Efficient Expression And Characterization Of Two Xylanases From Aspergillus Niger XZ-3S In Pichia Pastoris

BackgroudXylanase [EC3.2.1.8] is the most critical enzyme in the department of xylan degradation enzymes, which can degradate the xylan to xylose and low polyxyloses. In recent years, xylanase has very high economic value. xylanase has been widely used in environment, food, feed, energy, paper and other industries. Many microorganisms can produce xylanase, such as bacteria, fungi, actinomycetes and yeast, et al. Different sources and different kinds of xylanase shows different catalytic properties. Using various means to improve the enzyme production and reduce the production cost is the problem to be solved.ObjectiveTo clone the xylanase genes into the eukaryotic expression vector and efficient expresse them in Pichia pastoris. In addition, the enzymatic properties and hydrolytic products of the recombinant xylanase will be studied.Methods1. Construction of pichia pastoris engineering bacteria With xylanase gene xynZF-2 and xynZF-3 cloned from xylanase high-yield strains A.niger XZ-3S strain as the research object, restructured them to eukaryotic expression vector pPIC9K to construct recombinant plasmid. The recombinant vectors were transformed into Pichia pastoris GS115 and KM71 respectively by electroporation after linearization. Four high yield strain were got after G418 gradient and shake flask screening.2. Optimization of fermentation condition of restructuring xylanaseUsing single factor experiment, inoculation time, induction temperature, initial pH, methanol concentration, fermentation induction time were optimized. Then the response surface was carried on to determine the best fermentation conditions.3. The analysis of the enzymology properties of recombinant enzymesThe effects of temperature and pH on xylanase activity and stability ,and the influence of metal ions on xylanase activity were studied. The enzyme kinetics constants of the recombinant enzymes were determined, and the hydrolysis products were analyzed by thin-layer chromatography.Results1. Four kinds of xylanase engineering bacteria were built successfully. Four xylanase high-yield pichia pastoris strains, named GS 115/pPIC9K-2, S115/pPIC9K-3, KM71/pPIC9K-2, KM71/pPIC9K-3, were obtained by antibiotics screening and shaker screening.2. The best optimization scheme were obtained by single factor experiment analysis and L9(34) experiment. The optimal fermentation conditions of the GS 115/pPIC9K-2 were methanol concentration 1.0%, inoculation time 31 h, induction time 104 h, induction temperature 30℃, initial pH 4.0; KM71/pPIC9K-2 were methanol concentration 2.0%, inoculation time 26 h, induction time 132 h, induction temperature 30℃, fermentation starting pH 6.2; GS115/pPIC9K-3 were methanol concentration 2%, inoculation time 24 h, induction time 108 h, induction temperature 30℃, initial pH 6.3; KM71/pPIC9K-3 were methanol concentration 1.75%, inoculation time26 h, induction time 132 h, induction temperature 30℃, initial pH 6.5. Under the optimum conditions, the xylanase activity of the four recombinant strains could be 36523.2 U·mg-1、8654.28 U·mg-1、139.36 U·mg-1、 143.29 U·mg-1.3. The results of enzyme characterization experiment were as follows:the optimum temperature and pH of GS115/pPIC9K-2 were 45℃ and pH6.0; The optimum temperature and pH of KM71/pPIC9K-2 were 45℃ and pH 5.5; The optimum temperature and pH of KM71/pPIC9K-3 were 40℃ and pH 5.0; The optimum temperature and pH of GS115/pPIC9K-3 were 45℃ and pH 5.0. Fe3+, Mn2+, Pb2+, Gu2+ had obvious inhibitory effect on the enzyme, while Fe2+ and EDTA had different degrees of promoting function. The four recombinant xylanases had good temperature stability under temperature 50℃. Under pH 5.0-7.0, XynZF-2 was stability, while the enzyme activity of xynZF-3 was relatively stable between the pH 5.0-6.0.4. According to the results of thin-layer chromatography, the hydrolysis products of birchwood xylan hydrolysised by XynZF-2 were xylobiose(X2) and xylopentose(X5) towards birchwood xylan, so the recombinant xylanase was endo-xylanase.5. The enzyme kinetics constants of the four kinds xylanase were measured by double inverse mapping method respectively:Km were 1.5 mg·L-1,1.25 mg·L-1,3.56 mg·L-1 and 3.04 mg·L-1. Vmax were 2500 μmol·mL-1·min-1,2680 μmol·mL-1·min-1,1368 μmol·mL-1-min-1 and 1589 μmol·mL-1·min-1ConclusionsThe two xylanase gene cloned from Aspergillus niger XZ-3S were successfully heterologous expressied in Pichia pastoris GS115 and KM71. The enzyme production exprssed in Pichia pastoris was higher than the expression of prokaryotic cells. And the thermal stability and pH stability of the recombinant enzyme were better than that of prokaryotic expression recombinant enzyme.