CN101173226B - 一种巴斯德毕赤酵母重组菌及其应用 - Google Patents
一种巴斯德毕赤酵母重组菌及其应用 Download PDFInfo
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- CN101173226B CN101173226B CN2007101761669A CN200710176166A CN101173226B CN 101173226 B CN101173226 B CN 101173226B CN 2007101761669 A CN2007101761669 A CN 2007101761669A CN 200710176166 A CN200710176166 A CN 200710176166A CN 101173226 B CN101173226 B CN 101173226B
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- pichia pastoris
- glucanase gene
- pastoris phaff
- sequence
- dextranase
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Abstract
本发明公开了微生物领域中的一种巴斯德毕赤酵母重组菌及其应用。本发明所提供的巴斯德毕赤酵母重组菌，是将β-1，3-1，4-葡聚糖酶基因导入巴斯德毕赤酵母得到的。用本发明的巴斯德毕赤酵母重组菌生产β-1，3-1，4-葡聚糖酶，效率高，而且生产得到的β-1，3-1，4-葡聚糖酶具有活性高、酸稳定性好、催化pH值范围广等优点，其最适催化温度为40℃，最适催化pH为6.4。因此，由本发明重组菌生产的β-1，3-1，4-葡聚糖酶适于用作猪、鸡等单胃动物的饲料添加剂，也适合应用于酿酒工业中，以提高酿酒效率，提高产品质量，降低成本。
Description
技术领域
本发明涉及微生物领域中的一种巴斯德毕赤酵母重组菌及其应用。
背景技术
β-葡聚糖是大麦中的主要抗营养因子，存在于糊粉内胚层的细胞壁中，不能被非反刍动物体内消化酶所降解。水溶性β-葡聚糖具有形成胶状物质的特性，能增加胃肠道中食糜黏性，降低消化酶与底物的接触，增加消化道黏膜上不流动的水层厚度，进而抑制营养物质的消化和吸收，降低动物生产性能。同时，所形成的黏性排泄物造成了养殖场的环境污染。另外，在酿酒工业中，高比例的大麦β-1，3-1，4-葡聚糖会增加麦芽汁黏，降低过滤速度，进而在啤酒发酵过程会引起沉淀，若葡聚糖含量过高，则会在啤酒成品中也出现沉淀。
能够降解葡聚糖的内切葡聚糖酶可以分为三类，一是特异性β-1，3-1，4-葡聚糖酶(EC 3.2.1.73)，水解与β-1，3糖苷键相连的β-1，4糖苷键；二是内切β-1，4-葡聚糖酶(EC 3.2.1.4)，又称纤维素酶，水解β-1，4糖苷键；三是β-1，3-葡聚糖酶(EC 3.2.1.39)，又称昆布多糖酶。
特异性β-1，3-1，4-葡聚糖酶(EC 3.2.1.73)又称地衣酶，是一种内源β-葡聚糖酶，严格水解大麦β-葡聚糖(β-glucan from barley)和地衣多糖(lichenan)中的与β-1，3糖苷键相连的β-1，4糖苷键，主要水解产物是纤维二糖、三糖和四糖等多种寡糖，而对羧甲基纤维素(CMC，β-1，4葡聚糖)和昆布多糖(laminarin，β-1，3-葡聚糖)没有降解作用。但是真核与原核来源的β-1，3-1，4-葡聚糖酶对底物作用后的产物也不尽相同。
在麦类饲料中添加β-1，3-1，4-葡聚糖酶是解决β-葡聚糖抗营养作用的主要手段。Li等(1996)分别在无壳大麦-豆粕型和小麦-豆粕型日粮中添加2％的β-葡聚糖酶，试验结果表明，在无壳大麦-豆粕型日粮中添加与不添加相比，添加β-葡聚糖酶能增加总能、粗蛋白质、β-葡聚糖和所测定的多数氨基酸回肠消化率，能增加粪中总能、粗蛋白质和所有所测定的氨基酸消化率。在小麦-豆粕型日粮中添加β-葡聚糖与不添加相比，添加β-葡聚糖能增加粪中总能消化率。
Mathlouthi等在肉鸡小麦和大麦基础日粮中同时添加木聚糖酶和β-葡聚糖酶，显著降低了小肠内容物的黏性，改善了肉鸡的日增重、饲料采食量和饲料转化率。在没有改变pH条件下，小肠内容物养分消化率、表观代谢能也得到增加。同时，盲肠内容物中总兼性厌氧菌和大肠杆菌数量显著降低。因此推测，添加此酶制剂改善消化率，可能是通过减少小肠内容物黏性和阻止总兼性厌氧菌和大肠杆菌生长而发挥作用的。
Yu等在肉鸡日粮中用大麦替代传统型的玉米-豆粕型日粮中的玉米，同时添加β-葡聚糖酶，结果降低了肉鸡胃肠道相对重量，增加了生长阶段脂肪消化率和后期盲肠总挥发性脂肪酸含量，而对生长性能没有显著影响。
Ravindran等在使用四个品种大麦的肉鸡日粮中添加β-葡聚糖酶，改善了氮校正表观代谢能(AMEn)，改善了蛋白质和所有氨基酸的回肠表观消化率，18种氨基酸回肠表观消化率从5.1％提高至18.1％。
另外，在啤酒工业中，用内源β-1，3-1，4-葡聚糖酶代替麦芽酶，能够将未经发芽处理的谷类物质作为原料，而且还能通过降低麦芽汁黏性而减少滤过时间。
由此可见，β-葡聚糖酶在饲料添加及酿酒等工业中正发挥着很重要的作用。
发明内容
本发明的一个目的是提供一种巴斯德毕赤酵母重组菌，该重组菌能生产β-1，3-1，4-葡聚糖酶。
本发明所提供的巴斯德毕赤酵母重组菌，是将β-1，3-1，4-葡聚糖酶基因导入巴斯德毕赤酵母得到的。
其中，所述β-1，3-1，4-葡聚糖酶的氨基酸序列如序列表中的序列2所示。
所述β-1，3-1，4-葡聚糖酶基因的核苷酸序列是根据巴斯德酵母菌的密码子偏好性，优化枯草芽孢杆菌中β-1，3-1，4-葡聚糖酶基因核苷酸序列得到的，其核苷酸序列如序列表中的序列1所示。
所述β-1，3-1，4-葡聚糖酶基因是通过重组表达载体pPIC-glu-opt导入所述巴斯德毕赤酵母中的，其中，所述pPIC-glu-opt是将所述β-1，3-1，4-葡聚糖酶基因插入pPICz αA的多克隆位点得到的。
所述巴斯德毕赤酵母具体为巴斯德毕赤酵母X-33。
本发明的另一个目的是提供一种生产β-1，3-1，4-葡聚糖酶的方法。
本发明所提供的生产β-1，3-1，4-葡聚糖酶的方法，是发酵上述巴斯德毕赤酵母重组菌得到β-1，3-1，4-葡聚糖酶。
其中，所述发酵的温度可为28-30℃；所述发酵中发酵液的pH可为5.0-5.5。
所述发酵过程中流加甲醇，具体按照以下的方法流加：从开始发酵起算，41h-43h为菌的饥饿期，主要是为了消耗掉残存的甘油；44h开始诱导，44-46h按照0.3％初始发酵液体积/小时的流速流加甲醇；47h到48h按照0.57％初始发酵液体积/小时的流速流加甲醇；49h至发酵结束按照0.71％初始发酵液体积/小时的流速流加甲醇。
在上述发酵过程中还可进行搅拌，搅拌速度可为300-600转/分钟。
本发明所提供的巴斯德毕赤酵母重组菌能高效生产β-葡聚糖酶，用10升全自动发酵罐发酵本发明的巴斯德毕赤酵母重组菌，结果表明由该重组菌分泌表达的葡聚糖酶的酶活可达15000U/mL。而且由该重组菌生产的β-1，3-1，4-葡聚糖酶具有活性高、酸稳定性好、催化pH值范围广等优点，其最适催化温度为40℃，最适催化pH为6.4。因此，由本发明重组菌生产的β-1，3-1，4-葡聚糖酶适于用作猪、鸡等单胃动物的饲料添加剂，也适于酿酒工业中，以提高酿酒效率，提高产品质量和降低成本。
附图说明
图1为酵母重组菌X-33/pPIC-glu-opt传代培养过程中每代β-1，3-1，4-葡聚糖酶编码基因的PCR扩增产物的琼脂糖凝胶电泳图。
泳道M：Marker；泳道1、3、5-20：分别是1、3、5-20代培养物的PCR产物。
图2为酵母重组菌X-33/pPIC-glu-opt在发酵过程中的菌体生长曲线及酶活曲线。
图3为酵母重组菌X-33/pPIC-glu-opt在发酵过程中发酵液中β-1，3-1，4-葡聚糖酶的表达量的SDS-PAGE图。
泳道M：蛋白质Marker；泳道1-8：分别诱导12、24、36、48、60、72、84、96小时的蛋白表达量；泳道9：空白对照。
图4为β-1，3-1，4-葡聚糖酶的最适pH及pH稳定性的检测。
图5为β-1，3-1，4-葡聚糖酶的最适催化温度的测定。
图6为β-1，3-1，4-葡聚糖酶的温度稳定性的检测。
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。
实施例中所使用的各培养基的组成如下：
YPDS培养基的组成为：1％酵母提取物，2％蛋白胨，2％葡萄糖，2％琼脂，其余为水；所述YPDS培养基的pH为6.0。
BMGY培养基的组成为：1％酵母提取物，2％蛋白胨，1.34％YNB，4×10-5％生物素，1％甘油，100mM pH6.0磷酸盐缓冲液，其余为水；所述BMGY培养基的pH为6.0。
BMMY培养基的组成为：1％酵母提取物，2％蛋白胨，0.1mol/L pH6.0磷酸缓冲液，1.34％YNB，4×10-5％生物素，其余为水；所述BMMY培养基的pH为6.0。
其中，酵母提取物购自OXOID(Hampshire，England)公司，其货号为LP0021；蛋白胨购自OXOID(Hampshire，England)公司，其货号为LP0037；YNB是由Difco公司生产，经绿生源生物技术公司分装，100g包装；生物素由日本产，经绿生源生物技术公司分装，1g包装。
以上YPDS、BMGY和BMMY培养基中各物质的百分含量均为质量百分含量。
实施例1、巴斯德毕赤酵母重组菌X-33/pPIC-glu-opt的制备
一、β-1，3-1，4-葡聚糖酶基因序列的优化设计
根据GenBank Accession Number EU082110公开的β-1，3-1，4-葡聚糖酶基因序列，根据巴斯德毕赤酵母密码子偏好性，以高使用频率的密码子替代低使用频率密码子，重新设计β-1，3-1，4-葡聚糖酶基因序列，并保持成熟蛋白的氨基酸序列不变。为了方便克隆，在序列的5’和3’末端分别设计EcoRI和XbaI酶切位点。设计好的序列由上海生工生物工程有限公司进行全合成。优化后的序列如序列表中序列1所示，由该核苷酸序列编码的氨基酸序列如序列表序列2所示。
二、巴斯德毕赤酵母表达载体的构建、转化和重组菌的筛选
用限制性内切酶EcoRI和XbaI对步骤一中合成的基因序列进行双酶切，然后将其连入经相同酶切的载体pPICzαA(Invitrogen，USA)中α因子序列的下游，将连接产物转化大肠杆菌Top10感受态细胞，用含有25μg/mL抗生素Zeocin的LB抗性平板筛选出阳性克隆，提取质粒并进行测序，测序结果表明序列正确。将含有上述优化后的β-1，3-1，4-葡聚糖酶基因序列的巴斯德毕赤酵母表达载体命名为pPIC-glu-opt。
取10μL巴斯德毕赤酵母表达载体pPIC-glu-opt，将其用限制性内切酶Sac I在37℃下酶切24h，然后将酶切产物用无水乙醇沉淀，溶于10μL灭菌水中。加入80μL巴斯德毕赤酵母X-33感受态细胞，混匀，置于0.2cm电击杯中，冰浴5min，在2000伏电压(25μF)条件下电击，迅速加入1mL 1mol/L山梨醇，置于28℃静止培养2h。然后将培养物涂布于含抗生素Zeocin(100μg/mL)的YPDS平板上，28℃培养3-4d，直至长出清晰的菌落。
挑取阳性重组子，将其接种于含30mL BMGY培养基的250mL摇瓶中，在28℃、250-300rpm的条件下培养至OD600为2-6，然后室温下离心收集菌体，用BMMY液体培养基重悬菌体，使其OD600为1.0，再用终浓度为0.5％(V/V)的甲醇为唯一碳源进行诱导培养，培养48h，通过SDS-PAGE及酶活性测定筛选β-1，3-1，4-葡聚糖酶的表达量高且酶活较高的重组菌株，将其中一株取名为X-33/pPIC-glu-opt。
将上述巴斯德毕赤酵母重组菌X-33/pPIC-glu-opt传代培养20代，每代生长至对数生长期时提取总DNA，以5’-GGATTGTTTATGAGTTTGT-3’和5’-TTATTTTTTTSTATAGCGCA-3’为引物进行PCR扩增，鉴定外源基因的稳定性。扩增条件为：94℃1min，40℃30s，72℃1.5min，40个循环。实验重复3次，结果如图1所示。结果表明：传代培养中每代PCR扩增都能得到目的片段，说明被导入的β-1，3-1，4-葡聚糖酶基因在各代中都稳定存在。
实施例2、利用巴斯德毕赤酵母重组菌X-33/pPIC-glu-opt生产β-1，3-1，4-葡聚糖酶
在无菌条件下，将上述巴斯德毕赤酵母重组菌X-33/pPIC-glu-opt接种于装有200mL BMGY培养基的3 L摇瓶中，在28-30℃、280rpm下振摇12-24小时，得到OD600为3.0的种子培养液。然后配制4L BMGY培养基，装入10L自动控制发酵罐中，在121℃下灭菌后，冷却至28.5℃，加入200mL上述种子培养液，用氨水和磷酸调节pH至5.5，通过调节转速和空气流量控制溶氧大于20％。经测定，接入种子液23h后，甘油消耗完全(溶氧标记为100％)，进入流加25％(25g/100ml)甘油阶段，流速为60mL/h。流加17h后，停止流加甘油，3h后，甘油消耗完全(溶氧标记为100％)，进入甲醇流加阶段，流速为12mL/h，同时控制相对溶氧量为20％以上。3h后，将甲醇流速调到24mL/h，再过2h，将流速调到30mL/h，并保持到最后。制作发酵过程中菌体的生长曲线并测定酶活。采集诱导不同时间的发酵液，通过SDS-PAGE检测其中表达蛋白的量。
酶活的测定方法为：取1mL发酵液，离心收集得到0.5mL上清液。取1μl上清液用Na2HPO4-柠檬酸缓冲液稀释20万倍，取1mL稀释液与等体积底物(质量百分含量为0.8％的葡聚糖溶液)混合置于40℃水浴中反应30min，加入2.5mL DNS终止液，沸水浴煮5min，定容至12.5mL，测定酶反应液的540nm吸光度值(OD540)。
酶活单位(U)定义为：在40℃、pH值为6.4的条件下，每分钟从质量百分含量为0.8％的葡聚糖溶液中释放1μmol还原糖所需要的酶量为1个酶活力单位。
其中，酶活和OD540的换算关系如下：
E＝(OD540×0.85×1000×200000)/(180×30)
其中，E：1ml发酵液酶活，单位是U；
OD540：酶反应结束后，反应液的540nm吸光度值；
0.85：是OD540和葡萄糖浓度(mg/mL)的标准曲线斜率；
180：葡萄糖的分子量；
30：反应时间，单位是分钟。
实验设三次重复，发酵过程中的菌体生长曲线及酶活曲线见图2。结果表明：随着诱导时间的增加，所获得的β-1，3-1，4-葡聚糖酶的酶活逐渐增强。检测诱导96h即发酵140h时发酵液的酶活时，所测得的OD540为0.476±0.01(平均值±标准差)，经换算得到此时每毫升发酵液中的酶活为15000U±330U(平均值±标准差)。图2中的相对酶活是将诱导96 h(即发酵140h)的酶活计作100％。
SDS-PAGE的具体实验方法为：
分别采集诱导表达12h、24h、36h、48h、60h、72h、84h、96h的发酵液。取发酵液，离心取上清液，将上清液直接用12.5％SDS-PAGE分离。用考马斯亮蓝R-250染色过夜后，经脱色液(5％乙醇，10％冰醋酸)脱色至条带清晰。用试剂盒BCA ProteinAssay Kit(PIERCE，Lot# GB93715A)对蛋白质进行定量分析。
实验设三次重复，检测结果如图3所示。结果表明：甲醇诱导重组菌进行表达，获得了约30kDa的重组蛋白，与预期结果相符，且诱导表达96 h时的蛋白表达量最高，达到5mg/mL发酵液。
实施例3、β-1，3-1，4-葡聚糖酶的酶学性质分析
一、最适pH值和pH稳定性研究
最适pH值的确定：分别用pH值为4.0、5.0、5.5、6.0、6.5、7.0、7.5、8.0、9.0、10.0的磷酸氢二钠-柠檬酸缓冲液配制葡聚糖底物，于40℃反应温度下测定酶活力，确定该酶的最适pH值。酶活测定方法同实施例2。实验设3次重复，结果如图4所示。结果表明：此酶的催化pH范围比较广，从5.4至7.4，其中6.4为此酶的最适pH。图4中的相对酶活是将pH6.4条件下的酶活计作100％。
pH值稳定性的确定：将β-1，3-1，4-葡聚糖酶分别用pH值为4.0、5.0、5.5、6.0、6.5、7.0、7.5、8.0的磷酸氢二钠-柠檬酸的缓冲液和pH值为9.0、10.0的甘氨酸-NaOH的缓冲液处理60分钟后，于40℃反应温度下测定残留的酶活力，测定方法同上。实验设3次重复，结果如图4所示。结果表明：酶蛋白在pH 4-10的缓冲液中处理60分钟后，酶活性有不同程度的下降，但相对活性均在58％以上。酶蛋白在pH为6的条件下最稳定。
二、最适温度和温度稳定性研究
最适温度的确定：用100mM醋酸钠缓冲液配制葡聚糖底物，在pH值6.5的条件下分别测定20℃-80℃内不同温度下的酶活力，确定其最适反应温度，测定方法同上。实验设3次重复，结果如图5所示。结果表明：此酶在30-50℃的温度范围内都有比较高的活性，其最适反应温度为40℃。图5中的相对酶活是将40℃的酶活计作100％。
温度稳定性的确定：将β-1，3-1，4-葡聚糖酶分别在40℃、50℃、60℃、70℃、80℃保温不同时间，然后放入冰水中冷却，于40℃和pH值6.5的条件下测定酶活力，测定方法同上。实验设3次重复，结果如图6所示。结果表明：在40℃、50℃的温度下，此酶的稳定性比较高。图6中的相对酶活是将40℃处理0分钟的酶活计作100％。
三、金属离子和螯合剂(EDTA)对酶活力的影响
在磷酸氢二钠-柠檬酸盐缓冲液(最适pH值)中加入10mM的不同化合物(MgCl2、FeSO4、KI、MnSO4、Na2MoO4、CuSO4、ZnCl2、CaCl2、KCl、NaCl、EDTA、精氨酸、蔗糖)。β-1，3-1，4-葡聚糖酶在上述溶液中处理30min后，在最适温度40℃和最适pH值6.4的条件下测定酶活力，测定方法同上，以不添加金属离子和螯合剂等为空白对照。每个实验重复3次，结果如表1所示。结果表明：FeSO4、KI、NaCl、KCl、精氨酸(Arginine)明显增加酶的活力；而CuSO4、MnSO4、Na2MoO4、EDTA明显抑制酶的活力，其中CuSO4完全抑制酶的活性；ZnSO4、MgCL2、Na2SeO3、CaCl2和蔗糖(sucrose)对酶的活力基本无影响(与空白对照相比较，酶活性变化在5％以内的为基本无影响，提高5％以上的为明显增加酶活力，降低5％以上的为抑制酶活力)。表1中的相对酶活是将空白对照的酶活计作100％。
表1 金属离子和化学试剂对β-1，3-1，4-葡聚糖酶活性的影响
| 试剂 | 相对酶活<sup>a</sup>(％) |
| noneFeSO<sub>4</sub>CuSO<sub>4</sub>MnSO<sub>4</sub>ZnSO<sub>4</sub>KINa<sub>2</sub>SeO<sub>3</sub>Na<sub>2</sub>MoO<sub>4</sub>NaClKClMgCl<sub>2</sub>CaCl<sub>2</sub>EDTAArgininesucrose | 100106.34±4.28039.22±3.6496.08±0.86107.14±2.99102.52±2.1488.56±3.66113.73±2.28114.43±3.38101.82±2.53101.26±2.2585.15±3.57108.68±24399.02±1.73 |
a：平均值±标准差(n＝3)。所有反应均重复三次。
序列表
<160>2
<210>1
<211>657
<212>DNA
<213>人工序列
<220>
<223>
<400>1
gaattccaaa ctggtggttc cttcttcgag ccattcaact cctacaactc cggtttctgg 60
caaaaggcta acggttactc caacggtgac atgttcaact gcacttggag agctaacaac 120
gtttccgtta cttcctccgg tgagatgaga ttggctttga cttccccatc ctacaacaag 180
ttcgactgcg gtgagaacag atccgctcaa acttacggtt acggtttgta cgaggttaga 240
atgaagccag ctaagaacac tggtatcgtt tcctccttct tcacttacac tggtccaact 300
gacggtactc catgggacga gatcgacatc gagttcttgg gtaaggacac tactaaggtt 360
caattcaact actacactaa cggtgctggt aaccacgaga aggttgctga cttgggtttc 420
gacgctgcta acgcttacca cacttacgct ttcgactggc agccaaactc catcaagtgg 480
tacgttgacg gtcaattgaa gcacactgct acttcccaaa tcccaactac tccaggtaag 540
attatgatga acttgtggaa cggtatcggt gttgacgact ggttgggttc ctacaacggt 600
gttaacccat tgtacgctca ctacgactgg gttagataca ctaagaagta atctaga 657
<210>2
<211>214
<212>PRT
<213>枯草芽孢杆菌(Bacillus subtilis)
<400>2
Gln Thr Gly Gly Ser Phe Phe Glu Pro Phe Asn Ser Tyr Asn Ser Gly
1 5 10 15
Phe Trp Gln Lys Ala Asn Gly Tyr Ser Asn Gly Asp Met Phe Asn Cys
20 25 30
Thr Trp Arg Ala Asn Asn Val Ser Val Thr Ser Ser Gly Glu Met Arg
35 40 45
Leu Ala Leu Thr Ser Pro Ser Tyr Asn Lys Phe Asp Cys Gly Glu Asn
50 55 60
Arg Ser Ala Gln Thr Tyr Gly Tyr Gly Leu Tyr Glu Val Arg Met Lys
65 70 75 80
Pro Ala Lys Asn Thr Gly Ile Val Ser Ser Phe Phe Thr Tyr Thr Gly
85 90 95
Pro Thr Asp Gly Thr Pro Trp Asp Glu Ile AspIle Glu Phe Leu Gly
100 105 110
Lys Asp Thr Thr Lys Val Gln Phe Asn Tyr Tyr Thr Asn Gly Ala Gly
115 120 125
Asn His Glu Lys Val Ala Asp Leu Gly Phe Asp Ala Ala Asn Ala Tyr
130 135 140
His Thr Tyr Ala Phe Asp Trp Gln Pro Asn Ser Ile Lys Trp Tyr Val
145 150 155 160
Asp Gly Gln Leu Lys His Thr Ala Thr Ser GlnIle Pro Thr Thr Pro
165 170 175
Gly Lys Ile Met Met Asn Leu Trp Asn GlyIle Gly Val Asp Asp Trp
180 185 190
Leu Gly Ser Tyr Asn Gly Val Asn Pro Leu Tyr Ala His Tyr Asp Trp
195 200 205
Val Arg Tyr Thr Lys Lys
210
Claims (7)
1.一种巴斯德毕赤酵母重组菌，是将β-1，3-1，4-葡聚糖酶基因导入巴斯德毕赤酵母得到的；所述β-1，3-1，4-葡聚糖酶的氨基酸序列如序列表中的序列2所示。
2.根据权利要求1所述的巴斯德毕赤酵母重组菌，其特征在于：所述β-1，3-1，4-葡聚糖酶基因的核苷酸序列如序列表中的序列1所示。
3.根据权利要求1所述的巴斯德毕赤酵母重组菌，其特征在于：所述巴斯德毕赤酵母为巴斯德毕赤酵母X-33。
4.根据权利要求1、2或3所述的巴斯德毕赤酵母重组菌，其特征在于：所述β-1，3-1，4-葡聚糖酶基因通过重组表达载体pPIC-glu-opt导入所述巴斯德毕赤酵母中；所述pPIC-glu-opt是将所述β-1，3-1，4-葡聚糖酶基因插入pPICzαA的多克隆位点得到的。
5.一种生产β-1，3-1，4-葡聚糖酶的方法，是发酵权利要求1至4中任一所述巴斯德毕赤酵母重组菌得到β-1，3-1，4-葡聚糖酶。
6.根据权利要求5所述的方法，其特征在于：当发酵权利要求2所述的巴斯德毕赤酵母重组菌时，其发酵的温度为28.5℃，发酵液的pH为5.5。
7.根据权利要求6所述的方法，其特征在于：所述发酵过程中，从开始发酵起算，44h到46h，按照0.3％初始发酵液体积/小时的流速流加甲醇；47h到48h按照0.57％初始发酵液体积/小时的流速流加甲醇；49h至发酵结束按照0.71％初始发酵液体积/小时的流速流加甲醇。
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| WO2005003319A2 (en) * | 2003-07-02 | 2005-01-13 | Diversa Corporation | Glucanases, nucleic acids encoding them and methods for making and using them |
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| CN1485431A (zh) * | 2003-08-19 | 2004-03-31 | 中国农业科学院饲料研究所 | 表达β-1，3-1，4-葡聚糖酶的基因工程酵母菌株 |
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| 韦宇拓等.巴斯德毕赤酵母新型分泌表达载体构建.广西农业生物科学22 1.2003,22(1),第37页摘要. |
| 韦宇拓等.巴斯德毕赤酵母新型分泌表达载体构建.广西农业生物科学22 1.2003,22(1),第37页摘要. * |
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