Synthesis and secretion of a Bacillus circulans WL-12 1,3-1,4-beta-D-glucanase in Escherichia coli

Abstract

The synthesis and secretion of a 1,3-1,4-,B-D-glucanase were studied in different strains of Escherichia coli transformed with plasmids carrying the BaciUus circulans WL-12 1,3-1,4-j3-D-glucanase structural gene. This gene (named BGC) is contained within a 1.9-kilobase BamHI-HindM fragment and directs the synthesis in E. coli of an enzyme that specifically degrades lichenan. Only one active form of the enzyme was found when the gene was expressed in different E. coli strains. The electrophoretic pattern of this protein showed a molecular weight that was approximately the same as that of the mature 0-glucanase secreted from B. circulans suggesting that the processing of this protein may be similar in both species. As deduced from maxicell experiments, the Bacilus parental promoter directs the synthesis in E. coli. Pulse-chase experiments showed that the protein may be cotranslationafly processed.Species of the genus Bacillus synthesize a broad variety of extracellular hydrolytic enzymes with different activities, some of which have been extensively studied in view of their commercial importance (28). The advent of genetic engineering technology has focused interest on the production of such enzymes. On one hand, the characterization of the genetic systems controlling their synthesis may represent a crucial step for increasing their yield for industrial purposes. On the other hand, knowledge of the mechanisms of secretion of these enzymes may contribute to a much better understanding of the phenomenon of protein export in gram-positive bacteria in general and the use of bacilli as hosts for the secretion and efficient synthesis of foreign proteins in particular.Exocellular 1-glucanases are produced by many members of the Bacillaceae family. Among them, some act asglucan-endohydrolases, hydrolyzing mixed-linked 1-glucans containing 1,3-1-and 1,4-,B-linkages, such as lichenan and barley 1-glucan. The fact that these bacterial enzymes possess a substrate range similar to that of the lichenase of germinating barley has increased their potential value for the brewing industry as substitutes and supplements of malt enzymes to hydrolyze barley ,B-glucans during the brewing process. Genes coding for this kind of carbohydrolase have been cloned from different species'of'Bacillus such as B. subtilis and B. amyloliquefaciens; some of these genes have also been sequenced.Bacillus circulans WL-12 is a gram-positive microorganism isolated by Tanaka and Phaff (36) from soil samples and characterized by its ability to lyse a number of fungal and yeast cell walls. This organism synthesizes a number of carbohydrolases such as a-glucanases, chitinases, 1-xylanases, and 1-glucanases (13). Among the latter, 1,3-1-glucanases and 1,6-13-glucanases have been characterized (7,35 We have detected in culture supernatants of this microorganism an endo-1,3-1,4-,B-glucanase, which is not produced until the stationary phase of growth. In the present work we describe the cloning and expression of this B. circulans 1-glucanase gene in Escheri...

Mutational Analysis of the Major Loop of Bacillus1,3-1,4-β-d-Glucan 4-Glucanohydrolases

“…The enzyme has a strict cleavage specificity for ␤-1,4-glycosidic bonds in 3-O-substituted glucopyranose units (1,2). Genes encoding bacterial 1,3-1,4-␤-glucanases have been cloned and sequenced from different Bacillus species (3)(4)(5)(6)(7)(8)(9), Fibrobacter succinogenes (10), Ruminococcus flavofaciens (11), and Clostridium thermocellum (12). Together with 1,3-␤-glucanases ("laminarinases"), all bacterial 1,3-1,4-␤-glucanases share a high degree of sequence similarity and have been classified as members of family 16 of glycosylhydrolases (13,14).…”

Crystal structure of Bacillus licheniformis 1,3‐1,4‐β‐d‐glucan 4‐glucanohydrolase at 1.8 Å resolution

“…3.2.1.73) from different Bacillus strains have been cloned and sequenced [1][2][3][4][5][6][7]. These enzymes cleave fl-1,4-glycosidic bonds that are adjacent tofl-l,3-glycosidic bonds in mixed linked glucans.…”

Individual amino acids in the N‐terminal loop region determine the thermostability and unfolding characteristics of bacterial glucanases

“…These enzymes were found in several bacterid strains and in plants (Murphy et al, 1984;Fincher et al, 1986;Hofemeister et al, 1986;Boniss et d., 1990;Bueno et al, 1990;Gosalbes et al, 1991;Lloberas et al, 1991;Louw et al, 1993).…”