CN109679864B - 一种产转糖基活性β-半乳糖苷酶的菌株及用该酶生产低聚半乳糖的方法 - Google Patents
一种产转糖基活性β-半乳糖苷酶的菌株及用该酶生产低聚半乳糖的方法 Download PDFInfo
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- CN109679864B CN109679864B CN201811527058.6A CN201811527058A CN109679864B CN 109679864 B CN109679864 B CN 109679864B CN 201811527058 A CN201811527058 A CN 201811527058A CN 109679864 B CN109679864 B CN 109679864B
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P19/00—Preparation of compounds containing saccharide radicals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明公开了一种产转糖基活性β‑半乳糖苷酶的菌株及用该酶生产低聚半乳糖的方法,产转糖基活性β‑半乳糖苷酶的菌株分类命名为植物乳杆菌(Lactobacillus plantarum)YLBGNL‑S7,保藏在中国典型培养物保藏中心,保藏编号为:CCTCC M 2018397,保藏日期为2018年6月25日。本发明植物乳杆菌(Lactobacillus plantarum)YLBGNL‑S7菌株来源于奶酪,安全性及遗传稳定性好,生长快,培养简单,产β‑半乳糖苷酶水平高;以此菌为发酵菌株发酵生产β‑半乳糖苷酶的酶活可达1‑157U/mL;将得到的β‑半乳糖苷酶酶液添加到30%(w/v)乳糖溶液中催化反应合成低聚半乳糖,转化反应2‑24h,反应产物GOS得率可达43.40%(w/w),其中转移二糖和转移三糖分别为18.29%(w/w)和12.95%(w/w)。
Description
技术领域
本发明涉及食品生物技术领域,特别是一种产转糖基活性β-半乳糖苷酶的菌株及用该酶生产低聚半乳糖的方法。
背景技术
低聚半乳糖(Galacto-oligosaccharides,GOS)是一类不可被人体肠道消化吸收的功能性低聚糖。低聚半乳糖具有调整改善肠道菌群,促进人体健康的功效。低聚半乳糖对双歧杆菌等益生菌具有显著的增殖作用,其益生特性获得了广泛的临床验证,益生指数(PI)高达11.66,显著高于相同糖链单元低聚果糖(FOS)的PI值5.05。此外,低聚半乳糖还具有降低结肠癌风险,减少炎症,减少肠道病原体侵袭,提高宿主免疫力等功效。
低聚半乳糖的加工性能较好,具有低分子量水溶性膳食纤维的特点,热值较低,甜度为蔗糖的20-40%,对酸和热较稳定,作为一种天然存在的人乳寡糖(HMOS),低聚半乳糖可应用于婴儿配方奶粉,成为人乳寡糖(HMOS)在新生儿和婴儿配方奶粉中的替代品,是近年来倍受关注的功能性低聚糖。
低聚半乳糖的结构与人乳寡糖(HMOS)近似,由数量不等的半乳糖单元,以葡萄糖或半乳糖单元为还原性末端,通过β-(1-4),β-(1-6)、β-(1-3)等β-糖苷键构成的异质性低聚糖。结构分析鉴定发现的转移二糖、转移三糖、转移四糖形式的低聚半乳糖超过30种。不同结构及聚合度的低聚半乳糖,其益生特性也存在差异。
作为膳食补充剂的低聚半乳糖,商业化生产主要以各种原料形式的乳糖,利用微生物来源的β-半乳糖苷酶(EC3.2.1.23)通过酶法合成。国际上用于低聚半乳糖合成的工业化酶制剂产品仅仅局限于米曲霉(Aspergillus oryzae)、乳酸克鲁维酵母(Kluyveromyceslactis)和环状芽孢杆菌(Bacillus circulans)来源的β-半乳糖苷酶。
尽管已有上述不同来源β-半乳糖苷酶合成的低聚半乳糖产品上市,但酶法合成低聚半乳糖仍存不足:(1)低聚半乳糖的得率较低、乳糖转化率不高。目前商业化低聚半乳糖产品(丹麦Friesland Food Domo的和日本Yakult Honsha的/>)中实际上仍含有20%左右的乳糖和葡萄糖。为了获得高纯度的低聚半乳糖,酶法合成的低聚半乳糖还需经过色谱、膜分离或微生物发酵转化等方式进行下游纯化处理;(2)低聚半乳糖产品的同质化严重。酶的来源直接影响酶法合成低聚半乳糖的结构和益生特性,不同来源β-半乳糖苷酶合成低聚半乳糖产物谱存在显著差异,其低聚半乳糖产物主要成分的糖苷键类型以及聚合度也不同。
基于上述原因,筛选转糖基活性更高,转糖基反应产物益生指数更高的β-半乳糖苷酶成为近年的研究热点。国内外研究者先后从不同样品来源的巨大芽孢杆菌(Bacillusmegaterium)、成团肠杆菌(Enterobacter agglomerans)、脆壁克鲁维酵母(Kluyveromycesfragilis)、以及不同来源的婴儿双歧杆菌(Bifidobacterium infantis)、两岐双歧杆菌(Bifidobacterium bifidum)、罗伊氏乳杆菌(Lactobacillus reuteri)、戊糖乳杆菌(Lactobacillus pentosus)、保加利亚乳杆菌(Lactobacillus bulgaricus)、植物乳杆菌(Lactobacillus plantarum)、嗜热链球菌菌(Streptococcus thermophilus)等乳酸菌菌株中筛选具有转糖基活性的β-半乳糖苷酶。
然而,研究表明不同属、不同种及不同菌株来源β-半乳糖苷酶的活性及转糖基反应产物都存在差异。B.adolescentis来源的β-半乳糖苷酶(BgbⅡ)偏好形成β-(1-4)糖苷键的转半乳糖基产物,而不生成β-(1-6)糖苷键的转半乳糖基产物(Hinz et al.2004),B.bifidum来源的β-半乳糖苷酶(BgbⅡ)则明显偏好形成β-(1-6)糖苷键的转半乳糖基产物(Goulas et al.2009)。基于乳杆菌属菌株基因组学的研究也证明,乳杆菌属菌株基因组具有复杂的多样性(张和平等2016),不同环境来源的相同种属乳杆菌的代谢特点与其生活环境密切相关,特别是一些碳水化合物代谢相关蛋白的基因在乳杆菌属不同种菌株之间存在较大的差异。已有来源于人唾液L.plantarum WCFS1及来源于泡菜L.plantarum 70810合成GOS的报道,但来源于奶酪的乳源L.plantarum合成GOS的报道还未出现。
发明内容
本发明的目的是要解决现有技术中产转糖基活性β-半乳糖苷酶的菌株产酶水平较低低聚半乳糖产品存在的同质性问题,提供一种产转糖基活性β-半乳糖苷酶的菌株及用该酶生产低聚半乳糖的方法,以植物乳杆菌YLBGNL-S7产生的β-半乳糖苷酶可以较高的转化率,以乳糖为底物催化合成以转移二糖、转移三糖为主的低聚半乳糖产物。
为达到上述目的,本发明是按照以下技术方案实施的:
本发明的第一个目的是提供一种产转糖基活性β-半乳糖苷酶的菌株,其分类命名为植物乳杆菌(Lactobacillus plantarum)YLBGNL-S7,保藏在中国典型培养物保藏中心,保藏编号为:CCTCC M 2018397,保藏日期为2018年6月25日。
本发明的第二个目的是提供一种发酵生产β-半乳糖苷酶的方法,用上述产转糖基活性β-半乳糖苷酶的菌株,具体步骤如下:
(1)种子培养
种子培养基:乳糖5-20g,蛋白胨1-10g,牛肉浸粉1-10g,酵母浸粉1-5g,K2HPO4 5g,柠檬酸氢二铵2g,乙酸钠5g,MgSO4·7H2O 0.58g,吐温-80 1-4mL,MnSO4 0.25g,蒸馏水1000mL配制,pH 6.3,并于121℃下灭菌20min;
种子培养条件:将植物乳杆菌YLBGNL-S7于30-38℃、100-250rpm的转速震荡培养10-20h活化该菌株;
(2)发酵培养
发酵培养基:乳糖5-20g,蛋白胨1-10g,牛肉浸粉1-10g,酵母浸粉1-5g,MgSO4·7H2O 0.58g,吐温-80 1-4mL,MnSO4 0.25g,蒸馏水1000mL配制,pH6.3,121℃灭菌20min;
发酵条件:植物乳杆菌YLBGNL-S7的接种量为发酵培养基的1%-10%,在30-38℃、震荡转速100~300rpm发酵培养12-24h生产β-半乳糖苷酶;
(3)发酵后处理
发酵液经冷冻离心后收集湿菌体,用pH 6.0的0.1mol/L磷酸盐缓冲液制成菌悬液,然后于0℃冰浴条件下超声破碎,所得悬液即为β-半乳糖苷酶粗酶液,经检测酶活达到10-157U/mL。
进一步,一种发酵生产β-半乳糖苷酶的方法,用上述产转糖基活性β-半乳糖苷酶的菌株,具体步骤可以如下:
(1)种子培养
种子培养基:乳糖5-20g,蛋白胨1-10g,牛肉浸粉1-10g,酵母浸粉1-5g,K2HPO4 5g,柠檬酸氢二铵2g,乙酸钠5g,MgSO4·7H2O 0.58g,吐温-80 1-4mL,MnSO4 0.25g,蒸馏水1000mL配制,pH 6.3,并于121℃下灭菌20min;
种子培养条件:将植物乳杆菌YLBGNL-S7于30-38℃、100-250rpm的转速震荡培养10-20h活化该菌株;
(2)发酵培养
发酵培养基:乳糖5-20g,蛋白胨1-10g,牛肉浸粉1-10g,酵母浸粉1-5g,MgSO4·7H2O 0.58g,吐温-80 1-4mL,MnSO4 0.25g,蒸馏水1000mL配制,pH 6.3,121℃灭菌20min;
发酵条件:植物乳杆菌YLBGNL-S7的接种量为发酵培养基的1%-10%,在30-38℃、震荡转速100~300rpm发酵培养12-24h生产β-半乳糖苷酶;
(3)发酵后处理
发酵液离心收集湿菌体后,用含2.5μg/mL溶菌酶、0.4%吐温-80的磷酸盐缓冲液30℃透性化处理20分钟,所得细胞悬液作为β-半乳糖苷酶粗酶液,经检测酶活达到1-20U/mL。
作为本发明的优选方案,所述步骤(3)中菌悬液于0℃冰浴条件下超声破碎时,超声破碎每进行3s停8s,超声功率为125W,超声破碎的总时间为15min。
本发明的第三个目的是提供一种生产低聚半乳糖的方法,用上述发酵生产β-半乳糖苷酶的方法制得的β-半乳糖苷酶粗酶液,具体步骤如下:
取β-半乳糖苷酶粗酶液加入pH为6.0的磷酸氢二钠-柠檬酸钠缓冲液配制的30%(w/v)乳糖溶液中进行催化反应,反应条件:以干基计每克乳糖的酶用量为5-30U,反应温度为45-60℃,反应时间2-24h,得到含有低聚半乳糖的酶反应液。
与现有技术相比,本发明植物乳杆菌(Lactobacillus plantarum)YLBGNL-S7菌株安全,遗传稳定性好,生长快,培养简单,产β-半乳糖苷酶水平高;以此菌为发酵菌株,以乳糖为碳源、酵母浸粉、蛋白胨或牛肉浸粉为氮源及无机盐等组成的发酵培养基,发酵生产β-半乳糖苷酶,发酵后经酶活检测,发酵液中酶活可达10-157U/mL,远高于目前已报道的其他来源植物乳杆菌产β-半乳糖苷酶水平(<10U/mL)。将得到的β-半乳糖苷酶酶液添加到30%(w/v)乳糖溶液中催化反应合成低聚半乳糖,转化反应2-24h,反应产物GOS得率可达43.40%(w/w),其中转移二糖和转移三糖分别为18.29%(w/w)和12.95%(w/w),残余乳糖含量仅为11.48%(w/w)。目前国际上已报道的乳酸菌来源β-半乳糖苷酶合成低聚半乳糖GOS产物的得率在26.8%—47.6%之间,因此,本发明分离获得的植物乳杆菌(Lactobacillus plantarum)YLBGNL-S7的产β-半乳糖苷酶水平和β-半乳糖苷酶转糖基活性均具有明显优势。
附图说明
图1为产转糖基活性β-半乳糖苷酶的植物乳杆菌YLBGNL-S7的系统进化分析。
图2为薄层层析TLC分析复筛菌株的转糖基反应产物。
图3为不同反应时间生成低聚半乳糖GOS产物的TLC分析。
图4为产转糖基活性β-半乳糖苷酶的植物乳杆菌YLBGNL-S7所产转糖基活性β-半乳糖苷酶合成低聚半乳糖的HPLC分析。
具体实施方式
为使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步的详细说明。此处所描述的具体实施例仅用于解释本发明,并不用于限定发明。
本实施例提供的一种产转糖基活性β-半乳糖苷酶的菌株,其分类命名为植物乳杆菌(Lactobacillus plantarum)YLBGNL-S7,保藏在中国典型培养物保藏中心,保藏编号为:CCTCC M 2018397,保藏日期为2018年6月25日。该产转糖基活性β-半乳糖苷酶的植物乳杆菌(Lactobacillus plantarum)YLBGNL-S7具有如下生物学特征:
形态学特征:革兰氏阳性杆菌,不形成芽孢,电镜观察其大小为(0.5~1)μm×(1~2)μm,在MRS培养基上形成圆形菌落,边缘整齐,表面光滑,呈乳白色;
生理生化特征:能发酵葡萄糖、乳糖、蔗糖产酸,不能发酵阿拉伯糖、鼠李糖、木糖,淀粉水解试验、明胶水解试验、接触酶试验、硫化氢产生试验均为阴性。
本发明涉及的可产生较强转糖基活性β-半乳糖苷酶的植物乳杆菌(Lactobacillus plantarum)YLBGNL-S7的16S rRNA的核苷酸序列长度为1483碱基,如序列<210>所示,具体的核苷酸和氨基酸序列表为:
<210>1
<211>1483
<212>核苷酸序列
<213>植物乳杆菌(Lactobacillus plantarum YLBGNL-S7)
<220>
<221>misc_feature
<223>16S rDDNA基因核苷酸序列
<400>1
TCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTTACAGA。
本实施例的产β-半乳糖苷酶的植物乳杆菌(Lactobacillus plantarum)YLBGNL-S7是从新疆伊犁少数民族传统手工半干型奶酪中分离筛选获得,以下为本实施例的产β-半乳糖苷酶的植物乳杆菌(Lactobacillus plantarum)YLBGNL-S7的分离与筛选具体实施过程。
实施例1
产β-半乳糖苷酶的植物乳杆菌(Lactobacillus plantarum)YLBGNL-S7的分离。
在无菌条件下,将5g奶酪样品置于100ml灭菌生理盐水中,37℃摇床震荡4h充分打散均匀。用灭菌生理盐水将打散的样品溶液稀释为10-1、10-2、10-3、10-4、10-5、10-6梯度,各稀释度分别取100μL菌种的稀释液,均匀涂布于含2%碳酸钙的MRS平板上涂布,37℃培养24h。挑取有明显溶钙圈的单菌落用接种环在含有X-Gal和IPTG的MRS培养基平板上,37℃培养24h。挑取蓝色单菌落,在MRS平板上划线纯化,纯化三次。对分离菌株进行镜检,确定纯度,进行革兰氏染色并镜检区分革兰氏阳性菌和阴性菌。
MRS培养基平板(g/L):琼脂粉20,蛋白胨10,牛肉浸粉10,酵母浸粉5,乳糖20,K2HPO4 5,柠檬酸氢二铵2,乙酸钠5,MgSO4·7H2O 0.58,吐温-80 1(ml/L),MnSO4 0.25,pH6.3,121℃灭菌20min。
实施例2
产β-半乳糖苷酶的植物乳杆菌(Lactobacillus plantarum)YLBGNL-S7的分离的筛选。
将上述平板上的分离的蓝斑菌株分别接种至50ml产酶发酵培养基中,于37℃摇床培养14h,摇床转速为180转/分。培养结束后的发酵液于10000转/分离心5分钟收集细胞。用一定体积磷酸缓冲液洗涤2~3次后,按每50mg湿菌体悬浮于50μl pH7.0、50mmol/L磷酸钾缓冲液的比例,重悬细菌细胞,加入1/3体积的100μm酸洗玻璃珠,低温下震荡破碎细菌细胞,取50μl破碎后的细胞悬液加入300μL pH7.0磷酸钾缓冲盐配制的30%乳糖溶液,于50℃水浴反应6h,12000转/分离心5min,上清液即为转糖基反应产物。
分别对不同菌株的反应产物进行薄层层析(TLC),TLC分析采用活化过的硅胶铝板Silica gel 60No.553(Merck公司),用微量进样器点样,以正丁醇:乙醇:水=5:3:2为展层剂上行展层,层析至距离顶端1cm处,取出用吹风机吹干,然后以显色剂(苯胺一二苯胺一磷酸)喷雾,于80℃烘箱中烘烤10min,根据TLC板上对应低聚糖产物斑点的大小,筛选获得具有转糖基活性的乳酸菌菌株5株:YLBGNL-S5、YLBGNL-S6、YLBGNL-S7、YLBGNL-S8,YLBGNL-S9和YLBGNL-S10,结果详见图2所示,图2中:1为标准糖(葡萄糖、半乳糖和乳糖的混合物);2-7:依次为S5、S6、S7、S8、S9、S10菌株转糖基反应产物。
发酵培养基(g/L):蛋白胨5,牛肉浸粉10,酵母浸粉10,乳糖10,K2HPO42g、醋酸钠5、柠檬酸氢二铵2、MgSO4·7H2O 2、MnSO4·4H2O 0.05、吐温-801(ml/L),pH 6.3,121℃灭菌20min。
实施例3
进一步,对上述实施例2筛选得到的菌株YLBGNL-S7进行鉴定。
1)形态学观察和生理生化试验
将菌株YLBGNL-S7接种于营养琼脂培养基37℃培养24h,菌株YLBGNL-S7在MRS培养基上形成圆形菌落,边缘整齐,表面光滑,呈乳白色;菌体杆状,革兰氏染色阳性,扫描电镜观察显示其大小为(0.5~1)μm×(1~2)μm。菌株YLBGNL-S7的生理生化特征分析表明,该菌株能发酵葡萄糖、乳糖,不能发酵阿拉伯糖、鼠李糖、木糖,淀粉水解实验、明胶水解实验、硫化氢产生试验均为阴性。
2)16S rRNA的克隆及其序列分析
提取该菌株的基因组DNA,PCR扩增16S rRNA,扩增产物使用试剂盒进行纯化回收,纯化的目的产物与pMD19-T Cloning Vector进行连接,从转化成功的平板培养基上任意挑取白斑单菌落于LB液体培养基中,37℃培养箱中过夜培养,吸取1μl菌液扩增菌液PCR,菌液PCR产物用琼脂糖凝胶电泳进行鉴定,进而得到阳性克隆。提取得到的质粒使用M13ForwardPrimer,M13Reverse Primer引物进行序列分析,得到如上述的序列<210>。将测序结果提交于GenBank数据库(GenBank登录号为MH917109),BLAST进行在线序列同源性分析,比对结果显示,菌株YLBGNL-S7的16S rRNA基因序列与多株Lactobacillus plantarum的16S rRNA基因序列相似性均达到99%。从数据库获得相关种属的16S rRNA序列,建立系统发育树,进化距离的计算采用neighbor-joining method,在MEGA 6.0软件中用p-distances和Kimura-2parameter双参数法进行系统发育树的构建,选用bootstrap法评价进化树分支聚类的稳定性,重复1000次,分析结果如图1所示,YLBGNL-S7与Lactobacillus plantarum JCM13899(LC311069.1)、Lactobacillus plantarum DJ-04(KF929420.1)、Lactobacillusplantarum A16(MG754631.1)及Lactobacillus plantarum Ni344(AB601168.1)等菌株以100%的支持率聚成一簇,因此可将菌株YLBGNL-S7鉴定为植物乳杆菌Lactobacillusplantarum YLBGNL-S7。
实施例4
本实施例利用产转糖基活性β-半乳糖苷酶的菌株YLBGNL-S7发酵生产β-半乳糖苷酶的方法,具体步骤如下:
(1)种子培养
种子培养基:乳糖5-20g,蛋白胨1-10g,牛肉浸粉1-10g,酵母浸粉1-5g,K2HPO4 5g,柠檬酸氢二铵2g,乙酸钠5g,MgSO4·7H2O 0.58g,吐温-80 1-4mL,MnSO4 0.25g,蒸馏水1000mL配制,pH为6.3,并于121℃下灭菌20min;
种子培养条件:将植物乳杆菌YLBGNL-S7于30-38℃、100-250rpm的转速震荡培养10-20h活化该菌株;
(2)发酵培养
发酵培养基:乳糖5-20g,蛋白胨1-10g,牛肉浸粉1-10g,酵母浸粉1-5g,MgSO4·7H2O 0.58g,吐温-80 1-4mL,MnSO4 0.25g,蒸馏水1000mL配制,pH6.3,121℃灭菌20min;
发酵条件:植物乳杆菌YLBGNL-S7的接种量为发酵培养基的1%-10%,在30-38℃、震荡转速100~300rpm发酵培养12-24h生产β-半乳糖苷酶;
(3)发酵后处理
发酵液经冷冻离心后收集湿菌体,用pH 6.0的0.1mol/L磷酸盐缓冲液制成菌悬液,然后于0℃冰浴条件下超声破碎,超声破碎每进行3s停8s,超声功率为125W,超声破碎的总时间为15min;所得悬液即为β-半乳糖苷酶粗酶液;取50μL粗酶液与50μL 50mM pH 6.5的磷酸缓冲液配置的oNPG于一定温度下反应10min,反应结束后加入200μL 0.5M的碳酸钠溶液终止反应,静置5min后,肉眼可见显黄色,于420nm波长处测定吸光值,通过oNPG标准曲线法计算酶活大小;经检测酶活达到10-157U/mL。
实施例5
本实施例利用产转糖基活性β-半乳糖苷酶的菌株YLBGNL-S7发酵生产β-半乳糖苷酶的方法,具体步骤如下:
(1)种子培养
种子培养基:乳糖5-20g,蛋白胨1-10g,牛肉浸粉1-10g,酵母浸粉1-5g,K2HPO4 5g,柠檬酸氢二铵2g,乙酸钠5g,MgSO4·7H2O 0.58g,吐温-80 1-4mL,MnSO4 0.25g,蒸馏水1000mL配制,pH为6.3,并于121℃下灭菌20min;
种子培养条件:将植物乳杆菌YLBGNL-S7于30-38℃、100-250rpm的转速震荡培养10-20h活化该菌株;
(2)发酵培养
发酵培养基:乳糖5-20g,蛋白胨1-10g,牛肉浸粉1-10g,酵母浸粉1-5g,MgSO4·7H2O 0.58g,吐温-80 1-4mL,MnSO4 0.25g,蒸馏水1000mL配制,pH6.3,121℃灭菌20min;
发酵条件:植物乳杆菌YLBGNL-S7的接种量为发酵培养基的1%-10%,在30-38℃、震荡转速100~300rpm发酵培养12-24h生产β-半乳糖苷酶;
(3)发酵后处理
发酵液离心收集湿菌体后,用含2.5μg/mL溶菌酶、0.4%吐温-80的磷酸盐缓冲液30℃,80-100rpm震摇处理20min,所得透性化细胞悬液作为β-半乳糖苷酶粗酶液;取50μL粗酶液与50μL 50mM pH 6.5的磷酸缓冲液配置的oNPG于一定温度下反应10min,反应结束后加入200μL 0.5M的碳酸钠溶液终止反应,静置5min后,肉眼可见显黄色,于420nm波长处测定吸光值,通过oNPG标准曲线法计算酶活大小;经检测酶活达到1-20U/mL。
实施例6
本实施例提供一种用上述发酵生产β-半乳糖苷酶的方法制得的β-半乳糖苷酶粗酶液生产低聚半乳糖的方法,具体步骤如下:
取实施例4或实施例5制得的β-半乳糖苷酶粗酶液加入pH为6.0的磷酸氢二钠-柠檬酸钠缓冲液配制的30%(w/v)乳糖溶液中进行催化反应,反应条件:以干基计每克乳糖的酶用量为5-30U,反应温度为45-60℃,反应时间2-24h,得到含有低聚半乳糖的酶反应液。将转糖基反应液适度稀释,毛细管点样10μL在活化过的硅胶铝板Silica gel 60No.553(Merck公司)上点样,以正丁醇:乙醇:水=5:3:2为展层剂上行展层,层析至距离顶端1cm处,取出用吹风机吹干,然后以显色剂(苯胺一二苯胺一磷酸)喷雾,于80℃烘箱中烘烤10min,各种糖即显出不同颜色,与不同分子量的标准糖比较,结果见图3,图3中:1-2为反应时间2h;3-4为反应时间4h;5-6为反应时间8h;7-8为反应时间12h;9-10为反应时间16h;11-12为反应时间20h;13-14为反应时间24h,15为标准糖(葡萄糖、半乳糖和乳糖的混合物)。
三蒸水将反应液稀释至糖浓度为1%,用0.22μm滤膜过滤后HPLC进样分析,进样体积20μl,色谱柱为Hi-Plex Na Column 300mm×7.7mm(安捷伦),流动相为三蒸水,流速为0.3mL/min,柱温80℃,进样量20μL。结合TLC分析和HPLC分析,确定转糖基反应液中低聚半乳糖的得率达43.40%,其中转移二糖18.29%,转移三糖占12.95%,转移三糖以上的低聚糖含量占25.11%,,残余乳糖含量仅为11.48%(w/w),结果见图4,图4中,峰1为半乳糖、峰2为葡萄糖、峰3为乳糖和转移二糖、峰4为转移三糖、5-10为其他低聚半乳糖。
本发明的技术方案不限于上述具体实施例的限制,凡是根据本发明的技术方案做出的技术变形,均落入本发明的保护范围之内。
序列表
<110> 石河子大学
<120> 一种产转糖基活性β-半乳糖苷酶的菌株及用该酶生产低聚半乳糖的方法
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1483
<212> DNA
<213> 植物乳杆菌(Lactobacillus plantarum YLBGNL-S7)
<400> 1
tcaggacgaa cgctggcggc gtgcctaata catgcaagtc gaacgaactc tggtattgat 60
tggtgcttgc atcatgattt acatttgagt gagtggcgaa ctggtgagta acacgtggga 120
aacctgccca gaagcggggg ataacacctg gaaacagatg ctaataccgc ataacaactt 180
ggaccgcatg gtccgagttt gaaagatggc ttcggctatc acttttggat ggtcccgcgg 240
cgtattagct agatggtggg gtaacggctc accatggcaa tgatacgtag ccgacctgag 300
agggtaatcg gccacattgg gactgagaca cggcccaaac tcctacggga ggcagcagta 360
gggaatcttc cacaatggac gaaagtctga tggagcaacg ccgcgtgagt gaagaagggt 420
ttcggctcgt aaaactctgt tgttaaagaa gaacatatct gagagtaact gttcaggtat 480
tgacggtatt taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta 540
ggtggcaagc gttgtccgga tttattgggc gtaaagcgag cgcaggcggt tttttaagtc 600
tgatgtgaaa gccttcggct caaccgaaga agtgcatcgg gaaactggga aacttgagtg 660
cacaagagga cagtggaaac tccatgtgta gcggtgaaat gcgtagatat atggaagaac 720
accagtggcg aaggcggctg tctggtctgt aactgacgct gaggctcgaa aagtatgggt 780
agcaaacagg attagatacc ctggtagtcc ataccgtaaa cgatgaatgc taagtgttgg 840
agggtttccg cccttcagtg ctgcagctaa cgcattaagc attccgcctg gggagtacgg 900
ccgcaaggct gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt 960
ttaattcgaa gctacgcgaa gaaccttacc aggtcttgac atactatgca aatctaagag 1020
attagacgtt cccttcgggg acatggatac aggtggtgca tggttgtcgt cagctcgtgt 1080
cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tattatcagt tgccagcatt 1140
aagttgggca ctctggtgag actgccggtg acaaaccgga ggaaggtggg gatgacgtca 1200
aatcatcatg ccccttatga cctgggctac acacgtgcta caatggatgg tacaacgagt 1260
tgcgaactcg cgagagtaag ctaatctctt aaagccattc tcagttcgga ttgtaggctg 1320
caactcgcct acatgaagtc ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat 1380
acgttcccgg gccttgtaca caccgcccgt cacaccatga gagtttgtaa cacccaaact 1440
cggtggggta accttttagg aaccagccgc ctaaggttac aga 1483
Claims (5)
1.一种产转糖基活性β-半乳糖苷酶的菌株,其特征在于:其分类命名为植物乳杆菌(Lactobacillus plantarum)YLBGNL-S7,保藏在中国典型培养物保藏中心,保藏编号为:CCTCC M 2018397,保藏日期为2018年6月25日;
所述β-半乳糖苷酶的最适pH为6.0。
2.一种发酵生产β-半乳糖苷酶的方法,其特征在于,用如权利要求1所述的产转糖基活性β-半乳糖苷酶的菌株,具体步骤如下:
(1)种子培养
种子培养基:乳糖5-20g,蛋白胨1-10g,牛肉浸粉1-10g,酵母浸粉1-5g,K2HPO45g,柠檬酸氢二铵2g,乙酸钠5g,MgSO4·7H2O 0.58g,吐温-80 1-4mL,MnSO40.25g,蒸馏水1000mL配制,pH 6.3,并于121℃下灭菌20min;
种子培养条件:将植物乳杆菌YLBGNL-S7于30-38℃、100-250rpm的转速震荡培养10-20h活化该菌株;
(2)发酵培养
发酵培养基:乳糖5-20g,蛋白胨1-10g,牛肉浸粉1-10g,酵母浸粉1-5g,MgSO4·7H2O0.58g,吐温-80 1-4mL,MnSO40.25g,蒸馏水1000mL配制,pH 6.3,121℃灭菌20min;
发酵条件:植物乳杆菌YLBGNL-S7的接种量为发酵培养基的1%-10%,在30-38℃、震荡转速100~300rpm发酵培养12-24h生产β-半乳糖苷酶;
(3)发酵后处理
发酵液经冷冻离心后收集湿菌体,用pH 6.0的0.1mol/L磷酸盐缓冲液制成菌悬液,然后于0℃冰浴条件下超声破碎,所得悬液即为β-半乳糖苷酶粗酶液,经检测酶活达到10-157U/mL。
3.一种发酵生产β-半乳糖苷酶的方法,其特征在于,用如权利要求1所述的产转糖基活性β-半乳糖苷酶的菌株,具体步骤如下:
(1)种子培养
种子培养基:乳糖5-20g,蛋白胨1-10g,牛肉浸粉1-10g,酵母浸粉1-5g,K2HPO45g,柠檬酸氢二铵2g,乙酸钠5g,MgSO4·7H2O 0.58g,吐温-80 1-4mL,MnSO40.25g,蒸馏水1000mL配制,pH 6.3,并于121℃下灭菌20min;
种子培养条件:将植物乳杆菌YLBGNL-S7于30-38℃、100-250rpm的转速震荡培养10-20h活化该菌株;
(2)发酵培养
发酵培养基:乳糖5-20g,蛋白胨1-10g,牛肉浸粉1-10g,酵母浸粉1-5g,MgSO4·7H2O0.58g,吐温-80 1-4mL,MnSO40.25g,蒸馏水1000mL配制,pH 6.3,121℃灭菌20min;
发酵条件:植物乳杆菌YLBGNL-S7的接种量为发酵培养基的1%-10%,在30-38℃、震荡转速100~300rpm发酵培养12-24h生产β-半乳糖苷酶;
(3)发酵后处理
发酵液离心收集湿菌体后,用含2.5μg/mL溶菌酶、0.4%吐温-80的磷酸盐缓冲液30℃透性化处理20分钟,所得细胞悬液作为β-半乳糖苷酶粗酶液,经检测酶活达到1-20U/mL。
4.根据权利要求2所述的发酵生产β-半乳糖苷酶的方法,其特征在于:所述步骤(3)中菌悬液于0℃冰浴条件下超声破碎时,超声破碎每进行3s停8s,超声功率为125W,超声破碎的总时间为15min。
5.一种生产低聚半乳糖的方法,其特征在于,用权利要求2-4任一所述的发酵生产β-半乳糖苷酶的方法制得的β-半乳糖苷酶粗酶液,具体步骤如下:
取β-半乳糖苷酶粗酶液加入pH为6.0的磷酸氢二钠-柠檬酸钠缓冲液配制的30%(w/v)乳糖溶液中进行催化反应,反应条件:以干基计每克乳糖的酶用量为5-30U,反应温度为45-60℃,反应时间2-24h,得到含有低聚半乳糖的酶反应液。
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