Cloning and Expression of Cellulases from Rumen Bacteria in Escherichia Coli

Abstract

Lignocelluloses, whose major component is cellulose, represent the most abundant natural material on earth. The complete degradation of cellulose to glucose by cellulase complexes consisting of endocellulase, exocellulase and beta-glucosidase would provide a major source of food, fuel and chemical feedstacks [1]. In addition, the production of endocellulase in Lactobacillus plantarum in silage processing may assist the softening of cell walls making the substrate more readily available for lactic acid fermentation [2]. By using recombinant DNA technologies we have cloned and expressed an endoglucanase encoding gene from rumen bacteria Fibrobacter succinogenes SD35 in Escherichia coli. Dot blot hybridisation experiments revealed that this gene is different from the endoglucanase encoding gene in either Ruminococcus albus SY3 or Fibrobacter succinogenes BL2. Therefore, we have sequenced the cloned DNA and have studied the properties of the enzyme. The molecular weight of the enzyme determined by SDS-PAGE was approximately 51 Kdalton. Sudden viscosity decrease of CM-cellulose relative to reducing sugars released from CM-cellulose at the same time was the indication of endoglucanase activity. In addition, the enzyme showed some activity against lichenane and to a lesser extent, against xylane. It has a pH optimum of 6.0, and a temperature optimum of 37.5°C. The enzyme lost approximately one-third of its original activity at 55°C for 10 minutes. On the other hand, its activity was slightly stimulated in the presence of 4mM calcium chloride or magnesium chloride. As a result, it was concluded that the enzyme was a good candidate for expression in Lactobacillus plantarum in silage process.