Isolation Of Catechol-Produce Strain And Cloning Of Its Benzoate Dioxygenase Genes

A strain was isolated from soil for utilizing sodium benzoate to producecatechol, and identified as Pseudomonas sp. B3-1. The maximum yield ofcatechol reached 1.57 mg/ml after optimizing sunstrate contration, reactiontemperature and pH. The optimal substrate concentration was 3 mg/ml. Theoptimal temperature and pH for growth were 32℃and 6.0～7.0, respectively.Immobilized Pseudomonas sp. B3-1 cells were prepared by gelatin,sodium alginate and polyvinyl alcohol as supports, and applied for producingcatechol. Sodium alginate was Choosen for further study. The optimumimmobilized conditions were 3% sodium alginate, 4% (w/v)CaCl₂, wet cellweight 0.3g/ml, and 4h for immobilization.A DNA library of Pseudomonas sp. B3-1 containing about 20000 cloneswas constructed by cosmid vector pWEB::TNC. 14 clones were randomlychosen from the library and extracted their plasmids. The plasmids weredigested with BamHI and analyzed by agarose gel electrophoresis. The result showed that each of them harbored different foreign DNA fragments. And thelarge insert DNA fragment was 52 kb and the smallest was 31kb. The averagesize of the insert DNA fragment was 41.5 kb. The restriction analysis of 14plasmids from library revealed that the cloned DNA fragments were random andthe library had a high quanlity.45 positive clones were obtained from the library using function-basedscreening strategy of catechol. Benzoate genes benABCD were cloned frompositive clone Ben19. The ORF of gene benA was 1329 bp, which encodeda production of 442 amino acids, shared 96% identities and 99% homology toBenA of Pseudomonas entomophila str. L48 (accession No. AAX47023.1.); ThebenB harboring a 486 bp ORF, which encoded a protein of 161 amino acids,sharing 91% identities and 97% homology to BenB of Pseudomonasentomophila str. L48(GenBank accession No. AAX47024.1). The benCharboring a 1011 bp ORF, which encoded a protein of 336 amino acids,sharing 93% identities and 98% homology to BenC of Pseudomonasentomophila str. L48(GenBank accession No. AAX47025.1). The benDharboring a 762 bp ORF, which encoded a protein of 253 amino acids, sharing79% identities and 88% homology to BenC of Pseudomonas putida (GenBankaccession No. AAK52290.1.).