Cloning and Characterization of a Cold-Adapted Endo-1,5-α-L-arabinanase from Paenibacillus polymyxa and Rational Design for Acidic Applicability

摘要

AbnZl, with optimal pH of 6.0 and optimal temperature of 40 °C, is a cold-adapted endo-1,5-α-L-arabinanase encoded by the gene abnZ1 from Paenibacillus polymyxa 2.6. The specific activity of AbnZl remained 54.1% of maximum at 5 °C. To apply AbnZ1 in acidic conditions, three basic hsitidine (His) residues, His~(48), His~(218), and His~(297), around the catalytic domain were selected as mutation sites, which were replaced with Asp, Glu, Arg, and Lys, respectively, to yield 12 mutants, H48D/E/R/ K, H218D/E/R/K, and H297D/E/R/K. The optimum pH of mutant H218D shifted toward the acidic direction by 0.S unit, and the relative activity was enhanced from 20.4 to 55.7% at pH 5.0. Furthermore, the specific activity of H218D in optimal conditions was 82.6 U/mg versus that of wild type, 73.4 U/mg, and the K_m decreased from 11.9 to 7.1 mg/mL. This work provided an arabinanase candidate for juice clarification and pectin extraction.