| The plant cell wall consists mainly of cellulose, hemicellulose, lignin. Hemicellulose, as the second most abundant renewable organic carbon on earth, makes an important role in maintaining integrity of plant cell wall. Xylan is the predominant component of hemicelluloses. Complete breakdown of xylan requires a variety of hydrolytic enzymes cooperation, among them xylanase was a key enzyme in the xylanolytic enzyme system. In recent years, xylanase becomes more attractive due to its biotechnological applications in the food, animal feed, and pulp&paper industries. However, it is necessary keeping higher activity and better thermostability at high temperature for enzyme in industrial application. Therefore, it is a hotspot issue to look for xylanase with thermostability.The thermophilic fungi Humicola insolens Y1we used in this study was isolated from forest soil sample of Hebei, it could produce many kinds of cellulase and hemicellulase. Based on the degenerate primers and fusion primer and nested integrated PCR, four GH10xylanase were cloned from the gene of Humicola insolens Y1, named xynA, xynB, xynC, and xynD, respectively. They deduced amino acid sequences had the identity of71%～83%to known fungal xylanases and of≤56%with each other. The homology-modeled structures of the four xylanases displayed a classic (β/α)8barrel fold, and had more alkaline residues located on the protein surface. All of them consist of GH10catalytic domain, and XYNC had a family1CBM at the N terminus.After construction of the eukaryotic expression vector, the four xylanases were successfully expressed in Pichia pastoris GS115, and all of them were purified for enzyme characteristics study. The XYNA-D showed similar pH characteristics, and the maximal activity at pH6.0-7.0, the optimum temperature is70℃～80℃, all of them had good pH and temperature stability, they could catalyzed a variety of substrates, and resist to metal ions, chemical reagent and trypsin. Among them, XYNA’s optimum temperature was80℃, still maintaining more than50%of the maximal activity after pre-incubation in70℃for10min; The optimal pH was6.0, it was alkaline-tolerant, having70%of the maximal activity at pH9.0, and remained stable at pH3.0～10.0; even at5mmol/L SDS, XYNA can keep more than60%maximum activity; no activity was detected in the presence of CMC-Na and barlyβ-gluan; They were xylobiose and xylotriose in the hydrolysis products of XYNA, but no xylose. Three xylanase XYNB-D displayed the optimum tempreture was70℃, all of them stable at60℃for pre-incubation; XYNB had maximal activity at pH7.0, the others were6.0, they exhibited good stability within the neutral and alkaline ph range. Used birchwood xylan as substrate, the Km were1.6,1.1,2.1,1.9mM, and the Vmax. were974.4,306.8,287.7,482.7μmol/min/mg.Under simulated mashing conditions, addition of XYNA-B showed better performance on filtration acceleration and viscosity reduction than commercial enzyme. Especially for XYNA, it can increase filtration rate more than35.6%, and decrease viscosity about11.7%. In addition, comparing to the single enzyme, there was more enzyme activity when three enzymes were mixed together. It indicated that the xylanases we obtained were suitable for brewing industry.In conclusion, four new GH10xylanase cloned from Humicola insolens Y1, which were higher enzyme activity and stablity at high temperture, alkaline and neutral pH, and had better resistance to metal ions, had obvious effect in application experiment. Thus they represent good candidates for application in the brewing, baked food, and pulp and paper industry. |
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