CN114317500A - 木聚糖酶Scxyn5及其编码基因和应用 - Google Patents
木聚糖酶Scxyn5及其编码基因和应用 Download PDFInfo
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- CN114317500A CN114317500A CN202210155118.6A CN202210155118A CN114317500A CN 114317500 A CN114317500 A CN 114317500A CN 202210155118 A CN202210155118 A CN 202210155118A CN 114317500 A CN114317500 A CN 114317500A
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- Prior art keywords
- xylanase
- scxyn5
- ala
- recombinant
- gene
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- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 title claims abstract description 78
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 16
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Abstract
本发明涉及基因工程领域，具体涉及木聚糖酶Scxyn5及其编码基因和应用。其氨基酸序列如SEQ ID NO：1所示。本发明提供了自裂褶菌(Schizophyllum commune)的木聚糖酶Scxyn5，为极端耐盐/嗜盐的木聚糖酶，高盐浓度的催化体系能提高个木聚糖酶对玉米纤维胶的水解活性；应用木聚糖酶单酶水解玉米纤维胶能够制备具有不同分力量分布特点的阿拉伯木聚糖，为以玉米皮为原料工业化制备特定分子量的功能性阿拉伯木聚糖提供了基础。
Description
技术领域
本发明涉及基因工程领域，具体涉及木聚糖酶Scxyn5及其编码基因和应用。
背景技术
内切-β-1,4-木聚糖酶(β-1,4-xylanase,EC 3.2.1.8)是一种糖苷水解酶，能够催化水解木聚糖主链β-1,4-D-木糖苷键生成木寡糖，是木聚糖降解酶系中最重要的一类糖苷水解酶。根据催化结构域的序列保守性，木聚糖酶被归为糖苷水解酶(GH)第5、7、8、9、10、11、12、16、26、30、43、44、51、62家族。目前，已报道的木聚糖酶大多分布在GH10家族和GH11家族。嗜盐/耐盐性是酶的重要特性。嗜盐/耐盐木聚糖酶一般是指在高盐浓度下(0.5～4.5M)保持有催化活性的木聚糖酶。嗜盐/耐盐木聚糖酶在海产品、腐乳、面制品等高含盐量食品的加工生产中有很大的应用潜力。目前己报道的耐盐木聚糖酶多属于GH10家族，而属于GH11家族的耐盐木聚糖酶则非常少。
目前还未见在高盐体系中，应用GH10家族和GH11家族极端嗜盐/耐盐木聚糖酶水解玉米纤维胶，制备具有不同分子量分布特点的阿拉伯木聚糖的报道，而这些差异使阿拉伯木聚糖可能具有不同的理化性质和生物学功能。
发明内容
为了解决上述问题，提出并完成了本发明。
本发明的目的是提供一种来自裂褶菌(Schizophyllum commune)的木聚糖酶Scxyn5。
本发明的再一目的是提供编码上述木聚糖酶Scxyn5的基因。
本发明的再一目的是提供包含上述基因的重组载体。
本发明的再一目的是提供包含上述基因的重组菌株。
本发明的再一目的是提供上述木聚糖酶Scxyn5的应用。
根据本发明的来自裂褶菌(Schizophyllum commune)的木聚糖酶Scxyn5，其氨基酸序列如SEQ ID NO：1所示。
LPKRQTTGLSAHSRQTTGLNTIAQAAGLKYLGSATDNPELTDTHYVAILSDSSEFGQLTPGNSMKWDATEPTQGQFSFDNADAIVELAQNNSQLIRGHTCVWYSQLPSWVSNGSWDADSLNVAMTTHTSTVVDHFKGKIYSWDVVNEAFEDDGSFRQNVFYTTIGEDYIANAFKAARAADPDAKLYINDYNIEGTGAKADALYTFVSSLLNASVPIDGIGMQAHLIVGSVPTTIQENIARFTALGLEVALTELDIRMPVPAAEADLEQQKADYEAVVGACAAVEGCVGVTVWDYTDKYSWVPSVFDGYGAALPWDENLEKKPAYDGIVSGLGA。
根据本发明的木聚糖酶基因，编码上述木聚糖酶Scxyn5。
根据本发明的木聚糖酶基因，其核苷酸序列如SEQ ID NO：2所示。
CTCCCCAAGCGTCAGACCACTGGCCTCAGCGCCCACTCGCGGCAAACGACCGGCCTTAACACCATCGCGCAAGCCGCCGGCCTCAAGTACCTCGGCTCCGCGACGGACAACCCCGAATTGACGGACACGCACTACGTCGCGATCCTGAGCGACTCGAGTGAGTTTGGGCAGCTCACGCCGGGGAATAGTATGAAGTGGGACGCCACGGAGCCCACGCAGGGACAGTTCTCGTTCGACAATGCGGACGCGATCGTGGAGCTTGCTCAGAACAACAGCCAGCTCATTCGAGGTCACACCTGCGTTTGGTACAGTCAGCTGCCCAGCTGGGTCTCAAACGGGTCCTGGGACGCGGACTCGCTGAATGTGGCCATGACGACGCATACTTCGACGGTGGTCGATCATTTCAAGGGCAAAATATATAGCTGGGATGTAGTAAACGAGGCGTTTGAGGACGACGGCAGCTTCCGTCAGAACGTCTTCTACACAACCATCGGCGAGGATTACATCGCCAACGCGTTCAAAGCCGCCCGCGCGGCTGACCCTGATGCAAAACTTTATATCAACGACTACAACATCGAAGGCACCGGCGCCAAAGCCGACGCGCTCTACACCTTCGTCTCCTCCCTTCTCAACGCCTCCGTCCCCATCGACGGCATCGGCATGCAGGCGCACCTCATCGTCGGCTCTGTCCCAACGACCATCCAGGAGAACATCGCGCGCTTCACTGCTTTGGGCCTCGAGGTTGCGCTCACGGAGCTCGACATACGGATGCCTGTGCCCGCCGCCGAGGCGGATTTGGAGCAGCAGAAGGCGGATTACGAGGCCGTGGTGGGCGCGTGTGCGGCGGTGGAGGGGTGCGTGGGTGTGACGGTCTGGGATTATACGGATAAGTACTCCTGGGTTCCGAGTGTCTTCGATGGGTATGGAGCGGCTTTGCCGTGGGATGAGAACTTGGAAAAGAAGCCAGCTTACGACGGGATCGTGAGCGGCTTGGGTGCA。
本发明还提供了包含上述基因的重组载体。
本发明还提供了包含上述基因的重组菌株。
本发明还提供了上述嗜盐木聚糖酶Scxyn5的应用。
本发明提供了上述嗜盐木聚糖酶Scxyn5用于水解玉米纤维胶制备阿拉伯木聚糖的应用，其中，所述嗜盐木聚糖酶Scxyn5在高盐浓度下水解玉米纤维胶。
本申请发现结构复杂天然底物在高盐体系中的理化状态会发生改变，进而影响酶催化效率和酶解产物的特点。此外，高盐体系能够减少酶解底物制备低聚糖过程中的微生物污染和杂菌生长，因此不需要对底物和反应体系采取高温灭菌或加入抑菌剂等处理，能够降低成本、防止高温对生物活性物质的破坏、减少其他抑菌成分的使用。
本发明提供了自裂褶菌(Schizophyllum commune)的木聚糖酶Scxyn5，为极端耐盐/嗜盐的木聚糖酶，高盐浓度的催化体系能提高个木聚糖酶对玉米纤维胶的水解活性；应用木聚糖酶单酶水解玉米纤维胶能够制备具有不同分力量分布特点的阿拉伯木聚糖，为以玉米皮为原料工业化制备特定分子量的功能性阿拉伯木聚糖提供了基础。
附图说明
图1显示重组木聚糖酶Scxyn5的发酵液上清电泳图片；
图2显示重组木聚糖酶Scxyn5最适pH值；
图3显示重组木聚糖酶Scxyn5最适温度；
图4显示NaCl浓度对Scxyn5水解活性影响；
图5显示NaCl浓度对Scxyn5水解CFG效率影响；
图6显示4M的NaCl体系中重组木聚糖酶Scxyn5最适pH值；
图7显示4M的NaCl体系中重组木聚糖酶Scxyn5最适温度；
图8显示不同影响因素对重组木聚糖酶Scxyn5水解CFG的影响。
具体实施方式
试验材料和试剂
1、菌株及载体：表达宿主Pichiapastoris GS115，表达质粒载体pPIC9K为本实验室保存；
2、工具酶及生化试剂：内切酶购自New England Biolabs公司，重组酶购自全式金公司，桦木木聚糖购自Sigma公司，小麦阿拉伯木聚糖购自Megazyme公司；玉米皮购自河北广玉淀粉公司，其它都为国产分析纯试剂(均可从普通生化试剂公司购买得到)；
3、培养基：
(1)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，
(2)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)。
实施例1木聚糖酶Scxyn5编码基因的克隆
以玉米皮纤维为碳源发酵培养Schizophyllum commune，分离菌体提取mRNA进行转录组测序分析；根据测序结果克隆了Scxyn5编码基因(Xyn5-F：5′-CCGGAATTCCTCCCCAAGCGTCAGACCACT-3′；Xyn5-R：5′-CGCTAGCGGCCGCTGCACCCAAG CCGCTCAC-3′)，并构建了重组载体Scxyn5-pET28a。
实施例2木聚糖酶重组载体的构建
以S.commune sp.DB1来源的木聚糖酶Scxyn5-pET28a为模板，采用PCR反应克隆木聚糖酶基因Scxyn5片段，引物和反应条件如下：
Scxyn5-F：5′-CCGGAATTCCTCCCC AAGCGTCAGACCACT-3′；
Scxyn5-R：5′-CGCTAGCGGCCGCTGCACCCAAG CCGCTCAC-3′；
扩增产物分别经过EcoRⅠ和NotⅠ双酶切后直接与切开的pPIC9k质粒进行连接，转化TransI-T1感受态，挑取转化子测序验证。测序验证正确的转化子即为重组酵母表达质粒pPIC9K-Scxyn5。
实施例3重组表达木聚糖酶基因的毕赤酵母工程菌的构建
将重组表达载体pPIC9K-Scxyn5用内切酶SacI线性化并转化毕赤酵母GS115，获得重组酵母菌株GS115/pPIC9K-Scxyn5。
挑取阳性转化子转接于含5mL BMGY培养基的50mL三角瓶中，置于30℃，230rpm摇床培养36h；将发酵液3000g离心5min，弃上清后沉淀菌体用5mL的BMMY培养基重悬，再置于30℃，230rpm摇床诱导培养72h。如图1所示，发酵液取上清用于酶活性检测和SDS-PAGE电泳检测。
实施例4重组木聚糖酶的活性分析
木聚糖酶酶活力检测方法如下：200μL的酶解反应体系包含190μL底物和10μL适当的稀释酶液，在给定的温度和pH条件下保温反应10min，随后加入300μL的DNS试剂，沸水煮5min。样品冷却后测定540nm下的吸光值。1个酶活单位(U)定义为在给定的条件下，每分钟生成1μmol还原端所需的酶量。
1、重组木聚糖酶最适pH测定方法如下:
将实施例3纯化的重组木聚糖酶在不同的pH下酶促反应以测定其最适pH。底物桦木木聚糖用不同pH的缓冲液中(0.1mol/L Gly-HCl缓冲溶液，1.5-3.0；0.1mol/L柠檬酸缓冲液，3.0-7.0；0.1mol/LNaH2PO4-Na2HPO4缓冲液，6.5-8.0；0.1mol/LTris-HCl缓冲溶液，7.5-8.5；0.1mol/L Gly-NaOH缓冲溶液，8.5-10.5)50℃下进行木聚糖酶活力测定。结果表明，重组木聚糖酶Scxyn5最适反应pH大致在5.0-6.0左右(图2)。
2、重组木聚糖酶最适温度测定方法如下:
重组木聚糖酶最适温度的测定为在0.1mol/L柠檬酸缓冲液各自最适pH缓冲液体系及不同温度下进行酶促反应。结果表明,重组木聚糖酶Scxyn5最适温度在65-75℃左右(图3)。
实施例5NaCl浓度对酶活性的影响
NaCl浓度对酶活性的影响：以桦木木聚糖为底物，在含有不同浓度NaCl(1-5M)缓冲体系中和最佳pH、温度下，参照实施例3测定重组木聚糖酶的水解活性。以未加入NaCl反应体系酶活计100％，ScXyn5在含有1～5M的NaCl反应体系中，水解桦木木聚糖的酶活力分别提高了1.18、1.46、1.88、2、2.13倍(图4)。
实施例6NaCl浓度对重组木聚糖酶催化水解CFG酶活性的影响
1.玉米纤维胶制备
玉米纤维胶的提取步骤如下：
1)玉米皮干燥粉碎后过60目筛。称取50g玉米皮粉，加入500mL正己烷，搅拌2h后用布氏漏斗过滤。用适量无水乙醇清洗样品，过滤干燥后按1:10(w/v)固液比加入蒸馏水，调节pH为6.0，向体系中加入耐高温α-淀粉酶，95℃水浴下处理30min，降低水浴温度为55℃，加入糖化酶，反应30min，并利用I2-KI溶液检查淀粉水解情况。向上述处理处理液中加入中性蛋白酶，在水浴温度55℃，pH为7的条件下，反应1h后，加热至100℃灭酶5min，纱布过滤，滤渣用蒸馏水洗涤2遍，60℃烘干后备用。
2)称取1)中得到的50g样品，加入2g的NaOH和1.9g的Ca(OH)2，加入0.5L蒸馏水，混合后沸水浴1h。将反应后的溶液冷却至室温，6000×g离心20min后分离清A1和沉淀B1。
3)上清液A1加入3g的H2O2(30％，w/w)，调整溶液的pH为11.5，室温搅拌2h；将pH值调整到4.0，10000×g离心30min分离上清液；向上清液加入2倍体积无水乙醇，得到白色絮状沉淀物CFG1。
4)向沉淀B1加入适量蒸馏水，再加入30％的H2O2(按照干基的0.1％添加)，调pH至11.5，沸水浴1.5h。冷却后6000×g离心20min，分离上清液A2；调节A2的pH至4.0～4.5，再10000×g离心30min，分离上清液后加入2倍体积无水乙醇，得到白色絮状沉淀CFG2。
5)用蒸馏水溶解CFG1和CFG2后混合，在冷冻干燥获得玉米皮纤维胶CFG。
2.NaCl浓度对重组木聚糖酶催化水解CFG酶活性的影响
参照实施例3和实施例5测定NaCl浓度对重组木聚糖酶催化水解CFG的活性的影响：200μL的酶解反应体系包含180μL底物(用含有1～5M的NaCl的缓冲液溶解CFG底物)和20μL适当的稀释酶液，在最佳pH、温度条件下保温反应1h，随后加入300μL的DNS试剂，沸水煮5min。样品冷却后测定540nm下的吸光值，计算重组木聚糖酶的水解CFG生成还原端的量。如图5所示，以未加入NaCl反应体系酶活计100％，再用相同反应条件下，木聚糖酶在1～5M的NaCl反应体系中催化水解CFG时还原端生成量相对于0M的NaCl反应体系中还原端生成量做图，显示NaCl对木聚糖酶活性影响，ScXyn5在含有1～5M的NaCl反应体系中，水解CFG的还原端生成量分别提高了1.41、1.56、1.66、2.03、1.3倍(图5)。
3.木聚糖酶在高NaCl浓度体系水解CFG的最适催化条件
1)重组木聚糖酶在高NaCl浓度体系水解CFG最适pH测定方法如下:
配制含4M NaCl的不同pH缓冲液：0.1mol/L Gly-HCl缓冲溶液，pH 1.5-3.0；0.1mol/L柠檬酸缓冲液，pH 3.0-7.0；0.1mol/LNaH2PO4-Na2HPO4缓冲液，pH 6.5-8.0；0.1mol/LTris-HCl缓冲溶液，pH 7.5-8.5；0.1mol/L Gly-NaOH缓冲溶液，pH 8.5-10.5。用配好的不同pH缓冲液溶解CFG底物(1％，w/v)。将实施例3纯化的重组木聚糖酶在50℃条件下、不同的pH缓冲体系中，进行木聚糖酶活力测定，测定其最适pH。结果表明，重组木聚糖酶Scxyn5最适反应pH约为6.0(图6)。
2)重组木聚糖酶在高NaCl浓度体系水解CFG最适温度测定方法如下:
重组木聚糖酶最适温度的测定为在0.1mol/L柠檬酸缓冲液(含4M NaCl)最适pH缓冲液体系及不同温度下进行酶促反应。结果表明,重组木聚糖酶Scxyn5最适温度在40-50℃之间(图7)。
4.响应面优化实验
在单因素试验基础上，选择温度、pH值和NaCl浓度3个因素为考察对象，以相对还原末端产率为响应值，设计3因素3水平回归试验(表1)。
表1试验因素和水平
采用Design-Expert software 11.0Trial软件对表2数据进行多元回归分析，结果(图8)表明，重组木聚糖酶Scxyn5水解CFG的最佳条件为：温度40.82℃，pH值5.77，NaCl浓度3.25M。
表2响应面分析
5.高NaCl浓度下重组木聚糖酶催化水解CFG产物分析
参照上述最适反应条件，应用重组木聚糖酶Scxyn5在最适条件下水解CFG底物制备低聚糖，应用凝胶色谱仪(ELEOS System，Wyatt)分析水解产物分子量分布特征。计算结果如表3所示，重组木聚糖酶Scxyn5水解产物中70.8％阿拉伯木聚糖分子量在2.6-22KDa。
表3 CFG和重组木聚糖酶水解产物分子量分布特征
以上实施例仅用于解释本申请的技术方案，不限定本申请的保护范围。
序列表
<110> 国家粮食和物资储备局科学研究院
<120> 木聚糖酶Scxyn5及其编码基因和应用
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 333
<212> PRT
<213> 裂褶菌(Schizophyllum commune)
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Leu Ser Asp Ser Ser Glu Phe Gly Gln Leu Thr Pro Gly Asn Ser Met
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ctccccaagc gtcagaccac tggcctcagc gcccactcgc ggcaaacgac cggccttaac 60
accatcgcgc aagccgccgg cctcaagtac ctcggctccg cgacggacaa ccccgaattg 120
acggacacgc actacgtcgc gatcctgagc gactcgagtg agtttgggca gctcacgccg 180
gggaatagta tgaagtggga cgccacggag cccacgcagg gacagttctc gttcgacaat 240
gcggacgcga tcgtggagct tgctcagaac aacagccagc tcattcgagg tcacacctgc 300
gtttggtaca gtcagctgcc cagctgggtc tcaaacgggt cctgggacgc ggactcgctg 360
aatgtggcca tgacgacgca tacttcgacg gtggtcgatc atttcaaggg caaaatatat 420
agctgggatg tagtaaacga ggcgtttgag gacgacggca gcttccgtca gaacgtcttc 480
tacacaacca tcggcgagga ttacatcgcc aacgcgttca aagccgcccg cgcggctgac 540
cctgatgcaa aactttatat caacgactac aacatcgaag gcaccggcgc caaagccgac 600
gcgctctaca ccttcgtctc ctcccttctc aacgcctccg tccccatcga cggcatcggc 660
atgcaggcgc acctcatcgt cggctctgtc ccaacgacca tccaggagaa catcgcgcgc 720
ttcactgctt tgggcctcga ggttgcgctc acggagctcg acatacggat gcctgtgccc 780
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gcggcggtgg aggggtgcgt gggtgtgacg gtctgggatt atacggataa gtactcctgg 900
gttccgagtg tcttcgatgg gtatggagcg gctttgccgt gggatgagaa cttggaaaag 960
aagccagctt acgacgggat cgtgagcggc ttgggtgca 999
Claims (9)
1.一种木聚糖酶Scxyn5，其特征在于，其氨基酸序列如SEQ ID NO：1所示。
2.编码权利要求1所述木聚糖酶Scxyn5的基因。
3.根据权利要求2所述的基因，其特征在于，其核苷酸序列如SEQ ID NO：2所示。
4.包含权利要求2所述基因的重组载体。
5.包含权利要求2所述基因的重组菌株。
6.权利要求1所述木聚糖酶Scxyn5的应用。
7.权利要求1所述木聚糖酶Scxyn5在高盐浓度下水解木聚糖的应用。
8.权利要求1所述木聚糖酶Scxyn5用于水解玉米纤维胶制备阿拉伯木聚糖的应用，其中，所述木聚糖酶Scxyn5在高盐浓度下水解玉米纤维胶。
9.根据权利要求8所述的应用，其特征在于，所述木聚糖酶Scxyn5在高盐浓度下水解玉米纤维胶在含有1～5M的NaCl反应体系中水解玉米纤维胶。
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| CN114621987A (zh) * | 2022-02-22 | 2022-06-14 | 国家粮食和物资储备局科学研究院 | 一种制备具有不同分子量分布特点的阿拉伯木聚糖方法 |
| CN114621987B (zh) * | 2022-02-22 | 2024-05-17 | 国家粮食和物资储备局科学研究院 | 一种制备具有不同分子量分布特点的阿拉伯木聚糖方法 |
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