GENETIC IMPROVEMENT OF CHITINASE PRODUCTION BY SERRATIA MARCESCENS

Abstract

DNA libraries from Serratia marcescens QMB1466 were constructed in the plasmid pBR322 and the lambda vector Charon 4. The libraries were screened for endochitinase and chitobiase gene products. Endochitinase activity was to be detected as zones of clearing on chit in-impregnated agar plates. Chitobiase activity was detected by the hydrolysis of the chitobiose analogue p-nitrophenyl-N-acetyl-β-D-glucosaminide (NPGlu) by colonies or plaques. Direct screening for endochitinase activity was unsuccessful. One recombinant plasmid (pNPG) conferred activity against NPGlu on Escherichia coli. The activity was located in the periplasmic fraction, but this fraction did not hydrolyze chitobiose. Therefore, the cloned gene was a β-N-acetylhexosaminidase and not a chitobiase gene. Thirty recombinant phage which hydrolyzed NPGlu were isolated. Preliminary results of assays of phage lysates indicated that 9 of the recombinant phage had chitobiase and endochitinase activity, 4 had only chitobiase activity, 3 had only endochitinase activity, and 14 lacked both activities. Restriction analysis revealed that pNPG and all the chitobiase negative phage contained identical 2.6 and 1.3 kilobase EcoRI/SalI fragments. All chitobiase positive phage contained identical 1.8 and 1.2 kilobase EcoRI/SalI fragments. These results indicate that chitobiase, endochitinase, and β-N-acetylglucosaminidase genes have been cloned and that the chitobiase and endochitinase genes are linked.