食品科学 ›› 2020, Vol. 41 ›› Issue (2): 158-165.doi: 10.7506/spkx1002-6630-20181120-226

• 生物工程 • 上一篇    下一篇

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

郑义,张健,曹永强,赵笑,余志坚,陈超,杨贞耐   

  1. (1.北京食品营养与人类健康高精尖创新中心 北京工商大学,北京 100048;2.东君乳业(禹城)有限公司,山东 德州 253000)
  • 出版日期:2020-01-25 发布日期:2020-01-19
  • 基金资助:
    国家重点研发计划政府间国际科技创新合作重点专项(2017YFE0131800); 2019年研究生科研能力提升计划项目(19008001616)

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

ZHENG Yi, ZHANG Jian, CAO Yongqiang, ZHAO Xiao, YU Zhijian, CHEN Chao, YANG Zhennai   

  1. (1. Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University, Beijing 100048, China; 2. Dongjun (Yucheng) Dairy Co. Ltd., Dezhou 253000, China)
  • Online:2020-01-25 Published:2020-01-19

摘要: 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-5 β-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。

关键词: 藏灵菇, β-D-半乳糖苷酶, 发酵条件, 分离纯化, 酶学性质, 低聚半乳糖

Abstract: A strain, named ZX-5, with the ability to produce a high yield of β-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 of β-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 purified β-D-galactosidase exhibited the maximum catalytic activity at 35 ℃ and pH 6.0, and had good stability at 20–40 ℃ and pH 5.0–7.0. Mn2+ could promote the enzyme activity. When the β-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.

Key words: Tibetan kefir, β-D-galactosidase, fermentation conditions, isolation and purification, enzyme characterization, galacto-oligosaccharide

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