)LB培养基:1%NaCl,0.5%酵母提取物,1%蛋白胨,1%琼脂粉(固体);
2)YPD培养基:1%酵母提取物,2%蛋白胨,2%葡萄糖;
3)MD培养基:2%葡萄糖,1.34%YNB,0.00004%Biotin,1.5%琼脂糖;
4)BMGY培养基:1%酵母提取物,2%蛋白胨,1%甘油(V/V),1.34%YNB,0.00004%Biotin;
5)BMMY培养基:2%蛋白胨,1%酵母提取物,1.3%YNB,0.5%甲醇(V/V),0.004%Biotin。
4.本实施例中未做详细具体说明的分子生物学实验方法,均参照试剂盒和产品说明书或者《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。
实施例1热稳定性提高的葡聚糖酶突变体重组载体pPIC9r-A43P、pPIC9r-T59I、pPIC9r-G165K的制备。
将葡聚糖酶野生型(突变前)序列片段(去除信号肽)克隆到表达载体pPIC9r上,重组载体命名pPIC9r-Bisglu16b_ΔC;以此重组质粒为模板,通过点突变引物对其进行扩增,获得携带突变体序列的线形重组载体,命名为pPIC9r-A43P、pPIC9r-T59I、pPIC9r-G165K。
表1热稳定性提高突变体特异引物表
实施例2葡聚糖酶突变体的制备
1.葡聚糖酶突变体在毕赤酵母中的大量表达。
将获得的含有突变体基因的重组质粒pPIC9r-A43P、pPIC9r-T59I、pPIC9r-G165K采用内切酶BglⅡ线性化后转化毕赤酵母GS115感受态,获得重组酵母菌株GS115/A43P、GS115/T59I、GS115/G165K。取含有重组表达载体的单克隆酵母菌株,首先接种于30mL YPD培养基中,220rpm摇床培养48h后获得种子培养液,按1%接种量接种于含有300mL BMGY培养基的1L三角瓶中,30℃,220rpm摇床培养48h;培养液4000×g离心5min,弃上清,沉淀用200mL含有0.5%甲醇的BMMY培养基重悬,并再次置于30℃,220rpm条件下诱导培养。每隔12h补加1mL甲醇,同时取上清用于酶活性检测。
2.重组蛋白酶的纯化
收集摇瓶表达的重组葡聚糖酶上清液,采用5kDa膜包进行浓缩,同时用磷酸氢二钠-柠檬酸缓冲液(pH6.0,10mM)置换其中的培养基,最后发酵上清液浓缩至20mL。浓缩到一定倍数的重组葡聚糖酶,利用离子交换层析法进行纯化。具体地,取葡聚糖酶浓缩液5mL经预先用10mmol/L磷酸氢二钠-柠檬酸缓冲液(pH6.0)平衡过的HiTrap Q HP阴离子柱,然后用含有1mol/L NaCl的10mmol/L磷酸氢二钠-柠檬酸缓冲液(pH6.0)进行线性连续梯度洗脱,采用DNS法对梯度洗脱蛋白进行活性检测,利用SDS-PAGE对洗脱蛋白进行纯度检测。
实施例3重组热稳定性提高的葡聚糖酶突变体活性分析
采用二硝基水杨酸(DNS)法测定重组内切β-1,3-1,4-葡聚糖酶的基本酶学性质。具体方法如下:在pH4.0、55℃下,1mL的反应体系包含100μL酶液(电泳纯),900μL底物(0.5%的地衣多糖),反应10min后加入1.5mL DNS终止反应,沸水煮5min。冷却至室温后测定OD540值。内切β-1,3-1,4-葡聚糖酶活性单位定义:在给定的条件下,每分钟水解葡聚糖生成1μmoL还原糖所需的酶量为1个酶活单位(U)。
1.pH特性测定比较
将纯化好的突变体和野生型葡聚糖酶在不同的pH(1.0-6.5)下进行酶促反应,以测定其最适pH。底物地衣多糖用不同pH(1.0、1.5、2.0、2.5、3、3.5、4、4.5、5、5.5、6、6.5)的0.1mol/L磷酸氢二钠-柠檬酸缓冲液中55℃下进行酶活力测定;将酶液在不同pH(1-12)的缓冲液中37℃孵育1小时,测定剩余酶活以分析酶的pH稳定性。
结果如图2所示,野生型和突变体的最适反应pH相近分布在3.5-4.0之间;在pH稳定性方面:3个突变体在pH7-10之间的稳定性较野生型有不同程度提高。
2.最适温度和热稳定性测定比较
葡聚糖酶最适温度的测定方法为:在pH 4.0,0.1mol/L磷酸氢二钠-柠檬酸缓冲液缓冲液及不同温度(37-95℃)下进行酶促反应。将100μL纯化的野生型和突变体在60℃下分别处理一定时间(0-180分钟),处理时所有突变体和野生型的浓度为0.1mg/mL,在不同时间点取样后迅速置于冰上,并在55℃、pH 4.0的条件下测定剩余酶活,未处理的酶液作为对照。
结果如图3所示,野生型和3个突变体A43P、T59I、G165K的最适温度分别为55℃、60℃、60℃、55℃,在高温条件下(65-75℃)突变体A43P、T59I、G165K的相对酶活(5%-94%)明显高于野生型(0-16%);在热稳定性方面:突变体A43P、T59I、G165K在60℃下的半衰期较野生型(16分钟)延长34分钟、49分钟和24分钟。
3.葡聚糖酶动力学参数和比活测定比较
检测方法参照文献(Functional Analysis of a Highly Activeβ-Glucanasefrom Bispora sp.MEY-1Using Its C-terminally Truncated Mutant.10.1021/acs.jafc.8b01928)。首先测定反应的一级反应时间为5min。用不同浓度的地衣多糖(0.625,0.5,0.4,0.2,0.1,0.075和0.05mg/mL)为底物,在标准条件(55℃、pH4.0)下测定酶活性,计算出相应的反应速度,利用GraFit7软件计算Km值及Vmax。
在标准条件下,3个突变体的比活和催化效率较野生型相比没有明显降低;突变体G165K的比活较野生型提高62%。(见表2)。
表2以地衣多糖为底物野生酶和突变体的比活力和催化效率比较
序列表
<110> 鑫缘茧丝绸集团股份有限公司
江苏科技大学
<120> 一种热稳定性提高的葡聚糖酶突变体及其应用
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aaccagcaaa ctaccaatga catgaccctc cacactagtg aaggctgcac aatatccagc 420
agtggcgatt tctcgggctc gatagttagc accgactgct gggtcgatga ccccaaccaa 480
tccgacaatg aaggctgtca gatcactacg agcaataccg aaacttacgg ttccggtttt 540
aatgctaaca atggcggcgt ctatgcgacg gacttccaag acgccgctat cagcatctat 600
ttcttccccc gtggttccat accttcggac attacagacg gctctccaga cccgtccggc 660
tggggtacgc caattgcgca gttcacggat agcagctgtg acattcaaag ctatttcacc 720
gatttacaga tcgttttcga tacgacgttc tgtggacaat gggctggcaa cgtctggtca 780
agtggctctt gtgcctctgt ggcaagtacc tgcgacgact acgtggaaaa caacccggct 840
gccttcgtcg atgcatactg gtcgatcaac agtcttcagg tttattcggg aacctccaat 900
ggtcccatgc agaatgatac ttcgagcagc agctggggtc catctgcttc tgcaaatgtg 960
gcagtgtga 969
<210> 6
<211> 969
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
caatataccc ttcagcagga ttacatggca gacggcaact tttttagcca attttcattt 60
tgggataccg ccgaccctac agatggcttt gtggcttata aaaatgagac ttattgcata 120
gacaacgatc tcatcagcag ttccagcacg aacgtgcaga ttcgggtgga cagctccaat 180
gttacaccga atggacggcc tagtgttcgc attaccagca accagtcgta caatccaggc 240
acacttgtaa tcctggacct tgaacacatg ccaggtggca tctgcggtac ctggccagca 300
ttttggatgg ttgggccgaa ttggcccgac gatggggaaa tcgacatcat tgagggtgtc 360
aaccagcaaa ctaccaatga catgaccctc cacactagtg aaggctgcac aatatccagc 420
agtggcgatt tctcgggctc gatagttagc accgactgct gggtcgatga ccccaaccaa 480
tccgacaatg aaggctgtca gatcactacg agcaataccg aaacttacgg ttccggtttt 540
aatgctaaca atggcggcgt ctatgcgacg gacttccaag acgccgctat cagcatctat 600
ttcttccccc gtggttccat accttcggac attacagacg gctctccaga cccgtccggc 660
tggggtacgc caattgcgca gttcacggat agcagctgtg acattcaaag ctatttcacc 720
gatttacaga tcgttttcga tacgacgttc tgtggacaat gggctggcaa cgtctggtca 780
agtggctctt gtgcctctgt ggcaagtacc tgcgacgact acgtggaaaa caacccggct 840
gccttcgtcg atgcatactg gtcgatcaac agtcttcagg tttattcggg aacctccaat 900
ggtcccatgc agaatgatac ttcgagcagc agctggggtc catctgcttc tgcaaatgtg 960
gcagtgtga 969
<210> 7
<211> 873
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
caatataccc ttcagcagga ttacatggca gacggcaact tttttagcca attttcattt 60
tgggataccg ccgaccctac agatggcttt gtggcttata aaaatgagac ttattgcacc 120
gacaacgatc tcatcagcag ttccagcacg aacgtgcaga ttcgggtgga cagctccaat 180
gttacaccga atggacggcc tagtgttcgc attaccagca accagtcgta caatccaggc 240
acacttgtaa tcctggacct tgaacacatg ccaggtggca tctgcggtac ctggccagca 300
ttttggatgg ttgggccgaa ttggcccgac gatggggaaa tcgacatcat tgagggtgtc 360
aaccagcaaa ctaccaatga catgaccctc cacactagtg aaggctgcac aatatccagc 420
agtggcgatt tctcgaaaca atataccctt cagcaggatt acatggcaga cggcaacttt 480
tttagccaat tttcattttg ggataccgcc gaccctacag atggctttgt ggcttataaa 540
aatgagactt attgcaccga caacgatctc atcagcagtt ccagcacgaa cgtgcagatt 600
cgggtggaca gctccaatgt tacaccgaat ggacggccta gtgttcgcat taccagcaac 660
cagtcgtaca atccaggcac acttgtaatc ctggaccttg aacacatgcc aggtggcatc 720
tgcggtacct ggccagcatt ttggatggtt gggccgaatt ggcccgacga tggggaaatc 780
gacatcattg agggtgtcaa ccagcaaact accaatgaca tgaccctcca cactagtgaa 840
ggctgcacaa tatccagcag tggcgatttc tcg 873
<210> 8
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
attttgggat acccctgacc ctacagatgg c 31
<210> 9
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
aggggtatcc caaaatgaaa attggctaaa a 31
<210> 10
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tgagacttat tgcatcgaca acgatctcat c 31
<210> 11
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gatgcaataa gtctcatttt tataagccac a 31
<210> 12
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ggcgatttct cgaaatcgat agttagcacc g 31
<210> 13
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
tttcgagaaa tcgccactgc tggatattgt g 31
Claims (8)
1.一种热稳定性提高的葡聚糖酶突变体,其特征在于,所述突变体的氨基酸序列为SEQID NO:1所示的野生型葡聚糖酶的第43位的Ala突变为Pro,氨基酸序列如SEQ ID NO:2所示;第59位的Thr突变为Ile,氨基酸序列如SEQ ID NO:3所示;或第165位的Gly突变为Lys,氨基酸序列如SEQ ID NO:4所示。
2.编码权利要求1所述热稳定性提高的葡聚糖酶突变体的基因。
3.根据权利要求2所述基因,其特征在于,所述基因的核酸序列如SEQ ID NO:5所示。
4.根据权利要求2所述基因,其特征在于,所述基因的核酸序列如SEQ ID NO:6所示。
5.根据权利要求2所述基因,其特征在于,所述基因的核酸序列如SEQ ID NO:7所示。
6.包含权利要求2-5任一所述基因的重组表达载体。
7.包含权利要求6所述重组表达载体的重组酵母菌株。
8.权利要求6所述重组酵母菌株在制备热稳定性提高的葡聚糖酶中的应用。
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