Cloning and sequencing of a cellulase gene from Fibrobacter succinogenes SD35
F.succinogenes is one of the most active bacteria of the rumen in growth-related degradation of recalcitrant forms of cellulose. The bacterium has multienzyme cellulase complex encoded by multiple genes to degrade crystalline cellulose. A genomic library of F.succinogenes SD35, constructed in the EMBL3, was screened for cellulolytic activity and 2.7kbp DNA fragment expressing a endoglucanase (SD-END-1) was further subcloned into pUC18 and expressed in E.coli DH5 alpha. DNA hybridisation (dot blot) experiments revealed that this gene (sd-end-1) is different from the endoglucanase encoding gene in either Ruminococcus albus SY3 or F.succinogenes BL2. The 2.7 kbp DNA fragment carrying the sd-end-1 with an ORF of 1167 bp was fully sequenced. The alignment of the amino acid sequence of SD-END-1 (388 amino acids) with other multifunctional endoglucanases from F.succinogenes S85 (EG3), R.albus SY3 (EGA) and B.fibrisolvens (End1) revealed less than 25% identity. The molecular weight of SD-END-1 calculated from deduced amino acid sequence, 50,201 Da is in agreement with the molecular weight estimated by CMC-SDS-PAGE (49 kDa). Specific activity of SD-END-1 calculated by CMC, lichenan, xylan and avicel were 101, 41, 27 and 15mumol reducing sugar/min/mg protein. SD-END-1 has a pH optimum of 6.0 and a temperature optimum of 37.5°C. When the enzyme and substrate were incubated alone at various temperatures for 10 min and then assayed at 39°C for 30 min, 75% of its activity was lost at 60°C. On the other hand, its activity was slightly stimulated in the presence of 4 mM CaCl2 or MgCl2. A rapid and simple method for the isolation of lambda and a new plate for rapid screening of CMC+ recombinant E.coli cells were also developed. In addition, a new CMC-PAGE assay was developed and used as an alternative method to investigate whether SD-END-1 was subject to proteolysis in the E.coli DH5 alpha cells.