CN115216460B - 一种米黑根毛霉糖苷水解酶12家族木葡聚糖酶的制备方法及应用 - Google Patents
一种米黑根毛霉糖苷水解酶12家族木葡聚糖酶的制备方法及应用 Download PDFInfo
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- CN115216460B CN115216460B CN202110419851.XA CN202110419851A CN115216460B CN 115216460 B CN115216460 B CN 115216460B CN 202110419851 A CN202110419851 A CN 202110419851A CN 115216460 B CN115216460 B CN 115216460B
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- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 description 1
- 238000004383 yellowing Methods 0.000 description 1
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Abstract
本发明公开了一种米黑根毛霉糖苷水解酶12家族木葡聚糖酶的制备方法及应用。本发明通过将巴斯德毕赤酵母的FLD1基因和酿酒酵母的Sso1基因与目的蛋白质RmXEG12A共表达,构建出产木葡聚糖酶的毕赤酵母工程菌,提高RmXEG12A的分泌表达水平。RmXEG12A在5L发酵罐中高密度发酵,发酵液的酶活力可达25700U/mL。本发明提供的蛋白质RmXEG12A具有良好水解特性,高效水解富含木葡聚糖的罗望子粉和罗望子胶生成不同聚合度的木葡寡糖,将木葡寡糖制备益生元酸奶可增加酸奶的持水力,提升酸奶的品质。因此本发明所得木葡聚糖酶RmXEG12A在食品工业中具有重要的应用潜力和发展前景。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种米黑根毛霉糖苷水解酶12家族木葡聚糖酶的制备方法及应用。
背景技术
木葡聚糖是一类主链由D-吡喃葡萄糖残基以β-1,4-糖苷键相连形成,主链上60%-75%的葡萄糖残基O-6被α-D-吡喃木糖所取代形成的长链多聚糖。通常,木葡聚糖侧链的木糖基团进一步被β-D-1,2-半乳糖和α-D-1,2-岩藻糖取代,有时木葡聚糖中也存在极少量的***糖。木葡聚糖中主要含有葡萄糖、木糖和半乳糖,其残基比例约为4:3:1(Kiefer et al.Carbohydrate Research,1990,197:139-158)。由于木葡聚糖的侧链较复杂,一般采用***命名法区分木葡聚糖聚合物的各个单元(Fry et al.PhysiologiaPlantarum,1993,89:1-3)。未连接木糖的葡萄糖残基用G代表,连接一个木糖的葡萄糖残基用X代表,在木糖上又连接一个半乳糖的X用L代表,而在半乳糖上又连接一个果糖的L用F代表。木葡聚糖是一种半纤维素,存在于高等植物的初生细胞壁、胞间层和胶质层中,是植物初生细胞壁中主要组成成分之一。在双子叶植物初生壁中木葡聚糖约占干重的20-25%,单子叶植物和禾本科植物中约占2-5%(Benko et al.Enzyme and Microbial Technology,2008,43:109-114)。
木葡聚糖酶(EC3.2.1.151)是水解木葡聚糖的关键酶,能随机水解木葡聚糖主链中的β-1,4-糖苷键,生成木葡寡糖(Menon et al.Journal of Biotechnology 2010,148:233-239)。木葡聚糖酶在自然界分布广泛,植物、动物和微生物中均发现木葡聚糖酶,而微生物是木葡聚糖酶的主要来源。微生物产木葡聚糖酶具有很多优点,如产酶条件简单、产酶量大、来源稳定、提取方便、酶学性质多样等。因此,微生物来源的木葡聚糖酶广泛用于食品、洗涤等行业中(Rashmi and Siddalingamurthy.Biocatalysis andBiotransformation,2018,36:280-295)。
根据氨基酸序列的同源性,木葡聚糖酶可划分为糖苷水解酶5、12、16、44和74家族。一些木葡聚糖酶已用于制备木葡寡糖,如来自热纤维梭菌(Clostridiumthermocellum)(Martinez-Fleites et al.Journal of Biological Chemistry,2006,281:24922-24933)和嗜热单胞菌属(Thermomonospora sp.)的木葡聚糖酶可生产木葡寡糖(Pol et al.Extremophiles,2012,16:135-146)。为满足工业生产需求,一些木葡聚糖酶基因已成功在酵母中表达,但表达水平仍较低,如嗜热毁丝菌(Myceliophthorathermophila)VKPM F-244木葡聚糖酶酶活为118U/mL(Berezina et al.AppliedMicrobiology and Biotechnology,2017,101:5653-5666)和鹿皮色曲霉(Aspergilluscervinus)木葡聚糖酶酶活为1000U/mL(Rykov et al.Applied Microbiology andBiotechnology,2019,103:7553-7566)。因此,提高木葡聚糖酶的表达水平对其工业生产和应用具有重要意义。
功能性寡糖作为一种益生元具有调节肠道微生物菌群、提高机体免疫力和抗氧化能力的作用,在食品和饲料等工业有很大应用潜力(Zuniga et al.Current Issues inMolecular Biology,2021,40:49-80;饶时庭等.饲料研究,2020,9:129-131)。目前主要利用化学合成、酸水解及酶法水解等方法制备木葡寡糖。酶法制备木葡寡糖具有过程容易控制、反应条件温和、副反应少等优点,已经成为木葡寡糖制备中最常用的方法。制备木葡寡糖的原料主要为富含木葡聚糖的植物。目前,研究和工业化中所使用的木葡聚糖大部分来自于罗望子。其中,常用来制备木葡聚糖的原料是罗望子胶和罗望子粉,但是水解时所用的原料浓度较低(≤10%)(Martinez-Fleites et al.Journal of Biological Chemistry,2006,281:24922-24933;Satoh et al.Journal of the Japanese Society forHorticultural Science,2013,82:270-276;Pol et al.Extremophiles,2012,16:135-146;Matsuzawa et al.Applied Microbiology and Biotechnology,2020,104:8761-8773)。罗望子胶在食品中的应用较多,但是木葡寡糖多应用于调节植物生长,在食品中应用较少(Busato et al.Phytochemistry,2001,58:525-531;Mishra et al.Journal ofMaterials Chemistry,2009,19:8528-8536;Satoh et al.Journal of the JapaneseSociety for Horticultural Science,2013,82:270-276)。中国专利申请号CN201811546164.9中公开了一种即食型复合天然营养果冻的制作方法,在原料中加入0.5%-0.6%罗望子胶可提高果冻的营养价值,增加消费者的体验感。中国专利申请号CN201810702200.X中公开了一种速溶红茶的制作方法,在原料中加入35-45份罗望子胶(主料按重量份数计)可改善速溶茶的香气。因此,利用木葡聚糖酶水解富含木葡聚糖的原料,生产木葡寡糖,可以丰富我国的功能性寡糖市场,具有广阔的发展前景。
酸奶是一种以生牛(羊)奶为原料,经过巴氏杀菌后接种嗜热链球菌和保加利亚乳杆菌等发酵制成的乳制品。酸奶质地稠厚、口感丝滑、细腻、顺爽,具有优化肠道内菌群、调整肠道功能、提高免疫力、调节血糖和血脂等优点(Baspinar andCritical Reviewsin Food Science and Nutrition,2020;Donkor et al.International Dairy Journal,2007,17:1321-1331)。将益生元添加到酸奶中制备益生元酸奶能改善酸奶的理化性质,提高酸奶的功能活性,受到广泛关注(Meybodi et al.International Dairy Journal,2020,109:104793;Cruz et al.Journal of Food Engineering,2013,114:323-330;Das etal.LWT-Food Science and Technology,2019,108:69-80;Fazilah et al.Journal ofFunctional Foods,2018,48:387-399)。中国专利申请号CN201811327958.6中公开了一种低聚糖褐色酸奶的制作方法,在酸奶中添加1-3g/L低聚木糖,15-22g/L低聚果糖,18-26g/L低聚异麦芽糖制成益生元酸奶,该酸奶营养丰富,有益于人体健康,符合现代人对酸奶的消费要求。中国专利申请号CN201910267919.X中公开了一种益生元酸奶(石花菜酸奶)的制作方法,在复原乳中添加10-30份(w/v)石花菜琼胶寡糖,然后接种菌发酵得到益生元酸奶,该酸奶可以满足消费者对口感和健康的双重需求,易被消费者接受。其中添加琼胶寡糖还具有抑菌和防腐作用,有利于酸奶的贮藏和运输。目前,尚无利用木葡寡糖制作益生元酸奶的报道。
米黑根毛霉是一株能够分泌多种糖苷水解酶的嗜热丝状真菌,其分泌的木葡聚糖酶具有多种优良的酶学特性(Song et al.Applied Microbiology and Biotechnology,2013,97:10013-10024)。
发明内容
本发明的目的是提供一种米黑根毛霉糖苷水解酶12家族木葡聚糖酶的制备方法及应用。
第一方面,本发明要求保护一种生产木葡聚糖酶的方法。
本发明所要求保护的生产木葡聚糖酶的方法,可包括如下步骤:
(A)将来源于米黑根毛霉CAU432的木葡聚糖酶XEG12A的编码基因、来源于毕赤酵母的FLD1蛋白的编码基因、来源于酿酒酵母的Sso1蛋白的编码基因导入受体毕赤酵母,得到重组毕赤酵母。
其中,米黑根毛霉(Rhizomucor miehei)CAU432已保存在中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址:北京市朝阳区北辰西路1号院,中国科学院微生物研究所,邮编100101)。保藏日期:2011年6月21日。保藏编号为CGMCC No.4967。
(B)按照如下步骤对所述重组毕赤酵母进行发酵培养,从发酵产物中获得木葡聚糖酶XEG12A:
B1)基础发酵培养:将所述重组毕赤酵母接种于BSM培养基中培养,待甘油消耗完全进行甘油补料发酵阶段;
B2)甘油补料发酵阶段:在步骤B1)的基础上,将甘油以15-30mL/h/L(如30mL/h/L)起始发酵液的速度流加到发酵体系中,待菌体湿重达180-220g/L(如200g/L),停止流加甘油,饥饿0.5h-1h(如0.5h)然后进行甲醇补料诱导表达阶段;
B3)甲醇补料诱导表达阶段:在步骤B2)的基础上,将甲醇流加到发酵体系中,4h内使流速从3.6mL/h/L起始发酵液增加至10.9mL/h/L起始发酵液,然后维持10.9mL/h/L起始发酵液速度流加甲醇至发酵结束。
步骤(A)中,所述来源于米黑根毛霉CAU432的木葡聚糖酶XEG12A为如下任一所示蛋白质:
a1)SEQ ID No.1自N末端第21至242位氨基酸残基组成的蛋白质(SEQ ID No.1的第1-20位为信号肽);
a2)由SEQ ID No.1所示的氨基酸序列组成的蛋白质;
a3)将a1)或a2)所述的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有木葡聚糖酶活性的蛋白质;
a4)与a1)-a3)中任一所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且具有木葡聚糖酶活性的蛋白质;
a5)在a1)-a4)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
步骤(A)中,所述来源于毕赤酵母的FLD1蛋白为如下任一所示蛋白质:
b1)由SEQ ID No.2所示的氨基酸序列组成的蛋白质;
b2)将b1)所述的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同活性的蛋白质;
b3)与b1)-b2)中任一所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且相同活性的蛋白质;
b4)在b1)-b3)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
步骤(A)中,所述来源于酿酒酵母的Sso1蛋白为如下任一所示蛋白质:
c1)由SEQ ID No.3所示的氨基酸序列组成的蛋白质;
c2)将c1)所述的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同活性的蛋白质;
c3)与c1)-c2)中任一所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且相同活性的蛋白质;
c4)在c1)-c3)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
在上述蛋白质中,所述标签指利用DNA体外重组技术,与目的蛋白一起融合表达的一种多肽或者蛋白,以便于目的蛋白的表达、检测、示踪和/或纯化。所述蛋白标签可为Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。
在上述蛋白质中,同一性指氨基酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性,如NCBI主页网站的BLAST网页。例如,可在高级BLAST2.1中,通过使用blastp作为程序,将Expect值设置为10,将所有Filter设置为OFF,使用BLOSUM62作为Matrix,将Gap existence cost,Per residue gap cost和Lambda ratio分别设置为11,1和0.85(缺省值)并进行检索一对氨基酸序列的同一性进行计算,然后即可获得同一性的值(%)。
在上述蛋白质中,所述95%以上的同源性可为至少96%、97%、98%的同一性。所述90%以上的同源性可为至少91%、92%、93%、94%的同一性。所述85%以上的同源性可为至少86%、87%、88%、89%的同一性。所述80%以上的同源性可为至少81%、82%、83%、84%的同一性。
步骤(A)中,所述来源于米黑根毛霉CAU432的木葡聚糖酶XEG12A的编码基因是通过重组载体1的形式导入所述受体毕赤酵母中的。
进一步地,所述重组载体1为将所述来源于米黑根毛霉CAU432的木葡聚糖酶XEG12A的编码基因***到pPIC9K载体的多克隆位点(如EcoRI和NotI)后得到的重组质粒。
步骤(A)中,所述来源于毕赤酵母的FLD1蛋白的编码基因是通过重组载体2的形式导入所述受体毕赤酵母中的。
进一步地,所述重组载体2为将所述来源于毕赤酵母的FLD1蛋白的编码基因***到pPICZA载体的多克隆位点(如EcoRI和NotI)后得到的重组质粒。
步骤(A)中,所述来源于酿酒酵母的Sso1蛋白的编码基因是通过重组载体3的形式导入所述受体毕赤酵母中的。
进一步地,所述重组载体3为将所述来源于酿酒酵母的Sso1蛋白的编码基因***到pGAPZB载体的多克隆位点(如EcoRI和NotI)后得到的重组质粒。
在本发明中,所述毕赤酵母具体为毕赤酵母GS115。
步骤(A)中,所述来源于米黑根毛霉CAU432的木葡聚糖酶XEG12A的编码基因可为如下任一所示的DNA分子:
d1)SEQ ID No.4的自5′末端第61-729位所示的DNA分子(SEQ ID No.4的第1-60位为信号肽编码序列);
d2)SEQ ID No.4所示的DNA分子;
d3)在严格条件下与d1)或d2)限定的DNA分子杂交且编码所述来源于米黑根毛霉CAU432的木葡聚糖酶XEG12A的DNA分子;
d4)与d1)-d3)中任一限定的DNA序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且编码所述来源于米黑根毛霉CAU432的木葡聚糖酶XEG12A的DNA分子。
步骤(A)中,所述来源于毕赤酵母的FLD1蛋白的编码基因可为如下任一所示的DNA分子:
e1)SEQ ID No.5所示的DNA分子;
e2)在严格条件下与e1)限定的DNA分子杂交且编码所述来源于毕赤酵母的FLD1蛋白的DNA分子;
e3)与e1)-e2)中任一限定的DNA序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且编码所述来源于毕赤酵母的FLD1蛋白的DNA分子。
步骤(A)中,所述来源于酿酒酵母的Sso1蛋白的编码基因可为如下任一所示的DNA分子:
f1)SEQ ID No.6所示的DNA分子;
f2)在严格条件下与f1)限定的DNA分子杂交且编码所述来源于酿酒酵母的Sso1蛋白的DNA分子;
f3)与f1)-f2)中任一限定的DNA序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且编码所述来源于酿酒酵母的Sso1蛋白的DNA分子。
在上述DNA分子中,所述严格条件可为如下:50℃,在7%十二烷基硫酸钠(SDS)、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在50℃,2×SSC,0.1%SDS中漂洗;还可为:50℃,在7%SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在50℃,1×SSC,0.1%SDS中漂洗;还可为:50℃,在7%SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在50℃,0.5×SSC,0.1%SDS中漂洗;还可为:50℃,在7%SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在50℃,0.1×SSC,0.1%SDS中漂洗;还可为:50℃,在7%SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在65℃,0.1×SSC,0.1%SDS中漂洗;也可为:在6×SSC,0.5%SDS的溶液中,在65℃下杂交,然后用2×SSC,0.1%SDS和1×SSC,0.1%SDS各洗膜一次。
在上述DNA分子中,同一性指核苷酸序列的同一性。可使用国际互联网上的同源性检索站点测定核苷酸序列的同一性,如NCBI主页网站的BLAST网页。例如,可在高级BLAST2.1中,通过使用blastp作为程序,将Expect值设置为10,将所有Filter设置为OFF,使用BLOSUM62作为Matrix,将Gap existence cost,Per residue gap cost和Lambda ratio分别设置为11,1和0.85(缺省值)并进行检索一对核苷酸序列的同一性进行计算,然后即可获得同一性的值(%)。
在上述DNA分子中,所述95%以上的同源性可为至少96%、97%、98%的同一性。所述90%以上的同源性可为至少91%、92%、93%、94%的同一性。所述85%以上的同源性可为至少86%、87%、88%、89%的同一性。所述80%以上的同源性可为至少81%、82%、83%、84%的同一性。
步骤B1)中,接种于所述BSM培养基中的所述重组毕赤酵母为所述重组毕赤酵母的种子液。
进一步地,所述种子液按照包括如下步骤的方法制备得到:将步骤(A)所得的重组毕赤酵母接种于BMGY培养基,在30℃、200rpm振荡培养至发酵液在600nm处吸光值达到2-6(如600nm处吸光值为4)。
其中,所述BMGY培养基的组成如下:质量百分浓度为1%酵母提取物,质量百分浓度为2%蛋白胨,质量百分浓度为1.34%无氨基酵母氮源YNB,质量百分浓度为4×10-5%生物素,体积百分比为1%甘油,余量为100mM pH 6.0磷酸盐缓冲液。
步骤B1)中,将所述种子液接种于所述BSM培养基为接种于装有所述BSM培养基的发酵罐中,所述BSM培养基在所述发酵罐中的装液量为所述发酵罐容积的1.5/5。
步骤B1)中,将所述种子液接种于所述BSM培养基为接种于装有所述BSM培养基的发酵罐中(接种量为所述种子液为所述BSM培养基的1/10体积),所述BSM培养基在所述发酵罐中的装液量为所述发酵罐容积的1.5/5(如5-L发酵罐中装有1.5L所述BSM培养基)。
步骤B1)中,所述BSM组成如下:每1L所述BSM培养基中含有浓度为85g/100mL的磷酸26.7mL,甘油40g,CaSO4 0.93g,K2 SO4 18.2,MgSO4·7H2O 14.9g,KOH 4.13g,余量为水。
步骤B1)中,还包括加入PTM1溶液(毕赤酵母痕量金属盐溶液)的步骤。
进一步地,所述PTM1溶液的加入量为每1L起始发酵液中加入4.35mL所述PTM1溶液。
所述PTM1溶液溶剂为水,溶质及浓度如下:CuSO4·7H2O 6.0g/L,NaI 0.08g/L,MnSO4·H2O 3.0g/L,Na2MoO4·2H2O 0.2g/L,硼酸0.02g/L,CoCl2 0.5g/L,ZnCl2 20.0g/L,FeSO4·7H2O 65.0g/L,生物素0.2g/L,浓硫酸5.0mL/L。
步骤B1)中,所述培养的温度为30℃、pH为4.0、转速为600rpm。
步骤B2)中,所述甘油是溶于水中而形成的500g/L的甘油水溶液;所述甘油补料发酵阶段的培养温度为30℃、pH为4.0、控制溶氧大于20%(发酵时间4-6h)。
步骤B3)中,所述甲醇是无水甲醇;所述甲醇补料诱导表达阶段的培养温度为30℃、pH为6.0、控制溶氧大于20%,发酵时间6-7天(如7天)。
在本发明的具体实施方式中,步骤(B)中的所述发酵培养的时间具体为168小时(即7天)。
步骤(B)中,对所述重组毕赤酵母进行发酵培养后还包括如下步骤:将发酵液的上清液进行透析除盐(所用透析袋的截留分子量为3.5kDa),然后进行阴离子交换层析,用含0-500mM NaCl的20mM pH 7.0的MOPS缓冲液进行线性洗脱,收集得到纯化后木葡聚糖酶XEG12A。
进一步地,所述阴离子交换层析为Q-琼脂糖凝胶FF(Q-Sepharose FF)离子交换层析。
所述用含0-500mM NaCl的20mM pH 7.0的MOPS缓冲液进行线性洗脱的洗脱程序为:50min内洗脱液中NaCl的浓度从0线性升至500mM。
进行所述阴离子交换层析(Q-琼脂糖凝胶FF,Q-Sepharose FF离子交换层析)时,是使用20mM pH 7.0的MOPS缓冲液进行柱平衡的。进行所述阴离子交换层析(Q-琼脂糖凝胶FF,Q-Sepharose FF离子交换层析)时,上样流速为0.5mL/min。上样后先用20mM pH 7.0的MOPS缓冲液洗脱5个柱体积,然后再用含0-500mM NaCl的20mM pH 7.0的MOPS缓冲液进行线性洗脱。洗脱流速为1mL/min。
进行所述阴离子交换层析(Q-琼脂糖凝胶FF,Q-Sepharose FF离子交换层析)后还包括收集具有木葡聚糖酶活性的洗脱液的步骤,以及将具有酶活的洗脱液合并后用20mMpH 7.0的MOPS缓冲液再次透析的步骤。
第二方面,本发明要求保护利用前文第一方面所述方法制备得到的木葡聚糖酶XEG12A。
第三方面,本发明要求保护重组毕赤酵母。
本发明要求保护的重组毕赤酵母为前文第一方面中步骤(A)所述重组毕赤酵母。
第四方面,本发明要求保护如下任一所示应用:
P1、前文第二方面所述木葡聚糖酶XEG12A在制备木葡寡糖中的应用;
P2、前文第二方面所述木葡聚糖酶XEG12A在酶解富含木葡聚糖植物中的应用;
P3、前文第二方面所述木葡聚糖酶XEG12A在制备用于酶解富含木葡聚糖植物的产品中的应用;
P4、前文第二方面所述木葡聚糖酶XEG12A在制作益生元酸奶中的应用;
P5、权利要求7所述木葡聚糖酶XEG12A在食品工业中的应用;
P6、前文第三方面所述重组毕赤酵母在生产前文第二方面所述木葡聚糖酶XEG12A中的应用。
进一步地,P1中,所述木葡寡糖为2-10个葡萄糖、木糖和/或半乳糖聚合而成的木葡寡糖。
进一步地,P1中,所述制备木葡寡糖以木葡聚糖或富含木葡聚糖的植物(包括植物组织或器官)作为底物。
进一步地,所述植物为罗望子、苹果和/或蓝莓(包括这些植物的组织或器官)。
第五方面,本发明要求保护如下任一方法或利用所述方法制备所得产物:
Q1、一种水解罗望子粉的方法,包括如下步骤:用前文第二方面所述的木葡聚糖酶XEG12A水解罗望子粉,罗望子粉和所述木葡聚糖酶XEG12A的配比为1g罗望子粉:200-1000U所述木葡聚糖酶XEG12A(如1g罗望子粉:500U所述木葡聚糖酶XEG12A),于30-60℃(如55℃)水解8h,水解完成后,将反应体系沸水浴(如20min),离心(如11510g离心10min)取上清,得到如下三种高分子量为主的木葡寡糖:XXXG、XXLG(XLXG)和XLLG。
进一步地,所述罗望子粉溶于MOPS缓冲液或水。
Q2、一种水解罗望子胶的方法,包括如下步骤:用前文第二方面所述的木葡聚糖酶XEG12A水解罗望子胶,罗望子胶和所述木葡聚糖酶XEG12A的配比为1g罗望子胶:200-1000U所述木葡聚糖酶XEG12A(如1g罗望子胶:500U所述木葡聚糖酶XEG12A),于30-60℃(如55℃)水解8h,水解完成后,将反应体系沸水浴(如20min),离心(如11510g离心10min)取上清,得到如下三种高分子量为主的木葡寡糖:XXXG、XXLG(XLXG)和XLLG。
进一步地,所述罗望子胶溶于MOPS缓冲液或水。
Q3、一种促进棒状乳杆菌NRRL B-4391、短乳杆菌NRRL B-4527、干酪乳杆菌AS1.62、德氏乳杆菌NRRL B-548、鼠李糖乳杆菌AS 1.2466和/或两歧双歧杆菌NRRL B-41410增殖的方法,包括如下步骤:用前文第二方面所述的木葡聚糖酶XEG12A生产木葡寡糖,然后利用所述木葡寡糖促进棒状乳杆菌NRRL B-4391、短乳杆菌NRRL B-4527、干酪乳杆菌AS1.62、德氏乳杆菌NRRL B-548、鼠李糖乳杆菌AS 1.2466和/或两歧双歧杆菌NRRL B-41410增殖。
Q4、一种制备益生元酸奶的方法,包括如下步骤:用前文第二方面所述的木葡聚糖酶XEG12A生产木葡寡糖,然后利用所述木葡寡糖、鲜牛奶、蔗糖和嗜热链球菌(Streptococcus thermophilus)和保加利亚乳杆菌(Lactobacillus bulgaricus)制备得到益生元酸奶。
进一步地,所述方法包括如下步骤:将质量百分比为93-91%(如92.5-91%,再如92-91%,进一步如91.5-91%)的鲜牛奶、质量百分比为0-2%(不含0点)(如0.5-2%,再如1-2%,进一步如1.5-2%)的所述木葡寡糖和质量百分比为7%的蔗糖(三者质量百分比加和为100%)混合,灭菌(如巴氏灭菌)后接种所述嗜热链球菌(Streptococcusthermophilus)和所述保加利亚乳杆菌(Lactobacillus bulgaricus),所加入的两种菌在体系中的活菌含量均为1×106CFU/mL,然后依次进行发酵、破乳打冷、冷藏后熟,得到所述益生元酸奶。
更进一步地,所述发酵的条件为42℃发酵4h(pH为4.6左右)。所述破乳打冷为:350rpm破乳1min,打冷至20℃。所述冷藏后熟为:4℃后熟16h。
在Q3和Q4中,用前文第二方面所述的木葡聚糖酶XEG12A生产木葡寡糖的方法为Q1或Q2。
本发明通过将巴斯德毕赤酵母的FLD1基因和酿酒酵母的Sso1基因与目的蛋白质RmXEG12A共表达,构建出产木葡聚糖酶的毕赤酵母工程菌,提高RmXEG12A的分泌表达水平。RmXEG12A在5L发酵罐中高密度发酵,发酵液的酶活力可达25700U/mL。木葡聚糖酶的最适pH为7.0,最适温度为65℃,本发明提供的蛋白质RmXEG12A具有良好的水解特性,高效水解富含木葡聚糖的罗望子粉和罗望子胶,生成不同聚合度的木葡寡糖。木葡寡糖可以促进棒状乳杆菌NRRL B-4391、短乳杆菌NRRL B-4527、干酪乳杆菌AS1.62、德氏乳杆菌NRRL B-548、鼠李糖乳杆菌AS 1.2466和两歧双歧杆菌NRRL B-41410的增殖。将木葡寡糖加入酸奶中制得益生元酸奶,增加酸奶的持水力,提高乳杆菌和嗜热链球菌的活菌数,提升酸奶的品质。因此木葡聚糖酶RmXEG12A在食品工业中具有重要的应用潜力和发展前景。
附图说明
图1为重组载体pPIC9k-RmXEG12A的载体图谱。
图2为重组载体pPICZA-FLD1的载体图谱。
图3为重组载体pGAPZB-Sso1的载体图谱。
图4为木葡聚糖酶在5L发酵罐中高密度发酵的产酶历程图(A)和SDS-PAGE电泳图(B)。其中(■)酶活、(▲)蛋白浓度、(●)菌体湿重。泳道M:低分子量标准蛋白;泳道1-8:分别为诱导0、24、48、72、96、120、144、168h的发酵上清液。
图5为木葡聚糖酶纯化图,其中泳道标记1为粗酶液,泳道标记2为纯酶液。
图6为重组蛋白质RmXEG12A作为木葡聚糖酶的最适pH值测定曲线图。其中(▲)柠檬酸缓冲液(pH 3.0-5.5)、(◆)MES(pH 5.0-6.5)、(■)MOPS(pH 6.0-8.0)、(△)Tris-HCl(pH 7.0-9.0)、(□)Gly-NaOH(pH 8.5-11.0)。
图7为重组蛋白质RmXEG12A作为木葡聚糖酶的pH稳定性测定曲线图。其中(▲)柠檬酸缓冲液(pH 3.0-5.5)、(◆)MES(pH 5.0-6.5)、(■)MOPS(pH 6.0-8.0)、(△)Tris-HCl(pH 7.0-9.0)、(□)Gly-NaOH(pH 8.5-11.0)。
图8为重组蛋白质RmXEG12A作为木葡聚糖酶的最适温度测定曲线图。
图9为重组蛋白质RmXEG12A作为木葡聚糖酶温度稳定性测定曲线图。
图10为重组蛋白质RmXEG12A作为木葡聚糖酶水解罗望子粉薄层层析色谱分析图(G1:葡萄糖;G2:纤维二糖;G3:纤维三糖;G4:纤维四糖;木葡寡糖XXXG,XXLG/XLXG和XLLG标准品组成的混合物),其中1为水解罗望子粉。
图11为重组蛋白质RmXEG12A作为木葡聚糖酶水解罗望子胶薄层层析色谱分析图(G1:葡萄糖;G2:纤维二糖;G3:纤维三糖;G4:纤维四糖;木葡寡糖XXXG,XXLG/XLXG和XLLG标准品组成的混合物),其中1为水解罗望子胶。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。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。
以下实施例中的试验,均已设置三次重复实验,结果为三次实验结果的平均值。
木葡聚糖酶酶活测定采用3,5-二硝基水杨酸(DNS)法(Miller,AnalyticalChemistry,1959,31:426-428),具体测定步骤如下:(1)取100μL 0.5%(质量体积分数,即0.5g/100mL)木葡聚糖(tamarind seed)溶液(pH 7.0、50mM的MOPS缓冲液配制),然后加入50μL 50mM的pH 7.0MOPS缓冲液,混匀。65℃预热反应3min,加入50μL适当稀释的待测酶液,置于65℃恒温水浴中反应10min;(2)完成步骤(1)反应后,采用200μLDNS试剂(1%3,5-二硝基水杨酸,1%NaOH和0.2%苯酚,均为质量体积分数,即%表示1g/100mL),终止反应并与释放的还原糖反应,煮沸15min后加入200μL饱和酒石酸钾钠溶液,冷却后于540nm波长下测定吸光值,以葡萄糖作为标准。以葡萄糖作为标线,木葡聚糖酶的活力单位定义为:在上述反应条件下,每分钟反应生成1μmol葡萄糖所需要的酶量为一个酶活力单位(1U)。
比酶活力定义为每mg蛋白所具有的酶活力单位(U/mg)。
实施例1、重组木葡聚糖酶的制备
一、米黑根毛霉木葡聚糖酶基因的PCR扩增
木葡聚糖酶基因JQ901459的编码区为729bp,编码242个氨基酸。分别设计上游引物RmXEG12AF和下游引物RmXEG12AR。序列如下:
RmXEG12AF:5’-CTCAGGAATTCATTCCACTTGAAAAGCGCGC-3’(下划线为限制性内切酶EcoRI的酶切位点)
RmXEG12AR:5’-TGACTGCGGCCGCTTAGTTGACAGAGACCGAGTAAGC-3’(下划线为限制性内切酶NotI的酶切位点)
以米黑根毛霉CAU432(CGMCC No.4967)的cDNA为模板,PCR扩增所述蛋白的编码基因序列(SEQ ID No.4的第61-729位,编码SEQ ID No.1的第21-242位所示蛋白质。SEQ IDNo.1的第1-20位为信号肽,由SEQ ID No.4的第1-60位编码)。PCR反应程序为:94℃预变性10min;94℃变性30s,55℃退火30s,72℃延伸50s,循环30次;72℃后延伸5min。PCR产物经1%的琼脂糖凝胶电泳检测。对PCR产物回收。
二、重组木葡聚糖酶(RmXEG12A)的表达
1、EcoRI和NotI双酶切得到的PCR产物及质粒pPIC9k(为Novagen公司产品),用T4DNA连接酶进行,得到重组载体,转入DH5α中,涂布于含氨苄青霉素(100μg/mL)的LB平板(涂于LB Amp+板)上,挑取单菌落进行菌落PCR,将条带正确的菌落进行测序,比对后得到阳性克隆。将经测序验证正确后得到的重组载体命名为pPIC9k-RmXEG12A(图1)。
2、人工合成巴斯德毕赤酵母的FLD1基因(SEQ ID No.5,编码SEQ ID No.2所示蛋白质),通过无缝克隆将FLD1基因和经EcoRI和NotI双酶切后的载体pPICZA(Novagen公司)进行连接,经测序验证正确后得到重组表达载体pPICZA-FLD1(图2)。此外,酿酒酵母的Sso1基因(SEQ ID No.6,编码SEQ ID No.3所示蛋白质)通过化学合成,并***pGAPZB载体(Novagen公司)EcoRI和NotI酶切位点之间,经测序验证正确后得到重组表达载体pGAPZB-Sso1(图3)。
3、SalI线性化重组载体pPIC9k-RmXEG12A,SacI线性化重组载体pPICZA-FLD1以及AvrII线性化重组载体pGAPZB-Sso1通过醇沉回收,随后电击共同转化毕赤酵母GS115,涂布于MD/ZeocinTM平板,于30℃培养箱中倒置培养2-3d,直至长出菌落为止,得到重组菌。
4、用无菌水收集重组菌菌体,并将适当稀释的菌体涂布于浓度分别为1mg/mL、2mg/mL、3mg/mL、4mg/mL的G418筛选平板上,挑选出不同G418浓度下生长良好的单菌落,接种于5mL BMGY培养基(配方:质量百分浓度为1%的酵母提取物,质量百分浓度为2%的蛋白胨,质量百分浓度为1.34%的无氨基酵母氮源YNB,质量百分浓度为4×10-5%的生物素,体积百分比1%的甘油,余量为100mM pH 6.0磷酸盐缓冲液),置于30℃,200rpm摇床中,振荡培养,待OD600为2-6时,离心,弃上清,收集菌体转接至10mL BMMY培养基(用体积百分比0.5%的甲醇替换甘油)中,置于30℃,200rpm摇床中诱导表达。每隔24h加入甲醇至终浓度为体积百分比0.5%,诱导3d,按前述方法测定发酵液酶活。结果:得到产酶高达359U/mL的重组毕赤酵母(命名为重组菌甲),用于后续高密度发酵。
4、高密度发酵
参照文献“Picha Fermentation Process Guidelines(Version B,053002,Invitrogen)”的方法进行高密度发酵。发酵罐为5L发酵罐。种子培养基BMGY、基本培养基BSM(85%磷酸(85%磷酸是分析纯,%表示g/100mL)40mL,CaSO4 1.4g,K2SO4 27.3g,MgSO4·7H2O 22.4g,KOH 6.19g,甘油60g,加蒸馏水至1.5L)、甘油分批补料培养基和100%甲醇诱导培养基均参照文献中的方法配置。整个发酵过程分为种子液培养、基础培养、甘油补料发酵阶段和100%甲醇补料诱导表达阶段四个阶段。具体步骤如下:
1)种子培养:将步骤3中的重组菌甲接种到装有150mL BMGY培养基的500mL的三角瓶中,在30℃,200rpm摇床中培养24h以上,得到OD600为4的种子液。
2)基础培养:将1)中的种子液接种到5L灭菌后的发酵罐中(装有1.5L发酵基本培养基BSM),用氨水调节培养基pH 4.0,加入PTM1 4.35mL/L起始发酵液,种子液接种量10%(体积百分比),培养过程中温度为30℃,转速600rpm,待溶氧DO值迅速上升,甘油耗尽时,进入甘油补料发酵阶段。其中,PTM1配方:CuSO4·7H2O 6.0g,NaI 0.08g,MnSO4·H2O3.0g,Na2MoO4·2H2O 0.2g,硼酸0.02g,CoCl2 0.5g,ZnCl2 20.0g,FeSO4·7H2O 65.0g,生物素0.2g,浓硫酸5.0mL,加水至1L)。
3)甘油补料发酵阶段:甘油水溶液(甘油浓度为500g/L)流加速度为30mL/h/L起始发酵液。该阶段始终监测溶氧,保持溶氧在20%-70%。控制温度30℃、发酵液pH 4.0,待菌体湿重达200g/L,停止流加,整个过程发酵时间为6h。
4)100%甲醇补料诱导表达阶段:停止流加甘油后,饥饿0.5h使罐内甘油耗尽,调整发酵液pH至6.0、转速至800rpm,开始流加100%甲醇诱导产酶,4h内甲醇流加从3.6mL/h/L起始发酵液增加至10.9mL/h/L起始发酵液,然后维持10.9mL/h/L起始发酵液速度流加甲醇至发酵结束。监控罐内溶氧保持溶氧在20%-70%,控制温度30℃,发酵7天。发酵过程中取样测定菌体湿重、蛋白质含量和木葡聚糖酶活。测定的条件为:50mM MOPS缓冲液(pH7.0),反应温度为65℃(具体方法见上文)。
测定步骤如下:
1)菌体湿重:称取离心管质量,记为m1,在离心管中加入1mL发酵液,使用离心机11510×g离心3min,弃去上清液,将离心管及下层物质称重记为m2。
菌体湿重计算公式为:菌体湿重=m2-m1。
2)蛋白质含量:以牛血清蛋白为标准蛋白,采用Lowry法(Lowry et al.TheJournal of Biological Chemistry,1951,193(1):265-275)测定蛋白含量(mg/mL)。蛋白质含量为蛋白质总含量。
发酵过程中菌体湿重、蛋白质含量和木葡聚糖酶活力变化如图4中A所示,发酵过程中蛋白的SDS-PAGE电泳图如图4中B所示。高密度发酵168h时酶活力达到最高,发酵液的上清液中木葡聚糖酶活力为25700U/mL,蛋白浓度为5.1mg/mL,菌体湿重为426g/L。
三、重组木葡聚糖酶的纯化
Q-琼脂糖凝胶FF(Q-Sepharose FF)离子交换层析:将5mL发酵上清液装入透析袋(截流分子量为3.50kDa)并放入20mM MOPS(pH 7.0)中透析过夜,将透析好的酶液用11510×g离心5min。取上清液上样于用MOPS缓冲液(20mM,pH 7.0)平衡好的Q-琼脂糖凝胶FF(Q-Sepharose FF)层析柱(10×50mm)进行纯化,上样过程的流速为0.5mL/min。上样后,用相同缓冲液洗脱5个柱体积,之后用0-500mM NaCl溶液(20mM MOPS缓冲液,pH7.0)线性洗脱(50min内NaCl从0线性升至500mM),洗脱流速1mL/min,收集有酶活的部分,将得到的纯酶合并后用20mM MOPS(pH 7.0)透析4h备用。整个纯化过程见表1,SDS-PAGE结果见图5,其中泳道1为粗酶液,泳道2为经Q-Sepharose层析柱纯化后的纯酶。图5的结果表明,重组木葡聚糖酶显示单一分子量条带,对应分子量为21.9kDa,与预期结果一致。
表1、重组木葡聚糖酶的纯化
纯化步骤 | 总酶活(U) | 总蛋白(mg) | 比酶活(U/mg) | 回收率(%) | 纯化倍数 |
粗酶液 | 128500 | 25.5 | 5039.2 | 100 | 1.0 |
纯酶 | 117150 | 14.8 | 7915.5 | 91.2 | 1.6 |
表1中,回收率是纯酶的总酶活力占粗酶液总酶活力的百分含量。纯化倍数是纯酶的比酶活与粗酶的比酶活的比值。
实施例2、重组蛋白RmXEG12A作为木葡聚糖酶的酶学性质
各缓冲液具体如下:(▲)柠檬酸缓冲液(pH 3.0-5.5)、(◆)MES(2-(N-吗啉代)乙磺酸)缓冲液(pH 5.0-6.5)、(■)MOPS(3-吗啉丙磺酸)缓冲液(pH 6.0-8.0)、(△)Tris-HCl(三(羟甲基)氨基甲烷-盐酸)缓冲液(pH 7.0-9.0)、(□)Gly-NaOH(甘氨酸-氢氧化钠)缓冲液(pH 8.5-11.0)。以上缓冲液的浓度均为50mM。
一、重组木葡聚糖酶的最适pH值
以实施例1制备的纯化后重组木葡聚糖酶溶液为待测样品溶液,在上述缓冲液(pH3.0-11.0范围的不同缓冲体系)中稀释适当倍数,45℃下按照上述方法测定酶活。稀释时使用pH 3.0-11.0范围的不同缓冲体系稀释纯酶液,测定时使用对应稀释缓冲液。将实施例1制备的纯化后的重组木葡聚糖酶溶液作为待测样品溶液,测定其酶活力,方法如下:(1)取100μL0.5%(质量体积分数,即0.5g/100mL)木葡聚糖(tamarind seed)溶液(利用木葡聚糖和50mM pH 3.0-11.0范围的不同待测缓冲液配制),然后加入50μL 50mM的对应缓冲液,混匀。45℃预热反应3min,加入50μL适当稀释的待测酶液,置于45℃恒温水浴中反应10min;(2)完成步骤(1)反应后,采用200μL DNS试剂(1%3,5-二硝基水杨酸,1%NaOH和0.2%苯酚,均为质量体积分数,即1%3,5-二硝基水杨酸为1g/100mL 3,5-二硝基水杨酸),终止反应并与释放的还原糖反应,煮沸15min后加入200μL饱和酒石酸钾钠溶液,冷却后于540nm波长下测定吸光值,以葡萄糖作为标准。以葡萄糖作为标线,木葡聚糖酶的活力单位定义为:在上述反应条件下,每分钟反应生成1μmol葡萄糖所需要的酶量为一个酶活力单位(1U)。以最大值为100%。分别计算各pH下的相对酶活力,以最高酶活力为100%,计算各个pH值条件下木葡聚糖酶的相对活力,结果如图6所示,重组木葡聚糖酶的最适pH值为7.0。
二、重组木葡聚糖酶的pH稳定性
将实施例1制备的纯化后重组木葡聚糖酶溶液用上述缓冲液稀释适当倍数,置于40℃水浴中保温30min,再迅速取出并立即冰浴30min,按照具体实施方式下实施例1前所述方法测定残余的木葡聚糖酶活力。对照为未经上述处理(上述处理指的是先40℃水浴30min,再迅速冰浴30min)的重组木葡聚糖酶溶液的稀释液。以对照的酶活力为100%,计算用不同pH缓冲液处理后的酶的相对活力,结果如图7所示,该酶在pH 3.5-9.0范围内保持稳定,处理30min后残余酶活力仍然保持在80%以上。
三、重组木葡聚糖酶的最适温度
将实施例1制备的纯化后重组木葡聚糖酶溶液作为待测酶液,pH 7.0、50mM的MOPS稀释至适当倍数,在30℃-80℃范围内的不同温度下反应,然后按照具体实施方式下实施例1前所述方法测定木葡聚糖酶活力,以最高酶活力为100%,计算在各个反应温度下的相对活力。结果如图8所示,重组木葡聚糖酶的最适温度为65℃。
四、重组木葡聚糖酶的温度稳定性
将实施例1制备的纯化后重组木葡聚糖酶溶液用pH 7.0、50mM的MOPS稀释至适当倍数,置于不同温度下分别水浴30min(水浴温度为30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃或80℃),迅速取出并冰浴30min,按照具体实施方式下实施例1前所述方法测定残余的木葡聚糖酶活力。对照为未经上述步骤处理(上述处理指的是水浴30min,迅速冰浴30min)的重组木葡聚糖酶溶液稀释液。以对照的酶活力作为100%。上述各个温度处理后的待测酶液相对活力结果如图9所示,重组木葡聚糖酶在60℃以下稳定。
五、底物特异性的测定
将实施例1制备的纯化后重组木葡聚糖酶溶液作为待测酶液,测定不同底物的木葡聚糖酶活力,具体如下:(1)取100μL 0.5%(质量体积分数,即0.5g/100mL)底物溶液(pH7.0、50mM的MOPS缓冲液配制),然后加入50μL 50mM的pH 7.0MOPS缓冲液,混匀。65℃预热反应3min,加入50μL适当稀释的待测酶液,置于65℃恒温水浴中反应10min;(2)完成步骤(1)反应后,采用200μL DNS试剂(1%3,5-二硝基水杨酸,1%NaOH和0.2%苯酚,均为质量体积分数,即%表示1g/100mL),终止反应并与释放的还原糖反应,煮沸15min后加入200μL饱和酒石酸钾钠溶液,冷却后于540nm波长下测定吸光值,以葡萄糖作为标准。底物包括木葡聚糖(tamarind seed)、大麦β-葡聚糖、地衣多糖、昆布多糖、可得然多糖、微晶纤维素、桦木木聚糖、羧甲基纤维素钠(CMC)、槐豆胶(LBG)、普鲁兰糖和可溶性淀粉等,底物浓度均为0.5%(质量体积比,0.5g底物/100mL 50mM MOPS pH7.0缓冲液)。
以重组木葡聚糖酶对木葡聚糖(tamarind seed)的酶活力为100%,计算该酶对大麦β-葡聚糖、地衣多糖、昆布多糖、可得然多糖、微晶纤维素、桦木木聚糖、羧甲基纤维素钠(CMC)、槐豆胶(LBG)、普鲁兰糖和可溶性淀粉的相对活力和比酶活力。
结果见表2。重组木葡聚糖酶对木葡聚糖(tamarind seed)的比酶活力最高,为7915.5U/mg;其次为地衣多糖,比酶活力为110.8U/mg;对大麦β-葡聚糖的比酶活力最低,为87.1U/mg,对昆布多糖、可得然多糖、微晶纤维素、桦木木聚糖、羧甲基纤维素钠(CMC)、槐豆胶(LBG)、普鲁兰糖和可溶性淀粉均未表现出活性。
表2、重组木葡聚糖酶的底物特异性
底物 | 比酶活力(U/mg) | 相对活力(%) |
木葡聚糖(tamarind seed) | 7915.5±132.3 | 100 |
地衣多糖 | 110.8±4.0 | 1.4 |
大麦β-葡聚糖 | 87.1±4.1 | 1.1 |
昆布多糖 | - | - |
可得然多糖 | - | - |
微晶纤维素 | - | - |
桦木木聚糖 | - | - |
羧甲基纤维素钠 | - | - |
槐豆胶 | - | - |
普鲁兰糖 | - | - |
可溶性淀粉 | - | - |
注:“-”表示未检测到活性。
实施例3、利用重组木葡聚糖酶(RmXEG12A)水解罗望子粉生产木葡寡糖
称取15g罗望子粉,溶于100mL蒸馏水(蒸馏水也可换为pH 7.0、50mM的MOPS缓冲液),加入7500U重组木葡聚糖酶(RmXEG12A),在200rpm充分搅拌,置于55℃水解8h。水解完成后,将反应体系沸水浴20min,11510×g离心10min,取上清即为木葡寡糖粗糖液。木葡寡糖粗糖液经喷雾干燥后得到木葡寡糖粉(喷雾干燥条件:进风口温度180℃,出风口温度80℃)。用3,5-二硝基水杨酸法测定粗糖液中的还原糖含量,计算还原糖得率、水解率和产物得率。采用薄层层析色谱(TLC)定性分析粗糖液的组成。
还原糖得率、水解率、产物得率的计算公式分别为:
还原糖得率(%)=水解后体系中总还原糖质量/水解前加入原料质量×100;
水解率(%)=(1-水解液离心后沉淀干重/水解前加入原料质量)×100;
产物得率(%)=产物干重/水解前加入原料质量×100。
还原糖测定方法:(1)取150μL蒸馏水(蒸馏水也可换为pH 7.0、50mM的MOPS缓冲液),加入50μL适当稀释的待测木葡寡糖糖液,混匀。(2)完成步骤(1)后,采用200μL DNS试剂(1%3,5-二硝基水杨酸,1%NaOH和0.2%苯酚,均为质量体积分数),与释放的还原糖反应,煮沸15min后加入200μL饱和酒石酸钾钠溶液,冷却后于540nm波长下测定吸光值,以葡萄糖作为标准。
薄层层析色谱检测条件:展层剂为正丁醇:乙醇:水=2:1:1(体积比v/v),显色剂为甲醇:硫酸=95:5(体积比v/v)。标准对照为葡萄糖、纤维二糖、纤维三糖、纤维四糖、木葡寡糖XXXG,XXLG/XLXG和XLLG标准品组成的混合物。在薄层层析色谱分析板上点适量待测样本,放入展层剂展层两次后用显色剂完全浸湿吹干,130℃烘烤显色。
其中,葡萄糖、纤维二糖、纤维三糖和纤维四糖均为上海源叶科技有限公司产品,货号分别为B21882、B25328、S46082和S46724。木葡寡糖XXXG,XXLG/XLXG和XLLG标准品组成的混合物来自Megazyme公司产品,货号为O-XGHON。
结果显示,RmXEC12A水解罗望子粉的水解率为56.6%,还原糖得率为32.5%,产物得率为50.8%。薄层层析色谱分析结果见图10(M为标准对照,从上至下依次为葡萄糖、纤维二糖、纤维三糖、纤维四糖、木葡寡糖XXXG,XXLG/XLXG和XLLG标准品组成的混合物)。结果表明,当以罗望子粉木葡聚糖作为底物时,主要产生三种高分子量为主的木葡寡糖,分别为XXXG、XXLG(XLXG)和XLLG。
实施例4、利用重组木葡聚糖酶(RmXEG12A)水解罗望子胶生产木葡寡糖
称取20g罗望子胶,溶于100mL蒸馏水(蒸馏水也可换为pH 7.0、50mM的MOPS缓冲液),加入10000U重组木葡聚糖酶(RmXEG12A),在200rpm充分搅拌,置于55℃水解8h。水解完成后,将反应体系沸水浴20min,11510×g离心10min,取上清即为木葡寡糖粗糖液。木葡寡糖粗糖液经喷雾干燥后得到木葡寡糖粉(喷雾干燥条件:进风口温度180℃,出风口温度80℃)。用3,5-二硝基水杨酸法测定粗糖液中的还原糖含量,计算还原糖得率、水解率和产物得率。采用薄层层析色谱定性分析粗糖液的组成。还原糖得率、水解率和产物得率计算方法和薄层层析色谱条件同实施例3。
结果显示,RmXEC12A水解罗望子胶的水解率为74.3%,还原糖得率为35.0%,产物得率为68.7%。薄层层析色谱的实验结果见图11(M为标准对照,从上至下依次为葡萄糖、纤维二糖、纤维三糖、纤维四糖、木葡寡糖XXXG,XXLG/XLXG和XLLG标准品组成的混合物)。结果表明,当以罗望子胶木葡聚糖作为底物时,主要产生三种高分子量为主的木葡寡糖,分别为XXXG、XXLG(XLXG)和XLLG。
实施例5、益生菌增殖实验
本实施例使用的木葡寡糖为实施例3中的木葡寡糖。
一、木葡寡糖对益生菌乳杆菌增殖实验
(1)种子培养:将9株乳杆菌(中国农业大学,食品科学与营养工程学院,中国轻工业食品生物工程重点实验室保存,记载于“Li et al.Preparation,characterization,andprebiotic activity of mannooligosaccharides produced from cassia gum by aglycoside hydrolase family 134β-mannanase.Food Chemistry,2020,309:125709”一文)(表3)接种至商品化的MRS培养基中活化培养3次(MRS培养基配方:蛋白胨10g/L,牛肉膏10g/L,酵母提取物5g/L,柠檬酸氢二胺2g/L,葡萄糖20g/L,吐温80 1mL/L,乙酸钠5g/L,磷酸氢二钾2g/L,硫酸镁0.6g/L,硫酸锰0.25g/L,pH 6.4±0.2),11510×g离心5min收集菌体。将菌体重悬于无葡萄糖的MRS液体培养基中作为种子液。
(2)将9株乳杆菌种子液分别接种至无葡萄糖的MRS培养基中,作为对照组。同时用10g/L木葡寡糖替换MRS中葡萄糖,设为实验组,置于厌氧培养箱37℃培养48h,采用比浊法测定595nm波长下的吸光值OD595。
从表3可以看出,以木葡寡糖为唯一碳源的棒状乳杆菌NRRL B-4391生长量(OD595)为0.25、短乳杆菌NRRL B-4527为0.12、干酪乳杆菌AS 1.62为0.31、鼠李糖乳杆菌AS1.2466为0.07、德氏乳杆菌NRRL B-548为0.34,显著高于对照组。说明木葡寡糖可以促进棒状乳杆菌NRRL B-4391、短乳杆菌NRRL B-4527、干酪乳杆菌AS1.62、鼠李糖乳杆菌AS1.2466和德氏乳杆菌NRRL B-548的增殖。
表3、木葡寡糖对9株乳杆菌纯培养48h的增殖效果
注:标注不同的小写字母表示差异显著(P<0.05)。
二、木葡寡糖对益生菌双岐杆菌增殖实验
(1)种子培养:将5株双岐杆菌(中国农业大学,食品科学与营养工程学院,中国轻工业食品生物工程重点实验室保存,记载于“Li et al.Preparation,characterization,and prebiotic activity of mannooligosaccharides produced from cassia gum by aglycoside hydrolase family 134β-mannanase.Food Chemistry,2020,309:125709”一文)(表4)接种至商品化的MRS培养基中活化培养3次(MRS培养基配方:蛋白胨10g/L,牛肉膏10g/L,酵母提取物5g/L,柠檬酸氢二胺2g/L,葡萄糖20g/L,吐温80 1mL/L,乙酸钠5g/L,磷酸氢二钾2g/L,硫酸镁0.6g/L,硫酸锰0.25g/L,pH 6.4±0.2),11510×g离心5min收集菌体。将菌体重悬于无葡萄糖的MRS液体培养基中作为种子液。
(2)将5株双岐杆菌种子液分别接种至无葡萄糖的MRS培养基中,作为对照组。同时用10g/L木葡寡糖替换MRS中葡萄糖,设为实验组,置于厌氧培养箱37℃培养48h,采用比浊法测定595nm波长下的吸光值OD595。
从表4可以看出,木葡寡糖对两歧双歧杆菌NRRL B-41410的OD595为0.23,显著高于对照组-0.06。说明木葡寡糖可以促进两歧双歧杆菌NRRL B-41410的增殖。
表4、木葡寡糖对5株双岐杆菌纯培养48h的增殖效果
注:标注不同的小写字母表示差异显著(P<0.05)。
实施例6、利用木葡寡糖生产益生元酸奶
本实施例使用的木葡寡糖为实施例3中的木葡寡糖。木葡寡糖生产益生元酸奶由如下重量份的原料(1000份)制成:鲜牛奶930份-910份,木葡寡糖粉0份-20份,蔗糖70份(木葡寡糖的添加质量百分比(%)设置为:0、0.5、1、1.5、2%)。
益生元酸奶的制备方法具体包括以下步骤:
(1)混料:将鲜牛奶、木葡寡糖和蔗糖按照上述重量份混合均匀后于65℃预热至物料完全溶解,在40MPa下进行均质处理。
(2)灭菌:将步骤(1)得到的料液加热到95℃,巴氏杀菌5min。
(3)接种:待上述料液冷却至40℃后,在无菌环境下向料液中加入嗜热链球菌(Streptococcus thermophilus)和保加利亚乳杆菌(Lactobacillus bulgaricus)进行接种,所加入的两种菌在体系中的活菌含量均为1×106CFU/mL。
(4)发酵:将接种后的料液放于恒温发酵室中,在42℃条件下发酵4h(至pH 4.6左右)。
(5)破乳打冷:将发酵所得的益生元酸奶于350rpm破乳1min,打冷至20℃。
(6)冷藏后熟:将步骤(5)中打冷后的益生元酸奶放入冷藏库(4℃)后熟16h,即得到酸奶。
效果评价
1、感官评价:请20位有酸奶感官评价经验的人员对酸奶的口感、风味、色泽、粘稠度进行整体评价。
2、持水力:取后熟16h的酸奶进行测定,称取灭菌离心管质量,记为m1,在每个离心管中加入酸奶20g并称取总质量,记为m2,使用离心机3000rpm离心10min,弃去上清液,将离心管及下层物质称重记为m3。
持水力计算公式为:持水力(%)=(m3-m1)/(m2-m1)×100%。
3、活菌计数
按照国标GB4789.35-2016《食品微生物学检验乳酸菌检验》中方法进行活菌数检测,具体如下:
嗜热链球菌计数:选择2-3个连续的适宜稀释度,每个稀释度吸取1mL样品匀液于灭菌平皿内。稀释液移入平皿后,将冷却至48℃的MC培养基倾注入平皿约15mL,转动平皿使混合均匀。36℃±1℃需氧培养72h±2h,培养后计数。
乳杆菌计数:选择2-3个连续的适宜稀释度,每个稀释度吸取1mL样品匀液于灭菌平皿内。稀释液移入平皿后,将冷却至48℃的MRS琼脂培养基倾注入平皿约15mL,转动平皿使混合均匀。36℃±1℃厌氧培养72h±2h,培养后计数。
4、结果
木葡寡糖酸奶质地均匀、表面光滑、有光泽、颜色洁白无发黄现象,与对照组一致。发酵香气浓郁、无不良气味,与对照组一致。说明木葡寡糖可以应用于益生元酸奶的制作。
表5是木葡寡糖酸奶持水力的结果,从表5可以看出,添加木葡寡糖后酸奶的持水力(46.7%-48.8%)均高于未添加木葡寡糖酸奶的持水力(43.2%)。在木葡寡糖添加量为0-2%的范围内,酸奶的持水力随木葡寡糖添加量的增加而增大,说明添加木葡寡糖可以增加酸奶的持水力。在酸奶中加入木葡寡糖后,酸奶的凝胶结构得到增强,从而增强其对水分的截留效果,防止乳清洗出,改善酸奶的组织状态。
表5、木葡寡糖酸奶持水力
表6是木葡寡糖酸奶对乳杆菌和嗜热链球菌的活菌数影响。从表6可以看出,未添加木葡寡糖的酸奶中的活菌数为2.78×108CFU/mL,而添加木葡寡糖后酸奶中活菌数在3.38×108CFU/mL-4.56×108CFU/mL之间,显著高于未添加木葡寡糖的酸奶中的的活菌数。说明木葡寡糖的添加有效提高酸奶中乳杆菌和嗜热链球菌的活菌数,提高酸奶的品质。
表6、木葡寡糖酸奶对活菌的影响
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
<110> 中国农业大学
<120> 一种米黑根毛霉糖苷水解酶12家族木葡聚糖酶的制备方法及应用
<130> GNCLN210707
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Ser Asn Ala Val Tyr Ser Phe Val Ala Ala Asp Ser Leu Thr Ser Ile
180 185 190
Ser Ser Asp Leu Leu Pro Phe Leu Lys Tyr Leu Thr Ser Asn Gly Tyr
195 200 205
Ile Ser Ser Ser Gln Tyr Leu Arg Ala Val Gln Ala Gly Thr Glu Pro
210 215 220
Phe Val Gly Thr Asn Ala Lys Leu Thr Thr Thr Ala Tyr Ser Val Ser
225 230 235 240
Val Asn
<210> 2
<211> 379
<212> PRT
<213> Pichia pastoris
<400> 2
Met Ser Thr Glu Gly Gln Ile Ile Lys Cys Lys Ala Ala Val Ala Trp
1 5 10 15
Glu Ala Gly Lys Asp Leu Ser Ile Glu Glu Ile Glu Val Leu Pro Pro
20 25 30
Arg Ala His Glu Val Arg Val Lys Val Glu Phe Thr Gly Val Cys His
35 40 45
Thr Asp Ala Tyr Thr Leu Ser Gly Ala Asp Ala Glu Gly Ser Phe Pro
50 55 60
Val Val Phe Gly His Glu Gly Ala Gly Val Val Glu Ser Val Gly Glu
65 70 75 80
Gly Val Glu Ser Val Lys Val Gly Asp Ser Val Val Leu Leu Tyr Thr
85 90 95
Pro Glu Cys Arg Glu Cys Lys Phe Cys Leu Ser Gly Lys Thr Asn Leu
100 105 110
Cys Gly Lys Ile Arg Ala Thr Gln Gly Lys Gly Leu Leu Pro Asp Gly
115 120 125
Thr Ser Arg Phe Arg Cys Lys Gly Lys Asp Leu Phe His Tyr Met Gly
130 135 140
Cys Ser Ser Phe Ser Gln Tyr Thr Val Val Ala Asp Ile Ser Val Val
145 150 155 160
Lys Val Gln Asp Glu Ala Pro Lys Asp Lys Thr Cys Leu Leu Gly Cys
165 170 175
Gly Val Thr Thr Gly Tyr Gly Ala Ala Ile Asn Thr Ala Lys Ile Ser
180 185 190
Lys Gly Asp Lys Ile Gly Val Phe Gly Ala Gly Cys Ile Gly Leu Ser
195 200 205
Val Ile Gln Gly Ala Val Ser Lys Gly Ala Ser Glu Ile Ile Val Ile
210 215 220
Asp Ile Asn Asp Ser Lys Lys Ala Trp Ala Asp Gln Phe Gly Ala Thr
225 230 235 240
Lys Phe Val Asn Pro Thr Thr Leu Pro Glu Gly Thr Asn Ile Val Asp
245 250 255
Tyr Leu Ile Asp Ile Thr Asp Gly Gly Phe Asp Tyr Thr Phe Asp Cys
260 265 270
Thr Gly Asn Val Gln Val Met Arg Asn Ala Leu Glu Ser Cys His Lys
275 280 285
Gly Trp Gly Glu Ser Ile Ile Ile Gly Val Ala Ala Ala Gly Lys Glu
290 295 300
Ile Ser Thr Arg Pro Phe Gln Leu Val Thr Gly Arg Val Trp Arg Gly
305 310 315 320
Cys Ala Phe Gly Gly Ile Lys Gly Arg Thr Gln Met Pro Ser Leu Val
325 330 335
Gln Asp Tyr Leu Asp Gly Lys Ile Lys Val Asp Glu Phe Ile Thr His
340 345 350
Arg His Asp Leu Asp Asn Ile Asn Lys Ala Phe His Asp Met His Ala
355 360 365
Gly Asn Cys Ile Arg Ala Val Ile Thr Met His
370 375
<210> 3
<211> 290
<212> PRT
<213> Saccharomyces cerevisiae
<400> 3
Met Ser Tyr Asn Asn Pro Tyr Gln Leu Glu Thr Pro Phe Glu Glu Ser
1 5 10 15
Tyr Glu Leu Asp Glu Gly Ser Ser Ala Ile Gly Ala Glu Gly His Asp
20 25 30
Phe Val Gly Phe Met Asn Lys Ile Ser Gln Ile Asn Arg Asp Leu Asp
35 40 45
Lys Tyr Asp His Thr Ile Asn Gln Val Asp Ser Leu His Lys Arg Leu
50 55 60
Leu Thr Glu Val Asn Glu Glu Gln Ala Ser His Leu Arg His Ser Leu
65 70 75 80
Asp Asn Phe Val Ala Gln Ala Thr Asp Leu Gln Phe Lys Leu Lys Asn
85 90 95
Glu Ile Lys Ser Ala Gln Arg Asp Gly Ile His Asp Thr Asn Lys Gln
100 105 110
Ala Gln Ala Glu Asn Ser Arg Gln Arg Phe Leu Lys Leu Ile Gln Asp
115 120 125
Tyr Arg Ile Val Asp Ser Asn Tyr Lys Glu Glu Asn Lys Glu Gln Ala
130 135 140
Lys Arg Gln Tyr Met Ile Ile Gln Pro Glu Ala Thr Glu Asp Glu Val
145 150 155 160
Glu Ala Ala Ile Ser Asp Val Gly Gly Gln Gln Ile Phe Ser Gln Ala
165 170 175
Leu Leu Asn Ala Asn Arg Arg Gly Glu Ala Lys Thr Ala Leu Ala Glu
180 185 190
Val Gln Ala Arg His Gln Glu Leu Leu Lys Leu Glu Lys Ser Met Ala
195 200 205
Glu Leu Thr Gln Leu Phe Asn Asp Met Glu Glu Leu Val Ile Glu Gln
210 215 220
Gln Glu Asn Val Asp Val Ile Asp Lys Asn Val Glu Asp Ala Gln Leu
225 230 235 240
Asp Val Glu Gln Gly Val Gly His Thr Asp Lys Ala Val Lys Ser Ala
245 250 255
Arg Lys Ala Arg Lys Asn Lys Ile Arg Cys Trp Leu Ile Val Phe Ala
260 265 270
Ile Ile Val Val Val Val Val Val Val Val Val Pro Ala Val Val Lys
275 280 285
Thr Arg
290
<210> 4
<211> 729
<212> DNA
<213> Rhizomucor miehei
<400> 4
atgaagattt cccttctgac aaccgctgct gctctcttgt ttgcaaccgc atccactgct 60
attccacttg aaaagcgcgc cgacttttgt ggccaatggg ataccgctga agaagggccc 120
tataccattt acaacaactt gtggggtcaa gactctgcta cgagtggtca gcagtgcacc 180
ggtgtcgata gtcttagtgg aaacactctt gcttggcaca catcctggac ctgggctggt 240
ggacaataca atgtaaaatc gtacgccaac gctgctctct cttttactgc tacccgagtt 300
tctgatatct ccagcatccc cttcacttgg cattggtcct acactggctc tggtattgtt 360
gctaacgtcg ctttcgacct ctggacttcc tcctcgacct ctggtaacta tgagatcgaa 420
atcatggtct ggcttggtgc cttgggtggt gctggtcctc tcggaagcgc cgttggtacc 480
tttgagcaag ctggcactac ttggacactg tacagtggta gcaatggatc caacgccgtt 540
tacagcttcg ttgccgcaga cagcctcacc agtatctcga gcgacttgct tccattcttg 600
aaatatctca ccagcaatgg atacatctcc tcgtctcagt atcttcgtgc tgtccaagca 660
ggtacagaac cattcgttgg tacaaatgcc aagttaacta ccacagctta ctcggtctct 720
gtcaactaa 729
<210> 5
<211> 1140
<212> DNA
<213> pichia pastoris
<400> 5
atgtctaccg aaggtcaaat catcaaatgt aaggcagctg ttgcctggga ggcaggaaag 60
gatctctcta ttgaggagat tgaggttctt cctccaagag cccatgaagt tagagtgaaa 120
gtggaattca ctggtgtatg ccacactgat gcttacacgc tttctggtgc agatgcagag 180
ggaagtttcc ctgttgtgtt cggccatgaa ggtgctggtg ttgtcgagtc agttggagaa 240
ggtgttgagt ccgtgaaggt tggggattct gtagtgcttc tgtacactcc tgagtgcaga 300
gagtgcaagt tctgtctgtc tggtaagacg aacctctgtg gtaaaatcag agccacccag 360
ggtaaaggtt tgttaccaga cgggacttct cgtttccgtt gtaagggcaa ggatttgttt 420
cactatatgg gatgttcttc cttttctcaa tacactgtgg tggctgacat ctcagtggtt 480
aaagtccaag acgaagctcc taaggacaag acatgtctgt tgggttgtgg tgttaccaca 540
gggtacggtg ctgctatcaa cactgctaag atctctaagg gtgacaagat cggtgtgttt 600
ggtgctggat gtattggatt atctgtcatc caaggtgcag tttccaaagg tgcaagcgag 660
attattgtaa ttgacatcaa tgattcaaag aaggcatggg cggaccaatt tggtgcaact 720
aagtttgtca atcctacaac cttaccagaa ggtaccaata ttgttgacta cttgattgat 780
atcactgacg gaggctttga ctataccttc gactgtaccg gtaatgttca agtaatgaga 840
aatgcacttg aatcttgcca caagggttgg ggtgagtcga tcatcatcgg tgtcgctgct 900
gctggtaaag aaatctctac ccgtcctttc cagttggtta ctggcagagt ctggagagga 960
tgcgcctttg gaggtatcaa gggacgtact caaatgccat ctttggttca ggactatctt 1020
gatggtaaga ttaaagttga cgagtttatc acacacagac atgacctgga caacatcaac 1080
aaagcatttc atgacatgca tgctggaaac tgtattcgtg ctgtgattac tatgcactaa 1140
<210> 6
<211> 873
<212> DNA
<213> Saccharomyces cerevisiae
<400> 6
atgagttata ataatccgta ccagttggaa accccttttg aagagtcata cgagttggac 60
gaaggttcga gcgctatcgg tgctgaaggc cacgatttcg tgggcttcat gaataagatc 120
agtcaaatca atcgcgatct cgataagtac gaccatacca tcaaccaggt cgattctttg 180
cataagaggc tactgaccga agttaatgag gagcaagcaa gtcacttaag gcactccctg 240
gacaacttcg tcgcacaagc cacggacttg cagttcaaac tgaaaaatga gattaaaagt 300
gcccaaaggg atgggataca tgacaccaac aagcaagctc aggcggaaaa ctccagacaa 360
agatttttga agcttatcca ggactacaga attgtggatt ccaactacaa ggaggagaat 420
aaagagcaag ccaagaggca gtatatgatc attcaaccag aggccaccga agatgaagtt 480
gaagcagcca taagcgatgt agggggccag cagatcttct cacaagcatt gttgaatgct 540
aacagacgtg gggaagccaa gactgctctt gcggaagtcc aggcaaggca ccaagagtta 600
ttgaaactag aaaaatccat ggcagaactt actcaattgt ttaatgacat ggaagaactg 660
gtaatagaac aacaagaaaa cgtagacgtc atcgacaaga acgttgaaga cgctcaactc 720
gacgtagaac agggtgtcgg tcataccgat aaagccgtca agagtgccag aaaagcaaga 780
aagaacaaga ttagatgttg gttgattgta ttcgccatca ttgtagtcgt tgttgttgtc 840
gttgttgtcc cagccgttgt caaaacgcgt taa 873
Claims (12)
1.一种生产木葡聚糖酶的方法,包括如下步骤:
(A)将来源于米黑根毛霉CAU432的木葡聚糖酶XEG12A的编码基因、来源于毕赤酵母的FLD1蛋白的编码基因、来源于酿酒酵母的Sso1蛋白的编码基因导入受体毕赤酵母,得到重组毕赤酵母;
(B)按照如下步骤对所述重组毕赤酵母进行发酵培养,从发酵产物中获得木葡聚糖酶XEG12A:
B1)基础发酵培养:将所述重组毕赤酵母接种于BSM培养基中培养,待甘油消耗完全进行甘油补料发酵阶段;
B2)甘油补料发酵阶段:在步骤B1)的基础上,将甘油以15-30 mL/h/L起始发酵液的速度流加到发酵体系中,待菌体湿重达180-220 g/L,停止流加甘油,饥饿0.5-1 h左右,然后进行甲醇补料诱导表达阶段;
B3)甲醇补料诱导表达阶段:在步骤B2)的基础上,将甲醇流加到发酵体系中4 h内使流速从3.6 mL/h/L起始发酵液增加至10.9 mL/h/L起始发酵液,然后维持10.9 mL/h/L起始发酵液速度流加甲醇至发酵结束;
步骤(A)中,所述来源于米黑根毛霉CAU432的木葡聚糖酶XEG12A为SEQ ID No.1自N末端第21至242位氨基酸残基组成的蛋白质;
步骤(A)中,所述来源于米黑根毛霉CAU432的木葡聚糖酶XEG12A的编码基因是通过重组载体1的形式导入所述受体毕赤酵母中的;所述重组载体1为将所述来源于米黑根毛霉CAU432的木葡聚糖酶XEG12A的编码基因***到pPIC9K载体的多克隆位点后得到的重组质粒;
步骤(A)中,所述来源于毕赤酵母的FLD1蛋白的编码基因是通过重组载体2的形式导入所述受体毕赤酵母中的;所述重组载体2为将所述来源于毕赤酵母的FLD1蛋白的编码基因***到pPICZA载体的多克隆位点后得到的重组质粒;
步骤(A)中,所述来源于酿酒酵母的Sso1蛋白的编码基因是通过重组载体3的形式导入所述受体毕赤酵母中的;所述重组载体3为将所述来源于酿酒酵母的Sso1蛋白的编码基因***到pGAPZB载体的多克隆位点后得到的重组质粒;
步骤B1)中,还包括加入PTM1溶液的步骤;所述PTM1溶液的加入量为每1 L起始发酵液中加入4.35 mL所述PTM1溶液;
步骤B1)中,所述培养的温度为30℃、pH为4.0、转速为600 rpm;
步骤B2)中,所述甘油是溶于水中而形成的500 g/L的甘油水溶液;所述甘油补料发酵阶段的培养温度为30℃、pH为4.0、控制溶氧大于20%;
步骤B3)中,所述甲醇是无水甲醇;所述甲醇补料诱导表达阶段的培养温度为30℃、pH为6.0、控制溶氧大于20%,发酵时间6-7天。
2. 根据权利要求1所述的方法,其特征在于:步骤(A)中,所述来源于毕赤酵母的FLD1蛋白为由SEQ ID No.2所示的氨基酸序列组成的蛋白质。
3. 根据权利要求1所述的方法,其特征在于:步骤(A)中,所述来源于酿酒酵母的Sso1蛋白为由SEQ ID No.3所示的氨基酸序列组成的蛋白质。
4.根据权利要求1所述的方法,其特征在于:所述毕赤酵母为毕赤酵母GS115。
5. 根据权利要求1所述的方法,其特征在于:步骤(A)中,所述来源于米黑根毛霉CAU432的木葡聚糖酶XEG12A的编码基因为SEQ ID No.4的自5′末端第61-729位所示的DNA分子。
6. 根据权利要求1所述的方法,其特征在于:步骤(A)中,所述来源于毕赤酵母的FLD1蛋白的编码基因为SEQ ID No.5所示的DNA分子。
7. 根据权利要求1所述的方法,其特征在于:步骤(A)中,所述来源于酿酒酵母的Sso1蛋白的编码基因为SEQ ID No.6所示的DNA分子。
8.根据权利要求1所述的方法,其特征在于:步骤B1)中,接种于所述BSM培养基中的所述重组毕赤酵母为所述重组毕赤酵母的种子液;
所述种子液按照包括如下步骤的方法制备得到:将步骤(A)所得的重组毕赤酵母接种于BMGY培养基,在30℃、200 rpm振荡培养至发酵液在600 nm处吸光值达到2-6。
9.根据权利要求8所述的方法,其特征在于:步骤B1)中,将所述种子液接种于所述BSM培养基为接种于装有所述BSM培养基的发酵罐中,所述BSM培养基在所述发酵罐中的装液量为所述发酵罐容积的1.5/5。
10. 根据权利要求1-9中任一所述的方法,其特征在于:步骤(B)中,对所述重组毕赤酵母进行发酵培养后还包括如下步骤:将发酵液的上清液进行透析除盐,然后进行阴离子交换层析,用含0-500 mM NaCl的20 mM pH 7.0的MOPS缓冲液进行线性洗脱,收集得到纯化后木葡聚糖酶XEG12A。
11.根据权利要求10所述的方法,其特征在于:所述阴离子交换层析为Q-琼脂糖凝胶FF离子交换层析。
12. 根据权利要求11所述的方法,其特征在于:所述用含0-500 mM NaCl的20 mM pH7.0的MOPS缓冲液进行线性洗脱的洗脱程序为:50 min内洗脱液中NaCl的浓度从0线性升至500 mM。
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