The capacity for thermal tolerance is critical for industrial enzyme. In the past decade, great efforts have been made to endow wild-type enzymes with higher catalytic activity or thermostability using gene engineering and protein engineering strategies. In this study, a recently developed SpyTag/SpyCatcher system, mediated by isopeptide bond-ligation, was used to modify a rumen microbiota-derived xylanase XYN11-6 as cyclized and stable enzyme C-XYN11-6. After incubation at 60, 70 or 80 ℃ for 10 min, the residual activities of C-XYN11-6 were 81.53%, 73.98% or 64.41%, which were 1.48, 2.92 or 3.98-fold of linear enzyme L-XYN11-6, respectively. After exposure to 60-90°C for 10 min, the C-XYN11-6 remained as soluble in suspension, while L-XYN11-6 showed severely aggregation. Intrinsic and 8-anilino-1-naphthalenesulfonic acid (ANS)-binding fluorescence analysis revealed that C-XYN11-6 was more capable of maintaining its conformation during heat challenge, compared with L-XYN11-6. Interestingly, molecular cyclization also conferred C-XYN11-6 with improved resilience to 0.1-50 mmol/L Ca²⁺ or 0.1 mmol/L Cu²⁺ treatment. In summary, we generated a thermal- and ion-stable cyclized enzyme using SpyTag/SpyCatcher system, which will be of particular interest in engineering of enzymes for industrial application.
Animals ; Cyclization ; Endo-1,4-beta Xylanases ; chemistry ; metabolism ; Enzyme Stability ; Industrial Microbiology ; methods ; Microbiota ; Protein Engineering ; Rumen ; enzymology ; microbiology ; Temperature

Efficient utilization of cellulose and xylan is of importance in the bioethanol industry. In this study, a novel bifunctional xylanase/cellulase gene, Tcxyn10a, was cloned from Thermoascus crustaceus JCM12803, and the gene product was successfully overexpressed in Pichia pastoris GS115. The recombinant protein was then purified and characterized. The pH and temperature optima of TcXyn10A were determined to be 5.0 and 65-70 °C, respectively. The enzyme retained stable under acid to alkaline conditions (pH 3.0-11.0) or after 1-h treatment at 60 °C. The specific activities of TcXyn10A towards beechwood xylan, wheat arabinoxylan, sodium carboxymethyl cellulose and lichenan were (1 480±26) U/mg, (2 055±28) U/mg, (7.4±0.2) U/mg and (10.9±0.4) U/mg, respectively. Homologous modeling and molecular docking analyses indicated that the bifunctional TcXyn10A has a single catalytic domain, in which the substrate xylan and cellulose shared the same binding cleft. This study provides a valuable material for the study of structure and function relationship of bifunctional enzymes.
Cellulase ; Endo-1,4-beta Xylanases ; Enzyme Stability ; Hydrogen-Ion Concentration ; Molecular Docking Simulation ; Pichia ; Substrate Specificity ; Thermoascus

Thermophilic and alkalophilic xylanases have great potential in the pulp bleaching industry. In order to improve the thermal stability of an alkaline family 11 xylanase Xyn11A-LC, aromatic residues were introduced into the N-terminus of the enzyme by rational design. The mutant increased the optimum temperature by 5 degrees C. The wild type had a half-time of 22 min at 65 degrees C and pH 8.0 (Tris-HCl buffer). Under the same condition, the mutant had the half-time of 106 min. CD spectroscopy revealed that the melting temperature (T(m)) values of the wild type and mutant were 55.3 degrees C and 67.9 degrees C, respectively. These results showed that the introduction of aromatic residues could enhance the thermal stability of Xyn11A-LC.
Endo-1,4-beta Xylanases ; chemistry ; Enzyme Stability ; Hydrogen-Ion Concentration ; Protein Engineering ; Temperature

Xylanase is the key enzyme to degrade xylan that is a major component of hemicellulose. The enzyme has potential industrial applications in the food, feed, paper and flax degumming industries. The use of xylanases becomes more and more important in the paper industry for bleaching purposes. Xylanases used in the pulp bleaching process should be stable and active at high temperature and alkaline pH. Thermophilic and alkalophilic xylanases could be obtained by screening the wild type xylanases or engineering the mesophilic and neutral enzymes. In this paper, we reviewed recent progress of screening of the thermophilic and alkalophilic xylanases, molecular mechanism of thermal and alkaline adaptation and molecular engineering. Future research prospective was also discussed.
Endo-1,4-beta Xylanases ; chemistry ; Enzyme Stability ; Hot Temperature ; Hydrogen-Ion Concentration ; Paper ; Protein Engineering

A mesophilic xylanase from Aspergillus oryzae, abbreviated to AoXyn11A, belongs to glycoside hydrolase family 11. Using AoXyn11A as the parent, the thermotolerant hybrid xylanase, we constructed AEx11A by substituting its N-terminus with the corresponding region of a hyperthermostable family 11 xylanase, EvXyn11(TS). AoXyn11A- and AEx11A-encoding genes were expressed in Pichia pastoris GS115 separately, and effects of temperatures on expressed products were determined and compared. The optimum temperature (T(opt)) of AEx11A was 75 degrees C and its half-life at 70 degrees C (t1/2(70)) was 197 min, improved as compared with those (T(opt) = 50 degrees C, t1/2(70) = 1.0 min) of AoXyn11A. Homology modeling of the AEx11A's structure and comparison between structures of AEx11A and AoXyn11A revealed that one disulfide bridge (Cys5-Cys32) was introduced into AEx11A resulted from N-terminus substitution. To explore the effect of the disulfide bridge on the thermostability of AEx11A, it was removed from AEx11A by site-directed mutagenesis (C5T). Analytical results show that the T(opt) of the mutant AEx11A (AEx11A(C5T)) dropped to 60 degrees C from 75 degrees C of AEx11A, and its t1/2(70) and t1/2(80) also decreased to 3.0 and 1.0 min from 197 and 25 min.
Amino Acid Sequence ; Amino Acid Substitution ; Aspergillus oryzae ; enzymology ; Base Sequence ; Disulfides ; chemistry ; metabolism ; Endo-1,4-beta Xylanases ; biosynthesis ; chemistry ; genetics ; Enzyme Stability ; genetics ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; methods ; Pichia ; genetics ; metabolism ; Protein Engineering ; methods ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics

High-concentration sugars production from stover is an important perspective technology for the cellulosic ethanol industrialization. Fed-batch process is an effective way to achieve this goal in the fermentation industry. In this study, based on fed-batch process, high-concentration sugars were produced from pretreated corn stover by enzymatic hydrolysis. After being pretreated by the dilute sulphuric acid, the impacts of the ratio of solid raw material to liquid culture, the content of supplementary materials and the refilling time on the saccharification rate were investigated. Results showed that the initial ratio of solid raw material to liquid culture was 20% (W/V) and the initial concentrations of enzymes for xylanase, cellulose and pectinase were 220 U, 6 FPU, and 50 U per gram of substrates, respectively. After 24 hours and 48 hours, 8% pretreated corn stovers were added respectively together with the additions of xylanase (20 U) and cellulose (2 FPU) per gram of substrates. After 72 hours, the final concentration of reducing sugar was increased to 138.5 g/L from 48.5 g/L of the non fed-batch process. The rate of enzyme hydrolysis of the raw material was 62.5% of the thoretical value in the fed-batch process. This study demonstrated that the fed-batch process could significantly improve the concentration of reducing sugar.
Batch Cell Culture Techniques ; methods ; Carbohydrates ; analysis ; Cellulase ; metabolism ; Cellulose ; metabolism ; Endo-1,4-beta Xylanases ; metabolism ; Ethanol ; metabolism ; Fermentation ; Hydrolysis ; Plant Stems ; chemistry ; metabolism ; Zea mays ; chemistry

Besides the catalytic domain, some xylanases contained a non-catalytic domain which is named as carbohydrate binding module (CBM). CBM can be used to improve their binding-ability to insoluble substrates. We illustrated the importance of CBM by reviewing the source of CBMs, type of families, features of binding to insoluble substrates, specific amino acids involved in substrate-binding, linker peptides connecting the catalytic domain, and the effect of CBMs on xylanase thermostability. CBM is important for xylanase to break down complicate carbohydrates. Perspectives on engineering xylanase activity according to the characteristics of CBMs were given.
Binding Sites ; Carbohydrate Metabolism ; Catalysis ; Endo-1,4-beta Xylanases ; metabolism ; Multienzyme Complexes ; chemistry ; Substrate Specificity

We engineered a disulphide bridge between two adjacent double-layered beta-sheet at the N-terminal region of Trichoderma reesei endo-1,4-beta-xylanase II(XYN II) by site-directed mutagenesis. The native xylanase XYN-OU and the mutated xylanase XYN-HA12 (T2C, T28C and S156F) were separately expressed in Pichia pastoris. Both xylanases were purified and characterized. The optimum temperature of XYN-HA12 was increased from 50 degrees C to 60 degrees C, relative to XYN-OU. At 70 degrees C, the halftime of inactivation for XYN-OU and XYN-HA12 were 1 min and 14 min, respectively. The optimum pH of XYN-HA12 was 5.0, similar to XYN-OU. However, XYN-HA12 could retain over 50% activity from pH 3.0 to 10.0 at 50 degrees C for 30 min. As for XYN-OU, it could retain over 50% activity from the pH value 4.0 to 9.0 at 50 degrees C in 30 min. The result of the mutated xylanase indicated that constructed disulphide bridge could improve its thermostability at relatively higher temperature.
Amino Acid Substitution ; Disulfides ; chemistry ; metabolism ; Endo-1,4-beta Xylanases ; biosynthesis ; chemistry ; genetics ; Enzyme Stability ; genetics ; Mutagenesis, Site-Directed ; Pichia ; genetics ; metabolism ; Protein Engineering ; methods ; Recombinant Proteins ; biosynthesis ; chemistry ; Trichoderma ; enzymology ; genetics

The aims of this research were to isolate a Aspergillus niger strain with higher beta-glucosidase activity. We utilized the beta-glucosidase producing strain Aspergillus niger CGMCC 3.316 as the original strain to first obtain a mutant 3-3M through ultraviolet irradiation. Then we studied the conditions of protoplast release and regeneration for strain 3-3M. We treated the protoplasts of strain 3-3M via ultraviolet irradiation and obtained another isolated mutant 60B-3D. The strain 60B-3D showed much higher beta-glucosidase production than the original strain and 3-3M strain. The beta-glucosidase activity of strain 60B-3D was 23.4 IU/mL, with an improvement of 39% compared with the original strain, and 23% compared with strain 3-3M. We also studied the fermentation process of strain 60B-3D, and compared it with the original strain and strain 3-3M. We found the strain 60B-3D exhibited an improvement in xylanase production. The comparison results also showed that the strain 60B-3D secreted more protein. These results were beneficial for producing beta-glucosidase through this productive mutant.
Amino Acid Sequence ; Aspergillus niger ; enzymology ; genetics ; Endo-1,4-beta Xylanases ; biosynthesis ; Fermentation ; Molecular Sequence Data ; Mutagenesis ; Mutation ; Protoplasts ; enzymology ; beta-Glucosidase ; biosynthesis

OBJECTIVETo study the optimal medium composition for xylanase production by Aspergillus niger XY-1 in solid-state fermentation (SSF).

METHODSStatistical methodology including the Plackett-Burman design (PBD) and the central composite design (CCD) was employed to investigate the individual crucial component of the medium that significantly affected the enzyme yield.

RESULTSFirstly, NaNO(3), yeast extract, urea, Na(2)CO(3), MgSO(4), peptone and (NH(4))(2)SO(4) were screened as the significant factors positively affecting the xylanase production by PBD. Secondly, by valuating the nitrogen sources effect, urea was proved to be the most effective and economic nitrogen source for xylanase production and used for further optimization. Finally, the CCD and response surface methodology (RSM) were applied to determine the optimal concentration of each significant variable, which included urea, Na(2)CO(3) and MgSO(4). Subsequently a second-order polynomial was determined by multiple regression analysis. The optimum values of the critical components for maximum xylanase production were obtained as follows: x(1) (urea)=0.163 (41.63 g/L), x(2) (Na(2)CO(3))=-1.68 (2.64 g/L), x(3) (MgSO(4))=1.338 (10.68 g/L) and the predicted xylanase value was 14374.6 U/g dry substrate. Using the optimized condition, xylanase production by Aspergillus niger XY-1 after 48 h fermentation reached 14637 U/g dry substrate with wheat bran in the shake flask.

CONCLUSIONBy using PBD and CCD, we obtained the optimal composition for xylanase production by Aspergillus niger XY-1 in SSF, and the results of no additional expensive medium and shortened fermentation time for higher xylanase production show the potential for industrial utilization.