Characterization of an extremely thermostable but cold-adaptive β-galactosidase from the hyperthermophilic archaeon Pyrococcus furiosus for use as a recombinant aggregation for batch lactose degradation at high temperature.
β-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 β-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-β-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 β-galactosidase and β-glucosidase activity. The active inclusion bodies of β-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 β-galactosidase may be suitable for the hydrolysis of lactose in milk processing.
Hubei Biopesticide Engineering Research Centre, 8 Nanhu Avenue, Wuhan 430064, China. Electronic address: dq081@163.com.
Qing
Q
Dong
College of Life Sciences, Wuhan University, Luo-jia-shan, Wuhan 430072, China.
Xufan
X
Yan
Hubei Biopesticide Engineering Research Centre, 8 Nanhu Avenue, Wuhan 430064, China.
Minhui
M
Zheng
Hubei Biopesticide Engineering Research Centre, 8 Nanhu Avenue, Wuhan 430064, China.
Ziwen
Z
Yang
D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry* / Q000302:isolation & purification; D004795:Enzyme Stability; D004926:Escherichia coli; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007785:Lactose / Q000737:chemistry*; D008892:Milk / Q000737:chemistry / Q000378:metabolism; D009598:Nitrophenylgalactosides / Q000737:chemistry; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D001616:beta-Galactosidase / Q000737:chemistry* / Q000302:isolation & purification
D019843:Archaeal Proteins; C055012:2-nitrophenylgalactoside; D009598:Nitrophenylgalactosides; D001616:beta-Galactosidase; D007785:Lactose
Cold-adaptive enzyme; Inclusion body; Lactase; Pyrococcus furiosus; Thermophilic enzyme; β-Galactosidase