Development Of A High-yield Recombinant Strain For Xylanase Production And Characterization Of The Recombinant Enzyme

To improve xylanase production, the Xyn2 gene from Trichoderma reesei Rut C-30 was successfully expressed in Escherichia coli and Pichia pastoris, respectively. Meanwhile, a hybrid xylanase gene was constructed by fusion of the thermostabilizing domain (A2) from Thermotoga maritima XynA into the N-terminal region of Xyn2, and subsequently expressed in P. pastoris. Both the native and hybrid enzymes were fully characterized. The main results were listed as following:1. Cloning and sequencing of the Xyn2 geneThe Xyn2 gene encoding the main Trichoderma reesei Rut C-30 endo-β-1, 4-xylanase was amplified by PCR from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 573-bp open reading frame that encodes a 191-amino-acid propeptide. The cDNA fragment from T. reesei Rut C-30 is 99% identical to that of T. reesei QM6α, differing for only two base pair substitutions. The two substitutions are at nucleotides 43 and 272 of the Rut C-30 Xyn2 sequence, resulting in two amino acids (Try14/Glu91) in the proprotein being different from those in the proprotein (His14/Gly91) encoded by the QM6αXyn2 gene.2. Expression of the Xyn2 gene in Escherichia coliThe Xyn2 gene was cloned into plasmids pET28α(+) and pET30α(+), and successfully expressed in E. coli BL21 under the control of strong bacteriophage T7 transcription and translation signals. The molecular weight of the recombinant protein Xyn228 and Xyn230 were estimated by SDS-PAGE to be 25 kDa and 27 kDa, respectively. The two recombiannt proteins were purified by Ni²⁺-NTA affinity chromatography and enzyme activity assay verified the protein as a xylanase. Both Xyn228 and Xyn230 present a temperature optimum at 50℃and a pH optimum at 6.0. Compared with the native Xyn2, both of them had an improved thermostability and retained more than 60% of its activity after 30 mins incubating at 60℃.3. Expression of the Xyn2 gene in Pichia pastorisThe Xyn2 gene was cloned into the expression vector pPICZαA, and successfully expressed in P. pastoris. The desired P. pastoris strain produced as high as 300 U/mL β-xylanase under the control of the methanol incucible alcohol Oxidase 1 (AOX1) promoter. And the secreted protein PTX2 was estimated by SDS-PAGE as 21 kDa. The activity of PTX2 was highest at 60℃which was 5℃higher than native enzyme. In addition, the recombinant enzyme was active over a broad range of pH 3.0-4.0 with maximal activity at pH 6.0. PTX2 was quite stable at 50℃and reatined more than 90% of its activity after 30 mins incubation at this temperature. However, PTX2 was not stable at 60℃, only 30% activity retained. Using Oat spelts xylan and Birchwood xylan, the determined apparent K_m was 0.9 mg/mL and 1.2 mg/mL, respectively. The enzyme was highly specific towards xylan and analysis of the products from xylan degradation confirmed that the enzyme was an endo-xylanase with xylobiose and xylotriose as the main degradation products.4. Construction and expression of the thermostable xylanase gene in Pichia pastorisTo improve the thermostability of T. reesei Xyn2, the thermostabilizing domain (A2) of XynA from T. maritima has been engineered into the N-terminal region of the Xyn2 gene. The hybrid gene was successfully expressed in P. pastoris, and a level of 90 U/mL was achieved. The hybrid enzyme PTXC2 produced clearly increased both the thermostability and substrate-binding capacity compared to the corresponding enzyme PTX2. The temperature and pH optimum of PTXC2 were at 65℃and 6.0, respectively. The hybrid enzyme was more stable than PTX2 at 60℃, and retained more than 74% of its activity after 30 mins incubation at this temperature. Both the hybrid and native enzyme exhibited a low affinity towards cellulosic substrates. However, the binding capacity of PTXC2 toward oat spelt xylan has been improved. These results also suggested that the N-terminal domain (A2) is responsible for both thermostability and substrate-binding capacity of the T. maritima XynA.5. Production of recombinant xylanase by small-scale fermetation and primary evaluation of its potential for industrial applications.The high-density cultivation of PX-1 was performed in a 15-L automatic fermenter. The highest expression level of 560 U/mL was achieved after 78 h fermentation, which was almost doubled than we achieved in flasks. The culture supernatant was collected and used as a liquid enzyme source. To evaluate its potental for industrial applications, the liquid enzyme (PTX2) was supplemented into a wheat-based diet in weaned piglets. Both the performance and nutrient digestibilities was determined. Results showed that the average body weight gain increased 16.9% when piglets received a diet containing 500 U/kg PTX2 . There also was a positive (0.05