Gene Cloning And Expression Of Three Glycosyl Hydrolases From Bispora Antennata

Fungi of genera Bispora can secrete a variety of important glycoside hydrolases, includingxylanase, β-mannanase, β-glucanase etc, that are important enzymes for degradation of hemicelluloseand cellulose. In this study, Bispora antennata was selected as the microbial source for identification ofmore novel glycoside hydrolase genes. All the genes were expressed in Pichia pastoris, and therecombinant enzymes were purified and characterized.Through degenerate PCR and TAIL-PCR techniques, a xylanase gene xyn11，a-mannanase geneman5and a-glucanase gene cel5were cloned from B.antennata. Homology searches and sequencealignment analysis indicated that the deduced amino acid sequence of xyn11exhibited the highestidentity of74%with a family11endo-β-1,4-xylanase from Alternaria sp. HB186, multiple sequencealignment indicated that deduced Xyn11had9N-terminal residues more than its thermostable andthermophilic counterparts, deduced MAN5exhibited the highest amino acid sequence identity of58%to a-mannanase of glycoside hydrolase family5from Aspergillus aculeatus, and deduced Cel5had64%identity with the Botryotinia fuckeliana endo-β-1,4-glucanase. Further homology modeling andcatalytic residue analysis revealed that these genes had certain sequence and structure novelty.Recombinant Xyn11had an optimal pH at5.5, and retained30.0%of the maximal activity at pH3.0and8.0. Xyn11was stable at pH2.0–12.0, retaining more than85%relative activity. It exhibitedmaximal activity at35°C and remained21%of the activity at0°C. Xyn11had poor thermostability. Itretained81%of the initial activity at30°C for60min, but lost all the activity at40°C for30min.Xyn11was the first reported cold-active xylanase of glycosyl hydrolase family11. To study the effectsof the extra9N-terminal residues, Xyn11mutant version without the extra sequence, t-Xyn11, was thenconstructed and compared with Xyn11. Both enzymes showed similar properties in pH optimum,temperature optimum and pH stability, but varied in thermostability. After pre-incubation at40°C for20min, Xyn11retained28%of the initial activity but t-Xyn11was completely inactivated. The specificactivities of purified Xyn11and t-Xyn11towards birchwood xylan were203.8U/mg and214.5U/mg,respectively. The K_mand V_max values of Xyn11were1.65mg/ml and236.3μmol/min/mg, respectively,when using birchwood xylan as the substrate. With beechwood xylan as substrate, K_mand V_max valueswere1.73mg/ml and276.6μmol/min/mg, respectively.Recombinant Man5produced in P. pastoris showed optimal activity at pH6.0and retained21%activity at pH2.0and10.0. It had good stability over a broad range of pH values, retaining more than80%of activity after incubation at37°C for1h at pH4.0–11.0. The enzyme had a temperatureoptimum of70°C, and was stable at60°C, retaining72%relative activity after60min. The specificactivity, K_mand V_max values were288.9U/mg,1.33mg/ml and444μmol/min/mg, respectively, forlocust bean gum and135.5U/mg,1.17mg/ml and196μmol/min/mg, respectively, for konjac flour.Man5was highly resistant to trypsin and most metal ions.The recombinant Cel5showed optimal activity at pH5.0and55°C, and retained95%activityafter preincubation at37°C for60min. Cel had strong resistance against trypsin. The specific activity,K_mand V_max values were70.6U/mg,10.6mg/ml and560.2μmol/min/mg for barley-glucan and68.7 U/mg,3.2mg/ml and147.4μmol/min/mg for CMC-Na, respectively. Under simulated barley flourdegrading conditions, addition of Cel5（100U） to the chyme significantly reduced the viscosity by7.2%.In summary, three glycoside hydrolase genes have been cloned from B. antennata, and theirrecombinant enzymes had good properties. Of them, Xyn11represents a good material to study thefamily11cold active xylanase, and Man5and Cel5have good prospects for application in the feedindustries.