Armillariella Tabescens EJLY2098 MAN47 β-mannanase: In Vitro Directed Evolution And Strutural Analysis

Objective:Due to the special requirements of feeding, nutrition and feed processing mannanase must have two property. Firstly, mannanase should has good thermal stability and withstand pelleting temperature of at least 80℃.Secondly, mannanase should maintain high activity in acidic and neutral pH range.However, generally,p-mannanase has only one characteristic, enzyme with high temperature stability can not tolerate acidic environment, or enzyme can withstand the acidic environment will easily lose their activity at high temperatures.In order to obtainβ-mannanase with higher thermalstablility and acid tolerance which can be used in actual production,directed evolution is used to evolve theβ-mannanase gene from A. tabescens MAN47 EJLY2098.At the same time,We can find relation of structure and function inβ-mannanase by analyzing the sequence and structural of mutant.Methods:Firstly, error-prone PCR(EP-PCR) was used to induce mutations on A.tabescens EJLY2098 MAN47β-mannanase gene, Secondly, we cloned the mutated fragments into secreted expression vector pYCa, Then recombinant plasmids were transformed into S.cerevisiae BJ5465 after amplified and extracted inDH5a cells. Thirdly,we built a mutant database and screened mutants with acid tolerance or thermostability or high activity which would be taken as parent gene of DNA Shuffling.Then we searched mutants with higher thermalstablility and acid tolerance from DNA Shuffling database by facilitation and 96 deep wall plate culture screen.At last,we can get some information about bioinformatics ofβ-mannanase by sequence comparison and homologous model.Results:Through two cycles of EP-PCR and DNA Shuffling, we build a mutant database, Then we screened one optimum 1108 from about 104 mutants.The evoluted P-mannanase displayed both higher thermalstability and acid tolerance than wide type. The evoluted enzyme 1108 retained high activity after treatment at 90℃for 30 min, whereas, the wild type nearly lost activity under this condition. Meanwhile, the activity of 1108 under 80℃and acid treatment was 10 times and 5 times as wide type respectively. The sequence comparison illustrated that there were three nucleotide substitutionsT289/A289,A535/T535,T1085/C1085 whichcarried correspo-nding amino acid changes Ser97/Thr97,Ile179/Leul79,Val362/Ala362 According to homologous modeling by SWISS-MODEL Repository and amino acid anylysis,we found mutations locate near the catylyze core,So we speculate Ser97/Thr97 and Ee179/Leu179,Val362/Ala362 have something with acid tolerence/activity and thermalstablility respectively.