产低温β-半乳糖苷酶菌株的筛选及其酶学性质研究

Screening and Enzyme Properties of Psychrotrophic Bacterium Producing Cold-active β-galactosidase

从新疆高寒冻土、冷冻乳制品及生乳中分离到101株耐冷菌,利用低温β-半乳糖苷酶筛选模型,获得1株低温酶高产菌株L2004,根据其形态特征、生理生化特性,鉴定为短芽孢杆菌属(BrevibacillusShida,1996)。在以乳糖为主要碳源的发酵培养基中,L2004菌最适生长温度和最适产酶温度分别为20、15℃。该低温酶的最适pH值为6.5,最适作用温度为33℃,0℃相对酶活为总酶活的25%,在0～0℃范围内,具有较好的稳定性。不同金属离子对β-半乳糖苷酶活性的影响为:Mn2+>Mg2+>K+>Na+>Ca2+>Fe2+>Hg2+>Cu2+>Zn2+。Mn2+、Mg2+增强酶活,而Hg2+、Cu2+、Zn2+抑制酶活。该酶Km值为5.29mmol/L,具低温酶特性。

A strain L2004 was obtained by screening model of cold-active β -galactosidase from 101 strains isolated from the sample of permafrost, refrigeration dairy products under 4 ℃. Based on morphological, physiology and biochemistry properties, the strain 12004 was identified as belonging to the genus Brevibacillus. The growth characteristics of the psychrotrophic bacterium L 2004 were studied, and the results showed that the optimal temperature for growth and enzyme production of the strain is about 20 ℃ and 15 ℃ respectively. The maximum enzyme activity occurred at pH 9 for L2004, and the optimal temperature of β-galactosidase action was about 330Cwith ONPG as the substrate. β-galactosidase activity under 0 ℃ exhibited nearly 25% of the maximum activity, stimulated by Mg^2＋ and Mn^2＋, inhibited by Zn^2＋, Cu^2＋ and Hg^2＋. Moreover, K^＋ of the cold-active β - galactosidase activity for ONPG from strain L2004 is of found to be 52.9 mmol/L at 30 ℃Therefore the cold-active β- galactosidase of BrevibaciUus sp. L2004 has particular advantage for producing lactose-reduced dairy at low temperature. The strain L2004 gives a maximum β-galactosidase activity of 80 U/ml in shaking flask after 48 h cultivation at 20 ℃ and 200 r/min when growing in a medium containing tryptone 37.5 g/L, soya peptone 5 g/L, lactose 6 g/L, yeast extract 4.5 g/L, KH2PO4 2.5 g/L and NaC17.5 g/L.