摘要
通过水解活性和转糖基活性筛选,从实验室97株保存菌种中获得1株具有转糖基活性的β-半乳糖苷酶产生菌,克隆并序列分析了该菌株16S rDNA基因片断,GenBank收录号为DQ267829.综合其形态学特征、生理生化特征及16S rDNA序列同源性分析结果,将其鉴定为巨大芽孢杆菌(Bacillus megaterium)2-37-4-1.确定了该菌株β-半乳糖苷酶产酶培养基的碳源为乳糖1%,氮源为蛋白胨0.5%和酵母膏0.5%,培养条件为37℃摇床培养18 h;碳源实验证明,该菌株β-半乳糖苷酶产生为乳糖诱导型.利用薄层层析技术研究了pH值、乳糖底物浓度、反应温度和反应时间对该菌株β-半乳糖苷酶以乳糖为底物转糖基合成低聚半乳糖的影响,确定最适反应条件为pH7.5、50 mmol/L(磷酸缓冲液)配制的40%乳糖溶液,55℃反应24 h.转糖基反应产物高压液相色谱分析其组成为低聚半乳糖25.68%,双糖(包括乳糖和转移二糖)33.02%,葡萄糖26.37%和半乳糖14.92%.
One bacterium strain producing β-galactosidase with transgalactosylation activity was screened from 97 strains of microorganisms stored in our laboratory using hydrolysis activity on o-nitrophenyl-β-D-galactopyranoside followed by transgalactosylation activity on lactose. Phenotypic analyses including morphology and physiology characteristics and 16S rDNA sequence analyses were carried out. The GenBank accession number of the 16S rDNA was DQ267829, By making a comprehensive view on all the results of taxonomy, the strain was identified as Bacillus megaterium 2-37-4-1. The growth condition for production of β-galactosidare of B. megaterium 2-37-4-1 was investigated. The optimal carbon source and nitrogen source were 1% lactose and 0.5 % peptone plus 0.5% yeast extract, respectively. High yield of the enzyme was obtained at 37℃ for 18 h. Effects of pH, the concentration of lactose, reaction temperature and time on the enzyme-catalyzed transgalactosylation activity were studied and the reaction products were analyzed by thin layer chromatography （TLC） and high-performance liquid chromatography （HPLC）. In 50mM phosphate buffer （pH 7.5） containing 40% lactose, reacted at 55~C with shaking for 24 h, the transgalactosylation product was composed of 25.68% galacto-oligosaccharides （GOS）, 33.02% disaccharides including lactose and transgalctosylated disaccharides, 26.37% glucose and 14.92% galactose.
出处
《山东大学学报（理学版）》
CAS
CSCD
北大核心
2006年第1期133-139,共7页
Journal of Shandong University(Natural Science)
基金
国家"十五"科技攻关计划项目(2004BA713B04-06)
