Cloning of the Bacillus subtilis DLG beta-1,4-glucanase gene and its expression in Escherichia coli and B. subtilis

Abstract

The gene encoding beta-1,4-glucanase in Bacillus subtilis DLG was cloned into both Escherichia coli C600SF8 and B. subtilis PSL1, which does not naturally produce beta-1,4-glucanase, with the shuttle vector pPL1202. This enzyme is capable of degrading both carboxymethyl cellulose and trinitrophenyl carboxymethyl cellulose, but not more crystalline cellulosic substrates (L. M. Robson and G. H. Chambliss, Appl. Environ. Microbiol. 47:1039-1046, 1984). The beta-1,4-glucanase gene was localized to a 2-kilobase (kb) EcoRI-HindIII fragment contained within a 3-kb EcoRI chromosomal DNA fragment of B. subtilis DLG. Recombinant plasmids pLG4000, pLG4001a, pLG4001b, and pLG4002, carrying this 2-kb DNA fragment, were stably maintained in both hosts, and the beta-1,4-glucanase gene was expressed in both. The 3-kb EcoRI fragment apparently contained the beta-1,4-glucanase gene promoter, since transformed strains of B. subtilis PSL1 produced the enzyme in the same temporal fashion as the natural host B. subtilis DLG. B. subtilis DLG produced a 35,200-dalton exocellular beta-1,4-glucanase; intracellular beta-1,4-glucanase was undetectable. E. coli C600SF8 transformants carrying any of the four recombinant plasmids produced two active forms of beta-1,4-glucanase, an intracellular form (51,000 +/- 900 daltons) and a cell-associated form (39,000 +/- 400 daltons). Free exocellular enzyme was negligible. In contrast, B. subtilis PSL1 transformed with recombinant plasmid pLG4001b produced three distinct sizes of active exocellular beta-1,4-glucanase: approximately 36,000, approximately 35,200, and approximately 33,500 daltons. Additionally, B. subtilis PSL1(pLG4001b) transformants contained a small amount (5% or less) of active intracellular beta-1,4-glucanase of three distinct sizes: approximately 50,500, approximately 38,500 and approximately 36,000 daltons. The largest form of beta-1,4-glucanase seen in both transformants may be the primary, unprocessed translation product of the gene.

Cloning, sequencing and analysis of expression of a Butyrivibrio fibrisolvens gene encoding a -glucosidase

“…The cloning vector pEBl is an E. coli/Bacillus subtilis shuttle vector with positive selection for inserted DNA fragments in E. coli. pEBl was constructed by replacing the pBR322 fragment of pLPl202 (Robson & Chambliss, 1986;Ostroff & Pene, 1983) with pEcoR252, which is a derivative of pEcoR251 lacking the PstI site in the p-lactamase gene (P. Janssen, personal communication). The structure of pEcoR251 (a gift from M. Zabeau, Plant Genetic Systems, Gent, Belgium) is similar to other plasmids utilizing inactivation of the EcoRI endonuclease gene as a selective marker (e.g.…”

Cellulosimicrobium cellulans’dan β-1,3-Glukanaz Geninin Escherichia coli DH5α’da Klonlanması ve Ekspresyonu

“…was cloned and expressed in E. coli DH5α and the enzyme was produced intracellularly by the transformant 39 . Similarly, the Bacillus subtilis β-1,4-glucanase gene (a similar enzyme hydrolyzing β-1,4-linkages present in noncrystalline cellulosic substrates such as carboxymethyl cellulose and trinitrophenyl carboxymethyl cellulose) was cloned in E. coli expressing the intracellularly 40 . These reported results are supporting our thesis.…”

Cloning of two genes from Bacillus circulans WL-12 which encode 1,3- -glucanase activity

“…The molecular cloning and expression in E. coli of 1,3/1,4-pglucanases from both B . subtilis (Cantwell & McConnell, 1983;Robson & Chambliss, 1986) and B. amyloliqueJiciens (Borriss et al, 1985) have been described.…”