Molecular identification of a novel β-1,3-glucanase from alkaliphilic Nocardiopsis sp. strain F96

Abstract

Alkaliphilic Nocardiopsis sp. strain F96 produced three beta-1,3-glucanase isozymes of different molecular masses (BglF1, BglF2 and BglF3). The N-terminal amino acid sequences of BglFs indicated that these isozymes were the products of a single gene. The beta-1,3-glucanase gene (bglF) was cloned from the chromosomal DNA of strain F96. The bglF gene encoded a polypeptide of 270 amino acids including a signal sequence. The deduced amino acid sequence of mature BglF exhibited the highest homology to those of glycoside hydrolase (GH) family 16 beta-1,3-glucanases, suggesting that the enzyme belonged to the GH family 16. The mature region of bglF gene was functionally expressed in Escherichia coli. The optimum pH and temperature of purified recombinant BglF were pH 9.0 and 70 degrees C, respectively. This enzyme efficiently hydrolyzed insoluble beta-1,3-glucans and showed the highest activity toward a beta-1,3-1,4-glucan rather than beta-1,3-glucans. These results suggested that BglF would be a novel beta-1,3-glucanse. Mutational analysis revealed that Glu123 and Glu128 should be the catalytic residues of BglF.

Nocardiopsis species as potential sources of diverse and novel extracellular enzymes

“…This thermostable enzyme could be put to use for preparing high-fructose syrups. Such syrups are extensively preferred β-1,3-1,4-glucans rather than β-1,3-glucans; cloning and expression; crystal structure; construction and characterization of chimeras Masuda et al 2003Masuda et al , 2006Fibriansah et al 2006Fibriansah et al , 2007Koizumi et al 2007Koizumi et al , 2009 Alkali-tolerant thermostable inulinase Xylan bioconversion into xylose for different products Tsujibo et al 1990aTsujibo et al , 1991 used in the beverage industry as sweeteners for making soft drinks (Van der Maarel et al 2002). Another recent paper describes the isolation and immobilization of a thermostable α-amylase derived from a haloalkaliphilic marine isolate referred to as Nocardiopsis sp.…”

Additional Carbohydrate-Binding Modules Enhance the Insoluble Substrate-Hydrolytic Activity of β-1,3-Glucanase from AlkaliphilicNocardiopsissp. F96

“…Recombinant plasmids pET-BglF bearing the bglF gene from strain F96 16) and pET-B bearing the ChBD region of the chiJ gene from strain J813 21) were used. Plasmid pBG200 contains the bglH gene from B. circulans IAM1165.…”

“…15) It has been shown that strain F96 also produces an endo--1,3-glucanase. 16) The -1,3-glucanase gene (bglF) was cloned from the chromosomal DNA of strain F96. It encoded a polypeptide comprising a signal peptide (25 amino acids) and mature enzyme (245 amino acids).…”

Analysis of functional divergence within two structurally related glycoside hydrolase families

“…F96 (2HYK), 50 which exhibits an uncharacteristic preference for mixed linked b-1,3-1,4-glucans (typically lichenin). 51 Eighteen amino acid residue pairs with P* [ 20 were identified (Table II). As with other GH16 cluster comparisons, the N-terminal regions in the primary sequences of each subfamily are poorly aligned; in this case, consistent secondary structure agreement between P. tremula XTH and Nocardiopsis glucanase did not occur until the second major b-strand of each protein (XTH residues 51-61 and glucanase residues 88-95), suggesting that P* 5 2070 for the Gly44/Gly71 pair is a false positive.…”