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 model describing the solubilization of arabinoxylans and degradation by endo-xylanase random attacking during mashing was developed. The model was expected to predict the arabinoxylans concentration in wort at the settings of different initial value and mashing parameters for diminishing the negative effects of arabinoxylans on brewing. Results showed that the modeling errors range for the final concentration of arabinoxylans in wort was -9.5% to +13.6%. The model prediction accuracy for industrial scale mashing process was lower than that in laboratory scale. The errors were given 16.8% and 17.9%, respectively. The simulation results showed that arabinoxylans concentration was increased with the increase of mashing-in temperature, but it was decreased with prolonging the mashing-in time. The effect of initial arabinoxylans in malt on arabinoxylans concentration in wort was more remarkable than that of endo-xylanase activity in grist.
Endo-1,4-beta Xylanases ; metabolism ; Enzyme Activation ; Fermentation ; Models, Chemical ; Solubility ; Xylans ; metabolism

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.

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 can hydrolyze xylans into xylooligosaccharides and D-xylose, and has great prospect for applications in feed industry, paper and pulp industry, food industry and environment science. The study of xylanase had been started in 1960's. With the development and application of the new technologies, such as molecular biology, structural biology and protein engineering, many progresses have been made in the research of structures and functions of xylanase. This paper reviews the research progress and trend in the structure correlating with the important properties of xylanase. Analyses of three-dimensional structures and properties of mutants have revealed that glutamine and aspartic acid residues are involved in the catalytic mechanism. The thermostability of xylanase correlated with many factors, such as disulfide bridges, salt bridges, aromatic interactions, cotent of arginine and proline, and some multidomain xylanase have thermostability domains in N or C terminal. But no single mechanism is responsible for the remarkable stability of xylanase. The isoelectic points and reaction pH of xylanase are influenced by hydrophobicity and content of electric charges. Many researches had demonstrated that aromatic amino acid, histidine, and tryptophan play an important role in improving enzyme-substrate affinity. The researches of structures and functions of xylanase are of great significance in understanding the catalytic mechanism and directing the improvement of xylanase properties to meet the application requirement.
Catalysis ; Endo-1,4-beta Xylanases ; chemistry ; metabolism ; Enzyme Stability ; Protein Engineering ; Substrate Specificity

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

Microbial xylanases have received a great deal of attention in the last two decades for their potential applications in food, paper making and animal feed industries. Bacillus pumilus WL-11 was identified as a producer of alkane xylanase free of cellulase after screening soil samples of paper-making factories. The xylanase A (XylA) was purified to homogeneity from the culture filtrate of Bacillus pumilus WL-11 by (NH4) 2SO4 precipitation, CM-Sephadex and Sephadex G-75 chromatographies. The molecular mass of XylA is estimated to be 26.0 kD by SDS-PAGE and its isoelectric point is 9.5. The apparent Km is 16.6 mg/mL and V(max) is 1263 micromol/(min x mg) towards oat spelt xylan. XylA is optimally active between pH 7.2 and 8.0, and stable at pH 6.0 to 10.4. The enzyme is optimally active at 45 degrees C - 55 degrees C and stable at temperature below 45 degrees C, with its half time of activity of 35 min and 15 min at 55 degrees C and 60 degrees C respectively. HPLC analysis revealed that hydrolysis patterns of xylans from oat spelt, birch wood and beech wood by purified XylA were different. The XylA is determined to be an endo-beta-1,4-xylanase, as it generated mainly xylotriose and no xylose was detected among the three hydrolysates. XylA has strong hydrolytic activity towards the pentose in the hydrolysates of beech wood and birch wood xylans, but was not active to the pentose in the hydrolysate of oat spelt xylan. The crude WL-11 enzyme can efficiently hydrolyze oat spelt xylan to a series of xylo-oligosaccharides, suggesting its potential application in nutraceutical industry.
Bacillus ; classification ; enzymology ; Bacterial Proteins ; metabolism ; Culture Media ; Endo-1,4-beta Xylanases ; isolation & purification ; metabolism ; Substrate Specificity

The endo-1,4-xylanase gene from Bacillus pumilus HB030 was cloned into the Pichia pastoris expression vector, pPIC9k, the recombinant plasmid was named pHBM220. The digested recombinant plasmid pHBM220 was transformed into Pichia pastoris KM71, GS115, SMD1168, respectively. The recombinant Pichia pastoris KM71 (pHBM220), GS115 (pHBM220), SMD1168 (pHBM220) secreted functional endo-1,4-xylanase, and the enzymatic activities reached 10.80IU/mL, 11.63IU/mL, 9.68IU/mL, respectively. The temperature and pH optimum for the recombinant xylanase were 60 degrees C and pH5.5, respectively.
Bacillus ; enzymology ; genetics ; Electrophoresis, Polyacrylamide Gel ; Endo-1,4-beta Xylanases ; genetics ; metabolism ; Hydrogen-Ion Concentration ; Pichia ; genetics ; metabolism ; Temperature

After the cell enters into its programmed cell death, xylanases from grass plants gradually matured through its N-terminal and C-terminal sequence been cut by acid proteases several times. They could not be expressed by conventional protein expression system. Search the GenBank database, xynIII from a mutant of T. reesei QM9414(ATCC26921)was found. It is similar to grass plants' xylanase in their families and structures. It couldn't express in T. reesei QM9414, but its gene exist in genomic DNA as one copy. Through overlap-PCR method, 4 exons of xynIII were cloned, sequenced, spliced, and the whole cDNA of mature xynIII was acquired. The cDNA was inserted into pETBlue-2 vector and transformed into E. coli DE3 pLacI cell. Xyn III could be expressed in the transformed cell under the conditions of 37 degrees C, 1 mmol/L IPTG induced for 3h. Low temperature (15 degrees C), long time(64h) induction(0.2 mmol/L IPTG) could enhance xynIII activity.
Cloning, Molecular ; DNA, Complementary ; chemistry ; Endo-1,4-beta Xylanases ; genetics ; metabolism ; Polymerase Chain Reaction ; methods ; Trichoderma ; genetics

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