Bifidobacterium bifidum ATCC 29521 β-半乳糖苷酶的生物信息学分析、异源表达及其酶学性质
TS201.3; 本研究旨在为低聚半乳糖(galactooligosaccharide,GOS)的合成提供新的双歧杆菌酶源,对Bifidobacterium bifidum ATCC 29521的β-半乳糖苷酶(β-galATCC29521)进行生物信息学分析,并在Escherichia coli BL21(DE3)中实现异源表达.研究β-galATCC29521的异源表达条件和发酵优化条件、酶学性质及其在乳清体系合成GOS的应用.生物信息学分析表明β-galATCC29521属于糖苷水解酶家族2,不存在信号肽和跨膜区.发酵优化实验结果表明,E.coli BL21(DE3)-pET28a-β-...
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| Published in | 食品科学 Vol. 46; no. 6; pp. 98 - 107 |
|---|---|
| Main Authors | , , |
| Format | Magazine Article |
| Language | Chinese |
| Published |
江南大学食品学院,江苏 无锡 214122%江南大学食品学院,江苏 无锡 214122
2025
苏州朗邦营养科技有限公司,江苏 苏州 215011 |
| Subjects | |
| Online Access | Get full text |
| ISSN | 1002-6630 |
| DOI | 10.7506/spkx1002-6630-20240912-098 |
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| Abstract | TS201.3; 本研究旨在为低聚半乳糖(galactooligosaccharide,GOS)的合成提供新的双歧杆菌酶源,对Bifidobacterium bifidum ATCC 29521的β-半乳糖苷酶(β-galATCC29521)进行生物信息学分析,并在Escherichia coli BL21(DE3)中实现异源表达.研究β-galATCC29521的异源表达条件和发酵优化条件、酶学性质及其在乳清体系合成GOS的应用.生物信息学分析表明β-galATCC29521属于糖苷水解酶家族2,不存在信号肽和跨膜区.发酵优化实验结果表明,E.coli BL21(DE3)-pET28a-β-galATCC29521重组菌在含有10 g/L蔗糖、12 g/L胰蛋白胨、24 g/L酵母粉的培养基中,且0.05 mmol/L的异丙基-β-D-硫代半乳糖苷在27℃诱导培养16h后,酶活力可达318.03 U/mL.粗酶液经过镍柱亲和层析得到电泳纯蛋白,其最适pH值和最适温度分别为7.5和40℃.β-galATCC29521纯化酶在温度10～30℃和pH 6.5～9.0范围内表现出良好的稳定性.Mn2+和Mg2+可显著提高酶活性,Cu2+和Fe3+则显著抑制酶活性.以乳清为底物合成GOS时,该酶的产率为29.83%.研究结果表明β-galATCC29521在GOS的制备领域中具有潜在的应用价值. |
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| AbstractList | TS201.3; 本研究旨在为低聚半乳糖(galactooligosaccharide,GOS)的合成提供新的双歧杆菌酶源,对Bifidobacterium bifidum ATCC 29521的β-半乳糖苷酶(β-galATCC29521)进行生物信息学分析,并在Escherichia coli BL21(DE3)中实现异源表达.研究β-galATCC29521的异源表达条件和发酵优化条件、酶学性质及其在乳清体系合成GOS的应用.生物信息学分析表明β-galATCC29521属于糖苷水解酶家族2,不存在信号肽和跨膜区.发酵优化实验结果表明,E.coli BL21(DE3)-pET28a-β-galATCC29521重组菌在含有10 g/L蔗糖、12 g/L胰蛋白胨、24 g/L酵母粉的培养基中,且0.05 mmol/L的异丙基-β-D-硫代半乳糖苷在27℃诱导培养16h后,酶活力可达318.03 U/mL.粗酶液经过镍柱亲和层析得到电泳纯蛋白,其最适pH值和最适温度分别为7.5和40℃.β-galATCC29521纯化酶在温度10～30℃和pH 6.5～9.0范围内表现出良好的稳定性.Mn2+和Mg2+可显著提高酶活性,Cu2+和Fe3+则显著抑制酶活性.以乳清为底物合成GOS时,该酶的产率为29.83%.研究结果表明β-galATCC29521在GOS的制备领域中具有潜在的应用价值. |
| Abstract_FL | This study introduced a new source of bifidobacterial enzyme for the synthesis of galactooligosaccharide(GOS).We investigated the bioinformatics analysis,heterologous expression,enzymatic properties and application in the synthesis of GOS of a newly identified β-galactosidase(β-galATCC29521)derived from Bifidobacterium bifidum ATCC 29521.Bioinformatic analysis showed that β-galATCC29521 belonged to the glycoside hydrolase family 2,without a signal peptide or a transmembrane region.The optimal medium for β-galATCC29521 expression was composed of 10 g/L sucrose,12 g/L tryptone,and 24 g/L yeast powder,and after 16 h of culture induced by 0.05 mmol/L isopropyl-β-D-thiogalactopyranoside(IPTG)at 27℃,the activity of β-galATCC29521 was 318.03 U/mL.The crude enzyme was purified to electrophoretic homogeneity through Ni-IDA affinity chromatography.The optimal pH and temperature for β-galATCC29521 were 7.5 and 40℃,respectively.The purified enzyme was stable within the pH range of 6.5-9.0 and the temperature range of 10-30℃.Its activity was significantly enhanced in the presence of Mn2+and Mg2+but inhibited in the presence of Cu2+and Fe3+.When using whey as the substrate,the yield of GOS synthesized by β-galATCC29521 was 29.83%.The results indicated that β-galATCC29521 holds potential in the synthesis of GOS. |
| Author | 符长春 杨瑞金 仝艳军 |
| AuthorAffiliation | 江南大学食品学院,江苏 无锡 214122%江南大学食品学院,江苏 无锡 214122;苏州朗邦营养科技有限公司,江苏 苏州 215011 |
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| Author_FL | YANG Ruijin FU Changchun TONG Yanjun |
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| DocumentTitle_FL | Bioinformatics Analysis,Heterologous Expression and Enzymatic Properties of β-Galactosidase from Bifidobacterium bifidum ATCC 29521 |
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| Keywords | enzymatic properties β-半乳糖苷酶 β-galactosidase Bifidobacterium bifidum ATCC 29521 酶学性质 低聚半乳糖 发酵优化 galactooligosaccharide fermentation optimization |
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| Publisher | 江南大学食品学院,江苏 无锡 214122%江南大学食品学院,江苏 无锡 214122 苏州朗邦营养科技有限公司,江苏 苏州 215011 |
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| Snippet | TS201.3; 本研究旨在为低聚半乳糖(galactooligosaccharide,GOS)的合成提供新的双歧杆菌酶源,对Bifidobacterium bifidum ATCC 29521的β-半乳糖苷酶(β-galATCC29521)进行生... |
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| Title | Bifidobacterium bifidum ATCC 29521 β-半乳糖苷酶的生物信息学分析、异源表达及其酶学性质 |
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