Purification, Characterization, And Application Of Thermostable Xylanases From Malbranchea Cinnamomea

Lignocellulose degrading enzymes systems from microorganisms have attracted considerable research interest in recent years because of their potential applications in food, paper and pulp industries. The newly isolated thermophilic fungus, Malbranchea cinnamomea can produce diverse extracellular thermostable xylanases using lignocellulosic materials as carbon source. In the present dissertation, the production, purification, characterization and application of thermostable xylanases from M. cinnamomea S168were investigated. Meanwhile, production of xylooligosaccharide (XOS) from the autohydrolysis liquor of rice straw by enzymatic hydrolysis with thermostable xylanases were also studied. The mainly conclusions are as follows:Optimization of culture conditions for production of thermostable xylanases by M. cinnamomea S168using response surface methodologyFermentation conditions for xylanases production of M. cinnamomea S168was determined by response surface methodology (RSM). Single factor experiment was first adopted to determine the optimal carbon source and nitrogen source. Plackett-Burman design (PBD) was further adopted to screen the most important factors affecting the production of xylanases. Then, the Box-Behnken design (BBD) were adopted for further optimization, the optimum concentration levels and the relationships among these factors was found by quadratic regression model equation with Minitab statistic methods. The optimal fermentation conditions were obtained by Box-Behnken design and response surface analysis. Under these conditions, the xylanase acitivity reached646.0U/mL, which was increased by3.14folds compared with that (156.3U/mL) was obtained before optimization.Purification and characterization of McXyn32from M. cinnamomea S168A family10xylanase, McXyn32, from the thermophilic fungus M. cinnamomea S168was purified and characterized. McXyn32was most active at pH7.0and70℃, and it showed wide ranges of pH stability and excellent temperature stability. McXyn32showed highest specificity towards xylan but not towards other substrates. It hydrolyzed xylan to release mainly XOS, indicating that the xylanase was endoxylanases. These results demonstrate this xylanase has potential ability to produce XOS.Purification, characterization and cloning of McXyn44from M. cinnamomea S168Another xylanase, McXyn44, with a molecular mass of43.5kDa on SDS-PAGE was purified from M. cinnamomea S168. Amino acid sequences of eight internal peptide fragments of McXyn44were obtained by ESI-Q-TOF2. Seven peptides derived from McXyn44showed100%identity with some glycoside hydrolase (GH) family10xylanases, however, a peptide did not display any significant similarity with other xylanases, suggesting that McXyn44is a novel member of GH family10xylanases. The optimal pH and temperature of McXyn44were pH6.5and80℃, respectively. The enzyme showed a broad range of pH stability (pH3.5-10.5), and was stable up to70℃with90%retained after incubation for30min. The enzyme exhibited strict specificity for various xylans as substrates. McXyn44hydrolyzed xylans into XOS mainly. To clone the xylanase gene (McXyn44) of McXyn44, M. cinnamomea genomic DNA was used as the template, and the degenerate primers Xyn40DF and Xyn40DR were designed according to the conserved sequences of GGDVVAN and YYNDYNIEH, respectively, derived from the purified xylanase by MS using the CODEHOP algorithm. The full-length cDNA sequence of McXyn44was obtained by assembling5’and3’gene sequences. The3’-terminal sequence was obtained by rapid amplification of cDNA ends (RACE) using a SMART RACE cDNA Amplification Kit. The5’sequence was obtained by hiTAIL-PCR. The xylanasase gene(McXyn44) with a full length of1652bp was cloned from M. cinnamomea by PCR. There were two nontranslated regions (of103bp and210bp) at the5’and3’terminuses of the gene, respectively. McXyn44has an ORF of1191bp, encoding396amino acids. The N-terminal region of the deduced protein contains a predicted19-amino acid signal peptide. The mature protein has a predicted molecular mass of40kDa and a theoretical p1of4.37. Amino acid sequence alignment of McXyn44revealed the highest sequence identity of99%with a GH family10xylanase from M. pulchella.Production of XOS from rice straw by thermostable xylanases with hydrothermal processingThe main components were xylobiose and xylotriose of the liquid fraction of rice straw by McXyn25(from M. cinnamomea) hydrolysis. Under the optimized conditions (pH7.0,60℃,5U/mL and2h), the production of XOS was7.7%. A xylanase, PtXynA, from Paecilomyces thermophila was used to hydrolyze rice straw with hydrothermal for the production of XOS. The main products of hydrolysis were xylobiose and xylotriose, and the yield of XOS was8.7%. Xylobiose and xylotriose were the main product from rice straw (pretreated by strongly acidic electrolyzed water) using a two-stage process based on a hydrothermal pretreatment followed by XynBop hydrolysis. For enzymatic hydrolysis under optimum conditions, XOS yield with respect to rice straw was10.8%after1h. XOS syrup contained more than73.6%of xylobiose from rice straw (pretreated by distilled water) when the hydrolysis was carried out by XynBop under the optimized conditions. The purity of xylobiose was93.7%after the separation of the hydrolyzate by active carbon column chromatography.