Cloning and DNA sequencing of bgaA, a gene encoding an endo-beta-1,3-1,4-glucanase, from an alkalophilic Bacillus strain (N137)

Abstract

The gene bgaA encoding an alkaline endo-beta-1,3-1,4-glucanase (lichenase) from an alkalophilic Bacillus sp. strain N137, isolated in our laboratory, was cloned and expressed from its own promoter in Escherichia coli. The nucleotide sequence of a 1,416-bp DNA fragment containing bgaA was determined and revealed an open reading frame of 828 nucleotides. The deduced protein sequence consists of 276 amino acids and has a 31-amino-acid putative signal peptide which is functional in E. coli, in which the BgaA protein is located mainly in the periplasmic space. The lichenase activity of BgaA is stable between pH 6 and 12, it shows optimal activity at a temperature between 60 and 70 degrees C, and it retains 65% of its activity after incubation at 70 degrees C for 1 h. This protein is similar to some other lichenases from Bacillus species such as B. amyloliquefaciens, B. brevis, B. licheniformis, B. macerans, B. polymyxa, and B. subtilis. However, it has a lysine-rich region at the carboxy terminus which is not found in any other published lichenase sequence and might be implicated in the unusual biochemical properties of this enzyme. The location of the mRNA 5' end was determined by primer extension and corresponds to nucleotide 235. A typical Bacillus sigma A promoter precedes the transcription start site.

Soluble forms of YlCrh1p and YlCrh2p, cell wall proteins of Yarrowia lipolytica, have β‐1,3‐glycosidase activity

“…Identical amino acids are shaded in black and similar amino acids in grey. The box indicates the proposed catalytic domain of prokaryotic endo-β-1,3-1,4-glucanases (Tabernero et al, 1994) fuzzy band pattern disappeared and turned into discrete bands after the endo H treatment, the apparent molecular weights of the proteins were still larger than 90 kDa. In general, cell wall proteins with C-terminal Ser-and Thr-rich regions are heavily O-glycosylated (Klis et al, 2002).…”

“…In addition to the amino acid sequence similarity of YlCrh1p and Ylcrh2p to S. cerevisiae Crh1 and Crh2 proteins, YlCrh1p and YlCrh2p have a DE(I/L)DXE motif that is the hypothetical catalytic domain of S. cerevisiae Crh1p WT Ylcrh1 Ylcrh1Ylcrh2 Ylcrh2 10 5 10 4 10 3 10 2 10 1 10 5 10 4 10 3 10 2 10 1 10 5 10 4 10 3 10 2 and Crh2p (Rodriguez-Pena et al, 2000) and prokaryotic endo-β-1,3-1,4-glucanases (Tabernero et al, 1994). To assess whether the soluble YlC-rh1S and YlCrh2S proteins have glycosidase activity, we purified the proteins by His affinity chromatography.…”

Alkaliphiles — from an industrial point of view

“…The optimum temperature and pH for enzyme activity are 65-70°C and 8-10, respectively. Tabernero et al also reported the cloning and DNA sequencing of bgaA, a gene encoding an endo-(1,3-1,4)-/3-glucanase [94]. /3-Lactamases are important enzymes in antibiotic resistance mechanisms and chemotherapy as they can inactivate penicillins or cephalosporins by hydrolysis.…”

Structural and functional analysis of yeast Crh1 and Crh2 transglycosylases

“…In addition to the conserved catalytic site of the GH16 family, Crh1 and Crh2 show several structural features, such as a secretory signal peptide at the N-terminus, a family 18 chitin-binding domain [carbohydrate-binding module (CBM)18], which is missing in Crh1, a Ser-Thr-rich region and a GPI attachment signal, which is necessary for their anchoring to the plasma membrane [17]. Bacterial 1-3, 1-4-b-glucanases or lichenases, included in the GH16 family, hydrolyze linear b-glucans containing b-1,3 and b-1,4 linkages with a cleavage specificity for b-1,4 glycosidic bonds [18,19], whereas plant xyloglucan endotransglycosylases catalyze endotype hydrolysis of xyloglucan molecules and the linking of the newly generated reducing end to another xyloglucan molecule [20,21]. The catalytic mechanism for GH16 family was first deduced by 1 H-NMR monitoring for the endo-1,3-1,4-b-D-glucan glucanohydrolase from Bacillus licheniformis [22].…”