STABILITY AND PROPERTIES OF A THERMOSTABLE β-GALACTOSIDASE IMMOBILIZED ON CHTTIN

Thermostable β‐galactosidase from an E. coli transformant containing the enzyme gene from P. woesei was immobilized at pH 4.0 and a glutaraldehyde concentration of 10 mM on chitin isolated from shrimp Crangon crangon shells. These preparations had a specific activity of 43,000 U/g of chitin at 85C using ONPG as substrate. The optimum pH and temperature for immobilized β‐galactosidase activity were 5.2 and 93C. Immobilization shifts the optimum pH for the activity of the enzyme by 0.2 units towards the acid side. The immobilized enzyme is stable at temperatures close to the optimal value, and the residual activity for ONPG hydrolysis of the preparations incubated 5 h in 0.1 M phosphate citrate buffer (pH 5.4) at 90C and 100C was 70% and 40% of the initial value, respectively.     

ISOLATION AND SOME PROPERTIES OF THE THERMOSTABLE β-GALACTOSIDASE OF PYROCOCCUS WOESEI EXPRESSED IN ESCHERICHIA COLI

The enzyme with β-galactosidase activity from E. coli BL21(DE3) transformant containing the gene encoding enzyme from Pyrococcus woesei (DSM 3773) was isolated using cell extraction in 0.01 M phosphate buffer (pH 7.2), protein thermopredpitation at 85C, precipitation at acetone/extract ratio of 1:1 (v/v) and gel filtration on Sephadex G-200. The increase in the enzyme specific activity was determined using ONPG as substrate. The activity increased from 2.9 × 10³ U/mg protein to 37 × 10³ U/mg. Thermoprecipitation removed 78% of E. coli protein and retained 92% of the cell extract activity. The acetone precipitation and gel filtration applied in the next purification steps led to homogeneous enzyme with specific activity of 37,700 U/mg protein. The isolated enzyme had a half-life of 23 h and 9 h during incubation at 85C and 100C, respectively, in 0.1 M citrate-phosphate buffer (pH 5.4).