CN108795781A - 一种高产哈茨木霉α-1,3-葡聚糖酶的重组菌及其应用 - Google Patents
一种高产哈茨木霉α-1,3-葡聚糖酶的重组菌及其应用 Download PDFInfo
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- CN108795781A CN108795781A CN201710304433.XA CN201710304433A CN108795781A CN 108795781 A CN108795781 A CN 108795781A CN 201710304433 A CN201710304433 A CN 201710304433A CN 108795781 A CN108795781 A CN 108795781A
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- dextranases
- trichoderma
- trichoderma harzianum
- recombinant bacterium
- dextranase
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Abstract
本发明公开了一种高产哈茨木霉α‑1,3‑葡聚糖酶的重组菌及其应用。本发明对来自哈茨木霉CCM F‑340的α‑1,3‑葡聚糖酶基因进行密码子优化,采用里氏木霉cbh1强启动子及其信号肽序列和cbh1终止子,以pyr4基因为筛选标记基因构建了α‑1,3‑葡聚糖酶表达载体pSK‑mutAW,并在里氏木霉中实现了哈茨木霉α‑1,3‑葡聚糖酶的高效分泌表达。通过实验证明:重组表达的哈茨木霉α‑1,3‑葡聚糖酶能有效降解非水溶性葡聚糖mutan,发酵液酶活力单位可达到2.3U/ml,发酵液中α‑1,3‑葡聚糖酶含量约为68ug/ml,且本发明的发酵工艺和目标蛋白纯化工艺简单,非常适合工业生产及应用。
Description
技术领域
本发明属于生物技术领域,具体涉及一种高产哈茨木霉α-1,3-葡聚糖酶的重组菌及其应用。
背景技术
口腔牙菌斑是一种高度多样化和复杂的生物膜,牙菌斑是齿龈炎、牙周炎、龋齿等口腔疾病的重要病因。目前,常用的牙菌斑控制方法有机械法和药物法。机械法主要有刷牙和使用牙线、牙签等。药物法则是利用一些化学药物来去除或抑制牙菌斑的形成。目前市场上主要使用的化学药物为洗必泰。洗必泰具有很好的抗菌活性,但长期使用容易导致口腔中有益菌群的破坏和耐药菌的富集。
多糖是牙菌斑基质中的关键成分,可作为抗牙菌斑试剂的重要靶点,使用特异性多糖水解酶降解牙菌斑多糖基质,控制菌斑在牙面上的形成和聚集,是牙菌斑控制的又一途径。非水溶性葡聚糖(mutan)是由口腔链球菌分泌的糖基转移酶催化蔗糖形成的,非水溶性葡聚糖含有高比例的α-1,3糖苷键,因其水不溶性和粘附性,在致龋菌的粘附、牙菌斑的形成和阻碍牙菌斑中酸性物质的扩散中起着重要作用,是口腔变形链球菌致龋的重要毒力因子之一。α-1,3-葡聚糖酶(α-(1→3)-glucanase,EC3.2.1.84)能够特异性催化α-1,3糖苷键的水解,应用α-1,3-葡聚糖酶减少或清除牙菌斑的作用已见报道,是一种极具潜力的、科学安全的生物防龋新方法。α-1,3-葡聚糖酶来源于细菌和真菌,其中木霉属是α-1,3-葡聚糖酶的重要来源。在大多数菌中α-1,3-葡聚糖酶是诱导型表达的,通常在富含α-1,3糖苷键的葡聚糖存在的条件下表达。利用天然菌株发酵生产α-1,3-葡聚糖酶,存在目标蛋白产量低、诱导物来源受限及蛋白纯化工艺繁琐等问题。目前在不同表达系统中α-1,3-葡聚糖酶表达水平也普遍不高(见表1),Wiater A等人以原始哈茨木霉CCM F-340菌株进行摇瓶发酵,发酵液的最高酶活为0.82U/ml(Wiater A et al,2012),且需要专门制备硫磺菌子实体细胞壁提取物作为诱导物。Wiater A等人采用大肠杆菌表达系统表达哈茨木霉CCM F-340来源的α-1,3-葡聚糖酶,α-1,3-葡聚糖酶的酶活力仅为0.097U/mg(Wiater A et al,2011)。因此开发安全性高、发酵提纯工艺简单、成本低的新表达系统来高效表达α-1,3-葡聚糖酶是十分必要的。
表1、不同宿主表达α-1,3-葡聚糖酶的相关研究
发明内容
本发明的一个目的是提供一种重组菌。
本发明提供的重组菌是将哈茨木霉α-1,3-葡聚糖酶的编码基因导入宿主里氏木霉菌Trichoderma reesei,得到的菌。
上述重组菌中,所述哈茨木霉α-1,3-葡聚糖酶的编码基因是通过表达哈茨木霉α-1,3-葡聚糖酶的重组载体导入宿主里氏木霉菌。
上述重组菌中,所述表达哈茨木霉α-1,3-葡聚糖酶的重组载体为pSK-mutAW重组载体;
所述pSK-mutAW重组载体是将所述哈茨木霉α-1,3-葡聚糖酶的编码基因插入骨架载体的多克隆位点得到的;所述骨架载体具体为pSK-Lip,其含有大小为1904bp的cbh1启动子、大小为51bp的cbh1信号肽和大小为2243bp的Tcbh1终止子。
上述重组菌中,所述pSK-mutAW重组载体还包括筛选标记基因;所述筛选标记基因具体为pyr4基因。
上述重组菌中,所述pSK-mutAW重组载体为将序列3所示的DNA分子替换骨架载体pSK-Lip的EcoRI和SpeI酶切位点之间DNA序列,且保持骨架载体pSK-Lip的其他序列不变得到的载体。重组载体pSK-mutAW表达带有HIS标签的α-1,3-葡聚糖酶,带有HIS标签的α-1,3-葡聚糖酶的氨基酸序列如序列2所示。
上述重组菌中,所述哈茨木霉α-1,3-葡聚糖酶的编码基因的核苷酸序列为序列1。
上述重组菌中,所述里氏木霉菌为尿嘧啶缺陷型里氏木霉菌,所述尿嘧啶缺陷型里氏木霉菌具体为里氏木霉TU-6菌株。
本发明的另一个目的是提供上述重组菌的新用途。
本发明提供了上述重组菌在生产α-1,3-葡聚糖酶中的应用。
本发明还提供了上述重组菌在降解非水溶性葡聚糖中的应用。
本发明还提供了上述重组菌在提高α-1,3-葡聚糖酶酶活力中的应用。
本发明的最后一个目的是提供一种生产哈茨木霉α-1,3-葡聚糖酶的方法。
本发明提供的生产哈茨木霉α-1,3-葡聚糖酶的方法包括如下步骤:发酵培养上述重组菌,得到所述哈茨木霉α-1,3-葡聚糖酶。
上述方法中,所述培养的条件为28℃培养4-8天。
本发明的优势:
1、里氏木霉Trichoderma reesei是美国FDA认证的安全生产菌株,蛋白表达分泌能力强,某些突变株的胞外蛋白分泌量可达到100g/L,并且具有与高等哺乳动物相似的糖基化修饰系统,非常适合表达真核来源的药用、食品级蛋白。本发明选择里氏木霉作为表达宿主,高效分泌表达α-1,3-葡聚糖酶。
2、本发明对来自哈茨木霉CCM F-340的α-1,3-葡聚糖酶基因进行密码子优化,并将pyr4基因和优化后的α-1,3-葡聚糖酶基因mutAW构建在同一个载体上以提高转化子的阳性率,并且在pyr4两侧存在约1kb的cbh1终止子正向重复序列,在需要时可以实现pyr4基因的回敲,这有效地解决了里氏木霉中可用筛选标记少的问题,以便后续对菌株的进一步改造。
本发明对来自哈茨木霉CCM F-340的α-1,3-葡聚糖酶基因进行密码子优化,采用里氏木霉cbh1强启动子及其信号肽序列和cbh1终止子,以pyr4基因为筛选标记基因构建了α-1,3-葡聚糖酶表达载体pSK-mutAW,并在里氏木霉中实现了哈茨木霉α-1,3-葡聚糖酶的高效分泌表达。通过实验证明:重组表达的哈茨木霉α-1,3-葡聚糖酶能有效降解非水溶性葡聚糖mutan,发酵液酶活力单位可达到2.3U/ml,发酵液中α-1,3-葡聚糖酶含量约为68ug/ml,是哈茨木霉CCM F-340来源的α-1,3-葡聚糖酶的最高表达水平,且发酵工艺和目标蛋白纯化工艺简单,非常适合工业生产及应用。
附图说明
图1为pSK-mutAW质粒图。
图2为阳性转化子PCR验证。Lane1-8分别为转化子mu-1-转化子mu-8;Lane9为野生型里氏木霉TU-6。
图3为野生型里氏木霉TU-6和阳性转化子发酵液的SDS-PAGE图。Lane1-2:野生型里氏木霉TU-6;Lane3-4:阳性转化子mu-1;Lane5-6:阳性转化子mu-2;Lane7-8:阳性转化子mu-3;Lane9-10:阳性转化子mu-4。
图4为不同转化子发酵液的酶活。
图5为α-1,3-葡聚糖酶纯化样品SDS-PAGE图。Lane1:野生型里氏木霉TU-6发酵液;Lane2:阳性转化子mu-3发酵液;Lane3:α-1,3-葡聚糖酶纯化样品(阳性转化子mu-3发酵液的纯化样品)。
图6为α-1,3-葡聚糖酶平板检测。孔1:醋酸-醋酸钠缓冲液;孔2:α-1,3-葡聚糖酶纯化样品(阳性转化子mu-3发酵液的纯化样品);孔3:野生型里氏木霉T.reesei TU-6发酵液;孔4:阳性转化子mu-3发酵液。
图7为里氏木霉T.reesei TU-6中重组表达的α-1,3-葡聚糖酶最适温度。
图8为里氏木霉T.reesei TU-6中重组表达的α-1,3-葡聚糖酶最适pH。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中的定量试验,均设置三次重复实验,结果取平均值。
下述实施例中的大肠杆菌Escherichia coli strain Trans1-T1是北京全式金生物技术有限公司的产品,货号:CD501-03。
下述实施例中的里氏木霉TU-6菌株为尿嘧啶缺陷型TU-6菌株在文献“ZhangGuangtao,Lukas Hartl,Andre Schuster,et al.Gene targeting in a nonhomologousend joining deficient Hypocrea jecorina.Journal of Biotechnology,2009,139:146-151”中公开过,公众可从中国科学院微生物研究所获得。
下述实施例中的里氏木霉Trichoderma reesei QM9414菌株在文献“Chen F,ChenX Z,Su X Y,et al.An Ime2-like mitogen-activated protein kinase is involved incellulase expression in the filamentous fungus Trichodermareesei.Biotechnology Letters,2015,37(10):2055-2062”中公开过,公众可从中国科学院微生物研究所获得。
下述实施例中的质粒pSK-Lip在文献“Qin L N,Cai F R,Dong X R,etal.Improved production of heterologous lipase in Trichoderma reesei by RNAimediated gene silencing of an endogenic highly expressed gene.BioresourceTechnology,2012,109(2):116–122”中公开过,公众可从中国科学院微生物研究所获得。
下述实施例中的口腔变形链球菌Streptococcus mutans strain UA159(S.mutans UA159)在文献“Dragana William M.McShan,Robert E.McLaughlin,etal.Genome sequence of Streptococcus mutans UA159,a cariogenic dentalpathogen.Proceedings of the National Academy of Sciences of the United Statesof America,2002,99(22):14434-11439”中公开过,公众可从中国科学院微生物研究所获得。
下述实施例中的培养基的配方:
1、PDA培养基:葡萄糖20g、马铃薯200g、琼脂粉20g,去离子水定容至1L,自然pH,115℃高压蒸汽灭菌20min。
2、MM液体培养基:(NH4)2SO4 5g、KH2PO4 15g、MgSO4.7H2O 1.23g、CaCl2.2H2O 0.8g、FeSO4·7H2O 5mg、MnSO4·H2O 1.6mg、ZnSO4·7H2O 1.4mg、CoCl2 2mg,去离子水定容至1L,调pH值为5.3,115℃高压蒸汽灭菌20min。
3、LB液体培养基:蛋白胨10g、氯化钠10g、酵母提取物5g,去离子水定容至1L,121℃高压蒸汽灭菌20min。
4、脑心浸出液肉汤培养基(BHI)购自OXOID,使用时称取37g BHI粉末,用去离子水溶解并定容至1L,自然pH,121℃高压蒸汽灭菌。
5、10×YEPD培养基是将酵母提取物10%(质量分数)、蛋白胨20%(质量分数)、葡萄糖10%(质量分数)和水混匀得到的。
下述实施例中的试剂的配方:
1、铁氰化钾试剂:K3Fe(CN)6 0.4g/L、Na2CO3 20g/L,避光保存。
2、1.1M山梨醇溶液:称取20g山梨醇,用超纯水溶解并定容至100ml,115℃高压蒸汽灭菌20min。
3、50mM甘氨酸-盐酸缓冲液:
50mM甘氨酸溶液:称取1.5g甘氨酸,用去离子水溶解并定容至400ml;
50mM盐酸溶液:取620ul浓盐酸加入到400ul去离子水中,充分混匀;
pH为2.0和3.0的甘氨酸-盐酸缓冲液为将50mM甘氨酸溶液和50mM盐酸溶液分别按照体积比为50:53和50:12混匀得到的。按比例混匀后,用pH计测定混匀后溶液的实际pH,若实际pH和所需的pH有偏差,可进行微调。微调的具体操作为若溶液的实际pH低于所需的pH,则加入50mM甘氨酸溶液调pH至所需pH;若溶液的实际pH高于所需的pH,则加入50mM盐酸溶液调pH至所需pH。
4、50mM醋酸-醋酸钠缓冲液:
50mM醋酸溶液:取1.43ml冰醋酸加入到500ml去离子水中,充分混匀;
50mM醋酸钠溶液:称取2.05g无水醋酸钠,用去离子水溶解并定容至500ml;
pH为4.0、5.0和5.5的醋酸-醋酸钠缓冲液为将50mM醋酸溶液和50mM醋酸钠溶液分别按照体积比为41:9、15:35和5:45混匀得到的。按比例混匀后,用pH计测定混匀后溶液的实际pH,若实际pH和所需的pH有偏差,可进行微调。微调的具体操作为若溶液的实际pH低于所需的pH,则加入50mM醋酸钠溶液调pH至所需pH;若溶液的实际pH高于所需的pH,则加入50mM醋酸溶液调pH至所需pH。
5、50mM磷酸二氢钠-磷酸氢二钠缓冲液:
50mM磷酸二氢钠溶液:称取3.12g NaH2PO4.2H2O用去离子水溶解并定容至400ml;
50mM磷酸氢二钠溶液:称取7.16g Na2HPO4.12H2O用去离子水溶解并定容至400ml;
pH为6.0、7.0和8.0的磷酸二氢钠-磷酸氢二钠缓冲液为将50mM磷酸二氢钠溶液与50mM磷酸氢二钠溶液分别按照体积为88:12、39:61和5:95混匀得到的。按比例混匀后,用pH计测定混匀后溶液的实际pH,若实际pH和所需的pH有偏差,可进行微调。微调的具体操作为若溶液的实际pH低于所需的pH,则加入50mM磷酸氢二钠溶液调pH至所需pH;若溶液的实际pH高于所需的pH,则加入50mM磷酸二氢钠溶液调pH至所需pH。
实施例1、一种高效分泌表达哈茨木霉α-1,3-葡聚糖酶的重组菌及其在生产α-1,3-葡聚糖酶中的应用
一、哈茨木霉α-1,3-葡聚糖酶表达载体的构建
1、哈茨木霉α-1,3-葡聚糖酶基因的优化
根据里氏木霉密码子偏好性,对哈茨木霉CCM F-340的α-1,3-葡聚糖酶基因mutAW进行密码子优化,同时在编码的哈茨木霉α-1,3-葡聚糖酶的C端加入6His-tag便于后续的蛋白纯化,优化后的编码基因序列如序列1所示,其中序列1第1-111位为信号肽编码基因序列,第1903-1920位为6His-tag编码基因序列。
2、含有cbh1启动子和cbh1终止子载体骨架片段的制备
提取含有1904bp cbh1启动子、51bp cbh1信号肽、2243bp Tcbh1终止子序列的质粒pSK-Lip,采用EcoRI和SpeI对pSK-Lip质粒进行双酶切,得到骨架载体片段。
3、不含信号肽的mutAW基因片段的扩增
以合成的哈茨木霉α-1,3-葡聚糖酶基因质粒为模板,采用引物F-mutAW和R-mutAW进行PCR扩增,得到不含信号肽的mutAW基因片段。所用DNA聚合酶为高保真FastPfu(北京全式金生物技术有限公司,货号:AP221-02)。PCR扩增条件为:95℃预变性5min;95℃变性30s,60.5℃退火30s,72℃延伸1min30s,30个循环;最后72℃扩展延伸10min。
4、截短的cbh1终止子片段的扩增
以质粒pSK-Lip为模板,采用引物sTcbh1-F和sTcbh1-R进行PCR扩增,得到截短的cbh1终止子片段,其大小为1021bp,将其命名为sTcbh1。所用DNA聚合酶为高保真FastPfu(北京全式金公司)。PCR扩增条件为:95℃预变性5min;95℃变性30s,59℃退火30s,72℃延伸40s,30个循环;最后72℃扩展延伸10min。
5、pyr4的扩增
以里氏木霉QM9414基因组DNA为模板,采用引物pyr4-F和pyr4-R进行PCR扩增,得到含有pyr4基因启动子、编码区和终止子的pyr4基因表达盒,将其命名为pyr4。所用DNA聚合酶为高保真FastPfu(北京全式金公司)。PCR扩增条件为:95℃预变性5min;95℃变性30s,58℃退火30s,72℃延伸2min,30个循环;最后72℃扩展延伸10min。
表2、α-1,3-葡聚糖酶表达载体构建用到的引物
6、哈茨木霉α-1,3-葡聚糖酶表达载体pSK-mutAW的构建
采用对应的限制性核酸内切酶分别对不含信号肽的mutAW基因片段、sTcbh1和pyr4片段进行酶切,随后采用T4DNA连接酶(Thermo Scientific,货号:EL0011)将上述酶切片段进行连接,得到重组载体pSK-mutAW,其结构如图1所示。
对重组载体pSK-mutAW进行测序验证,结果表明:表达载体pSK-mutAW为将序列3所示的DNA分子替换骨架载体pSK-Lip的EcoRI和SpeI酶切位点之间DNA序列,且保持骨架载体pSK-Lip的其他序列不变得到的载体。重组载体pSK-mutAW表达带有HIS标签的α-1,3-葡聚糖酶,带有HIS标签的α-1,3-葡聚糖酶的氨基酸序列如序列2所示。序列3第1-1812位为带有HIS标签的α-1,3-葡聚糖酶的编码基因、第1813-2828位为sTcbh1片段的核苷酸序列、第2829-5212位为pyr4片段的核苷酸序列。
二、高效分泌表达哈茨木霉α-1,3-葡聚糖酶的重组菌的构建
1、大量提取质粒pSK-mutAW,用AseI(NEB,货号:R0526S)对质粒pSK-mutAW进行线性化,用普通DNA产物纯化试剂盒(天根生化科技有限公司,货号:DP204)对酶切产物进行纯化,并用ddH2O洗脱,随后用真空离心浓缩仪将线性化DNA产物浓缩至约1ug/ul,-20℃保存备用;
2、取新鲜培养的平板(d=3.5cm)上的里氏木霉TU-6孢子(每个转化用2-3个平板),用适量无菌水洗涤孢子制成孢子悬液,200目筛子过滤除去残余的菌丝,得到孢子液;
3、将孢子液移至无菌离心管中,3000g,4℃离心4min,弃去上清,收集孢子沉淀;
4、用1.1M预冷的山梨醇溶液洗涤孢子沉淀,3000g,4℃离心4min,弃去上清,收集沉淀;
5、重复步骤4两次;
6、用100ul 1.1M预冷的山梨醇重悬孢子沉淀,得到重悬后孢子液;冰上放置30min;
7、打开电转仪,设置电转参数为:1.8kV;800Ω;25uF;
8、将线性化质粒加入到上述重悬后孢子液中(线性化质粒样品的体积不得超过20ul),轻轻混匀,随后将含有线性化质粒的孢子液转移至预冷的电转杯中,进行电击;
9、电击结束后立即加入900ul预冷的1.1M山梨醇溶液,轻轻混匀,得到电转后的孢子液,冰上放置;
10、用1.1M山梨醇溶液将10×YEPD稀释10倍,得到1×YEPD;取5ml的1×YEPD加入到电转后的孢子液中,30℃孵育过夜,得到重组菌;
11、将重组菌涂布于里氏木霉基础培养基(MM)+2%葡萄糖+1.1M山梨醇+0.1%TritonX-100平板上,28℃培养。
12、重组菌的筛选与鉴定
待转化子长出后,转接至PDA平板上,培养7-9天,待孢子成熟后,刮取孢子,取少量孢子液采用UniversAll TM Tissue Extraction/PCR kit试剂盒(Yeastern Biotech,货号:FYU001-100P和FYU002-5ML)进行PCR验证,采用的引物为P1-mutAW和Tcbh1-mutAW(引物序列如表3)。选取经PCR验证为阳性的转化子,进行孢子液的梯度稀释,涂布于MM+2%葡萄糖+0.1%TritonX-100平板上,待菌长出后,挑至PDA平板上,培养7-9天,待孢子成熟后,采用上述方法再次进行PCR验证。
表3、转化子PCR鉴定引物
鉴定结果如图2所示,共筛选得到8株阳性转化子,分别将其命名为mu-1、mu-2、mu-3、mu-4、mu-5、mu-6、mu-7和mu-8。选取阳性转化子mu-1、mu-2、mu-3和mu-4用于下述发酵试验。
三、重组菌在高效分泌表达哈茨木霉α-1,3-葡聚糖酶中的应用
1、预培养
将筛选出的阳性转化子(mu-1、mu-2、mu-3和mu-4)的孢子液接种至MM+2%葡萄糖培养基中,接种后孢子浓度为105-106个/ml,28℃,200rpm培养36h。
2、发酵培养
收集预培养36h后的菌丝体,用MM培养基洗去残余的葡萄糖,随后将菌转接至50mlMM+1%Avicel(PH-101,SIGMA公司,货号:11365-1KG)培养基中,每瓶培养基接种2g菌体,28℃,200rpm培养4-8天,离心收集发酵液,通过观察发酵125h时α-1,3-葡聚糖酶的表达情况。并以出发菌株TU-6为对照。
TU-6和阳性转化子发酵液SDS-PAGE图如图3所示。从图中可以看出:阳性转化子mu-1、mu-2、mu-3和mu-4的发酵液中均得到大小为67kDa的α-1,3-葡聚糖酶。
3、α-1,3-葡聚糖酶的纯化
发酵结束后,离心收集发酵液,采用镍柱对哈茨木霉α-1,3-葡聚糖酶(阳性转化子mu-3发酵液)进行纯化,得到纯化后的α-1,3-葡聚糖酶酶液(α-1,3-葡聚糖酶纯化样品)。纯化过程中采用的Binding Buffer为含20mM咪唑的PBS(pH7.2),Elution buffer为含有0.5M咪唑的PBS(pH7.2)。纯化后的蛋白样品采用超滤管进行超滤浓缩,并将蛋白样品中的Buffer更换为50mM醋酸-醋酸钠缓冲液(pH5.5),4℃保存。纯化后的α-1,3-葡聚糖酶酶液的SDS-PAGE图如图5所示。
4、蛋白浓度的测定
采用Folin-酚试剂法测纯化后的α-1,3-葡聚糖酶酶液蛋白浓度(北京索莱宝Lowry法蛋白浓度测定试剂盒,货号:PC0030-1000),纯化后的α-1,3-葡聚糖酶酶液蛋白浓度为262.2ug/ml。
实施例2、哈茨木霉α-1,3-葡聚糖酶的活性测定及酶学性质研究
一、哈茨木霉α-1,3-葡聚糖酶的活性测定
1、非水溶性葡聚糖mutan的制备
(1)将S.mutans UA159以1/100接种量接种至BHI培养基中,37℃厌氧条件下静置培养;
(2)将过夜培养的S.mutans UA159以1/50接种量转接至新鲜的BHI培养基,37℃厌氧条件下静置培养24h;
(3)12,000g离心10min,除去菌体,收集上清液,并用0.22um无菌滤器过滤上清液;
(4)然后向上清液加入蔗糖至终浓度为3%,混匀,37℃放置48h;
(5)12,000g离心30min,弃去上清液,收集沉淀,并用无菌水反复洗涤沉淀,所得物质即为mutan,冷冻干燥,研磨成粉末,-20℃保存备用。
2、标准曲线的制作
取160ul不同浓度的葡萄糖标准品溶液,加入1.2ml铁氰化钾试剂,混匀,85℃水浴反应15min,反应结束后立即冷却,以水为空白对照,测量420nm处的吸光值(A420)。进行3次独立重复实验,结果取平均值。以葡萄糖浓度为横坐标,A420为纵坐标,绘制标准曲线。
3、α-1,3-葡聚糖酶酶活测定
分别检测对照菌株TU-6和各阳性转化子的发酵液的α-1,3-葡聚糖酶酶活,具体步骤如下:
(1)将0.5g步骤1制备的mutan与10ml浓度为50mM醋酸-醋酸钠缓冲液(pH5.5)混匀,得到质量分数为5%的mutan悬液,膨胀1h后使用;
(2)实验组:取100ul 5%的mutan悬液分别与300ul稀释至合适浓度的对照菌株TU-6发酵液及各阳性转化子的发酵液混匀,45℃孵育15min;然后加入100ul 0.4M NaOH,混匀,终止反应;
对照组:取100ul 5%的mutan悬液,45℃孵育15min;加入100ul 0.4M NaOH混匀后,分别加入300ul稀释至合适浓度的对照菌株TU-6发酵液及各阳性转化子的发酵液;
(3)12000rpm离心5min,吸取上清液,用0.22um滤膜过滤上清液,收集滤液;
(4)取160ul滤液,加入1.2ml铁氰化钾试剂,混匀,85℃水浴反应15min,反应结束后立即冷却,以水为空白对照,测量420nm处的吸光值,每个样品做三个重复,结果取平均值。1个单位α-1,3-葡聚糖酶酶活定义为在45℃,pH5.5条件下单位时间内释放相当于1umol葡萄糖的还原糖所需的酶量。
对照菌株TU-6和各阳性转化子的发酵液酶活检测结果如图4所示。结果表明:对照菌株TU-6的发酵液几乎检测不到α-1,3-葡聚糖酶酶活,不同阳性转化子发酵液酶活范围为1.7U/ml-2.3U/ml,其中,mu-3转化子的发酵液酶活较其它转化子略高一些。选用阳性转化子mu-3的发酵液进行镍柱纯化,纯化得到的α-1,3-葡聚糖酶纯化样品按照上述酶活测定方法测定酶活。结果表明:α-1,3-葡聚糖酶纯化样品的α-1,3-葡聚糖酶比活为33.6U/mg。阳性转化子mu-3的发酵液中α-1,3-葡聚糖酶含量约为68ug/ml。
二、α-1,3-葡聚糖酶平板检测法
1、用匀浆器将质量分数为5%的mutan悬液在4℃条件下匀化10min,得到匀化后的mutan悬液;
2、取1g琼脂糖加入100ml的50mM醋酸-醋酸钠缓冲液(pH5.5),得到质量分数为1%的琼脂糖,115℃高压蒸汽灭菌20min;
3、待1%的琼脂糖溶液冷却至50℃左右时,加入匀化后的mutan悬液,混匀,使mutan终浓度为0.2%,并将15ml的质量分数为1%的琼脂糖溶液加入各培养皿中(d=9cm),待琼脂糖凝固后,用打孔器打孔并向样品孔内分别加入醋酸-醋酸钠缓冲液、α-1,3-葡聚糖酶纯化样品、T.reesei TU-6发酵液和mu-3发酵液,平板于37℃保温过夜。
结果如图6所示。从图中可以看出:α-1,3-葡聚糖酶纯化样品(孔2)和mu-3发酵液样品(孔4)周围有清晰可见的透明圈产生,而醋酸-醋酸钠缓冲液(孔1)和T.reesei TU-6发酵液样品(孔3)周围无明显透明圈。平板检测法直观地表明了阳性转化子mu-3的发酵液和α-1,3-葡聚糖酶纯化样品均有酶活,而对照菌株TU-6的发酵液没有检测出α-1,3-葡聚糖酶酶活,这与上述采用分光光度法测定α-1,3-葡聚糖酶酶活的结果相一致。
二、酶学性质研究
1、最适温度测定
将纯化后的α-1,3-葡聚糖酶酶液分别在30℃、40℃、45℃、50℃、60℃和70℃下按步骤一中的酶活测定方法测定酶活,每个温度条件设三个重复实验,结果取平均值,酶活最高点对应的温度即为该酶的最适反应温度。
结果如图7所示。α-1,3-葡聚糖酶的最适温度为45℃。
2、最适pH测定
配制如下不同pH的缓冲液:50mM甘氨酸-盐酸缓冲液(pH2.0-3.0)、50mM醋酸-醋酸钠缓冲液(pH4.0-5.5)和50mM磷酸二氢钠-磷酸氢二钠缓冲液(pH6.0-8.0)。分别以不同pH的缓冲液为溶剂配制质量分数为5%的mutan悬液,并用不同pH的缓冲液稀释酶液,按步骤一中的酶活测定方法在不同pH条件下测定酶活并绘制相对活性曲线,每个pH条件设三个重复实验,结果取平均值,酶活最高点对应的pH即为该酶的最适反应pH。
结果如图8所示。从图8可以看出:该酶的最适反应pH为5.5,而且该酶在pH5.0-6.0条件下活性相差不大。
序列表
<110>中国科学院微生物研究所 中国科学院大学
<120>一种高产哈茨木霉α-1,3-葡聚糖酶的重组菌及其应用
<160>3
<210>1
<211>1923bp
<212>DNA
<213>人工序列
<220>
<223>
<400>1
atgctggggg tttttcgccg actgcgtctt ggtgccctcg ccgcggcggc cctttcgtca 60
cttggtagcg ctgcgccggc gaacgtggct atccgcagcc ttgaggagcg agcttcgagc 120
gccgaccgtc tcgtgttttg ccactttatg attggcattg tgggcgatcg tggctccagc 180
gctgattacg atgatgatat gcaacgcgcc aaggccgcgg gtattgatgc ctttgcgctg 240
aatattggtg tggacggcta cacagatcag cagctgggct acgcctatga ttcggctgat 300
cgcaacggca tgaaggtctt catctccttc gactttaatt ggtggtcacc tggcaatgcc 360
gtgggcgttg gccaaaagat cgcccagtac gctaaccgcc cagcccagct gtacgttgac 420
aatcgtccct ttgcctcgtc tttcgccggc gatggcctgg acgtcaacgc tctgcggtct 480
gcagccggta gcaatgtcta ctttgttcca aacttccacc ccggacaaag ctccccttcg 540
aacatcgacg gagcgcttaa ctggatggcc tgggataacg atggcaacaa caaggcccca 600
aaacccggcc agactgttac cgtcgcagat ggggacaacg cctacaagaa ctggctcggc 660
ggaaaaccct acttggcgcc ggtctccccc tggttcttta cccatttcgg accagaggtt 720
tcgtactcga aaaactgggt cttcccgggc ggcccgctca tctacaaccg atggcagcaa 780
gttctgcagc agggcttccc catggttgaa attgtcacat ggaacgatta cggcgagagc 840
cactacgtcg ggcccctcaa gtcgaagcac tttgatgacg gcaacagcaa gtgggttaac 900
gatatgccgc atgatgggtt cctggacctg tccaaaccat ttatcgcggc ttacaagaac 960
cgtgacaccg atatctccaa gtacgtgcaa aacgaacaac ttgtgtactg gtaccgacgg 1020
aaccttaagg ccctggactg cgatgcgaca gacaccactt cgaaccgccc tgccaacaac 1080
ggctccggca actacttcat gggccggcct gatggatggc agaccatgga cgacaccgtt 1140
tatgttgccg ccctgctcaa gaccgccggc tccgtgaccg tgacctcggg cggcacgacc 1200
cagacttttc aggcaaatgc cggcgcaaac ctgttccaga tcccagcatc aattggccag 1260
cagaagttcg cacttactcg caacggtcaa accgtcttct cgggtacgtc cctcatggat 1320
attacgaacg tttgtagctg cggcatctac aactttaacc cgtatgtcgg cactattccc 1380
gctggctttg acgaccccct gcaggccgac ggcctcttta gcctcaccat tggactccac 1440
gtcacgacgt gccaggctaa gcccagcctg ggcactaacc cccctgtcac gagcggaccc 1500
gtgtccagct tgcccgccag ctctacgacg cgcgcgagct cccctccacc ggtctcaagc 1560
acccgggtct cgtcaccgcc cgtgagctcc ccgcctgtct ctaggacaag ctctacccct 1620
ccgccagcta gctcgacccc gccctccggt caggtctgtg ttgcaggcac cgttgcggac 1680
ggggagtcag gcaactacat tggactctgc cagttctcct gcaactatgg ctactgtccg 1740
cctggcccct gcaaatgcac cgccttcgga gctcccatca acccccccgc tagcaacggt 1800
cgtaacggtt gccctctccc cggcgaggga gacggttatc tgggactctg ctctttcagc 1860
tgcaaccaca actactgccc gccgactgcc tgccagtatt gccaccatca ccaccaccat 1920
tag 1923
<210>2
<211>603
<212>PRT
<213>人工序列
<220>
<223>
<400>2
Ala Ser Ser Ala Asp Arg Leu Val Phe Cys His Phe Met Ile Gly Ile
1 5 10 15
Val Gly Asp Arg Gly Ser Ser Ala Asp Tyr Asp Asp Asp Met Gln Arg
20 25 30
Ala Lys Ala Ala Gly Ile Asp Ala Phe Ala Leu Asn Ile Gly Val Asp
35 40 45
Gly Tyr Thr Asp Gln Gln Leu Gly Tyr Ala Tyr Asp Ser Ala Asp Arg
50 55 60
Asn Gly Met Lys Val Phe Ile Ser Phe Asp Phe Asn Trp Trp Ser Pro
65 70 75 80
Gly Asn Ala Val Gly Val Gly Gln Lys Ile Ala Gln Tyr Ala Asn Arg
85 90 95
Pro Ala Gln Leu Tyr Val Asp Asn Arg Pro Phe Ala Ser Ser Phe Ala
100 105 110
Gly Asp Gly Leu Asp Val Asn Ala Leu Arg Ser Ala Ala Gly Ser Asn
115 120 125
Val Tyr Phe Val Pro Asn Phe His Pro Gly Gln Ser Ser Pro Ser Asn
130 135 140
Ile Asp Gly Ala Leu Asn Trp Met Ala Trp Asp Asn Asp Gly Asn Asn
145 150 155 160
Lys Ala Pro Lys Pro Gly Gln Thr Val Thr Val Ala Asp Gly Asp Asn
165 170 175
Ala Tyr Lys Asn Trp Leu Gly Gly Lys Pro Tyr Leu Ala Pro Val Ser
180 185 190
Pro Trp Phe Phe Thr His Phe Gly Pro Glu Val Ser Tyr Ser Lys Asn
195 200 205
Trp Val Phe Pro Gly Gly Pro Leu Ile Tyr Asn Arg Trp Gln Gln Val
210 215 220
Leu Gln Gln Gly Phe Pro Met Val Glu Ile Val Thr Trp Asn Asp Tyr
225 230 235 240
Gly Glu Ser His Tyr Val Gly Pro Leu Lys Ser Lys His Phe Asp Asp
245 250 255
Gly Asn Ser Lys Trp Val Asn Asp Met Pro His Asp Gly Phe Leu Asp
260 265 270
Leu Ser Lys Pro Phe Ile Ala Ala Tyr Lys Asn Arg Asp Thr Asp Ile
275 280 285
Ser Lys Tyr Val Gln Asn Glu Gln Leu Val Tyr Trp Tyr Arg Arg Asn
290 295 300
Leu Lys Ala Leu Asp Cys Asp Ala Thr Asp Thr Thr Ser Asn Arg Pro
305 310 315 320
Ala Asn Asn Gly Ser Gly Asn Tyr Phe Met Gly Arg Pro Asp Gly Trp
325 330 335
Gln Thr Met Asp Asp Thr Val Tyr Val Ala Ala Leu Leu Lys Thr Ala
340 345 350
Gly Ser Val Thr Val Thr Ser Gly Gly Thr Thr Gln Thr Phe Gln Ala
355 360 365
Asn Ala Gly Ala Asn Leu Phe Gln Ile Pro Ala Ser Ile Gly Gln Gln
370 375 380
Lys Phe Ala Leu Thr Arg Asn Gly Gln Thr Val Phe Ser Gly Thr Ser
385 390 395 400
Leu Met Asp Ile Thr Asn Val Cys Ser Cys Gly Ile Tyr Asn Phe Asn
405 410 415
Pro Tyr Val Gly Thr Ile Pro Ala Gly Phe Asp Asp Pro Leu Gln Ala
420 425 430
Asp Gly Leu Phe Ser Leu Thr Ile Gly Leu His Val Thr Thr Cys Gln
435 440 445
Ala Lys Pro Ser Leu Gly Thr Asn Pro Pro Val Thr Ser Gly Pro Val
450 455 460
Ser Ser Leu Pro Ala Ser Ser Thr Thr Arg Ala Ser Ser Pro Pro Pro
465 470 475 480
Val Ser Ser Thr Arg Val Ser Ser Pro Pro Val Ser Ser Pro Pro Val
485 490 495
Ser Arg Thr Ser Ser Thr Pro Pro Pro Ala Ser Ser Thr Pro Pro Ser
500 505 510
Gly Gln Val Cys Val Ala Gly Thr Val Ala Asp Gly Glu Ser Gly Asn
515 520 525
Tyr Ile Gly Leu Cys Gln Phe Ser Cys Asn Tyr Gly Tyr Cys Pro Pro
530 535 540
Gly Pro Cys Lys Cys Thr Ala Phe Gly Ala Pro Ile Asn Pro Pro Ala
545 550 555 560
Ser Asn Gly Arg Asn Gly Cys Pro Leu Pro Gly Glu Gly Asp Gly Tyr
565 570 575
Leu Gly Leu Cys Ser Phe Ser Cys Asn His Asn Tyr Cys Pro Pro Thr
580 585 590
Ala Cys Gln Tyr Cys His His His His His His
595 600
<210>3
<211>5212bp
<212>DNA
<213>人工序列
<220>
<223>
<400>3
gcttcgagcg ccgaccgtct cgtgttttgc cactttatga ttggcattgt gggcgatcgt 60
ggctccagcg ctgattacga tgatgatatg caacgcgcca aggccgcggg tattgatgcc 120
tttgcgctga atattggtgt ggacggctac acagatcagc agctgggcta cgcctatgat 180
tcggctgatc gcaacggcat gaaggtcttc atctccttcg actttaattg gtggtcacct 240
ggcaatgccg tgggcgttgg ccaaaagatc gcccagtacg ctaaccgccc agcccagctg 300
tacgttgaca atcgtccctt tgcctcgtct ttcgccggcg atggcctgga cgtcaacgct 360
ctgcggtctg cagccggtag caatgtctac tttgttccaa acttccaccc cggacaaagc 420
tccccttcga acatcgacgg agcgcttaac tggatggcct gggataacga tggcaacaac 480
aaggccccaa aacccggcca gactgttacc gtcgcagatg gggacaacgc ctacaagaac 540
tggctcggcg gaaaacccta cttggcgccg gtctccccct ggttctttac ccatttcgga 600
ccagaggttt cgtactcgaa aaactgggtc ttcccgggcg gcccgctcat ctacaaccga 660
tggcagcaag ttctgcagca gggcttcccc atggttgaaa ttgtcacatg gaacgattac 720
ggcgagagcc actacgtcgg gcccctcaag tcgaagcact ttgatgacgg caacagcaag 780
tgggttaacg atatgccgca tgatgggttc ctggacctgt ccaaaccatt tatcgcggct 840
tacaagaacc gtgacaccga tatctccaag tacgtgcaaa acgaacaact tgtgtactgg 900
taccgacgga accttaaggc cctggactgc gatgcgacag acaccacttc gaaccgccct 960
gccaacaacg gctccggcaa ctacttcatg ggccggcctg atggatggca gaccatggac 1020
gacaccgttt atgttgccgc cctgctcaag accgccggct ccgtgaccgt gacctcgggc 1080
ggcacgaccc agacttttca ggcaaatgcc ggcgcaaacc tgttccagat cccagcatca 1140
attggccagc agaagttcgc acttactcgc aacggtcaaa ccgtcttctc gggtacgtcc 1200
ctcatggata ttacgaacgt ttgtagctgc ggcatctaca actttaaccc gtatgtcggc 1260
actattcccg ctggctttga cgaccccctg caggccgacg gcctctttag cctcaccatt 1320
ggactccacg tcacgacgtg ccaggctaag cccagcctgg gcactaaccc ccctgtcacg 1380
agcggacccg tgtccagctt gcccgccagc tctacgacgc gcgcgagctc ccctccaccg 1440
gtctcaagca cccgggtctc gtcaccgccc gtgagctccc cgcctgtctc taggacaagc 1500
tctacccctc cgccagctag ctcgaccccg ccctccggtc aggtctgtgt tgcaggcacc 1560
gttgcggacg gggagtcagg caactacatt ggactctgcc agttctcctg caactatggc 1620
tactgtccgc ctggcccctg caaatgcacc gccttcggag ctcccatcaa cccccccgct 1680
agcaacggtc gtaacggttg ccctctcccc ggcgagggag acggttatct gggactctgc 1740
tctttcagct gcaaccacaa ctactgcccg ccgactgcct gccagtattg ccaccatcac 1800
caccaccatt agatcgatag ctccgtggcg aaagcctgac gcaccggtag attcttggtg 1860
agcccgtatc atgacggcgg cgggagctac atggccccgg gtgatttatt ttttttgtat 1920
ctacttctga cccttttcaa atatacggtc aactcatctt tcactggaga tgcggcctgc 1980
ttggtattgc gatgttgtca gcttggcaaa ttgtggcttt cgaaaacaca aaacgattcc 2040
ttagtagcca tgcattttaa gataacggaa tagaagaaag aggaaattaa aaaaaaaaaa 2100
aaaacaaaca tcccgttcat aacccgtaga atcgccgctc ttcgtgtatc ccagtaccac 2160
ggcaaaggta tttcatgatc gttcaatgtt gatattgttc ccgccagtat ggctccaccc 2220
ccatctccgc gaatctcctc ttctcgaacg cggtagtggc gcgccaattg gtaatgaccc 2280
atagggagac aaacagcata atagcaacag tggaaattag tggcgcaata attgagaaca 2340
cagtgagacc atagctggcg gcctggaaag cactgttgga gaccaacttg tccgttgcga 2400
ggccaacttg cattgctgtc aagacgatga caacgtagcc gaggaccgtc acaagggacg 2460
caaagttgtc gcggatgagg tctccgtaga tggcatagcc ggcaatccga gagtagcctc 2520
tcaacaggtg gccttttcga aaccggtaaa ccttgttcag acgtcctagc cgcagctcac 2580
cgtaccagta tcgaggattg acggcagaat agcagtggct ctccaggatt tgactggaca 2640
aaatcttcca gtattcccag gtcacagtgt ctggcagaag tcccttctcg cgtgcgagtc 2700
gaaagtcgct atagtgcgca atgagagcac agtaggagaa taggaacccg cgagcacatt 2760
gttcaatctc cacatgaatt ggatgactgc tgggcagaat gtgctgcctc caaaatcctg 2820
cgaagcttct ggcagacttg tgtgtatcat tcaccctatt tctgcttcat agtacatgta 2880
ctgtacctga acggctcaac cgctatttac gactcttatt tttttgtggc gttggtcacg 2940
tttgccagct gttgtccgtc tttctagggc tcctcaaact tgacctgacc gagctccctt 3000
tctggacccg gtgggcttca cttccagctg ctgagcgacc tgagccgaac atcctcagtc 3060
cttgtccagc gcaattcatt ttctttcctt ttcttttttt ttattccttt ctttactttt 3120
attctctctt tttctcctct tcctcttctt cttctttctc ctcctcctcc atatcctcac 3180
tctcgtctcc ctcattacta ccctctcggc tcctcaggtc caccaaccct cccgcaccca 3240
aacctctgcc gctgaaaccc attcggtggt cgccgttttt tttttttttt ttttctcacc 3300
cccaaagtcg caatatcggg tatcgccgcc ggcattgaat cgccttctcc gctagcatcg 3360
actactgctg ctctgctctc gttgccagcg ctgctcccta gaattttgac caggggacga 3420
gcccgacatt aaagcaactc cctcgcctcg agacgactcg gatcgcacga aattctccca 3480
atcgccgaca gttcctactc ctcttcctcc cgcacggctg tcgcgcttcc aacgtcattc 3540
gcacagcaga attgtgccat ctctctcttt tttttccccc cctctaaacc gccacaacgg 3600
caccctaagg gttaaactat ccaaccagcc gcagcctcag cctctctcag cctcatcagc 3660
catggcacca cacccgacgc tcaaggccac cttcgcggcc aggagcgaga cggcgacgca 3720
cccgctgacg gcttacctgt tcaagctcat ggacctcaag gcgtccaacc tgtgcctgag 3780
cgccgacgtg ccgacagcgc gcgagctgct gtacctggcc gacaagattg gcccgtcgat 3840
tgtcgtgctc aagacgcact acgacatggt ctcgggctgg gacttccacc cggagacggg 3900
cacgggagcc cagctggcgt cgctggcgcg caagcacggc ttcctcatct tcgaggaccg 3960
caagtttggc gacattggcc acaccgtcga gctgcagtac acgggcgggt cggcgcgcat 4020
catcgactgg gcgcacattg tcaacgtcaa catggtgccc ggcaaggcgt cggtggcctc 4080
gctggcccag ggcgccaagc gctggctcga gcgctacccc tgcgaggtca agacgtccgt 4140
caccgtcggc acgcccacca tggactcgtt tgacgacgac gccgactcca gggacgccga 4200
gcccgccggc gccgtcaacg gcatgggctc cattggcgtc ctggacaagc ccatctactc 4260
gaaccggtcc ggcgacggcc gcaagggcag catcgtctcc atcaccaccg tcacccagca 4320
gtacgagtcc gtctcctcgc cccggttaac aaaggccatc gccgagggcg acgagtcgct 4380
cttcccgggc atcgaggagg cgccgctgag ccgcggcctc ctgatcctcg cccaaatgtc 4440
cagccagggc aacttcatga acaaggagta cacgcaggcc tgcgtcgagg ccgcccggga 4500
gcacaaggac tttgtcatgg gcttcatctc gcaggagacg ctcaacaccg agcccgacga 4560
tgcctttatc cacatgacgc ccggctgcca gctgcccccc gaagacgagg accagcagac 4620
caacggatcg gtcggtggag acggccaggg ccagcagtac aacacgccgc acaagctgat 4680
tggcatcgcc ggcagcgaca ttgccattgt gggccggggc atcctcaagg cctcagaccc 4740
cgtagaggag gcagagcggt accgatcagc agcgtggaaa gcctacaccg agaggctgct 4800
gcgatagggg agggaaggga agaaagaagt aaagaaaggc atttagcaag aagggggaaa 4860
agggagggag gacaaacgga gctgagaaag agctcttgtc caaagcccgg catcatagaa 4920
tgcagctgta tttaggcgac ctctttttcc atcttgtcga tttttgttat gacgtaccag 4980
ttgggatgat ggatgattgt accccagctg cgattgatgt gtatctttgc atgcaacaac 5040
acgcgatggc ggaggcgaac tgcacattgg aaggttcata tatggtcctg acatatctgg 5100
tggatctgga agcatggaat tgtatttttg atttggcatt tgcttttgcg cgtggaggga 5160
acatatcacc ctcgggcatt tttcatttgg taggatggtt tggatgcagt tg 5212
Claims (10)
1.一种重组菌,是将哈茨木霉α-1,3-葡聚糖酶的编码基因导入宿主里氏木霉菌Trichoderma reesei,得到的菌。
2.根据权利要求1所述的重组菌,其特征在于:所述哈茨木霉α-1,3-葡聚糖酶的编码基因是通过表达哈茨木霉α-1,3-葡聚糖酶的重组载体导入宿主里氏木霉菌。
3.根据权利要求2所述的重组菌,其特征在于:所述表达哈茨木霉α-1,3-葡聚糖酶的重组载体为pSK-mutAW重组载体;
所述pSK-mutAW重组载体是将所述哈茨木霉α-1,3-葡聚糖酶的编码基因插入骨架载体的多克隆位点得到的。
4.根据权利要求3所述的重组菌,其特征在于:
所述pSK-mutAW重组载体还包括筛选标记基因;
或,所述筛选标记基因具体为pyr4基因。
5.根据权利要求1-3中任一所述的重组菌,其特征在于:
所述哈茨木霉α-1,3-葡聚糖酶的编码基因的核苷酸序列为序列1。
6.根据权利要求1-5中任一所述的重组菌,其特征在于:所述里氏木霉菌为尿嘧啶缺陷型里氏木霉菌。
7.权利要求1-6中任一所述的重组菌在生产α-1,3-葡聚糖酶中的应用;
或,权利要求1-6中任一所述的重组菌在降解非水溶性葡聚糖中的应用。
8.权利要求1-6中任一所述的重组菌在提高α-1,3-葡聚糖酶酶活力中的应用。
9.一种生产哈茨木霉α-1,3-葡聚糖酶的方法,包括如下步骤:发酵培养权利要求1-6中任一所述的重组菌,得到所述哈茨木霉α-1,3-葡聚糖酶。
10.根据权利要求9所述的方法,其特征在于:所述培养的条件为28℃培养4-8天。
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CN105176849A (zh) * | 2015-10-20 | 2015-12-23 | 中国科学院微生物研究所 | 一种高产扬奇青霉α-半乳糖苷酶的里氏木霉菌株的制备方法及其应用 |
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WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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