Use of bacilli for overproduction and secretionof endo-β-1,4-glucanase and β-glucosidase encoded by cloned genes

Abstract

A substantial overproduction and secretion of endo-$\beta$-1,4-glucanase via a shuttle vector, pCK98, carrying a Bacillus subtilis gene coding for this enzyme was allowed in $\underline{Bacillus}$ $\underline{subtilis}$ and $\underline{B.}$ $\underline{megaterium}$. Activities of the exocellular enzymes produced by $\underline{B.}$ $\underline{subtilis}$ RM125 (pCK98) and $\underline{B.}$ $\underline{megaterium}$ (pCK98) were found to be higher than that produced by the original gene donor $\underline{B.}$ $\underline{subtilis}$ strain by 52- and 43-fold, respectively. Most of the enzyme was produced during the exponential growth period and the production was not repressed by glucose or cellobiose. The plasmid was stable in $\underline{B.}$ $\underline{megaterium}$ but not in $\underline{B.}$ $\underline{subtilis}$. The production of $\beta$-glucosidase via new shuttle vectors having a $\underline{Alcaligenes}$ $\underline{faecalis}$ gene encoding this enzyme was less effectively allowed in $\underline{Bacillus}$ and $\underline{E.}$ $\underline{coli}$ cells even though the gene was put on amplifiable plasmids. Contrary to the endo-$\beta$-1, 4-glucanase gene, the $\beta$-glucosidase gene was comparatively unstable in Bacillus and E. coli cells. On the analysis of plasmids isolated from resultant transformed cells, intactness, deletion, rearrangement or modification of $\beta$-glucosidase gene portion in shuttle plasmids were all found. The concomitant production of glucose from carboxymethyl cullulose (CMC) with $\beta$-glucosidase and endo- $\beta$-1,4-glucansase complex produced by $\underline{Bacillus}$ or $\underline{E.}$ $\underline{coli}$ transformants was observed depending on the comparatively long incubation period. Additionally, a novel procedure using polysaccharides and dyes for the rapid detection and comparison of various polysaccharase activities was established. The starch, CMC, lichenan, laminarin, or pustulan as substrates, together Congo Red...