CN116103167A - 一种异常威克汉姆酵母菌及其分离方法和应用 - Google Patents
一种异常威克汉姆酵母菌及其分离方法和应用 Download PDFInfo
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Abstract
本发明提出一种异常威克汉姆酵母菌(Wickerhamomyces anomalus)LLF‑2101,保藏单位为中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC NO.22295,并将其应用于分离甘油葡萄糖苷。其能够大幅缩减甘油葡萄糖苷的分离工序,分离操作简单方便,并且得到的产品纯度较高,极大提高了甘油葡萄糖苷的分离效率,适合大规模生产操作。
Description
技术领域
本发明涉及微生物技术领域,具体涉及一种异常威克汉姆酵母菌。
背景技术
甘油葡萄糖苷(Glucosylglycerol,C9H18O8)是糖苷类化合物,由一分子葡萄糖基和一分子甘油组成。自然界中存在6种不同的甘油葡萄糖苷分子构型,其中具有生物活性的是2-α构型。一些异养细菌和光合蓝藻可以在盐胁迫条件下特异性地合成和积累2-α构型的甘油葡萄糖苷作为相容性溶质,可以保护细胞免受渗透压、热、干旱和紫外线等恶劣环境的破坏,具有保湿、抗氧化、抗衰老以及细胞修复等功效。
目前,现有技术中的合成甘油葡萄糖苷的方法存在多种,主要包括化学合成、微生物合成和酶催化合成。虽然现阶段已有多种甘油葡萄糖苷的合成方式,但已报道的甘油葡萄糖苷下游分离方法还比较少。酶催化合成甘油葡萄糖苷在工业化生产中更容易实现,但从酶反应体系中分离出纯度较高的甘油葡萄糖苷极为困难,尤其是对催化过程产生的果糖,催化残留的蔗糖等杂糖的去除;其分离成本高、去除率低,对甘油葡萄糖苷的工业化生产及应用有很大的限制。若要真正实现工业化生产,必须找到一种高效的分离方法。
发明内容
为克服现有技术的不足,本发明提出一种异常威克汉姆酵母菌,能够大幅缩减甘油葡萄糖苷的分离工序,分离操作简单方便,并且得到的产品纯度较高,极大提高了甘油葡萄糖苷的分离效率,适合大规模生产操作。
为实现上述目的,本发明的一种异常威克汉姆酵母菌,异常威克汉姆酵母菌为(Wickerhamomyces anomalus)LLF-2101,保藏单位为中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC NO.22295。
进一步地,异常威克汉姆酵母菌单菌落呈乳白色凸起,圆球形,不透明,菌落表面光滑、湿润、粘稠,有乙醇香味,细胞呈卵圆形,革兰氏染色后在光学显微镜下观察细胞呈紫色,通过出芽生殖的方式繁殖
进一步地,异常威克汉姆酵母菌具有如SEQ No.1所示的核苷酸序列。
进一步地,异常威克汉姆酵母菌在YPD培养基中,在8~36h处于对数生长阶段,在摇瓶中比生长速率(μ)最高达到0.26,在36~40h生长明显放缓,在40~60h菌体密度趋于稳定,测得最大OD600为40.3;异常威克汉姆酵母菌对乳糖、D-半乳糖、L-阿拉伯糖的利用能力较差,麦芽糖、蔗糖、果糖、甘油、葡萄糖做碳源时适宜该菌株生长,酵母粉、鱼粉蛋白胨、胰蛋白胨为优质氮源;异常威克汉姆酵母菌的最适培养温度为31℃,最适生长pH为6,在最适培养条件下,摇瓶中用YPD培养基培养24h即可使该酵母OD600达到35。
本发明提出一种异常威克汉姆酵母菌的分离方法,包括以下步骤:
S1:在无菌条件下将甘油葡萄糖苷生产环境中产生的不明菌液稀释涂布于无菌的YPD培养基平板上,在28℃培养箱中静置培养;
S2:挑出具有典型性酵母菌菌落特征的单菌落,划线法分离纯化2-3次,记录菌落特征,同时挑取菌落制成玻片,400倍显微镜下观察细胞形态特征;
S3:通过革兰氏染色观察细胞形态;
S4:利用真菌基因组DNA提取试剂盒方法提取酵母基因组DNA,对5.8S-ITS rDNA扩增,进行酶切图谱比对,并测序分析,将测序得到的基因序列与NCBI数据库中已有序列进行BLAST比对,与异常威克汉姆酵母菌株(Wickerhamomyces anomalus)相似性达到99%以上,确定其为异常威克汉姆酵母。
本发明的一种异常威克汉姆酵母菌应用于分离甘油葡萄糖苷。
本发明的异常威克汉姆酵母菌能够大幅缩减甘油葡萄糖苷的分离工序,分离操作简单方便,并且得到的产品纯度较高,极大提高了甘油葡萄糖苷的分离效率,适合大规模生产操作。
附图说明
下面结合附图对本发明作进一步描写和阐述。
图1是异常威克汉姆酵母菌在YPD固体培养基平板上的菌落形态;
图2是异常威克汉姆酵母菌在光学显微镜下观察到的细胞形态特征;
图3是异常威克汉姆酵母菌经革兰氏染色后在光学显微镜下观察到的细胞形态特征;
图4是酵母细胞5.8S-ITS rDNA扩增凝胶电泳图(M:5000bp DNA Marker、1:PCR扩增所得5.8S-ITS rDNA基因片段);
图5是异常威克汉姆酵母菌的生长曲线;
图6是不同碳源对异常威克汉姆酵母菌生长影响的统计图;
图7是不同氮源对异常威克汉姆酵母菌生长影响的统计图;
图8是不同温度对异常威克汉姆酵母菌生长影响的统计图;
图9是不同pH对异常威克汉姆酵母菌生长影响的统计图;
具体实施方式
下面将结合附图、通过对本发明的优选实施方式的描述,更加清楚、完整地阐述本发明的技术方案。
异常威克汉姆酵母菌(Wickerhamomyces anomalus)LLF-2101,保藏单位为中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC NO.22295,保藏时间为2021年6月24日。
实施例1:异常威克汉姆酵母菌的菌株筛选和鉴定
S1:YPD培养基分离筛选与显微观察:在无菌条件下将甘油葡萄糖苷生产环境中产生的不明菌液稀释涂布于无菌的YPD培养基平板上,在28℃培养箱中静置培养;挑出具有典型性酵母菌菌落特征的单菌落,划线法分离纯化2-3次,记录菌落特征,如图1所示,同时挑取菌落制成玻片,400倍光学显微镜下观察细胞形态特征,如图2所示。
S2:菌株的革兰氏染色:将菌液稀释后涂布在载玻片上,经火焰固定,加结晶紫溶液染色1min,水洗;加碘液媒染1min,水洗;加脱色液10~30s,至无紫色脱落为止,水洗;加番红复染1min,水洗;在光学显微镜下观察细胞形态特征,如图3所示。
S3:异常威克汉姆酵母菌株的分子生物学鉴定:利用真菌基因组DNA提取试剂盒方法提取酵母基因组DNA,并使用ITS序列的通用引物ITS1:5’-TCCGTAGGTGAACCTGCGG-3’和ITS4:5’-TCCTCCGCTTATTGATATGC-3’对ITS1、5.8S和ITS2的全长ITS区域序列进行特异性扩。PCR反应条件:94℃预变性4min;94℃变性30s,55℃退火30s,72℃延伸1.5min,30个循环;72℃延伸10min;4℃保温。扩增产物在2%琼脂糖凝胶电泳下为单一条带,无非特异扩增现象,如图4所示;条带长约600bp,送生工生物工程有限公司测序得到589bp的序列信息。将测序得到的基因序列与NCBI数据库中已有的序列进行BLAST比对,结果表明与异常威克汉姆酵母模式菌株(Wickerhamomyces anomalus)相似性达到99%以上,因此确定其为异常威克汉姆酵母。
本发明中筛选所得的异常威克汉姆酵母菌具有以下微生物学特征:单菌落呈乳白色凸起,圆球形,不透明,菌落表面光滑、湿润、粘稠,有乙醇香味。细胞在400倍光学显微镜下呈卵圆形,革兰氏染色后在光学显微镜下观察异常威克汉姆酵母细胞呈紫色,通过出芽生殖的方式繁殖。
异常威克汉姆酵母菌的ITS基因序列为:
CTCTGTCTCCTGATTTGAGGTCAACTTTTAGTTTATTGTTGTTAAGCCGAGCCTAAAATACTTCTAAACCTGCCTAGCTGATATAACGAGTTGGAAGAACCTAATACATTATTTCAGAAAGACTGCTTATTAGTACACTCTTGCTAAGTCAATATTTCAAGTTAACCCTTGACAGAGTATCACTCAATACCAAACCCGAAGGTTTGAGAGAGAAATGACGCTCAAACAGGCATACCCTCTGGAATACCAGAGGGTGCAATGTGCGTTCAAAGATTCGATGATTCACGAAAATCTGCAATTCACAATACGTATCGCATTTCGCTGCGTTCTTCATCGTTGCGAGAACCAAGAGATCCGTTGTTGAAAGTTTTGAAGATTTTAATTTTTGTTAAAAATTTTCATGACTATTGGTTAAAGGTTTTAACATTAAAAAAAATGTGTTTAGACCTTTGGGCAGTAAGCCAGGCTCACCACCCAAAGCAAAGTTCAAAAAAACTAGACAATGTGTGTAAGGTTTATCGCCGCGCAATTAAGCGCTGGCAATAGAATACTATAATGATCCTTCCGCAGGCCCCCTCAACCGGAAAAG。
实施例2:异常威克汉姆酵母菌的生长曲线测定
将异常威克汉姆酵母以2%(v/v)的接种量接种到无菌的YPD培养基中;在28℃下振荡培养60h,以灭菌的未接种的YPD培养基作为空白对照,每隔4小时取样测定OD600值。实验重复三次,结果取其平均值,记录数据并绘制该异常威克汉姆酵母菌株的生长曲线。如图5所示,在YPD培养基中,异常威克汉姆酵母菌株在8~36h处于对数生长阶段,在摇瓶中比生长速率(μ)最高达到0.26;在36~40h生长明显放缓,在40~60h细胞菌体密度趋于稳定,测得最大OD600值为40.3。
实施例3:异常威克汉姆酵母菌的生理生化检验结果
(1)碳源单因素试验
在无碳源的YPD培养基的基础上,分别以20g/L葡萄糖、蔗糖、乳糖、甘油、麦芽糖、D-山梨醇、海藻糖、D-半乳糖、可溶性淀粉、L-阿拉伯糖、果糖为碳源,在28℃、200rpm条件下培养异常威克汉姆酵母菌24h,于600nm波长下测定菌悬液的OD值,选择最优碳源。如图6所示,麦芽糖、蔗糖、果糖、甘油、葡萄塘等均适合作为碳源,而异常威克汉姆酵母菌对乳糖、D-半乳糖、L-阿拉伯糖等的利用能力较差。
(2)氮源单因素试验
在无氮源的YPD培养基的基础上,分别以10g/L胰蛋白胨、鱼粉蛋白胨、酵母粉、氯化铵、硫酸铵和草酸铵为氮源,在28℃、200rpm条件下培养异常威克汉姆酵母菌24h,于600nm波长下测定菌悬液的OD600值,选择最优氮源。如图7所示,酵母粉、鱼粉蛋白胨、胰蛋白胨均为可利用氮源,在摇瓶中培养24h OD600即可达到24以上
实施例4:异常威克汉姆酵母菌最适培养条件测定
(1)最适培养温度测定
在灭菌的YPD液体培养基上,将异常威克汉姆酵母菌在温度为22~37℃条件下培养24h,测定OD600值。结果如图8所示,最适培养温度为31℃。
(2)最适培养pH测定
在灭菌的YPD液体培养基上,将异常威克汉姆酵母菌在pH为4~7条件下培养24h,测定OD600值。结果如图9所示,在pH为6时,摇瓶YPD培养基培养24h的OD600值可达34.8,因此确定最适培养pH为6。
实施例5:异常威克汉姆酵母菌对果糖的消耗能力
在31℃条件下培养300mL含异常威克汉姆酵母菌的培养液,培养20h后,8000rpm离心10min,弃掉上清,每50mL异常威克汉姆酵母培养液约得到1.5g的湿菌泥。分别配50、100、150、200、250、300g/L果糖溶液50mL,向不同浓度的果糖溶液中分别加入约1.5g异常威克汉姆酵母湿菌泥,在31℃、200rpm条件下分别培养48h和96h,使用高效液相色谱仪检测剩余果糖浓度,检测结果如表1所示。
表1异常威克汉姆酵母菌株对果糖的消耗能力
初始果糖浓度(g/L) | 50 | 100 | 150 | 200 | 250 | 300 |
48h后剩余果糖浓度(g/L) | 0 | 0 | 39 | 102 | 155 | 216 |
96h后剩余果糖浓度(g/L) | 0 | 0 | 36 | 90 | 143 | 211 |
由表1可知,异常威克汉姆酵母菌对初始果糖浓度在100g/L以内时消耗较快,48h内即可消耗完全。初始果糖浓度大于150g/L时,1.5g异常威克汉姆酵母湿菌泥不能完全消耗果糖,且初始果糖浓度越高,96h内异常威克汉姆酵母消耗的果糖越少。初始果糖浓度在150~300g/L之间时,48h和96h剩余果糖浓度相差不超过12g/L,可见48h即可确定酵母对果糖的消耗能力。
实施例6:异常威克汉姆酵母菌对甘油葡萄糖苷分离能力的验证
酶催化反应制备的甘油葡萄糖苷溶液中,含有蔗糖、果糖、葡萄糖等杂糖,果糖占所有杂质组分的50%以上。分别取高活性干酵母、酿酒酵母、毕赤酵母及异常威克汉姆酵母菌,在相同菌量的条件下,比较四种酵母对蔗糖、甘油、葡萄糖、果糖、甘油葡萄糖苷的消耗能力。糖类初始浓度均为50g/L,加入相同菌量,在31℃、200rpm条件下发酵48h,结果如表2所示。
表2不同种类酵母的耗糖能力
由表2可知,大多数酵母菌株不仅能消耗蔗糖、果糖、葡萄糖,同时会消耗甘油葡萄糖苷,只有本发明的异常威克汉姆酵母菌在消耗蔗糖、果糖、葡萄糖的同时,不消耗甘油葡萄糖苷。
实施例7:异常威克汉姆酵母菌在甘油葡萄糖苷分离的应用
包括以下步骤:
S1:将含异常威克汉姆酵母菌的培养液在31℃下振荡培养24h,培养结束后在4℃条件下8000rpm离心10min,弃掉上清,留下异常威克汉姆酵母菌的菌泥;
S2:取100mL甘油葡萄糖苷溶液,其中含有约105g/L的甘油葡萄糖苷、70g/L的甘油、6g/L的蔗糖、100g/L的果糖、15g/L的葡萄糖。将约5g异常威克汉姆酵母湿菌泥重悬于甘油葡萄糖苷溶液中,31℃,200rpm条件下发酵60h;
S3:发酵结束后甘油葡萄糖苷溶液中蔗糖、果糖、葡萄糖等均耗尽,再进行过滤除菌。加入硅藻土助滤剂在滤纸或无纺布上形成1~2cm的滤饼,加少量溶剂湿润后,将发酵结束后的甘油葡萄糖苷溶液倒入滤饼层进行过滤,即可制得高纯度的甘油葡萄糖苷溶液;
S4:将制备的高纯度的甘油葡萄糖苷溶液除盐、脱色后,在50℃条件下进行旋蒸浓缩,即可制得高浓度和高纯度的甘油葡萄糖苷溶液。
综上所述,本发明的异常威克汉姆酵母菌能够大幅缩减甘油葡萄糖苷的分离工序,分离操作简单方便,并且得到的产品纯度较高,极大提高了甘油葡萄糖苷的分离效率,适合大规模生产操作。
上述具体实施方式仅仅对本发明的优选实施方式进行描述,而并非对本发明的保护范围进行限定。在不脱离本发明设计构思和精神范畴的前提下,本领域的普通技术人员根据本发明所提供的文字描述、附图对本发明的技术方案所作出的各种变形、替代和改进,均应属于本发明的保护范畴。本发明的保护范围由权利要求确定。
Claims (6)
1.一种异常威克汉姆酵母菌,其特征在于,所述异常威克汉姆酵母菌为(Wickerhamomyces anomalus)LLF-2101,保藏单位为中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC NO.22295。
2.根据权利要求1所述的一种异常威克汉姆酵母菌,其特征在于,所述异常威克汉姆酵母菌单菌落呈乳白色凸起,圆球形,不透明,菌落表面光滑、湿润、粘稠,有乙醇香味,细胞呈卵圆形,革兰氏染色后在光学显微镜下观察细胞呈紫色,通过出芽生殖的方式繁殖。
3.根据权利要求1所述的一种异常威克汉姆酵母菌,其特征在于,所述异常威克汉姆酵母菌的ITS基因序列为SEQ No.1。
4.根据权利要求1所述的一种异常威克汉姆酵母菌,其特征在于,所述异常威克汉姆酵母菌在YPD培养基中,在8~36h处于对数生长阶段,在摇瓶中比生长速率(μ)最高达到0.26,在36~40h生长明显放缓,在40~60h菌体密度趋于稳定,测得最大OD600为40.3;所述异常威克汉姆酵母菌对乳糖、D-半乳糖、L-阿拉伯糖的利用能力较差,麦芽糖、蔗糖、果糖、甘油、葡萄糖做碳源时适宜该菌株生长,酵母粉、鱼粉蛋白胨、胰蛋白胨为优质氮源;所述异常威克汉姆酵母菌的最适培养温度为31℃,最适生长pH为6,在最适培养条件下,摇瓶中用YPD培养基培养24h即可使该酵母OD600达到35。
5.根据权利要求1所述一种异常威克汉姆酵母菌的分离方法,其特征在于,包括以下步骤:
S1:在无菌条件下将甘油葡萄糖苷生产环境中产生的不明菌液稀释涂布于无菌的YPD培养基平板上,在28℃培养箱中静置培养;
S2:挑出具有典型性酵母菌菌落特征的单菌落,划线法分离纯化2-3次,记录菌落特征,同时挑取菌落制成玻片,400倍显微镜下观察细胞形态特征;
S3:通过革兰氏染色观察细胞形态;
S4:利用真菌基因组DNA提取试剂盒方法提取酵母基因组DNA,对5.8S-ITS rDNA扩增,进行酶切图谱比对,并测序分析,将测序得到的基因序列与NCBI数据库中已有序列进行BLAST比对,与异常威克汉姆酵母菌株(Wickerhamomyces anomalus)相似性达到99%以上,确定其为异常威克汉姆酵母。
6.根据权利要求1所述的一种异常威克汉姆酵母菌的应用,其特征在于,将其应用于分离甘油葡萄糖苷。
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