Properties Of Xylanase Inhibitor Protein From Wheat And Its Influence On Efficacy Of Exogenous Xylanases

?-1,4-endo xylanase can alleviate the unfavorable effects of xylan in cereals,such as barley,wheat and wheat bran.However,high levels of xylanase inhibitor protein in wheat decrease xylanase activity added into wheat processing,resulting in incomplete xylanase catalytic action.In this study,we investigated the inhibition of inhibitory proteins to resistant and sensitive xylanases.Detailed results are as follows:(1)Purification and inhibitory properties of wheat inhibitory protein in wheat seed.In different cereals,there are higher xylanase inhibitor activity in wheat,rye,buckwheat and green wheat,but lower activity in oats and barley;the inhibitor activity cannot be detected in rice,corn and sorghum.The xylanase inhibitory protein from wheat variety Xiaoyan 22 was purified.The results showed that the inhibitor has a molecular mass of approx 29 kDa.Its N-terminal amino acid sequence is not similar to those of other xylanase inbititors,however,with higher similarity to chitinases in barley.The protein had no chitinase activity,and strongly inhibited the activity of Aspergillus niger GH11 family xylanases,but insensitive to GH10 family xylanases.Therefore,these properties indicated that this inhibitory protein probablly be a novel XIP-type xylanase inhibitor.The optimum reaction temperature and time between the inhibitor protein and GH11 family xylanase were 30 ? and 30 min,respectively.The half-inactivation temperature of the inhibitory protein was 74 ?,and the inhibitory protein had good thermal stability.(2)Cloning,expression and inhibitory properties of wheat inhibitory protein gene.We cloned two XIP-type inhibitor genes(xip-9023 and xip-366)from wheat(cv.Zhengmai 9023 and Zhengmai 366)immature embryo using RT-PCR method.Compared to previously cloned XIP-I,amino acid sequence of xip-9023 was 100%identical and xip-366 was different in one amino acids,which indicated that there were slight differences in XIP gene among different wheat cultivars.XIP-9023 expressed in Pichia pastoris showed the optimal inhibition pH and temperature were 7 and 40 ?,respectively.Inhibition of the inhibitor to xylanase reached the maximum in 40 min.When the xylanase activity was inhibited by 50%,the molar ratio of inhibitor protein to xylanase was 26:1.The inhibitor was active to various fungal xylanases tested,and it was found for the first time that a bacterial xylanase produced by Paenibacillus sp.from cow rumen could be strongly inhibited by XIP-9023.(3)Construction and screening of resistant xylanase mutants.30 single point mutants were designed based on the interaction site of GH10 xylanase and XIP inhibitor.By analyzing the short peptides structure of bacterial and plant GH10 xylanase,which were important to substrate binding,the whole sequences of short peptides were substituted into the corresponding position of Aspergillus niger GH10 xylanase,and finally 22 mutants were obtained.However,the activity of most of xylanase mutant were significantly decreased or even completely lost.Since the XIP inhibitory protein was a competitive inhibitor,it competed with the substrates for binding to the same sites in xylanase.Therefore,it may not be a viable mutation strategy to obtain a XIP-resistant GH10 family xylanase by mutation of the substrate-binding sites,which can bind to the substrates but not to inhibitory protein.(4)Screening and characterization of xylanases resistant to XIP inhibitor proteins.The xylanase-producing strains in our laboratory and the xylanases products in the market were studied.It was found that the crude xylanases from fungi were inhibited by XIP-9023 and the inhibition rate varied from 24%to 70%,which was consistent with the known results.For the first time,we found that the xylanase from Streptomyces sp.was strongly inhibited by XIP-9023,and the xylanase from rumen anaerobic fungus Neocallimastix sp.had obvious resistance to XIP-923 inhibitory protein,with 6%inhibition rate.The purified Neocallimastix sp.xylanase had strongly risistance to XIP inhibitor at equivalent molar ratio.The results of mass spectrometry analysis were consistent with sequence of Q9UV68,Q01426,and Q69IG9 xylanase in UniProt database.Only the synthesized Q69IG9 xylanase XYN3 desmontrated xylanase activity.XIP-9023 had no inhibition to recombinant xylanase XYN3 in different molar proportion,thus the xylanase XYN3 was finally identified as the resistant xylanase.The optimum reaction conditions for the resistant enzyme XYN3 were pH5.4 and 60 ?,respectively.The xylanase activity of resistant enzyme XYN3 at pH 3.6 remained 72%of maximum activity,and above 80%after incubation at 40 ? for 6h,so the resistant enzyme XYN3 had better acid adaptability and thermal stability.The recombinant resistant xylanase XYN3 was aligned with A.niger sensitive xylanase(UniProt accession no.P55328)and Penicillium funiculosum sensitive xylanase(UniProt accession number Q9HFH0),and it was found that the amino acid sequence of the "thumb" part of resistant xylanase XYN3 was mutated from GTS to GGSE.(5)Enzymatic hydrolysis of different proportions of wheat-corn mixture by resistant xylanase XYN3.In the proportions of 20%-60%of wheat,the reducing sugar yield of resistant xylanase XYN3 was always higher than that of sensitive xylanase at 42? or 39?,which indicated that the resistant xylanase was more effective than the sensitive xylanase in degrading wheat xylan.When the proportion of wheat was 20%,the total hydrolysis sugar content by resistant xylanase was 3.239 g/L,8.36%higher than that of sensitive xylanase,and especially the xylobiose content increased by 45%.When the wheat proportion was 40%,xylooligosaccharides and xylose in xylan degradation products increased by 6.5%,of which the xylobiose content in the enzymatic hydrolyzate of resistant xylanase XYN3 increased by 89%than that of sensitive xylanase.The amount of reducing sugar produced by the resistant xylanase XYN3 at 39 ? was also higher than that of the sensitive xylanase when adding the pig digestive juice in the enzymolysis reaction system,with the maximum increase of 23%.Therefore,the addition of resistant xylanase XYN3 in feed wheat had a better effect than the sensitive xylanase.