CLONING, SEQUENCING AND ANALYSIS OF THE GGH-A GENE ENCODING A 1,4-BETA-D-GLUCAN GLUCOHYDROLASE FROM MICROBISPORA-BISPORA

The ggh-A gene, encoding a 1,4-beta-D-glucan glucohydrolase/beta-gluco sidase, of Microbispora bispora (Mb) was subcloned and expressed from a 4.0-kb XhoI DNA fragment. The nucleotide sequence of this fragment w as determined. Analysis of the sequence revealed one open reading fram e (ORF) which encodes a 986-amino-acid (aa) protein with a calculated molecular weight of 107 510. The ggh-A ORF has features typical of an actinomycete gene including high GC content (70.5%) and corresponding biased codon usage. Comparison of the aa sequence of the Mb 1,4-beta-D -glucan glucohydrolase (Mbggh-A) with other glycosidases reveals high overall homology to several beta-glucosidases and a 1,4-beta-D-glucan glucohydrolase belonging to the glycosyl hydrolase family 3. The aa se quence alignments of Mbggh-A and beta-glucosidases show that the activ e site region potentially involves two Asp residues. The aa sequence h omology studies revealed a potential two-domain structure for Mbggh-A and other beta-glucosidases. Furthermore, Mbggh-A has localized homolo gy to a cellulose-binding domain present in some xylanases. This repor t is significant, as, to date, 1,4-beta-D-glucan glucohydrolases have rarely been reported, though they are assumed to have a critical role in cellulolysis.