转糖基β-半乳糖苷酶产生菌Enterobacter agglomerans B1:筛选鉴定、发酵产酶及其合成低聚半乳糖的研究  

Enterobacter agglomerans B1 producing β-galactosidase with transglycosylation activity:screening,identification,fermentation conditions,and galacto-oligosaccharides synthesis

作　　者：卢丽丽[1] 肖敏[1] 徐晓东[1] 

机构地区：[1]山东大学微生物技术国家重点实验室,山东大学国家糖工程技术研究中心,济南250100

出　　处：《微生物学报》2008年第1期38-44,共7页Acta Microbiologica Sinica

基　　金：国家"十五"科技攻关计划项目(2004BA713B04-06);国家"863计划"(2006AA10Z338)~~

摘　　要：从土壤中筛选获得一株具有转糖基活性的β-半乳糖苷酶产生菌,综合其形态学特征、生理生化特征及16SrDNA序列同源分析结果,将其鉴定为成团肠杆菌(Enterobacter agglomerans)B1。通过单因子试验和正交试验,对B1菌株产转糖基β-半乳糖苷酶的培养条件进行了优化。最佳培养基主要组份为:乳糖1%,酵母粉1%,蛋白胨0.5%;发酵条件为:初始pH7.5,发酵温度25℃,发酵时间26h。在该培养条件下产酶量为9.7U/mL。利用薄层层析技术研究了pH、温度、底物浓度和反应时间对该菌株全细胞以乳糖为底物生成低聚半乳糖的影响,确定最适反应条件为:pH7.5缓冲液配制的30%乳糖溶液;50℃反应12h。最优化反应的转糖基产物经HPLC、TLC和MS分析,确定低聚半乳糖产量为40.7%,组分为转移二糖、三糖和四糖。Galacto-oligosaccharides （GOS） are promising non-digestible oligosaccharides recognized as prebiotics. Commercial GOS containing galactose as subunit, are synthesized from lactose using the galactosyl-transferase activity of β-galactosidase. A strain producing β-galactosidase with transglycosylation activity was screened from the soil. Phenotypic analysis including morphology and physiology characteristics and 16S rDNA sequence analysis were carried out. Based on taxonomy results, the strain was identified as Enterobacter agglomerans B1. Medium and fermentation conditions were optimized by single factor and orthogonal experiments. The enzyme reached 9.7 U/mL in the medium （pH 7.5） containing 1% lactose, 1% yeast extract, and 0.5% peptone when cultured at 25℃ for 26 h. Effects of pH, temperature, lactose concentration, and reaction time on transgalactosylation by whole cells were studied. Yield of GOS reached 40.7% in 30% lactose （pH 7.5） at 50℃ for 12 h, as analyzed by HPLC and TLC. The results of an MS analysis showed that GOS were composed of di, tri-, and tetrasaccharides.

关 键 词：成团肠杆菌 鉴定 转糖基β-半乳糖苷酶 发酵条件 低聚半乳糖 

分 类 号：Q93[生物学—微生物学]