Structure Elements For Inhibitor Protein Resistance Of Xylanse Xyn1B And Its Application Experiment

The presence of a xylanase inhibitory protein in cereals theoretically has an adverse effect on the action of extra xylanase during the processing of cereal feeds or grains,and xylanase,which is resistant to protein inhibitors,has advantages in application..A GH11-family xylanase Xyn1B(Gen Bank accession number: ACG68418.1)derived from Neocallimastix sp.GMLF1 that is not inhibited by the XIP-I inhibitory protein was obtained in the early stage of the study group.Currently,the GH11 family fungus xylanase anti-XIP was obtained.The mechanism of-I is not yet clear.This study will analyze the structural basis of Xyn1B's anti-XIP-I inhibitory activity,and at the same time,in order to confirm the application advantages of this resistance enzyme,this resistant enzyme and sensitive enzyme were added to the preparation process of wort to compare the two to maltose maltose.The influence ofThrough experiments,the main conclusions of this study are as follows:(1)Determine the corresponding resistance-determining structure by sensitively mutating Xyn1 B.Comparison of the amino acid sequence of Xyn1 B with the GH11 family xylanase of the identified sequence revealed differences in the "GTS" sequence between the "GGSE" sequence at the thumb structure of Xyn1 B and the corresponding position of the GH11 family xylanase sensitive to XIP-I.obvious.Therefore,in this study,the deletion of Gly,Ser,and Glu and the mutation of GGSE?GTS were performed on the thumb structure of Xyn1 B,ie the mutants GSE,GGE,GGS,and GTS were constructed;meanwhile,in order to exclude the interference of the electrostatic interactions of adjacent amino acids,the mutants were Based on the GTS,mutations of non-conserved amino acids in the thumb structure were constructed and the mutant TA was constructed.In addition,the thumb structure of Xyn1 B was completely replaced by the thumb structure of the Penicillium funiculosum xylanase XYNC,and a mutant Qing was constructed.Anti-inhibitory assays were performed on the above mutants and it was found that the constructed mutants were not eliminated by anti-inhibition,indicating that the thumb is not a necessary and sufficient condition for Xyn1 B to acquire resistance.Although the nature of the anti-XIP-I of each mutant did not change,the enzymological properties of the mutant mutant enzyme were found to be more prominent.The Xyn1 B Km was 3.77 mg/ml and the Kcat value was 935.85 s-1.Compared to Xyn1 B,the Kcat value of GTS was increased 1.5-fold;the Km of GSE was reduced by 2.7-fold,and the Kcat/Km was increased by 1.3-fold.(2)Xyn1B has no more than 30% homology with the identified GH11 family fungal xylanase sequences,so the crystal structure of the analytically-coupled Penicillium funiculosum xylanase XYNC bound to XIP-I(PDB Accession No.1TE1)as a reference.The amino acids that interact with XIP-I in the Loop?3-?4 and Loop?4-?5 domains of XYNC were sequentially introduced into the mutant(see Appendix 1)for the purpose of allowing these amino acids to interact with the XIP-I.In the interaction,mutants B3B4,B4B5,and YE were constructed.It was found that the mutants B3B4 and B4B5 still did not eliminate the resistance and the YE mutant was inactive,indicating that the amino acids corresponding to Loop?3-?4 and Loop?4-?5 of XYNC in Xyn1 B did not play a decisive role in the resistance of Xyn1 B.effect.From the inactivation of the YE mutant,we speculate that the reason for the resistance of Xyn1 B is the result of the amino acid interaction in the above mutation region.(3)In the maltosylation system,compare the enzyme activity of the resistant enzyme and sensitive enzyme in the equal activity unit with or without the addition of XIP-I,ie,detect the resistant enzyme Xyn1 B and the resistant enzyme + XIP-I.Change of xylose release and wort clarity under the treatment of sensitive enzyme Th Xyn1 and sensitive enzyme + XIP-I.The results showed that the amount of xylose released from the resistant enzyme was increased by 0.17 g/L when XIP-I was added and no XIP-I was added,and the xylose released by the sensitive enzyme with and without XIP-I.Change of 0.635 g/L.The advantage of Xyn1 B was also reflected in the clarity of wort.There was no significant difference in wort clarity between the addition of XIP-I and XIP-I in the resistant enzyme,while the sensitive enzyme was added with XIP-I and no XIP-I.The clarity of wort changes by 31%.The experimental results in this study provide theoretical basis for the modification of other GH11 family fungal xylanases.The advantages demonstrated by the first application of Xyn1 B to maltosylation established the basis for its application.