Screening And Identification Of Pullulanase-producing Bacteria And Directed Evolution And Overexpression Of Pullulanase

Pullulanase is one of the family GH13 a-amylases.It typically hydrolyzes the ?-1,6-glucocidic linkages of pullulan,starch or branched oligosaccharides in an endo-way to produce maltotriose or liner oligosaccharides,respectively.It also can hydrolyze the smallest unit of branched chain of oligosaccharides into maltotriose,which accelerate saccharification and increases the utilization rate of starch during its conversion.This enzyme plays a very significant role in the food,chemistry and medical industries,because they can degrade different saccharides with other glycoside hydrolases producing various products.And it has a broad prospect of development and application.In this paper,screening of pullulan enzyme producing bacteria,cloning,heterologous expression of pullulanase genes and directed evolution of pullulanase were carried out,in order to obtain efficient expression of pullulanase and enhance its acidic stability and activity.The main work of this paper is as follow.First of all,screen of pullulanase-producing bacteria was performed through plates containing neutral red as the indicator and pullulan as sole carbon source.A strain,designated strain GST4,which can produce pullulanase was isolated.Strain GST4 was classified as genus Tumebacillus based on both 16S rDNA identification and morphological,physiological-biochemical test.The genome sequence draft was obtained using the Illumina HiSeq 2500 next-generation DNA platform and all kinds of starch hydrolysis enzyme were successfully annotated.The availability of the first genome sequence of an organism belonging to the genus Tumebacillus helps to clarify the evolutionary status and ecological role of the genus Tumebacillus,and provides a genetic basis for analysis of the acid tolerance and starch-hydrolyzing activity of T.flagellatus GST4.Then pullulanase gene pulB from T.flagellatus GST4 was cloned and expressed successfully in E.coli..And PulB had been purified to homogeneity by Ni-chelating affinitychromatography from cell free culture supernatant and characterized.The optimal temperature of purified PulB is 50?,its optimal pH value is 5.0 and activity remains stable within the acidic range of pH 4.0?6.0.PulB displayed typical Michaelis-Menten kinetics,where its Km is 16.28±0.03mg.mL-1 and Vmax is 22.05±0.02 ?mol·min-1·-mg-1,respectively,when used pullulan as substrate.It typically hydrolyze pullulan and amylopectin to produce liner oligosaccharides or maltotriose.GenBank blastx results show that the encoding protein of PulB had the highest identity(54%)with cyclomaltodextrinase from Thermicanus aegyptius.PulB is a new starch/pullulan hydrolase,which has not yet been reported.This work can make preparation of pullulanase and genes resource for further studies.Due to the need for pullulanase with high specific activity and stability at acidic pH,which are critical parameters,for example,for the use in starch saccharification process,we have enhanced pullulanase from Bacillus naganoensis ATCC53909 and PulA324 was available in our Lab.The genes coding for the wildtype PulA324 was subjected to error-prone PCR.And for the screening of mutants,we developed a novel,reliable assay suitable for high throughput screening based on the two phase screening system.To screen simultaneously for improved activity at low pH and improved specific activity,two-dimensional screening at two different pH(pH5.0 and pH4.0)values was carried out with 96-well plate.After four rounds of mutation,one mutant PulA12(E256N&D520R)was obtained.It has an optimal activity at pH 4.8,which remain the same optimum pH as the wildtype,but its tolerance towards low pH was enhanced,resulting in a 2.75-fold higher acidic stability at pH4.0 than the wildtype,and the residual activity of PulA12 are still around 75 percent of the original,while the wildtype has reduced to 20%after they were kept under conditions of 4?,pH 4.0 for 5.5 hours.The sequencing and aligment of the amino acid sequences between this mutant and the wildtype reveals the mutation of the highly conserved residue D520 and E256.In addition,to select the residues to be mutated,rational design mutation strategy was adopted based on the homologous modeling analysis,combined with the calculation of the pKa values of acidic and alkaline amino acid groups,three residues E326,N680 and D570,which close to the triple active-site center of PulA324,were designated to site-directed saturation mutagenesis/site-directed mutagenesis.An other mutant N7-6 possesses a wild-type-like pH profile,but possesses a 2-fold higher acidic stability ability was obtained.Sequencing results reveal that Asparagine(N)of position 680 was replaced with Aspartic acid(D).Finally,the mutant pulullanase gene were inserted to expression vector of pWB980 and transformed into the host of Bacillus subtilis WB600 to build engineering strains.And secretive recombinant pullulanase was successfully expressed in WB600/pWB980-pulM,the extracellular pullulanase activity reached 348 U/mL after the optimization parameters of fermentation,which is the the highest level compared to that reported in the domestic literature.