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The mulberry branch can be used as a renewable biomass resource for the production of biofuels since it contains a significant amount of lignocellulose. However, the rapid deactivation of enzymes during lignocellulose degradation in a high-temperature environment leads to high costs and low efficiency of enzymatic saccharification. In this study, xylanase was modified by rational design, and xylanase and cellulase were used to synergistically degrade mulberry branches pretreated with seawater. The fermentable sugar yield and degree of synergy (DS) were determined. The dominant mutant S21Y/N318W of Hortaea werneckii xylanase Hwxyl10A was obtained with improved thermostability (T 50 increased by 12 °C, t 1/2 is 17-fold that of WT). Meanwhile, the specific activity at 75 °C increased from 1470 to 1901 U/mg. Under the condition of seawater immersion pretreatment, the highest yield of fermentable sugar was 313.5 μmol/g, and the highest DS was 1.57 after enzymatic hydrolysis of mulberry branches by cellulase and S21Y/N318W at 50 °C. Therefore, the biotransformation of mulberry branches reducing sugar has been significantly improved, which provides an efficient method of biomass saccharification for biofuel production. [Display omitted] • An efficient xylanase with improved properties was obtained by rational design technology. • The thermal stability of mutants were improved by enhancing the rigidity of flexible region. • The specific activity at 75 °C of xylanase mutant S21Y/N318W was increased by 29%. • The half-life of xylanase mutant S21Y/N318W was prolonged by 17 times. • The degradation was strengthened by the synergistic catalysis of S21Y/N318W and cellulase. [ABSTRACT FROM AUTHOR]

Background: Xylanase, an important accessory enzyme that acts in synergy with cellulase, is widely used to degrade lignocellulosic biomass. Thermostable enzymes with good catalytic activity at lower temperatures have great potential for future applications in the feed and fuel industries, which have distinct demands; however, the potential of the enzymes is yet to be researched. Results: In this study, a structure-based semi-rational design strategy was applied to enhance the low-temperature catalytic performance of Bispora sp. MEY-1 XYL10C_∆N wild-type (WT). Screening and comparisons were performed for the WT and mutant strains. Compared to the WT, the mutant M53S/F54L/N207G exhibited higher specific activity (2.9-fold; 2090 vs. 710 U/mg) and catalytic efficiency (2.8-fold; 1530 vs. 550 mL/s mg) at 40 °C, and also showed higher thermostability (the melting temperature and temperature of 50% activity loss after 30 min treatment increased by 7.7 °C and 3.5 °C, respectively). Compared with the cellulase-only treatment, combined treatment with M53S/F54L/N207G and cellulase increased the reducing sugar contents from corn stalk, wheat bran, and corn cob by 1.6-, 1.2-, and 1.4-folds, with 1.9, 1.2, and 1.6 as the highest degrees of synergy, respectively. Conclusions: This study provides useful insights into the underlying mechanism and methods of xylanase modification for industrial utilization. We identified loop2 as a key functional area affecting the low-temperature catalytic efficiency of GH10 xylanase. The thermostable mutant M53S/F54L/N207G was selected for the highest low-temperature catalytic efficiency and reducing sugar yield in synergy with cellulase in the degradation of different types of lignocellulosic biomass. Highlights: Key sites were proved to promote low-temperature catalytic efficiency of xylanase. Thermostable mutant with improved catalytic efficiency at 40 °C was obtained. Synergistic hydrolysis was dramatically improved between the mutant and cellulase. Agricultural wastes were converted into fermentable sugars by enzymolysis. [ABSTRACT FROM AUTHOR]

Xylanase is widely used for the degradation lignocellulosic biomass as an important accessory enzyme. But productive hydrolysis of the recalcitrant lignocellulose also requires for applicable pretreatment with environmental friendliness. Meanwhile, the complicated industrial processes also demand for catalytically efficient enzymes with more resistance to both pH and heating conditions, which barely exist for the wild types. In this study, the dominant mutant H51K/Q118A from Gloeophyllum trabeum derived GtXyn10 (WT) was obtained by directed evolution. Compared to the WT, apart from the impressive pH resistance from pH 1.0–9.0, the dominant mutant H51K/Q118A also exhibited higher specific activity (2.5-fold; 2510 vs. 1010 U/mg), higher catalytic efficiency (2.4-fold; 460 vs. 190 mL/s‧mg), and better thermostability (with t 1/2 at 80 °C extend by 10 min). When 'Seawater + Feton' pretreatment was applied, the lignin clearance rate reached 62.5%, 86.6% higher than that of feton pretreatment (33.5%). After pretreatment of 'Seawater + Feton', Compared with the cellulase-only treatment (159 μmol/g), synergistic hydrolysis with both H51K/Q118A and cellulase (236 μmol/g) increased the fermentable sugar yields from bagasse by 48.4%. In combination with the optimized pretreatment and synergistic hydrolysis of the modified xylanase and cellulase, the fermentable sugar production for green bio-energy was realized with efficiency. [Display omitted] • An efficient xylanase with improved properties was obtained by directed evolution. • Steric hindrance elimination was a possible mechanism for catalytic enhancement. • 'Seawater + Feton' pretreatment promoted the removal of lignin in bagasse. • Synergistic hydrolysis (H51K/Q118A + cellulase) realized the optimal degradation. • Biomass utilization for fermentable sugars was optimized by combined strategies. [ABSTRACT FROM AUTHOR]

Extremophilic xylanases have attracted great scientific and industrial interest. In this study, a GH10 xylanase-encoding gene, Xyl10E, was cloned from Bispora sp. MEY-1 and expressed in Pichia pastoris GS115. Deduced Xyl10E shares the highest identities of 62% and 57% with characterized family GH10 xylanases from Talaromyces leycettanus and Penicillium canescens (structure 4F8X), respectively. Xyl10E was most active at 93 to 95°C and pH 4.0, retained more than 75% or 48% of the initial activity when heated at 80°C or 90°C for 30 min, respectively, and hardly lost activity at pH 1.0 to 7.0, but was completely inhibited by SDS. Two residues, A160 and A161, located on loop 4, were identified to play roles in catalysis. Mutants A160D/E demonstrated higher affinity to substrate with lower Km values, while mutants A161D/E mainly displayed elevated Vmax values. All of these mutants had significantly improved catalytic efficiency. According to the molecular dynamics simulation, the mutation of A160E was able to affect the important substrate binding site Y204 and then improve the substrate affinity, and the mutation of A161D was capable of forming a hydrogen bond with the substrate to promote the substrate binding or accelerate the product release. This study introduces a highly thermophilic fungal xylanase and reveals the importance of loop 4 for catalytic efficiency. [ABSTRACT FROM AUTHOR]

[Display omitted] • Mutant from XYL10C_ΔN with improved catalytic activity was obtained through protein engineering. • Synergistic hydrolysis with the engineered xylanase and cellulase excelled other enzyme treatments. • Seawater immersion was helpful for biomass hydrolysis as chemical pretreatment. • Ramulus mori was converted into fermentable sugars with lower energy consumption. Biorefinery of Ramulus mori with lower energy consumption through improved enzyme and pretreatment strategies was reported. Directed evolution and saturation mutagenesis were used for the modification of xylanase, the yield of fermentable sugars and the degree of synergy (DS) were determined for different pretreatment (seawater/non-seawater) and enzyme treatment groups (xylanase/cellulase/co-treatment). The dominant mutant I133A/Q143Y of Bispora sp. xylanase XYL10C_ΔN was obtained with improved specific activity (1860 U/mg), catalytic efficiency (1150 mL/s∙mg) at 40 °C, and thermostability (T 50 increased by 7 °C). With the pretreatment of seawater immersion, the highest yield of fermentable sugars for Ramulus mori at 40 °C reached 199 μmol/g when hydrolyzed with cellulase and I133A/Q143Y, with the highest DS of 2.6; this was 4.5-fold that of the group hydrolyzed by cellulase alone with non-seawater pretreatment. Thus, bioconversion of reducing sugar from Ramulus mori was improved significantly at lower temperatures, which provides an efficient and energy-saving way for biofuel production. [ABSTRACT FROM AUTHOR]

[Display omitted] • An efficient bagasse biorefinery with low energy consumption was realized. • A thermostable xylanase mutant was engineered for catalysis under mild conditions. • Seawater following steam explosion promotes the enzymatic hydrolyzation of bagasse. • Synergistic hydrolysis of high activity xylanase and cellulase was optimal. Lignocellulosic biorefining for producing biofuels poses technical challenges. It is usually conducted over a long time using heat, making it energy intensive. In this study, we lowered the energy consumption of this process through an optimized enzyme and pretreatment strategy. First, the dominant mutant M137E/N269G of Bispora sp. MEY-1 XYL10C_ΔN was obtained by directed evolution with high catalytic efficiency (970 mL/s∙mg) and specific activity (2090 U/mg) at 37 °C, and thermostability was improved (T 50 increased by 5 °C). After pretreatment with seawater immersion following steam explosion, bagasse was co-treated with cellulase and M137E/N269G under mild conditions (37 °C), the resulting highest yield of fermentable sugars reached 219 µmol/g of bagasse, 46% higher than that of the non-seawater treatment group, with the highest degree of synergy of 2.0. Pretreatment with seawater following steam explosion and synergistic hydrolysis through high activity xylanase and cellulase helped to achieve low energy degradation of lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

Background: Xylanase is one of the most extensively used biocatalysts for biomass degradation. However, its low catalytic efficiency and poor thermostability limit its applications. Therefore, improving the properties of xylanases to enable synergistic degradation of lignocellulosic biomass with cellulase is of considerable significance in the field of bioenergy. Results: Using fragment replacement, we improved the catalytic performance and thermostability of a GH10 xylanase, XylE. Of the ten hybrid enzymes obtained, seven showed xylanase activity. Substitution of fragments, M3, M6, M9, and their combinations enhanced the catalytic efficiency (by 2.4- to fourfold) as well as the specific activity (by 1.2- to 3.3-fold) of XylE. The hybrids, XylE-M3, XylE-M3/M6, XylE-M3/M9, and XylE-M3/M6/M9, showed enhanced thermostability, as observed by the increase in the T50 (3–4.7 °C) and Tm (1.1–4.7 °C), and extended t1/2 (by 1.8–2.3 h). In addition, the synergistic effect of the mutant xylanase and cellulase on the degradation of mulberry bark showed that treatment with both XylE-M3/M6 and cellulase exhibited the highest synergistic effect. In this case, the degree of synergy reached 1.3, and the reducing sugar production and dry matter reduction increased by 148% and 185%, respectively, compared to treatment with only cellulase. Conclusions: This study provides a successful strategy to improve the catalytic properties and thermostability of enzymes. We identified several xylanase candidates for applications in bioenergy and biorefinery. Synergistic degradation experiments elucidated a possible mechanism of cellulase inhibition by xylan and xylo-oligomers. [ABSTRACT FROM AUTHOR]

Background: Improving the hydrolytic performance of hemicellulases to degrade lignocellulosic biomass is of considerable importance for second-generation biorefinery. Xylanase, as the crucial hemicellulase, must be thermostable and have high activity for its potential use in the bioethanol industry. To obtain excellent xylanase candidates, it is necessary to understand the structure–function relationships to provide a meaningful reference to improve the enzyme properties. This study aimed to investigate the catalytic mechanism of a highly active hyperthermophilic xylanase variant, XYL10C-ΔN, for hemicellulose degradation. Results: By removing the N-terminal 66 amino acids, the variant XYL10C-ΔN showed a 1.8-fold improvement in catalytic efficiency and could hydrolyze corn stover more efficiently in hydrolysis of corn stover; however, it retained similar thermostability to the wild-type XYL10C. Based on the crystal structures of XYL10C-ΔN and its complex with xylobiose, Glu175 located on loop 3 was found to be specific to GH10 xylanases and probably accounts for the excellent enzyme properties by interacting with Lys135 and Met137 on loop 2. Site-saturation mutagenesis confirmed that XYL10C-ΔN with glutamate acid at position 175 had the highest catalytic efficiency, specific activity, and the broadest pH-activity profile. The functional roles of Glu175 were also verified in the mutants of another two GH10 xylanases, XylE and XynE2, which showed increased catalytic efficiencies and wider pH-activity profiles. Conclusions: XYL10C-ΔN, with excellent thermostability, high catalytic efficiency, and great lignocellulose-degrading capability, is a valuable candidate xylanase for the biofuel industry. The mechanism underlying improved activity of XYN10C-ΔN was thus investigated through structural analysis and functional verification, and Glu175 was identified to play the key role in the improved catalytic efficiency. This study revealed the importance of a key residue (Glu175) in XYN10C-ΔN and provides a reference to modify GH10 xylanases for improved catalytic performance. [ABSTRACT FROM AUTHOR]

A xylanase gene of GH 10, Tlxyn10A , was cloned from Talaromyces leycettanus JCM12802 and expressed in Pichia pastoris . Purified recombinant Tl Xyn10A was acidic and hyperthermophilic, and retained stable over the pH range of 2.0–6.0 and at 90 °C. Sequence analysis of Tl Xyn10A identified seven residues probably involved in substrate contacting. Three mutants ( Tl Xyn10A_P, _N and _C) were then constructed by substituting some or all of the residues with corresponding ones of hyperthermal Xyl10C from Bispora sp. MEY-1. Tl Xyn10A_P with mutations at subsites +2 to +4 exhibited improved specific activity (by 0.44-fold) and pH stability (2.0–10.0). Molecular dynamics simulation analysis indicated that mutations E229I and F232E probably weaken the substrate affinity at subsites +3 to +4, and G149D may introduce a new hydrogen bond. These modifications altogether account for the improved performance of Tl Xyn10A_P. Moreover, Tl Xyn10A_P was able to hydrolyze wheat straw persistently, and has the application potentials in various industries. [ABSTRACT FROM AUTHOR]

A xylanase gene of glycoside hydrolase family 10, Gt Xyn10, was cloned from Gloeophyllum trabeum CBS 900.73 and expressed in Pichia pastoris GS115. Purified recombinant Gt Xyn10 exhibited significant activities to xylan (100.0%), lichenan (11.2%), glucan (15.2%) and p -nitrophenol-β-cellobiose (18.6%), demonstrated the maximum xylanase and glucanase activities at pH 4.5–5.0 and 75 °C, retained stability over the pH range of 2.0–7.5 and at 70 °C, and was resistant to pepsin and trypsin, most metal ions and SDS. Multiple sequence alignment and modeled-structure analysis identified a unique Gly48 in Gt Xyn10, and site-directed mutagenesis of Gly48 to Lys improved the temperature optimum up to 80 °C. Under simulated mashing conditions, Gt Xyn10 (80 U) reduced the mash viscosity by 12.8% and improved the filtration rate by 31.3%. All these properties above make Gt Xyn10 attractive for potential applications in the feed and brewing industries. [ABSTRACT FROM AUTHOR]

[Display omitted] • Enzyme-polymer conjugates were first applied in W/W droplets for biocatalysis. • The effect of dimensionless numbers (We , Ca) on droplet size in jetting pattern were analyzed. • The power-law relationship between droplet size and velocity ratio of two-phase was clarified. • The K m value of microfluidic immobilized enzymatic was 1/2 of that in the batch reactor. • Xylanase embedded in the d-phase was reused 10 times with a residual activity above 60%. To gain a high yield of xylooligosaccharides (XOS) at a low cost, W/W droplet-based microfluidic system combined with immobilized xylanase was constructed. Jetting-regime under the high viscosity of both two phases was correlated with the Ca and We numbers. W/W droplets were obtained with controllable sizes when the flow velocities of dispersed and continuous phases were in the range of 0.29–0.48 µL/min and 0.52–0.80 µL/min respectively. The droplet size could be expressed as a power-law relationship with the velocity ratio of two phases. The maximum yield of XOS was 75.0% within 10 min. K m value was reduced to a half compared with batch reactors. Xylanase-polymer conjugates were reused for 10 times, with a residual activity over 60%. The results confirmed that the combination of enzyme-polymer conjugates and W/W droplet-based microfluidic system is a low-cost and high catalytic efficiency method for biocatalysis. [ABSTRACT FROM AUTHOR]

[Display omitted] • Carrier-free immobilized xylanase was firstly constructed by ATRP "grafting-from" method. • Polymer grafting effectively increased the thermal stability (90 °C) of xylanase by 20 %. • The recycling of XYL-PNIPAAm conjugates was increased by 5 times (>60 %). • Over 99.95 % XOS in the DP range of 1–3 was produced from mulberry bark. In the past few years, xylooligosaccharides produced from lignocellulosic biomass have attracted great attention. Enzyme catalysis is widely used because of its mild reaction conditions. In order to solve the shortcomings of poor stability and high cost of enzyme, a carrier-free enzyme immobilization technology was introduced. For this purpose, protein-polymer conjugates, kinds of carrier-free immobilized enzymes, were first established by growing poly (N -isopropylacrylamide) chains on xylanase using a grafting-from method, to obtain reusable and high-value xylanase. SDS-PAGE and circular dichroism spectra showed the successful preparation of xylanase-PNIPAAm conjugates, and the integrity of the protein secondary structure. The remaining activity of the xylanase-PNIPAAm conjugates after heat treatment (90 °C) for 60 min was approximately 20 % higher than that of the free xylanase, and a higher pH stability was obtained in the pH range of 2−8. The remaining activity of xylanase-PNIPAAm conjugates was more than 60 % after recycling 5 times. In addition, the mulberry bark synergistic treated by xylanase-PNIPAAm conjugates and cellulase was studied. More than 99.95 % yield of XOS in the degree of polymerization range of 1–3 was produced from mulberry bark. Overall, the construction of xylanase-PNIPAAm conjugates provides a novel method to produce xylooligosaccharides from waste biomass. [ABSTRACT FROM AUTHOR]