Secretion and processing of the Bacillus subtilis endo‐β‐1,3‐1,4‐glucanase in Escherichia coli

Abstract

The endo-beta-1,3-1,4-glucanase enzyme of Bacillus subtilis C120, when synthesized in Escherichia coli, is located mainly in the cytoplasm, but enzyme activity is also detected in the periplasmic space and in the extracellular medium. The proportion recovered in the extracellular medium is not altered by changes in the levels of synthesis of the enzyme. Lysis of E. coli cells is ruled out as the cause of the secretion by the normal localization of beta-galactosidase, an intracellular protein. However, beta-lactamase, which is normally found in the periplasmic space, is detected in the extracellular medium of E. coli transformants containing beta-glucanase plasmids, suggesting that the presence of beta-glucanase in the cell alters the permeability of the outer membrane. The beta-glucanase proteins found in the extracellular medium, the periplasmic space and the cytoplasm have the same electrophoretic mobilities as the secreted enzyme of B. subtilis. Amino-terminal sequencing has shown that the beta-glucanase enzyme in the intracellular fraction of E. coli is processed at a site two amino acids distant from the processing site used in B. subtilis.

Two beta-glycanase genes are clustered in Bacillus polymyxa: molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase

“…This finding suggests that the signal peptide is recognized and the protein is processed, at least in part, by E. coli cells. In the clone containing pBPL5800, xylanase together with ,B-lactamase (9) and alkaline phosphatase (33) activities were found extracellularly in a significant proportion (i.e., 35% for xylanase); isocitrate dehydrogenase (30) activity remained cytoplasmic in this clone, possibly because of the presence in this clone of lichenase activity that can alter the permeability of the outer membrane, as suggested for the B. subtilis lichenase (17). The lichenase activity was found in the three fractions, extracellular medium, periplasm, and cytoplasm, in similar proportions in clones harboring pBPL1006 or pBPL5800.…”

Cloning and DNA sequencing of bgaA, a gene encoding an endo-beta-1,3-1,4-glucanase, from an alkalophilic Bacillus strain (N137)

“…These enzymes, which hydrolyze (1-4)-3 linkages in 3-0-substituted ,B-D-glucanopyranosyl residues, are unable to hydrolyze repeating sequences of (-1,3or (-1,4-linked glucans such as laminarin or carboxymethylcellulose (CMC), respectively. Genes encoding these carbohydrolases have been cloned from different species of Bacillus such as B. subtilis (8), B. amyloliquefaciens (4), B. macerans (6), B. circulans (7), B. polymyxa (13), B. licheniformis (21), and B. brevis (21a), and most of these genes have been cloned and expressed in heterologous hosts such as Escherichia coli, B. subtilis, and yeasts (7,9,12,15). Several hybrid enzymes have been obtained from B. amyloliquefaciens and B. macerans endo P-(1,3)-(1,4)-glucanases by using the PCR and extension of several overlapping segments.…”

alpha-Amylase of Clostridium thermosulfurogenes EM1: nucleotide sequence of the gene, processing of the enzyme, and comparison of other alpha-amylases

“…The results indicated that the leader peptide was cleaved at exactly the same site in E. coli as in C. thermosulfurogenes EM1, between two alanine residues. It has been reported that the B. subtilis endo-3-1,3-1,4-glucanase is processed in E. coli at a site two amino acid residues distant from the processing site used in B. subtilis (6).…”

“…Aliquots of the concentrated ot-amylase fraction were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12). ox-Amylase was still active under these conditions (6) and was identified by running two parallel lanes of the gel. Proteins of one lane were stained with Serva Blue G (Serva GmbH, Heidelberg, Federal Republic of Germany).…”