A Gene Encoding a Novel Multidomain β-1,4-Mannanase from Caldibacillus cellulovorans and Action of the Recombinant Enzyme on Kraft Pulp

Abstract

Genomic walking PCR was used to obtained a 4,567-bp nucleotide sequence from Caldibacillus cellulovorans. Analysis of this sequence revealed that there were three open reading frames, designated ORF1, ORF2, and ORF3. Incomplete ORF1 encoded a putative C-terminal cellulose-binding domain (CBD) homologous to members of CBD family IIIb, while putative ORF3 encoded a protein of unknown function. The putative ManA protein encoded by complete manA ORF2 was an enzyme with a novel multidomain structure and was composed of four domains in the following order: a putative N-terminal domain (D1) of unknown function, an internal CBD (D2), a ␤-mannanase catalytic domain (D3), and a C-terminal CBD (D4). All four domains were linked via proline-threonine-rich peptides. Both of the CBDs exhibited sequence similarity to family IIIb CBDs, while the mannanase catalytic domain exhibited homology to the family 5 glycosyl hydrolases. The purified recombinant enzyme ManAd3 expressed from the cloned catalytic domain (D3) exhibited optimum activity at 85°C and pH 6.0 and was extremely thermostable at 70°C. This enzyme exhibited high specificity with the substituted galactomannan locust bean gum, while more substituted galacto-and glucomannans were poorly hydrolyzed. Preliminary studies to determine the effect of the recombinant ManAd3 and a recombinant thermostable ␤-xylanase on oxygen-delignified Pinus radiata kraft pulp revealed that there was an increase in the brightness of the bleached pulp.The use of hemicellulases in the manufacture of kraft pulp has been shown to promote pulp bleaching, and in most application studies the workers have focused on using xylanases (24,47,48). Using mannanases was not studied until recently, when it was shown that mannanases, acting in combination with xylanases, enhance enzyme-aided bleaching of pulps in modified kraft processes (10, 40).1,4-␤-Mannans, which are some of the major constituents of hemicellulose, are hydrolyzed to mannose by endo-acting ␤-mannanases and exo-acting ␤-mannosidases. However, due to the complexity of the polysaccharide and the presence of side chain sugars, additional enzymes, including ␣-galactosidase, ␤-glucosidase, and acetylmannan esterase, are required to completely hydrolyze mannans (34). Extremely thermophilic bacteria and hyperthermophilic archaea are known to produce several types of thermostable glycosyl hydrolases (38). Few thermostable ␤-mannanases from thermophilic bacteria have been characterized and sequenced (14,(17)(18)(19)33), while no archaeal ␤-mannanase has been described so far. The mannanases that have been sequenced belong to either glycosyl hydrolase family 5 or glycosyl hydrolase family 26 (22).Huang et al. (X. P. Huang, J. A. Hudson, F. A. Rainey, P. D. Nichols, and H. W. Morgan, submitted for publication) described a new thermophilic, spore-forming, aerobic bacterium related to the bacilli that was able to grow on crystalline cellulose, and they named this organism Caldibacillus cellulovorans. Their isolate was related to members of the genus Al...

The chitinolytic system of Lactococcus lactis ssp. lactis comprises a nonprocessive chitinase and a chitin‐binding protein that promotes the degradation of α‐ and β‐chitin

“…These nonhydrolytic proteins are classified as family 33 CBMs [3,18], but, with one exception [19], they occur as individual proteins rather than as auxiliary domains in hydrolytic enzymes. Genome analyses indicate that secreted family 33 CBPs are produced by most chitin-degrading microorganisms [17], but only a few have been characterized biochemically.…”

A novel thermostable multidomain 1,4-β-xylanase from ‘Caldibacillus cellulovorans’ and effect of its xylan-binding domain on enzyme activity

“…The effects of temperature, pH and thermostability on the activity of XynAd1\2 and XynAd2 were determined as described by Sunna et al (2000b). Universal buffer was used for determination of optimal pH for activity and accordingly, all the pH values were adjusted at 90 mC and 70 mC.…”

“…The mixtures were incubated at 70 mC (XynAd1\2) or 60 mC (XynAd2) for 3 h. Products from enzymic hydrolysis were extracted and desalted as described previously . The ability of the purified enzymes to hydrolyse short xylooligosaccharides was also investigated as described by Sunna et al (2000b). Sugars liberated from xylan and xylooligosaccharide hydrolysis at 70 mC (XynAd1\2) or 60 mC (XynAd2) for 3 h were separated on silica gel plates as described previously (Sunna et al, 2000b).…”

The PT/S-Box of Modular Cellulase AcCel12B Plays a Key Role in the Hydrolysis of Insoluble Cellulose

“…The PT/S-box is composed of two axisymmetric regions of similar length in the case of AcCel12B. Other sources of PT-boxes, such as Cel5A and GuxA from A. cellulolyticus 11B [14] and mannanase from C. cellulovorans [13], are also separated into two or several axisymmetric regions by alanines or other amino acids; however, the length of each region is different. Hence, we defined the PT-box1, (PT) 2 (PS) 4 P, as one PT/S-box unit and then redesigned the LR of AcCel12B to contain 0, 1, 2 and 3 PT/S-box units (Figure 1).…”

“…For example, the xylanase Cex from Cellulomonas fimi contains a short PS-box [12]. The mannanase from Caldibacillus cellulovorans contains PS-boxes of three different lengths [13]. The endocellulase Cel5A from Acidothermus cellulolyticus 11B had a PT-box of approximately 40 amino acids, whereas the endoglucanase (GuxA) of GH12 from the same strain contained PS-boxes of three different lengths [14].…”