Abstract

The alkalophilic Bacillus sp. C-125 isolated from soil produced two types of xylanase (xylanase N and xylanasea) in the culture media. Xylanase N had an optimum at pH 7.0, and xylanase A showed a very broad pH optimum (pH 6 to 10). The gene for xylanase A was cloned in E. coli by using the plasmid pBR322. A plasmid pCX311, having two HindIII fragments (2.6 and 2.0 Kb) in a Hindlll site of pBR322, was isolated. More than 70% of the pCX 311-borne xylanase was found in the culture broth. Two HindIII fragments in pCX311 were essential not only for the xylanase production but also for the excretion of periplasmic proteins. The production of the enzyme required Na+, K+ or Li+. The enzyme produced in the culture broth was stable and no significant decrease in activity was observed after 24 h cultivation. Furthermore, the activity of pCX311-borne xylanase detected in the culture broth was higher than that of the xylanase produced by the alkalophilic Bacillus sp. strain C-125. The enzymatic properties of this xylanase were almost the same as those of xylanase A. The cloned 4.6 kb fragment may contain some components that make the outer membrane of E. coli permeable, thus allowing the excretion of some proteins, such as xylanase, and β-lactamase. However, alkaline phosphatase was not excreted into the culture broth.

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