产β-D-半乳糖苷酶马克斯克鲁维酵母的分离鉴定及其产酶特性

Isolation, Identification and Enzymatic Characteristics of Kluyveromyces marxianus Producing β-D-Galactosidase

从藏灵菇中分离筛选到的一株高产β-D-半乳糖苷酶菌株ZX-5,经ITS DNA序列分析,鉴定为马克斯克鲁维酵母。对产酶培养基的最佳碳源和氮源优化结果为半乳糖2.0%,胰蛋白胨1.0%;产酶优化条件:温度30℃,培养基初始pH 6.5,装液量30%,转速100 r/min,接种量2.0%,发酵36 h。粗酶液酶活力为2.60 U/mL;经硫酸铵分级沉淀和DEAE离子交换层析,获得纯化酶的比活力为157.35 U/mg。酶最适反应温度35℃,最适pH 6.0,在20~40℃和pH 5.0~7.0的范围内酶的稳定性较好;Mn2+对酶的活性有促进作用。利用菌株ZX-5β-D-半乳糖苷酶分解乳糖并合成低聚半乳糖(galacto-oligosaccharide,GOS),在35℃、乳糖质量浓度60 g/100 mL、酶浓度1.0 U/mL条件下,乳糖水解率达68.34%(50 h),GOS产率达34.70%(40 h),具有潜在的应用前景。

A strain,named ZX-5,with the ability to produce a high yield ofβ-D-galactosidase was isolated from traditional Tibetan kefir,and identified as Kluyveromyces marxianus by DNA internal transcribed spacer(ITS)sequence analysis.We found 2.0%galactose and 1.0%tryptone to be the optimal carbon and nitrogen sources,respectively.The optimal fermentation conditions were determined as follows:temperature 30℃,initial medium pH 6.5,medium volume 30%,shaking speed 100 r/min,inoculum size 2.0%and fermentation time 36 h.The activity ofβ-D-galactosidase produced under these conditions was 2.60 U/mL.Furthermore,the crude enzyme solution was purified by ammonium sulfate fractionation and DEAE-Sepharose fast flow ion-exchange chromatography to a specific activity of 157.35 U/mg.The purifiedβ-D-galactosidase exhibited the maximum catalytic activity at 35℃and pH 6.0,and had good stability at 2040℃and pH 5.07.0.Mn 2+could promote the enzyme activity.When theβ-D-galactosidase produced by strain ZX-5 was used to decompose lactose and produce galacto-oligosaccharide under the conditions of 35℃,lactose concentration of 60 g/100 mL and enzyme concentration of 1.0 U/mL,the hydrolysis rate of lactose was 68.34%(after 50 h)and the yield of galacto-oligosaccharide was 34.70%(after 40 h),showing good potential application prospects.