Gene Mining,Characterization And Molecular Modification Of Thermostable Pullulanase

Pullulanase(EC 3.2.1.41)was first discovered by Bender and Wallenfels during the cultivation of Aerobacterium aerogenes,named after its ability to hydrolyze pullulan.It can hydrolyze ?-1,6-glycoside bond in pullulan,starch and amylopectin.This characteristic makes pullulanases play a very important role in the industrial processes related to starch hydrolysis.Among them,the most important application of pullulanases is the saccharification process of starch.Its industrial application conditions are pH 4.5-5.5,temperature 55-65?(or higher),and the duration of action is 48-60 hours.Most of the reported type I pullulanases can not adapt to this industrial condition.Some of them have poor heat resistance,and some of them have very low or even inactivated activity at pH 5.5 or lower.Even commercial B.acidopulluliticus pullulanase has obvious short plate,and its half-life at 60? is only 34.9 min,which does not have enough heat resistance.Therefore,in this study,we aimed to obtain the pullulanase with excellent performance,combined with the existing pullulanase sequences information to mine the potential pullulanase sequence in the database of NCBI.Three potential pullulanase sequences were recombined and expressed in E.coli.The enzymatic properties of the three pullulanases were evaluated comprehensively,and the best pullulanases were selected for molecular modification,which further improved their heat resistance and catalytic efficiency.In addition,in order to avoid the influence on our experimental results caused by the variety of enzyme activity measurement timing reaction system in the literatures,and to make the determination of enzyme activity more convenient and quick in the experimental process,the study also optimized the activity determination method of pullulanase.(1)A more suitable enzyme activity determination system was developed.The effects of three DNS reagents,two reaction volume ratios and boiling time were evaluated.The DNS-Ghose reagent with high sensitivity and stability was selected for the follow-up experiments;the Mod method with low detection limit and high sensitivity was selected for the follow-up experiments;the boiling for 2 min was selected for color development,which greatly shortened the experiment time.(2)Three potential pullulanase sequences were found and successfully expressed in E.coli recombinant expression system.Using the reported pullulanase sequence as template,blast analysis was carried out in NCBI database,and three protein sequences in the analysis results were selected.The basic information analysis,phylogenetic tree analysis,homology analysis,conserved region analysis and characteristic site analysis were carried out for these three selected sequences.In order to further verify the enzymatic properties,the expression system of the three selected sequences was constructed and induced by IPTG.SDS-PAGE results showed that when the final concentration of IPTG was 1 mM and the induction time was 6 h,the expression of the target protein was high,which could meet the requirements of subsequent experiments.(3)A pullulanase suitable for starch saccharification was obtained.Three pullulanase proteins were isolated and purified.The effects of pH,temperature,pH stability,thermostability,effects of metal ions and chemical reagents,kinetic parameters and hydrolysates of different substrates were determined.The enzymatic properties of these three enzymes were compared,and their advantages and disadvantages were discussed in detail.The optimum pH values of these three enzymes are all in the industrial application range,of which PulF has the highest heat resistance and PulC has the highest specific enzyme activity.As far as we know,PulF is the one with the best heat resistance among the pullulanases with the most suitable pH and industrial requirements.(4)A mutant of pullulanase with high heat tolerance and high catalytic efficiency was obtained.Compared with commercial B.acidopulluliticus pullulanase,PulF still has a short plate with low specific activity.Based on sequence alignment and protein structure analysis,six non-conservative amino acid residues within 10 angstroms of catalytic sites were selected for site directed mutation.The effects of pH on enzyme activity,temperature on enzyme activity,thermostability and kinetic parameters of the mutants were measured.The heat tolerance of the six mutants was significantly improved,and the catalytic efficiency of V481 C was greatly improved,its specific enzyme activity was nearly twice that of wild type enzyme.