| Xylan is the most abundant, naturally occurring hemicellulosic polysaccharide. Endoxylanases play a key role in the biodegradation of xylan. Extremophilic xylanases, such as thermophilic, halophilic and basophilic xylanases, could be applied in a wide variety of industrial processes, including food processing, papermaking, hightemperature pelleting of feed, and washing. Therefore it is of great significance for the development and modification of extremophilic xylanase. The present study focused on the characterization of Xyn AGN16, a glycosyl hydrolase family 10(GH10) multidomain xylanase obtained from Arthrobacter sp. GN16 isolated from the feces of the Grus nigricollis. The thermo-salt adaptation of the xylanase is modified combining the method of ep PCR and DNA family shuffling. The main results were as follows:1 Heterologous expression and characterization of xylanase Xyn AGN16The multidomain xylanase Xyn AGN16 consists of a catalytic domain of GH10(Xyn AGN16L), a polysaccharide deacetylases(PD) domain, a carbohydrate binding domain, a catalytic domain of GH10, and a hyalin repeat domain, in order from the Nterminus. Xyn AGN16 and its truncated derivatives Xyn AGN16 L and Xyn AGN16Lpd(Xyn AGN16 L plus PD domain) were successfully expressed in Escherichia coli and characterized. Analyses of biochemical characterizations revealed that three xylanases were typical low-temperature-active enzymes that showed 15% relative activities at 0 °C and thermolabilities at temperatures of more than 37 °C. Xyn AGN16 L and Xyn AGN16 Lpd retained lower than 35% xylanase activity at 30%(w/v) Na Cl. Comparisons of the substrate specificity and kinetics of Xyn AGN16 L and Xyn AGN16 Lpd revealed that the PD domain could not effectively improve the activity of esterase, however, it could improve the affinity and catalytic efficiency.2 Modification of Xyn AGN16L’s thermo-salt adaptationXyn AGN16 L and three GH10 endoxylanase from previous work were used as the four parents in ep PCR and DNA family shuffling. 10,000 mutants were randomly selected from plates and subjected to be screened at 70 oC in the presence of 15%(w/v) Na Cl. As a result, the thermo-salt activities of 24 mutants were changed. The two mutants, s27b05 and s02b12, were homologous to Xyn AGN16 L and selected for subsequent analyses.3 Biochemical characterization of mutantsMutants s02b12 and s27b05 and wild-type Xyn AGN16 L were purified and characterized. The results revealed that mutants s27b05 and s02b12 really increased and decreased thermo-salt adaptation, respectively.(1) The thermal activity of purified s27b05 was apparently optimal at 55 oC, and the enzyme showed 34.6% and 43.2% activities at 0 oC and 80 oC, respectively. While wild type Xyn AGN16 L was optimal at 45 oC, and showed 20.0% and 1.7% at 0 oC and 80 oC, respectively. s27b05 exhibited 87% of the initial activity after incubation at 37 oC for 1 h, whereas Xyn AGN16 L exhibited 47% of the initial activity. s27b05 and Xyn AGN16 L retained 53.1% and 34.6% xylanase activity at the concentration of 30%(w/v) Na Cl, respectively, and 92.6% and 73.5% of the initial activity after incubation with 30%(w/v) Na Cl at 37 °C for 60 min, respectively.(2) Thermostability of s02b12 decreased rapidly at 37 °C, the half-life of the enzyme at 37 °C was less than 5 min. In the presence of 10.0 m M Ca Cl2, the s02b12 and Xyn AGN16 L remained appropriately 64.3% and 106.0% activities, respectively.In summary, this study revealed a multidomain xylanase Xyn AGN16 with lowtemperature-activity and moderate tolerance to salt, and found that its PD domain had the function of improving the affinity and catalytic efficiency of the enzyme toward xylans. Combining the methods of ep PCR and DNA family shuffling, two mutants each increased and decreased thermo-salt adaptation of Xyn AGN16 L were obtained. This study led to broaden the scope of the application potential of Xyn AGN16 L, and offer foundation for the study on the molecular mechanism of thermo-salt adaptation of the GH10 xylanase. |
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