| Xylanase and Xylosidase, two major components of xylanolytic enzymes, have the potential to be applied in the process of lignocellulose degredation, since they have excellent synergy with cellulases to increase glucose production. To solve these problems such as low productivity and low enzyme activity of xylanase and xylosidase, the present thesis focused on the respective overexpression of the xylanase and xylosidase with heterologous expression by constructing a recombinant Pichia pastoris, in which production conditions and enzymatic properties were studied.The high-yield recombinant P. pastoris with a high xylanase and xylosidase was obtained by two-step electroporation, respectively. According to the codon usage frequency of highly expressed genes in P. pastoris, the Aspergillus niger xylanase(XynC) and Thermomyces lanuginosus xylosidase(Xyl43) gene codon were firstly optimized. Then, we constructed two recombinants P. pastoris containing the pPIC9 K expression vector, respectively, labled as the sigle-plasmid recombinant P. pastoris(P. pastoris GS115-S-XynC and P. pastoris GS115-SXyl43). Subsequently, the second expression vector pPICZαA harboring the same target gene was transformed into the sigle-plasmid recombinant P. pastoris with the high enzyme activity, respectively. Finally, two double-plasmid recombinant P. pastoris(P. pastoris GS115-D-XynC and P. pastoris GS115-D-Xyl43) with a high enzyme activity were obtained.Basic enzyme properties of XynC were analyzed as below: the XynC molecular weight 35.5 kDa, the optimal reaction temperature 55 °C, the optimum pH 5.0, kinetic parameters Km = 3.5 g·L-1 and Vmax = 2327 μmol·min-1·mg-1, excellent thermostability at below 50 °C and good pH stability(pH 4.5–7.0). And the basic enzyme property of Xyl43 was detected as follows: the Xyl43 molecular weight 51.5 kDa, the optimal reaction temperature 55 °C, the optimum pH 7.0 with a good stability(pH 6.0–9.5), and kinetic parameters Km = 2.93 mmol·L-1 and Vmax = 157.9 μmol·min-1·mg-1. The XynC activity was enhanced by low Mg2+ concentration, and the Xyl43 activity was improved by the Ba2+ and Mn2+. However, Cu2+ and organic solvent(ethanol, isopropyl alcohol and n-butyl alcohol, etc.) showed an obvious inhibition on XynC and Xyl43. In addition, the detergents, Tween20 and Tween80 also exhibited a positive role on XynC and Xyl43 enzyme activity.The XynC and Xyl43 production was carried out at a shake-flask level with an optimization of some key fermentation conditions by one-factor test, including the composition of fermentation medium, initial induction time(seed age), inoculum size, initial pH, methanol supply, incubation temperature, agitation and accessory carbon source and additives. Then, some key factors(intial pH, methanol supply, incubation temperature and accessory carbon source) were further optimized by orthogonal experiment. Under optimized fermentation conditions, the maximum XynC and Xyl43 activity reached 415 U·m L-1(10 d) and 62 U·m L-1(6 d),with 0.39 g·L-1and 0.76 g·L-1 of the protein content, respectively.The XynC production was further investigated in a 5-L fermenter. The initial inductioncell density of the double-plasmid recombinant P. pastoris was optimized for the fed-batch cultivation. The XynC reached a maximum productivity of 1.58 g·L-1 with an initial cell density of OD600 = 300, at 108 h of methanol induction, meantime the XynC activity was up to 1660 U·m L-1, about 4 folds that in the shake-flask fermentation. At the same initial cell density(OD600 = 300), Xyl43 reached 222.2 U·m L-1 at 96 h(methanol induction), with protein concentration of 2.36 g·L-1, which was 2.6 folds that at the shake-flask level. Accordingly, the xylanase(XynC) and xylosidase(Xyl43) are of promise to be expressed in P. pastoris GS115 with a high production level, which can supply an important foundation for their industrial production and application. |
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