Exploration of Two Pectate Lyases from Caldicellulosiruptor bescii Reveals that the CBM66 Module Has a Crucial Role in Pectic Biomass Degradation

Abstract

Pectin deconstruction is the initial step in breaking the recalcitrance of plant biomass by using selected microorganisms that encode for pectinolytic enzymes. Pectate lyases that cleave α-1,4-galacturonosidic linkage of pectin are widely used in industries, such as paper making and fruit softening. However, reports on pectate lyases with good thermostability are few. Here, two pectate lyases (CbPL3 and CbPL9) from a hyperthermophilic bacterium Caldicellulosiruptor bescii, respectively, belonged to family 3 and family 9 polysaccharide lyases, were investigated. Biochemical properties on two CbPLs were shown similarly under optimized conditions of 80°C–85°C and pH 8–9. However, the degradation products from pectin and polygalacturonic acids (pGA) were different. A family-66 carbohydrate-binding module (CbCBM66), located in the N-terminus of two CbPLs, shares 100% amino acid identity. CbCBM66-truncated mutant of CbPL9 showed lower activities than the wild-type, whereas the CbPL3 with CbCBM66 knock-out portion was reported with enhanced activities, thereby revealing the different effect of CbCBM66. Prediction by I-TASSAR server revealed CbCBM66 is structurally close to BsCBM66 from Bacillus subtilis, however, COFACTOR and COACH programs speculated that the substrate-binding sites between CbCBM66 and BsCBM66 are different. Furthermore, substrate-binding assay indicated that the catalytic domains in two CbPLs had strong affinities on pectate-related substrates, but CbCBM66 showed weak interaction with a number of lignocellulosic carbohydrates. Finally, scanning electron microscope (SEM) analysis and total reducing sugar assay showed that the two enzymes could improve the saccharification of switchgrass. The two CbPLs are impressive sources for degradation of plant biomass. Importance Thermophilic proteins could be implemented in diverse industrial applications. We sought to characterize two pectate lyases, CbPL3 and CbPL9, from a thermophilic bacterium Caldicellulosiruptor bescii. The two enzymes share high optimum temperature, low optimum pH, and good thermostability at evaluated temperature. A family-66 carbohydrate binding module (CbCBM66) was identified in two CbPLs with sharing 100% amino acid identity. Deletion of CbCBM66 dramatically decreased the activity of CbPL9, but increase the activity and thermostability of CbPL3, suggesting the different roles of CbCBM66 in two enzymes. Moreover, the degradation products by two CbPLs were different. These results revealed these enzymes could represent a potential pectate lyase for applications in paper and textile industries.

Chemical and nutritional characteristics, and microbial degradation of rapeseed meal recalcitrant carbohydrates: A review

“…Pectate disaccharide-lyase ( Table 4 ) has been reported in Caldicellulosiruptor bescii ( 81 ), Bacteroides thetaiotaomicron ( 82 ), Bacteroides ovatus ( 83 ), Bacillus pumilus BK2 ( 84 ), Eubacterium eligens, Faecalibacterium prausnitzii ( 85 ), and Monoglobus pectinilyticus ( 41 ). Pectate lyase ( Table 4 ) has been reported in Caldicellulosiruptor bescii ( 81 ), genus Bacillus: Bacillus subtilis, B. licheniformis, B. cereus, B. circulans, B. pasteurii, B . amyloliquefaciens , and B. pumilus ( 86 , 87 ), Paenibacillus sp.…”

“…( 88 ), Clostridium cellulovorans ( 89 ), Streptomyces thermocarboxydus ( 90 ), Bacteroides thetaiotaomicron ( 82 ), Bacteroides ovatus ( 83 ), Eubacterium eligens, Faecalibacterium prausnitzii ( 85 ), and Monoglobus pectinilyticus ( 41 ). Endo-polygalacturonase ( Table 4 ) has been reported in Caldicellulosiruptor bescii ( 81 ), Bacteroides thetaiotaomicron ( 82 ), Bacteroides ovatus ( 83 ), Eubacterium eligens, Faecalibacterium prausnitzii ( 85 ), Monoglobus pectinilyticus ( 41 ), and Bifidobacterium longum subsp. longum ( 91 ).…”

“…Rhamnogalacturonan lyase [-L-rhamnopyranosyl-(1 → 4)-alpha-D-galactopyranosyluronate endolyase (EC 4.2.2.23)] cleaves the α-1,4 linkages of RG-I resulting in a double bond in the on-reducing GalA residue ( 130 ). Rhamnogalacturonan lyase and hydrolase ( Table 4 ) have been reported in Caldicellulosiruptor bescii ( 81 ), Bacteroides thetaiotaomicron ( 82 ), Bacteroides ovatus ( 83 ), Bacillus subtilis ( 92 ), Bacillus licheniformis ( 93 ), Cellvibrio japonicus ( 94 ), Clostridium cellulolyticum ( 95 ), Bacillus licheniformis DSM13 ( 93 ), Pseudomonas cellulose ( 94 ), Penicillium chrysogenum ( 96 ), and Monoglobus pectinilyticus ( 41 ).…”

Characterization of Two Novel Rumen-Derived Exo-Polygalacturonases: Catalysis and Molecular Simulations

“…Pectin, the most complex of these polysaccharides, gives plants structural rigidity and resistance to attack by insects and pathogens [ 1 , 2 ]. Pectin mainly comprises homogalacturonan regions (also known as “smooth” regions) consisting of D-galacturonic acid units, linked by α-1,4 glycosidic bonds, and substituted homogalacturonan regions (also known as “hairy” regions) such as xylogalacturonan, rhamnogalacturonan I, and rhamnogalacturonan II, which account for 15–30% of the pectin in fruits and vegetables [ 3 , 4 ]. Complete hydrolytic degradation of pectin requires a series of functional pectic enzymes, because of its complexity and heterogeneity.…”