Construction and characterization of chimeric chitinase by homologous recombination

Abstract

Nowadays, chitinases play important roles in chitooligosaccharides production which are applied and used in many fields. Chitinase 60 from Serratia sp. TU09 (Chi60), Chitinases 66 from Bacillus licheniformis SK-1 (Chi66) and Chitinase B from Serratia marcescens (ChiB) which belong to family 18 of glycosylhydrolase have been intensively studied. In this work, chimeric chitinase that recombined Chi60 and Chi66 with ChiB have been constructed via in vivo homologous recombination. HRCD66B, chimeras that were constructed from recombination between CatDChi66 and ChiB, are screened by LB agar plate containing ampicillin and CCMM broth containing ampicillin. Six chimeras with chimeric chitinase genes were observed and only 4 of them with gene size of 1.67, 1.68, 1.34 and 1.76 kb have in frame recombination and could be translated to amino acid sequences. HRCHI60B, chimeras constructed from recombination between Chi60 and ChiB were also screened by the same method as HRCD66B screening. Eighty seven chimeras were observed and have been grouped into 5 groups based on chimeric gene size. Only 3 of them with gene size of 1.49, 1.55 and 2.0 kb have in frame recombination and could be translated to amino acid sequences. Homologous recombination is mostly found to occur at conserved region encoding for active site motif of family 18 chitinases. These chimeras were expressed and assayed for chitinolytic activity on colloidal chitin, PNAC and chitooligosaccharides but no activity was observed.