Li Yumei, Lu Lili, Wang Hongmei, Xu Xiaodong, Xiao Min
State Key Laboratory of Microbial Technology, National Glycoengineering Research Center, Shandong University, Jinan 250100, People's Republic of China.
Appl Environ Microbiol. 2009 Sep;75(18):5938-42. doi: 10.1128/AEM.00326-09. Epub 2009 Jul 17.
A novel gene encoding transglycosylating beta-galactosidase (BGase) was cloned from Penicillium expansum F3. The sequence contained a 3,036-bp open reading frame encoding a 1,011-amino-acid protein. This gene was subsequently expressed on the cell surface of Saccharomyces cerevisiae EBY-100 by galactose induction. The BGase-anchored yeast could directly utilize lactose to produce galactooligosaccharide (GOS), as well as the by-products glucose and a small quantity of galactose. The glucose was consumed by the yeast, and the galactose was used for BGase expression, thus greatly facilitating GOS synthesis. The GOS yield reached 43.64% when the recombinant yeast was cultivated in yeast nitrogen base-Casamino Acids medium containing 100 g/liter initial lactose at 25 degrees C for 5 days. The yeast cells were harvested and recycled for the next batch of GOS synthesis. During sequential operations, both oligosaccharide synthesis and BGase expression were maintained at high levels with GOS yields of over 40%, and approximately 8 U/ml of BGase was detected in each batch.
从扩展青霉F3中克隆出一个编码转糖基化β-半乳糖苷酶（BGase）的新基因。该序列包含一个3036 bp的开放阅读框，编码一个1011个氨基酸的蛋白质。随后通过半乳糖诱导，该基因在酿酒酵母EBY-100的细胞表面表达。固定有BGase的酵母能够直接利用乳糖生产低聚半乳糖（GOS），以及副产物葡萄糖和少量半乳糖。葡萄糖被酵母消耗，半乳糖用于BGase表达，从而极大地促进了GOS的合成。当重组酵母在含有100 g/升初始乳糖的酵母氮源-酪蛋白氨基酸培养基中于25℃培养5天时，GOS产量达到43.64%。收获酵母细胞并循环用于下一批GOS合成。在连续操作过程中，寡糖合成和BGase表达均保持在高水平，GOS产量超过40%，且每批检测到约8 U/ml的BGase。
