A novel GH6 cellobiohydrolase from Paenibacillus curdlanolyticus B-6 and its synergistic action on cellulose degradation

Abstract

We recently discovered a novel glycoside hydrolase family 6 (GH6) cellobiohydrolase from Paenibacillus curdlanolyticus B-6 (PcCel6A), which is rarely found in bacteria. This enzyme is a true exo-type cellobiohydrolase which exhibits high substrate specificity on amorphous cellulose and low substrate specificity on crystalline cellulose, while this showed no activity on substitution substrates, carboxymethyl cellulose and xylan, distinct from all other known GH6 cellobiohydrolases. Product profiles, HPLC analysis of the hydrolysis products and a schematic drawing of the substrate-binding subsites catalysing cellooligosaccharides can explain the new mode of action of this enzyme which prefers to hydrolyse cellopentaose. PcCel6A was not inhibited by glucose or cellobiose at concentrations up to 300 and 100 mM, respectively. A good synergistic effect for glucose production was found when PcCel6A acted together with processive endoglucanase Cel9R from Clostridium thermocellum and β-glucosidase CglT from Thermoanaerobacter brockii. These properties of PcCel6A make it a suitable candidate for industrial application in the cellulose degradation process.

A Novel Application Potential of GH6 Cellobiohydrolase CtCel6 from Thermophilic Chaetomium thermophilum for Gene Cloning, Heterologous Expression and Biological Characterization

“…4). As previously reported, some cellobiohydrolases of GH6 were detected to hydrolyze CMC-Na, as well as PASC, which were defined as the exo-/endo-type cellobiohydrolases (Wang et al, 2013;Baramee et al, 2016). In exo-/endo-type cellobiohydrolases, the flexible tunnel-like active sites are the reason that causing exo-type action connected with endo-type action.…”

“…Similar conclusion was reported on GH9 endoglucanase CenC from Clostridium thermocellum and β-glucosidase Bgl4 from Penicillium funiculosum (Haq et al, 2015;Ramani et al, 2015), owing to bivalent metallic cations could increase the stability and cause conformational changes of catalytic center of cellulose. The observation of substrate specificity showed that CtCel6 displayed high activity towards β-D-glucan with 1.27 U/mg, which was higher than two cellobiohydrolase (CBHI, CBHII) from Trichoderma viride (Song et al, 2010), but lower than cellobiohydrolase PcCel6A from Paenibacillus curdlanolyticus (Baramee et al, 2016). PASC could also be hydrolyzed by CtCel6 as showed in Table 2.…”

Chemical Pretreatment-Independent Saccharifications of Xylan and Cellulose of Rice Straw by Bacterial Weak Lignin-Binding Xylanolytic and Cellulolytic Enzymes

“…The processive endo-␤-1,4-glucanase CtCel9R, from C. thermocellum, produced cellotetraose as a primary hydrolysis product and slowly but further hydrolyzed it to smaller oligosaccharides, while the ␤-glucosidase TbCglT, from T. brockii, was capable of rapidly converting cellobiose and short-chain cellooligosaccharides to glucose. These two enzymes demonstrated high thermostability and high glucose tolerance (19). Since the biorefineries generally employ high temperatures (2), these two enzymes were selected for glucose production from the pretreated rice straws, including XRS, ARS, and SXRS.…”

“…strain LD-1 (35) and Pleurotus ostreatus (36), with commercial Trichoderma viride cellulase ONOZUKA 3S and Aspergillus niger cellulase Y-NC, resulted in 18.6% and 32.0% glucose yields, respectively, which were lower than the values obtained by saccharification of all of the treated rice straws by the combination of CtCel9R and TbCglT (Table 3). These results may be ascribed to higher glucose tolerance and thermostability (19) and lower affinity for lignin of bacterial cellulases CtCel9R and TbCglT than fungal cellulases, and these factors together may have shown their best performance in cellulose saccharification. Therefore, the combination of CtCel9R and TbCglT is a great formulation in the saccharification of cellulose to generate glucose.…”

“…To gain insight into its potential as an enzymatic pretreatment using PcAxy43A, the structural change of the rice straw after the saccharification was observed by scanning electron microscopy (SEM). Nonspecific adsorption onto lignin was also tested for PcAxy43A, PcXyn10C, and PcXyn11A, two cellulolytic enzymes, including Clostridium thermocellum endoglucanase Cel9R (CtCel9R) and Thermoanaerobacter brockii ␤-glucosidase CglT (TbCglT) (19), and the commercial cellulase Celluclast 1.5 L (Novozymes, Copenhagen, Denmark), prepared from Trichoderma reesei ATCC 26921. Furthermore, we evaluated the saccharification of cellulose in xylanolytic enzyme-treated rice straw (XRS), steam-xylanolytic enzyme-treated rice straw (SXRS), and ammonia-treated rice straw (ARS) by CtCel9R, TbCglT, combinations of these, or the commercial cellulase Celluclast 1.5 L. This work may be very beneficial in producing xylose from xylan in untreated rice straw in one step without chemical pretreatment by a single protein, PcAxy43A from P. curdlanolyticus B-6, and offers a new environmentfriendly pretreatment that not only removes xylan from rice straw to improve the cellulose saccharification process but also produces xylose for many applications.…”