CN113699136B - 一组动物体温下高催化活力β-1,3-1,4-葡聚糖酶突变体及其应用 - Google Patents

一组动物体温下高催化活力β-1,3-1,4-葡聚糖酶突变体及其应用 Download PDF

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CN113699136B
CN113699136B CN202110629186.7A CN202110629186A CN113699136B CN 113699136 B CN113699136 B CN 113699136B CN 202110629186 A CN202110629186 A CN 202110629186A CN 113699136 B CN113699136 B CN 113699136B
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游帅
葛研
谢晨
张温馨
王雪
陈奕文
张伊欣
王俊
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Abstract

一组动物体温下高催化活力β‑1,3‑1,4‑葡聚糖酶突变体及其应用，包括BisGlu16B‑D213A、BisGlu16B‑D253A和BisGlu16B‑D213A/D253A突变体。三个突变体在65℃下的半衰期分别较野生型延长11min、28min和30min。动物肠道体温环境在37℃左右，酶在37℃下的催化活力提高有利于该酶在动物肠道中更好的发挥降解功能；且三种葡聚糖酶突变体的热稳定性均比野生型酶有不同程度的提高，这样的性质能够使得酶能够更好的抵抗高温制粒导致的酶失活。这样一种在动物体温下催化活力高、热稳定性优良的酸性葡聚糖酶在饲料加工、啤酒酿造及寡糖生产等领域中具有极大的应用潜力。

Description

一组动物体温下高催化活力β-1,3-1,4-葡聚糖酶突变体及其 应用
技术领域
本发明涉及基因工程、蛋白质工程领域，具体涉及一组动物体温下高催化活力β-1,3-1,4-葡聚糖酶突变体及其应用。
背景技术
纤维素类物质包括纤维素、半纤维素和木质素，是自然界中常见的、最丰富的可再生资源。纤维素是由D-吡喃式葡萄糖以β-1,4糖苷键连接而成的大分子聚合物，也是植物细胞壁的主要成分。半纤维素一般指自然界植物细胞壁中除果胶和纤维素类成分之外的其他聚糖类物质，主要包括葡聚糖、半乳甘露聚糖和半乳葡甘露聚糖。其中，β-葡聚糖是一类非淀粉葡萄糖聚合物，其包含通过β-糖苷键连接的β-葡萄糖残基。作为结构元素，β-葡聚糖广泛存在于植物细胞壁、谷物种子及一些真菌、藻类中，其中大麦和燕麦中的β-葡聚糖含量最高，分别可达2-20g/100g干重(65％可溶)和3-8g/100g干重(82％可溶)(Khoury，10.1155/2012/851362)。
β-葡聚糖酶是内切β-1,3-1,4-葡聚糖酶和外切β-1,3-1,4-葡聚糖酶的总称。他作用域葡聚糖的β-1,3和β-1,4糖苷键，使β-葡聚糖分解成寡糖。在饲料工业中，β-葡聚糖酶通过降解β-葡聚糖，改善单胃动物肠道内容物，降低食糜粘度，促进营养物质的消化与吸收，提高饲料利用率(Beckmann 10.1002/jobm.200510107)。在啤酒加工业中，β-葡聚糖酶能够高效地将麦芽中的β-葡聚糖水解为葡萄糖和寡糖，摧毁细胞壁，使细胞内容物大量释放，提高麦芽汁得率，降低醪液粘度从而缩短糖化醪过滤时间，所获得麦汁清亮，使制得的啤酒色泽浅，泡沫均匀持久(宫春波等，2002，广州食品工业科技)。
针对饲料业、啤酒酿造业等食品工业的需求，因此，获得具有优良特性的β-葡聚糖酶仍具有重大意义。
发明内容
解决的技术问题：本发明提供一组动物体温下高催化活力β-1,3-1,4-葡聚糖酶突变体及其应用。
技术方案：一组动物体温下高催化活力β-1,3-1,4-葡聚糖酶突变体，包括BisGlu16B-D213A、BisGlu16B-D253A和BisGlu16B-D213A/D253A突变体，所述BisGlu16B-D213A的核苷酸序列如SEQ ID NO.1所述；BisGlu16B-D253A的核苷酸序列如SEQ ID NO.2所示；BisGlu16B-D213A/D253A的核苷酸序列如SEQ ID NO.3所示；其中，所有突变位点的标号使用未去信号肽时的序列。
BisGlu16B-D213A的氨基酸序列如SEQ ID NO.4所示；BisGlu16B-D253A的氨基酸序列如SEQ ID NO.5所示；BisGlu16B-D213A/D253A的氨基酸序列如SEQ ID NO.6所示，其中，所有突变位点的标号使用未去信号肽时的序列。
一种重组载体，含有上述任一核苷酸序列。
一种重组菌株，含有上述的重组载体。
上述重组菌株在制备饲料添加剂中的应用。
有益效果：本发明提供一种性质优良的、适合于动物体温条件下催化半纤维素降解的葡聚糖酶突变体。该葡聚糖酶突变体最适pH在2-4.5之间，在pH1-12之间，突变体D213A/D253A的稳定性均优于野生型。在最适温度适应性方面，三个突变体的最适温度均为60℃，但37℃下的相对酶活，突变体比野生型分别提高96％、1.4倍和89％，且在高温70℃下，突变体的相对酶活比野生型分别提高3.3倍、70％和1.1倍。在37℃下，当以燕麦葡聚糖为底物时，三个突变体BisGlu16B-D213A、BisGlu16B-D253A和D213A/D253A的比活分别比野生型BisGlu16B提高78％、52％和90％，催化效率分别比野生型提高5％、16％和58％；当以地衣多糖为底物时，三个突变体的比活分别比野生型提高49％、43％和9％，催化效率分别比野生型提高55％、45％和1.1倍；当以昆布多糖为底物时，三个突变体的比活分别比野生型提高36％、25％和93％，催化效率与野生型无明显差别。其中组合突变体D213A/D253A催化活力提升最为明显，较野生型比活最高提93％(昆布多糖为底物)，催化效率最高提高1.1倍(地衣多糖为底物)。在热稳定性方面，三个突变体在65℃下的半衰期分别较野生型延长11min、28min和30min。动物肠道体温环境在37℃左右，酶在37℃下的催化活力提高有利于该酶在动物肠道中更好的发挥降解功能；且三种葡聚糖酶突变体的热稳定性均比野生型酶有不同程度的提高，这样的性质能够使得酶能够更好的抵抗高温制粒导致的酶失活。这样一种在动物体温下催化活力高、热稳定性优良的酸性葡聚糖酶在饲料加工、啤酒酿造及寡糖生产等领域中具有极大的应用潜力。
附图说明
图1为高催化活力葡聚糖酶突变体与野生型的蛋白纯化；
图2为40℃下高催化活力葡聚糖酶突变体与野生型的最适pH；
图3为40℃下高催化活力葡聚糖酶突变体与野生型的pH稳定性；
图4为高催化活力葡聚糖酶突变体与野生型的最适温度；
图5为50℃下高催化活力葡聚糖酶突变体与野生型的热稳定性。
具体实施方式
下面结合附图和具体实施例对本发明作进一步描述。
1、菌株及载体：表达宿主Pichia pastoris GS115，表达质粒载体pPIC9r实验室自备；
2、酶类及其它生化试剂：Taq酶购自全式金公司，内切酶购自Fermentas公司，连接酶购自Promaga公司，燕麦葡聚糖购自Sigma公司；其它都为国产分析纯试剂(均从国药集团购买)；
3、培养基：
(1)LB培养基：0.5％酵母提取物，1％蛋白胨，1％NaCl，pH 7.0；
(2)YPD培养基：1％酵母提取物，2％蛋白胨，2％葡萄糖；
(3)MD固体培养基：2％葡萄糖，1.5％琼脂糖，1.34％YNB，0.00004％Biotin；
(4)MM固体培养基：1.5％琼脂糖，1.34％YNB，0.00004％Biotin，0.5％甲醇；
(5)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；
(6)BMMY培养基：1％酵母提取物，2％蛋白胨，1.34％YNB，0.00004％Biotin，0.5％甲醇(V/V)；
(7)BisGlu16B的原始核苷酸序列如SEQ ID NO.7所示。
实施例1高催化活力葡聚糖酶突变体的构建
以来源于GH16家族葡聚糖酶BisGlu16B为出发材料，采用易错PCR的方法对葡聚糖酶基因进行随机突变,引物如表1所示。具体参数如下：使用TaqDNA聚合酶的工作浓度上调至正常PCR的2倍，Mg2+浓度上调为正常PCR的4.7倍，将dNTP的浓度提高至正常水平的5倍(易错PCR研究进展及应用[J].2013,27(05):607-612.)。
实施例2高催化活力葡聚糖酶突变体的制备
将表达载体pPIC9r进行双酶切(EcoR I+Not I)，同时将编码高催化活力葡聚糖酶突变体的基因双酶切(EcoR I+Not I)，再将酶切后编码成熟高催化活力葡聚糖酶突变体的基因片段与表达载体pPIC9r连接，获得含有高催化活力葡聚糖酶突变体基因的重组质粒并转化毕赤酵母GS115，获得重组酵母菌株，利用高通量筛选平板针对酶37℃下的活力进行筛选，获得比野生型活力高的突变体，并对突变体进行基因测序，引物如表1，并通过定点突变方法构建组合突变体，引物见表1。
将活力提高的重组GS115菌株，接种于300mL BMGY培养基的1L三角瓶中，置于30℃，220rpm摇床培养48h；后将培养液3000g离心5min，弃上清，沉淀用100mL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0.5mL甲醇，使菌液中的甲醇浓度保持在0.5％，同时取上清用于酶活检测。
表1易错PCR引物及组合突变构建引物
Figure GDA0003321849770000041
实施例3重组高催化活力葡聚糖酶突变体和野生型的活性分析
一、β-1,3-1,4-葡聚糖酶的酶活力测定
根据Yang等人先前所报道的方法(Yang 10.1021/jf800303b)，以1％(w/v，g/mL)的燕麦β-葡聚糖作为底物，测定β-1,3-1,4-葡聚糖酶的活力。具体方法如下：在给定的pH、温度条件下，1mL的反应体系包括100μL酶液，900μL底物，反应10min，加入1.5mL DNS终止反应，沸水煮5min。冷却后540nm测定OD值。β-1,3-1,4-葡聚糖酶活力单位(U)定义为在上述条件下，每分钟生成1μmol葡萄糖当量还原糖所需的酶量。
二、重组高催化活力葡聚糖酶突变体和野生型的性质测定
1、重组高催化活力葡聚糖酶突变体和野生型的最适pH测定如下:
将实施例2纯化的重组高催化活力葡聚糖酶突变体和野生型(图5)在不同的pH下进行酶促反应以测定其最适pH。用不同pH的0.1mol/L柠檬酸-磷酸氢二钠缓冲液稀释底物(燕麦葡聚糖)并在60℃下进行葡聚糖酶活力测定。
结果(图1)表明，重组高催化活力葡聚糖酶突变体和野生型的最适反应pH值在2-4.5之间，且在pH1.0-6.5范围内有相同的作用趋势(图2)。符合不改变最适pH值提高较低温度下比活力的目的。
2、重组高催化活力葡聚糖酶突变体和野生型的最适温度测定如下:
重组的高催化活力葡聚糖酶突变体和野生型的最适温度测定为在0.1mol/L柠檬酸-磷酸氢二钠缓冲液(pH 3.5)缓冲液体系及不同温度下进行酶促反应。酶促反应最适温度测定结果(图3)表明,重组高催化活力葡聚糖酶突变体与野生型的最适温度在50℃-65℃之间，彼此相差不明显。但在40℃下的催化活力分别是野生型的1.75倍、2.22倍和1.71倍。
3、重组高催化活力葡聚糖酶突变体和野生型在65℃的热稳定性测定如下:
将高催化活力葡聚糖酶突变体和野生型在65℃处理一定时间后热稳定性逐渐降低，但所有突变体的热稳定性均优于野生型。通过拟合曲线可以发现，突变体BisGlu16B-D213A较野生型酶半衰期延长11min，而突变体BisGlu16B-D253A的半衰期延长至58min，组合突变BisGlu16B-D213A/D253A的效果最佳，半衰期延长至60min，是野生型的两倍。
4、重组高催化活力葡聚糖酶突变体和野生型的动力学测定如下：
在最适条件下，分别用燕麦葡聚糖、地衣多糖和昆布多糖三种底物测定高催化活力葡聚糖酶和野生型的动力学参数和比活力，结果如表2所示。
其中，以燕麦葡聚糖为底物，在最适条件下，BisGlu16B-D213A的Km和Vm为2.44mg/mL和7200μmol/min·mg。BisGlu16B-D253A的Km和Vm分别为2.21mg/mL和7100μmol/min·mg。BisGlu16B-D213A/D253A的Km和Vm分别为1.76mg/mL和7600μmol/min·mg。野生型Km和Vm分别为1.51mg/mL和4200μmol/min·mg。
以地衣多糖为底物，在最适条件下，BisGlu16B-D213A的Km和Vm为2.37mg/mL和5900μmol/min·mg。BisGlu16B-D253A的Km和Vm分别为2.41mg/mL和5500μmol/min·mg。BisGlu16B-D213A/D253A的Km和Vm分别为1.07mg/mL和3500μmol/min·mg。野生型Km和Vm分别为2.19mg/mL和3600μmol/min·mg。
以昆布多糖为底物，在最适条件下，BisGlu16B-D213A的Km和Vm为1.12mg/mL和4000μmol/min·mg。BisGlu16B-D253A的Km和Vm分别为1.04mg/mL和3600μmol/min·mg。BisGlu16B-D213A/D253A的Km和Vm分别为1.59mg/mL和5200μmol/min·mg。野生型Km和Vm分别为0.87mg/mL和3000μmol/min·mg。
三种突变体较野生型的催化效率和比活均有很大程度上的提高，以燕麦葡聚糖为底物，三种突变体的kcat/Km分别为野生型的1.05、1.15和1.57倍，比活则为1.78、1.68和1.90倍；以地衣多糖为底物时，三种突变体的kcat/Km分别为野生型的1.54、1.45和2.09倍，比活则为1.48、1.42和1.08倍；以昆布多糖为底物时，三种突变体的比活为野生型的1.35、1.25和1.92倍。
综合来看，三种突变体不仅在动物体温条件下的酶活力有了大幅提升，在热稳定性方面也要优于野生型葡聚糖酶。而组合突变的叠加优势较弱，但是在一定程度上，突变体BisGlu16B-D213A/D253A的热稳定性要更优于BisGlu16B-D213A和BisGlu16B-D253A。
表2 BisGlu16B及其突变体在最适条件下对不同底物的动力学和比活
Figure GDA0003321849770000061
序列表
<110> 江苏科技大学
<120> 一组动物体温下高催化活力β-1,3-1,4-葡聚糖酶突变体及其应用
<140> 2021106291867
<141> 2021-06-04
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1350
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
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 tctcgggctc gatagttagc accgactgct gggtcgatga ccccaaccaa 480
tccgacaatg aaggctgtca gatcactacg agcaataccg aaacttacgg ttccggtttt 540
aatgctaaca atggcggcgt ctatgcgacg gacttccaag ccgccgctat 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
gcagtgccgt catcggtacg tgccattgtc ggtggctctg gatcagcagc cagctccact 1020
acatttgcga tctccactaa atctgctcca ttccccgtcg ggaactcaac ttccgtcgtt 1080
ggaactactg gcgccagttc gaatggcgca tgggctgcta tagtcacggg aacgggacct 1140
attggagttg ctcaagaaac tagcgtttcc gctgcttcag cagctcccag cagcccagcg 1200
gaggcaactc ctgcatctag cgtagctggg gcgcaatctt ggaactggca gtctcacgcg 1260
tggggcaatc ataatcatca cgaaccctcc gcagcagcct tgaaaaggca tctgagacat 1320
cacaagagac acgggccagg gaggctttga 1350
<210> 2
<211> 1350
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
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 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 ccattcaaag 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
gcagtgccgt catcggtacg tgccattgtc ggtggctctg gatcagcagc cagctccact 1020
acatttgcga tctccactaa atctgctcca ttccccgtcg ggaactcaac ttccgtcgtt 1080
ggaactactg gcgccagttc gaatggcgca tgggctgcta tagtcacggg aacgggacct 1140
attggagttg ctcaagaaac tagcgtttcc gctgcttcag cagctcccag cagcccagcg 1200
gaggcaactc ctgcatctag cgtagctggg gcgcaatctt ggaactggca gtctcacgcg 1260
tggggcaatc ataatcatca cgaaccctcc gcagcagcct tgaaaaggca tctgagacat 1320
cacaagagac acgggccagg gaggctttga 1350
<210> 3
<211> 1350
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
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 tctcgggctc gatagttagc accgactgct gggtcgatga ccccaaccaa 480
tccgacaatg aaggctgtca gatcactacg agcaataccg aaacttacgg ttccggtttt 540
aatgctaaca atggcggcgt ctatgcgacg gacttccaag ccgccgctat cagcatctat 600
ttcttccccc gtggttccat accttcggac attacagacg gctctccaga cccgtccggc 660
tggggtacgc caattgcgca gttcacggat agcagctgtg ccattcaaag 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
gcagtgccgt catcggtacg tgccattgtc ggtggctctg gatcagcagc cagctccact 1020
acatttgcga tctccactaa atctgctcca ttccccgtcg ggaactcaac ttccgtcgtt 1080
ggaactactg gcgccagttc gaatggcgca tgggctgcta tagtcacggg aacgggacct 1140
attggagttg ctcaagaaac tagcgtttcc gctgcttcag cagctcccag cagcccagcg 1200
gaggcaactc ctgcatctag cgtagctggg gcgcaatctt ggaactggca gtctcacgcg 1260
tggggcaatc ataatcatca cgaaccctcc gcagcagcct tgaaaaggca tctgagacat 1320
cacaagagac acgggccagg gaggctttga 1350
<210> 4
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Gln Tyr Thr Leu Gln Gln Asp Tyr Met Ala Asp Gly Asn Phe Phe Ser
1 5 10 15
Gln Phe Ser Phe Trp Asp Thr Ala Asp Pro Thr Asp Gly Phe Val Ala
20 25 30
Tyr Lys Asn Glu Thr Tyr Cys Thr Asp Asn Asp Leu Ile Ser Ser Ser
35 40 45
Ser Thr Asn Val Gln Ile Arg Val Asp Ser Ser Asn Val Thr Pro Asn
50 55 60
Gly Arg Pro Ser Val Arg Ile Thr Ser Asn Gln Ser Tyr Asn Pro Gly
65 70 75 80
Thr Leu Val Ile Leu Asp Leu Glu His Met Pro Gly Gly Ile Cys Gly
85 90 95
Thr Trp Pro Ala Phe Trp Met Val Gly Pro Asn Trp Pro Asp Asp Gly
100 105 110
Glu Ile Asp Ile Ile Glu Gly Val Asn Gln Gln Thr Thr Asn Asp Met
115 120 125
Thr Leu His Thr Ser Glu Gly Cys Thr Ile Ser Ser Ser Gly Asp Phe
130 135 140
Ser Gly Ser Ile Val Ser Thr Asp Cys Trp Val Asp Asp Pro Asn Gln
145 150 155 160
Ser Asp Asn Glu Gly Cys Gln Ile Thr Thr Ser Asn Thr Glu Thr Tyr
165 170 175
Gly Ser Gly Phe Asn Ala Asn Asn Gly Gly Val Tyr Ala Thr Asp Phe
180 185 190
Gln Ala Ala Ala Ile Ser Ile Tyr Phe Phe Pro Arg Gly Ser Ile Pro
195 200 205
Ser Asp Ile Thr Asp Gly Ser Pro Asp Pro Ser Gly Trp Gly Thr Pro
210 215 220
Ile Ala Gln Phe Thr Asp Ser Ser Cys Asp Ile Gln Ser Tyr Phe Thr
225 230 235 240
Asp Leu Gln Ile Val Phe Asp Thr Thr Phe Cys Gly Gln Trp Ala Gly
245 250 255
Asn Val Trp Ser Ser Gly Ser Cys Ala Ser Val Ala Ser Thr Cys Asp
260 265 270
Asp Tyr Val Glu Asn Asn Pro Ala Ala Phe Val Asp Ala Tyr Trp Ser
275 280 285
Ile Asn Ser Leu Gln Val Tyr Ser Gly Thr Ser Asn Gly Pro Met Gln
290 295 300
Asn Asp Thr Ser Ser Ser Ser Trp Gly Pro Ser Ala Ser Ala Asn Val
305 310 315 320
Ala Val Pro Ser Ser Val Arg Ala Ile Val Gly Gly Ser Gly Ser Ala
325 330 335
Ala Ser Ser Thr Thr Phe Ala Ile Ser Thr Lys Ser Ala Pro Phe Pro
340 345 350
Val Gly Asn Ser Thr Ser Val Val Gly Thr Thr Gly Ala Ser Ser Asn
355 360 365
Gly Ala Trp Ala Ala Ile Val Thr Gly Thr Gly Pro Ile Gly Val Ala
370 375 380
Gln Glu Thr Ser Val Ser Ala Ala Ser Ala Ala Pro Ser Ser Pro Ala
385 390 395 400
Glu Ala Thr Pro Ala Ser Ser Val Ala Gly Ala Gln Ser Trp Asn Trp
405 410 415
Gln Ser His Ala Trp Gly Asn His Asn His His Glu Pro Ser Ala Ala
420 425 430
Ala Leu Lys Arg His Leu Arg His His Lys Arg His Gly Pro Gly Arg
435 440 445
Leu
<210> 5
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Gln Tyr Thr Leu Gln Gln Asp Tyr Met Ala Asp Gly Asn Phe Phe Ser
1 5 10 15
Gln Phe Ser Phe Trp Asp Thr Ala Asp Pro Thr Asp Gly Phe Val Ala
20 25 30
Tyr Lys Asn Glu Thr Tyr Cys Thr Asp Asn Asp Leu Ile Ser Ser Ser
35 40 45
Ser Thr Asn Val Gln Ile Arg Val Asp Ser Ser Asn Val Thr Pro Asn
50 55 60
Gly Arg Pro Ser Val Arg Ile Thr Ser Asn Gln Ser Tyr Asn Pro Gly
65 70 75 80
Thr Leu Val Ile Leu Asp Leu Glu His Met Pro Gly Gly Ile Cys Gly
85 90 95
Thr Trp Pro Ala Phe Trp Met Val Gly Pro Asn Trp Pro Asp Asp Gly
100 105 110
Glu Ile Asp Ile Ile Glu Gly Val Asn Gln Gln Thr Thr Asn Asp Met
115 120 125
Thr Leu His Thr Ser Glu Gly Cys Thr Ile Ser Ser Ser Gly Asp Phe
130 135 140
Ser Gly Ser Ile Val Ser Thr Asp Cys Trp Val Asp Asp Pro Asn Gln
145 150 155 160
Ser Asp Asn Glu Gly Cys Gln Ile Thr Thr Ser Asn Thr Glu Thr Tyr
165 170 175
Gly Ser Gly Phe Asn Ala Asn Asn Gly Gly Val Tyr Ala Thr Asp Phe
180 185 190
Gln Asp Ala Ala Ile Ser Ile Tyr Phe Phe Pro Arg Gly Ser Ile Pro
195 200 205
Ser Asp Ile Thr Asp Gly Ser Pro Asp Pro Ser Gly Trp Gly Thr Pro
210 215 220
Ile Ala Gln Phe Thr Asp Ser Ser Cys Ala Ile Gln Ser Tyr Phe Thr
225 230 235 240
Asp Leu Gln Ile Val Phe Asp Thr Thr Phe Cys Gly Gln Trp Ala Gly
245 250 255
Asn Val Trp Ser Ser Gly Ser Cys Ala Ser Val Ala Ser Thr Cys Asp
260 265 270
Asp Tyr Val Glu Asn Asn Pro Ala Ala Phe Val Asp Ala Tyr Trp Ser
275 280 285
Ile Asn Ser Leu Gln Val Tyr Ser Gly Thr Ser Asn Gly Pro Met Gln
290 295 300
Asn Asp Thr Ser Ser Ser Ser Trp Gly Pro Ser Ala Ser Ala Asn Val
305 310 315 320
Ala Val Pro Ser Ser Val Arg Ala Ile Val Gly Gly Ser Gly Ser Ala
325 330 335
Ala Ser Ser Thr Thr Phe Ala Ile Ser Thr Lys Ser Ala Pro Phe Pro
340 345 350
Val Gly Asn Ser Thr Ser Val Val Gly Thr Thr Gly Ala Ser Ser Asn
355 360 365
Gly Ala Trp Ala Ala Ile Val Thr Gly Thr Gly Pro Ile Gly Val Ala
370 375 380
Gln Glu Thr Ser Val Ser Ala Ala Ser Ala Ala Pro Ser Ser Pro Ala
385 390 395 400
Glu Ala Thr Pro Ala Ser Ser Val Ala Gly Ala Gln Ser Trp Asn Trp
405 410 415
Gln Ser His Ala Trp Gly Asn His Asn His His Glu Pro Ser Ala Ala
420 425 430
Ala Leu Lys Arg His Leu Arg His His Lys Arg His Gly Pro Gly Arg
435 440 445
Leu
<210> 6
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Gln Tyr Thr Leu Gln Gln Asp Tyr Met Ala Asp Gly Asn Phe Phe Ser
1 5 10 15
Gln Phe Ser Phe Trp Asp Thr Ala Asp Pro Thr Asp Gly Phe Val Ala
20 25 30
Tyr Lys Asn Glu Thr Tyr Cys Thr Asp Asn Asp Leu Ile Ser Ser Ser
35 40 45
Ser Thr Asn Val Gln Ile Arg Val Asp Ser Ser Asn Val Thr Pro Asn
50 55 60
Gly Arg Pro Ser Val Arg Ile Thr Ser Asn Gln Ser Tyr Asn Pro Gly
65 70 75 80
Thr Leu Val Ile Leu Asp Leu Glu His Met Pro Gly Gly Ile Cys Gly
85 90 95
Thr Trp Pro Ala Phe Trp Met Val Gly Pro Asn Trp Pro Asp Asp Gly
100 105 110
Glu Ile Asp Ile Ile Glu Gly Val Asn Gln Gln Thr Thr Asn Asp Met
115 120 125
Thr Leu His Thr Ser Glu Gly Cys Thr Ile Ser Ser Ser Gly Asp Phe
130 135 140
Ser Gly Ser Ile Val Ser Thr Asp Cys Trp Val Asp Asp Pro Asn Gln
145 150 155 160
Ser Asp Asn Glu Gly Cys Gln Ile Thr Thr Ser Asn Thr Glu Thr Tyr
165 170 175
Gly Ser Gly Phe Asn Ala Asn Asn Gly Gly Val Tyr Ala Thr Asp Phe
180 185 190
Gln Ala Ala Ala Ile Ser Ile Tyr Phe Phe Pro Arg Gly Ser Ile Pro
195 200 205
Ser Asp Ile Thr Asp Gly Ser Pro Asp Pro Ser Gly Trp Gly Thr Pro
210 215 220
Ile Ala Gln Phe Thr Asp Ser Ser Cys Ala Ile Gln Ser Tyr Phe Thr
225 230 235 240
Asp Leu Gln Ile Val Phe Asp Thr Thr Phe Cys Gly Gln Trp Ala Gly
245 250 255
Asn Val Trp Ser Ser Gly Ser Cys Ala Ser Val Ala Ser Thr Cys Asp
260 265 270
Asp Tyr Val Glu Asn Asn Pro Ala Ala Phe Val Asp Ala Tyr Trp Ser
275 280 285
Ile Asn Ser Leu Gln Val Tyr Ser Gly Thr Ser Asn Gly Pro Met Gln
290 295 300
Asn Asp Thr Ser Ser Ser Ser Trp Gly Pro Ser Ala Ser Ala Asn Val
305 310 315 320
Ala Val Pro Ser Ser Val Arg Ala Ile Val Gly Gly Ser Gly Ser Ala
325 330 335
Ala Ser Ser Thr Thr Phe Ala Ile Ser Thr Lys Ser Ala Pro Phe Pro
340 345 350
Val Gly Asn Ser Thr Ser Val Val Gly Thr Thr Gly Ala Ser Ser Asn
355 360 365
Gly Ala Trp Ala Ala Ile Val Thr Gly Thr Gly Pro Ile Gly Val Ala
370 375 380
Gln Glu Thr Ser Val Ser Ala Ala Ser Ala Ala Pro Ser Ser Pro Ala
385 390 395 400
Glu Ala Thr Pro Ala Ser Ser Val Ala Gly Ala Gln Ser Trp Asn Trp
405 410 415
Gln Ser His Ala Trp Gly Asn His Asn His His Glu Pro Ser Ala Ala
420 425 430
Ala Leu Lys Arg His Leu Arg His His Lys Arg His Gly Pro Gly Arg
435 440 445
Leu
<210> 7
<211> 1350
<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 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
gcagtgccgt catcggtacg tgccattgtc ggtggctctg gatcagcagc cagctccact 1020
acatttgcga tctccactaa atctgctcca ttccccgtcg ggaactcaac ttccgtcgtt 1080
ggaactactg gcgccagttc gaatggcgca tgggctgcta tagtcacggg aacgggacct 1140
attggagttg ctcaagaaac tagcgtttcc gctgcttcag cagctcccag cagcccagcg 1200
gaggcaactc ctgcatctag cgtagctggg gcgcaatctt ggaactggca gtctcacgcg 1260
tggggcaatc ataatcatca cgaaccctcc gcagcagcct tgaaaaggca tctgagacat 1320
cacaagagac acgggccagg gaggctttga 1350
<210> 8
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
aaaagagagg ctgaagctta cgtagaattc 30
<210> 9
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
catgtctaag gcgaattaat tcgcggccgc 30
<210> 10
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
acggacttcc aagccgccgc tatcagcatc 30
<210> 11
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ggcttggaag tccgtcgcat agacgccgcc 30
<210> 12
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gatagcagct gtgccattca aagctatttc 30
<210> 13
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ggcacagctg ctatccgtga actgcgcaat 30

Claims (5)

1.一种动物体温下高催化活力β-1,3-1,4-葡聚糖酶突变体，其特征在于，所述突变体为BisGlu16B-D213A、BisGlu16B-D253A或BisGlu16B-D213A/D253A突变体；所述BisGlu16B-D213A的氨基酸序列如SEQ ID NO.4所示；BisGlu16B-D253A的氨基酸序列如SEQ ID NO.5所示；BisGlu16B-D213A/D253A的氨基酸序列如SEQ ID NO.6所示，其中，所有突变位点的标号使用未去信号肽时的序列。
2.编码权利要求1所述葡聚糖酶突变体的多核苷酸，其特征在于，编码所述BisGlu16B-D213A突变体的多核苷酸序列如SEQ ID NO.1所示；编码 BisGlu16B-D253A突变体的多核苷酸序列如SEQ ID NO.2所示；编码 BisGlu16B-D213A/D253A突变体的多核苷酸序列如SEQID NO.3所示；其中，所有突变位点的标号使用未去信号肽时的序列。
3.一种重组载体，含有权利要求2所示的任一多核苷酸序列。
4.一种重组菌株，含有权利要求3所述的重组载体。
5.权利要求4所述重组菌株在制备饲料添加剂中的应用。
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US6501008B1 (en) * 1998-06-10 2002-12-31 The Regents Of The University Of California Maize endo-1,3;1,4-β-glucanase nucleic acid
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