Cloning、expression And Characterization Of The Xylanase Inhibitor XIP From Wheat

Xylanase inhibitor played a defensive role in plant growth,defense against the pathogens and pests. And it had an important affect on the fields of the xylanase application. In the experiment the gene xip encoding the mature protein of xylanase inhibitor obtained from Wheat variety Zhengmai 9023 was cloned,and then expressed in Escherichia coli and Pichia pastoris to investigate the characterizations of the recombinant xylanase inhibitor,the results are as follows:(1)The clone of xip gene : The RNA was extracted from the kernel’s immature embryo of Zhengmai 9023, The xip gene(Gen Banknumber:KC342667)was cloned using the RT-PCR technology.(2)The expression of the xip in Escherichia coli: The xip was inserted into the expression vector of p ET21a(+) which was expressed successfully in Escherichia coli and transformed into E.coli BL21(DE3) by inducing with IPTG at 28℃.The inhibitor was not detected.(3)The expression of the xip in Pichia pastoris : The xip gene was inserted into the expression vector of p PIC9 K and transformed into Picha pastoris GS115. Induced with methanol in 30 ℃, the concentration of methanol was 0.5%, the inhibitory activity reached the highest(71.88 IA) six days after the induction,the concentration of protein was 121.03μg/ml. The results of protein mass spectra sequencing confirmed that the protein is the XIP xylanase inhibitor.(4) The characterizations analysis recombinant xylanase inhibitor: the optimum p H was p H7, the stability also was the best at p H7; the optimum temperature was 40 ℃, the protein was stable in the range of 30-50 ℃, and the inhibitory activity sharply declined after 60 ℃;The optimum reaction time was 40 min;the inhibitory activity increased with the increase of dose of the inhibitor, the inhibitory activity had no change up to 500μl; the molar ratio of inhibitor and xylanase is 26/1 when the inhibitory activity percentage reached up to 50%; the inhibitor had inhibitory activity on both fungal and bacteria xylanase, and can inhibit Aspergillus Niger F/10 and G / 11 xylanase; the type of inhibition was competitive inhibition.