| β-1,4-xylanases(EC 3.2.1.8) catalyze the hydrolysis of xylan(the major constituent of hemicellulose) and play a significant role in nature by recycling hemicellulose. They are widely applied in animal feed and food industries and in biobleaching. However, the yield of xylanases" is one of the factors limiting the economic feasibility of the process. Recombinant DNA techniques offer the means to enhance protein production. In this study, the cDNA encoding xylanase ANX of Aspergillus niger ?6 and xylanase TFX gene of Thermomonospora fusca TF were expressed in Pachia pastoris GS115 , and the recombinants of P. pastoris 9KANXE01, 9KANXE02 and 9KTFXE were screened. The biochemical characteristics of products from 9KANXE01 and 9KTFXE were analysed. The main results were as follow:The cDNA fragment containing the ANX-coding region was amplified by Polymerase Chain Reaction (PCR) from pGEM?T easy ANX Vector by using pyrobes?DNA Polymerase and a pair of primes(5ANXE04/3ANXE04) which were designed by analyzing the restriction site mapping of ANX cDNA and the multiple cloning site(MCS) of vector pPIC9K , so introducing EcoRI site at 3-end and NotI site at 5-end of the gene. The resulting PCR product (659bp) was subcloned into pGEM?T easy Vector, the positive clone was double digested by EcoRI and NotI , the DNA insert was ligated into the EcoRI-Notl-cut site of pPIC9K, constructing recombinant vector pPIC9K-ANXE. The pPIC9K-ANXE linearized by restriction enzyme Bgl II was transformed into P. pastoris GS115. By clones selection and activity assays of supernatant xylanase, two P. pastoris recombinants (9KANXE01 and 9KANXE02) secreting xylanase ANX were obtained. Their xylanase activities were 291U/ml and 188.5U/ml, respectively. Xylanase molecular weight from 9KANXE01 was 23.0KDa. The recombinant 9KTFXE secreting xylanase TFX was carried out by using the procedures of 9KANXE01 as described above, but the pPIC9K-TFXE was linearized by restriction enzyme Sad. The9KTFXE xylanase activity was 195.5U/ml, and protein molecular weight was 38.8KDa. The clearing zones on RBB-plate produced by xylanases of 9KANXE01, 9KANXE02 and 9KTFXE were obvious.The pH activity curves of the both xylanases showed that optimum pH of 9KANXE01 xylanase was 5.0, and the substantial amounts of activities found at pH3.0, 6.0 and 7.0 were 92.0%, 95.7% and 93.2% of maximum activity at pH 5.0, respectively; but its activity declined to 74.7% at pH4.0. As to 9KTFXE xylanase, the optimum pH was 5.0, and the activity appeared another peak at pH9.0, a drastic decline of activity was detected at pHs below the optimum pH. The effect of temperatures on 9KANXE01 and 9KTFXE xylanases demonstrated that the optimum temperatures of 9KANXE01 and 9KTFXE xylanases were at 50C and 60 C, respectively. More than 90% activity of Both enzymes could be measured between 50C and 60C, but decreased drastically at 70 C and 80 C, only remained 47.8%, 37.7% of 9KANXE01 xylanase and 21.0%, 2.9% of 9KTFXE xylanase, respectively. In addition, the results of enzyme thermostability indicated that xylanase activities of 9KANXE01 and 9KTFXE remained 100%, 89.5%, 89.5% and 97.7%, 97.7%, 93.2% after incubating at 50C for lmin, 3min and 5min, and 89.6%, 50.8%, 50.8% and 97.7%, 88.6%, 75.0% after incubating at 60C for lmin, 3min and 5min, respectively. If both enzymes were incubated at 70C or 80 C for 1 min, their activities dropped quickly(except for 9KTFXE heated at 70 C), but were scarcely affected for an extended period.
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