Directed Evolution Of Alkali Resistance Endoglucanase

China is a large agricultural country,and the annual output of straw is more than 900 million tons.As an available biomass resource,straw has multi-level applications in the field of agricultural,industrial and light chemical industry.Rational utilization of the cheap resources,and its effective conversion in different areas,have important significance to agriculture and industry to our country.The straw mainly consists of cellulose,hemicelluloses and lignin,among which cellulose accounted for a large proportion.Cellulase is one of the main enzymes of hydrolytic in straw degradation,it is mainly consist of three kinds of enzymes,including endoglucanase,exoglucanase and glucanase,Endoglucanase is an important component of cellulose.Through directional modification of endoglucanase gene,the recombinant cellulose is expected to be obtained,so as to enhance the enzyme activity or tolerance,and to lay the preliminary foundation for later straw degradation.At the early stage of thisexperiment,17 strains of cellulase producing bacteria and 4 strains of fungi were screened by Congo red screening method from Jilin Agricultural University.Comparing the ratio of hydrolysis circle diameter and colony diameter,4 strains with the larger ratio were determined,HZ79,Tx-25,Rh-52,and Zj-2 respectively.CMC activity and FPA activity of these 4 strains were determined by shaking flask fermentation.Finally,the HZ79 strain with the highest enzyme activity was selected as the starting strain for further study.The strain of HZ79 was identified as Bacillus amyloliquifaciens through the16 SrDNA technology combined with gyrA technology and physiological and biochemical identification.The endoglucanase gene was successfully cloned from the genome of Bacillus amyloliquefaciens.The key point of the directional modification technology to modify the endoglucanase gene is to establish a reasonable method of high throughput screening.In this study,by using the Qpix 420 strain selection system of MD to select transformants which were inoculated in 96 well microtiter plates,and the enzyme activity was determined by 96 microtiter plates.To replace conventional DNS enzyme activity assay method.It is proved that the stability of the method is consistent with the traditional DNS enzyme activity assay method.And its accuracy is better than the traditional enzyme activity assay method.This research uses error prone PCR to directional transform the endoglucanase.Through the early optimization of error-prone PCR reaction system,the concentration of Mn2+ is 0.5mmol/L,the concentration of Mg2+ is 7mmol/L,and the PCR reaction cycles is 35 cycles.Structured.Error prone PCR products obtained by error prone PCR reaction are connected with plasmid vector pET32 a and transformed into E.coli BL21(DE3),pick mutants containing the desired fragment and construct a mutant library.By using Qpix 420 strain selection system,target fragment mutants are inserted in to 96 well microtiter plates.After different treatment,the CMC enzyme activity in different mutants is determinated.After two rounds of screening of error prone PCR,we acquired a mutant strain HZ79-eglep,and the enzyme activity is increased about 17% compared with the original mutant strain.The optimum pH value varies from 5.6 to 6.5,and its enzyme activity can remin more than 70% in the range of pH 7.0-9.0.After DNA sequencing,we found that 6 bases have changed in mutant sequence,and 3 amino acid change,that is 140 th by Gln to Glu,222 th by the Ala to Thr,399 th by Asn to His respectively.The secondary structure of endoglucanase protein was predicted by ANTHEPROT5.0.The results showed that the protein secondary structure of endoglucanase was consistent with the original,and the physical and chemical properties did not change significantly.The tertiary structure of endoglucanase protein was predicted by Swiss-model simulation of endoglucanase.The predicted results were analyzed by Deep View(Swiss-PdbViewer)software to simulate the tertiary structure information.The results showed that the endoglucanase have the same polypeptide backbone as the original enzyme,and the three-dimensional structure essentially in agreement with original enzyme.Three amino acid mutations only make the structure of the enzyme protein slightly changed.The connection mode of hydrogen bonds in the space have changed,making the entire spatial structure become looser,and rigidity reduce.It is conducive to the substrate combine with the active site of the enzyme to improve the catalytic activity of enzyme.