Insights into the Catalytic Mechanism of a Novel XynA and Structure-Based Engineering for Improving Bifunctional Activities.

Xylan and cellulose are the two major constituents of numerous types of lignocellulose. The bifunctional enzyme that exhibits xylanase/cellulase activity has attracted a great deal of attention in biofuel production. Previously, a thermostable GH10 family enzyme (XynA) from Bacillus sp. KW1 was found to degrade both xylan and cellulose. To improve bifunctional activity on the basis of structure, we first determined the crystal structure of XynA at 2.3 Å. Via molecular docking and activity assays, we revealed that Gln250 and His252 were indispensable to bifunctionality, because they could interact with two conserved catalytic residues, Glu182 and Glu280, while bringing the substrate close to the activity pocket. Then we used a structure-based engineering strategy to improve xylanase/cellulase activity. Although no mutants with increased bifunctional activity were obtained after much screening, we found the answer in the N-terminal 36-amino acid truncation of XynA. The activities of XynA_ΔN36 toward beechwood xylan, wheat arabinoxylan, filter paper, and barley ß-glucan were significantly increased by 0.47-, 0.53-, 2.46-, and 1.04-fold, respectively. Furthermore, upon application, this truncation released more reducing sugars than the wild type in the degradation of pretreated corn stover and sugar cane bagasse. These results showed the detailed molecular mechanism of the GH10 family bifunctional endoxylanase/cellulase. The basis of these catalytic performances and the screened XynA_ΔN36 provide clues for the further use of XynA in industrial applications.

A novel glycoside hydrolase family 42 enzyme with bifunctional ß-galactosidase and α-L-arabinopyranosidase activities and its synergistic effects with cognate glycoside hydrolases in plant polysaccharides degradation.

GH42 enzymes are potential candidates for bifunctional ß-galactosidase/α-L-arabinopyranosidase. A novel GH42 enzyme (BaBgal42A) from Bacillus was identified, the recombinant BaBgal42A hydrolyzed not only ß-D-galactopyranosidic bonds in pNP-ß-D-galactopyranoside, oNP-ß-D-galactopyranoside, lactose, galactan, and arabinan but also α-L-arabinopyranosidic linkages in pNP-α-L-arabinopyranoside, wheat arabinoxylan and galactan. The Km values of BaBgal42A for pNP-ß-D-galactopyranoside and pNP-α-L-arabinopyranoside were 2.76 and 16.23 mM, respectively. Investigation of cooperative activities of BaBgal42A with cognate enzymes revealed that BaBgal42A showed obvious synergy with an endo-ß-1,4-galactanase (BaGal53A) in the decomposition of galactan, supplementing BaBgal42A resulted in a 0.56-fold increase in the release of reducing sugars; BaBgal42A also exhibited a little synergy with its cognate endoxylanase (BaXynA)/α-L-arabinofuranosidase (BaAraA) in hydrolyzing wheat arabinoxylan/arabinan, addition of BaBgal42A released 12.7%/7.8% more reducing sugars than that produced by BaXynA/BaAraA alone. Moreover, BaBgal42A is a cold-adapted enzyme, exhibiting 28-46% of the maximal activity at the range of 5-20 °C and its activity was slightly stimulated by addition of Na+, K+, or Ca2+ at low concentrations. This study not only expands the diversity within GH42 family, but also provides new insights into the role of microbial GH42 enzymes, which would contribute to its potential application in polysaccharides degradation and milk lactose hydrolysis.

A novel thermostable GH10 xylanase with activities on a wide variety of cellulosic substrates from a xylanolytic Bacillus strain exhibiting significant synergy with commercial Celluclast 1.5 L in pretreated corn stover hydrolysis.

BACKGROUND: Cellulose and hemicellulose are the two largest components in lignocellulosic biomass. Enzymes with activities towards cellulose and xylan have attracted great interest in the bioconversion of lignocellulosic biomass, since they have potential in improving the hydrolytic performance and reducing the enzyme costs. Exploring glycoside hydrolases (GHs) with good thermostability and activities on xylan and cellulose would be beneficial to the industrial production of biofuels and bio-based chemicals. RESULTS: A novel GH10 enzyme (XynA) identified from a xylanolytic strain Bacillus sp. KW1 was cloned and expressed. Its optimal pH and temperature were determined to be pH 6.0 and 65 °C. Stability analyses revealed that XynA was stable over a broad pH range (pH 6.0-11.0) after being incubated at 25 °C for 24 h. Moreover, XynA retained over 95% activity after heat treatment at 60 °C for 60 h, and its half-lives at 65 °C and 70 °C were about 12 h and 1.5 h, respectively. More importantly, in terms of substrate specificity, XynA exhibits hydrolytic activities towards xylans, microcrystalline cellulose (filter paper and Avicel), carboxymethyl cellulose (CMC), cellobiose, p-nitrophenyl-ß-d-cellobioside (pNPC), and p-nitrophenyl-ß-d-glucopyranoside (pNPG). Furthermore, the addition of XynA into commercial cellulase in the hydrolysis of pretreated corn stover resulted in remarkable increases (the relative increases may up to 90%) in the release of reducing sugars. Finally, it is worth mentioning that XynA only shows high amino acid sequence identity (88%) with rXynAHJ14, a GH10 xylanase with no activity on CMC. The similarities with other characterized GH10 enzymes, including xylanases and bifunctional xylanase/cellulase enzymes, are no more than 30%. CONCLUSIONS: XynA is a novel thermostable GH10 xylanase with a wide substrate spectrum. It displays good stability in a broad range of pH and high temperatures, and exhibits activities towards xylans and a wide variety of cellulosic substrates, which are not found in other GH10 enzymes. The enzyme also has high capacity in saccharification of pretreated corn stover. These characteristics make XynA a good candidate not only for assisting cellulase in lignocellulosic biomass hydrolysis, but also for the research on structure-function relationship of bifunctional xylanase/cellulase.