UNESCO Chinese Center of Marine Biotechnology and Institute of Marine Biodiversity and Evolution, Ocean University of China, Yushan Road, No. 5, Qingdao 266003, China.
Appl Biochem Biotechnol. 2012 Feb;166(3):599-611. doi: 10.1007/s12010-011-9451-4. Epub 2011 Nov 16.
In order to isolate β-galactosidase overproducers of the psychrotolerant yeast Guehomyces pullulans 17-1, its cells were mutated by using nitrosoguanidine (NTG). One mutant (NTG-133) with enhanced β-galactosidase production was obtained. The mutant grown in the production medium with 30.0 g/l lactose and 2.0 g/l glucose could produce more β-galactosidase than the same mutant grown in the production medium with only 30.0 g/l lactose while β-galactosidase production by its wild type was sensitive to the presence of glucose in the medium. It was found that 40.0 g/l of the whey powder was the most suitable for β-galactosidase production by the mutant. After optimization of the medium and cultivation conditions, the mutant could produce 29.2 U/ml of total β-galactosidase activity within 132 h at the flask level while the mutant could produce 48.1 U/ml of total β-galactosidase activity within 144 h in 2-l fermentor. Over 77.1% of lactose in the whey powder (5.0% w/v) was hydrolyzed in the presence of the β-galactosidase activity of 280 U/g of lactose within 9 h while over 77.0% of lactose in the whey was hydrolyzed in the presence of β-galactosidase activity of 280 U/g of lactose within 6 h. This was the first time to show that the β-galactosidase produced by the psychrotolerant yeast could be used for hydrolysis of lactose in the whey powder and whey.
为了分离耐冷酵母 Pullulan 17-1 的β-半乳糖苷酶过表达菌,使用亚硝胍(NTG)对其细胞进行诱变。获得了一个产β-半乳糖苷酶能力增强的突变体(NTG-133)。该突变体在含有 30.0 g/l 乳糖和 2.0 g/l 葡萄糖的生产培养基中生长时,比在仅含有 30.0 g/l 乳糖的生产培养基中生长时能产生更多的β-半乳糖苷酶,而其野生型的β-半乳糖苷酶生产对培养基中葡萄糖的存在敏感。结果发现,40.0 g/l 的乳清粉是该突变体生产β-半乳糖苷酶的最适条件。优化培养基和培养条件后,该突变体在摇瓶水平可在 132 h 内产生 29.2 U/ml 的总β-半乳糖苷酶活性,而在 2-L 发酵罐中可在 144 h 内产生 48.1 U/ml 的总β-半乳糖苷酶活性。在 280 U/g 乳糖的β-半乳糖苷酶活性存在下,乳清粉(5.0% w/v)中超过 77.1%的乳糖在 9 h 内被水解,在 280 U/g 乳糖的β-半乳糖苷酶活性存在下,乳清中超过 77.0%的乳糖在 6 h 内被水解。这是首次表明耐冷酵母产生的β-半乳糖苷酶可用于水解乳清粉和乳清中的乳糖。