Characterization of an extremely thermostable but cold-adaptive 尾-galactosidase from the hyperthermophilic archaeon Pyrococcus furiosus for use as a recombinant aggregation for batch lactose degradation at high temperature

详细信息    查看全文

• 作者：Qing Dong¹ ; ^{dq081@163.com" class="auth_mail} ; Xufan Yan² ; Minhui Zheng¹ ; Ziwen Yang¹
• 关键词：Thermophilic enzyme ; Cold-adaptive enzyme ; 尾-Galactosidase ; Lactase ; Pyrococcus furiosus ; Inclusion body
• 刊名：Journal of Bioscience and Bioengineering
• 出版年：June 2014
• 年：2014
• 卷：117
• 期：6
• 页码：706-710
• 全文大小：930 K

文摘

尾-Galactosidase (lactase), which catalyzes the hydrolysis of lactose into glucose and galactose, is one of the most important enzymes used in dairy processing. In this study, a gene that encoded an extremely thermostable 尾-galactosidase from Pyrococcus furiosus (Pflactase) was cloned and expressed in Escherichia coli BL21. The recombinant enzyme was purified by heat treatment and Ni-NTA affinity chromatography. The enzyme displayed optimal activity at 90°C and pH 7.0 in phosphate buffer. The specific activity of the recombinant enzyme on o-nitrophenyl-尾-d-galactopyranoside was 10.2 U/mg at 0°C and 130.0dU/mg at 90°C. The half-lives of the enzyme were 31423.4, 8168.3, 4017.7, 547.4, 309.6, and 203.5 min at 70°C, 80°C, 85°C, 90°C, 95°C, and 100°C, respectively. The recombinant enzyme exhibited both 尾-galactosidase and 尾-glucosidase activity. The active inclusion bodies of 尾-galactosidase were easily isolated by nonionic detergent treatment and directly used for lactose conversion in a repetitive batch mode. More than 54% (90°C) or 88% (10°C) of the original enzyme activity was retained after 10 conversion cycles under optimum conditions. These results suggest that the recombinant thermostable 尾-galactosidase may be suitable for the hydrolysis of lactose in milk processing.