Cloning And Expression Of Endoxylanase Inhibitor TAXI-Ⅰ In E.coli And The Properties Of The Recombinant TAXI-Ⅰ

β-1, 4-xylanases (EC 3.2.1.8) can 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, substances which inhibit the hydrolytic activity of endoxylanases in cereals affect the functionality and performance of the enzymes. In this study, the Triticum aestivum xylanase inhibitor I (TAXI-I) was cloned and expressed by Escherichia coli BL21. Xylanase inhibitor activities against xylanase were analysed with a purpose to provide a theoretical basis for an accurate screening of xylanase. The main results were as follows:The Triticum aestivum xylanase inhibitor I (TAXI-I, AJ438880) gene was cloned according to secquence information in GenBank. The fragment containing the TAXI-I region was amplified by Polymerase Chain Reaction (PCR) from pUCm-T TAXI Vector by using pyrobes^TM DNA Polymerase and a pair of primes 5TAXI02E/3TAXI02E which were designed by analyzing the restriction site mapping of TAXI and the multiple cloning site (MCS) of vector pET-30a(+), so introducing EcoRI site at 3-end and XhoI site at 5-end of the gene. The resulting PCR product (1175bp) was subcloned into pUCm-T Vector, the positive clone was double digested by EcoRI and XhoI, the DNA insert was ligated into the EcoRI-XhoI-cut site of pET-30a(+), constructing recombinant vector pET-30a(+)-TAXIE. The recombinant pET-30a (+)-TAXIE was transformed into Escherichia coli BL21 and the recombinants were obtained.The specificity of TAXI-I indicated that xylanase inhibitor had a molecular weight of 46.7KDa, and the activity was at its lowest at 20℃. The inhibitor activity ascended fast during 30min at 20℃, while its activity had no significant diversification in 30min. The activity increased gently after being treated during 0℃-70℃for 30min. TAXI-I inhibit different endoxylanases to varying extents. High inhibition was found against fungal endoxylanases(ANX and TrXA) as well as low inhibition against bacterial endoxylanases (TFX,BtXA and MHX). In addition, the results indicated that xylanase activities of ANX and TFX remained 15.8% and 88.4%, respectively. So TFX xylanase is the optimum screen in this experiment.