Overexpression And Molecular Modification Of Starch Debranching Enzymes

Starch debranching enzymes, which catalyze hydrolysis of α-1,6-glucosidic linkages inamylopectin, can be divided into two classes: pullulanase （EC3.2.1.41） and isomaylase（EC3.2.1.68）. The major difference between pullulanase and isoamylase is that they possessdifferent substance specificity. They can be used in the production of glucose, fructose,maltose, cyclodextrins, and alcohol. In order to promote the application of the enzymes instarch industry, there are some problems that need to be overcome. Firstly, the pullulanasesneed to be modified to enhance its thermostability. And more attention should be putted intothe development of isoamylase which prefers substrates with high molecular weight. Lastly,the yields of the starch debranching enzymes need to be improved.In the present study, the gene encoding pullulanase from Bacillus deramificans wasoverexpressed in Escherich coli BL21（DE3）. Then Asp437and Asp503were selected astargets for site-directed mutagenesis to enhance the catalytic efficiency and thermostability.And two-stage glycerol feeding strategy and N-terminal truncated mutagenesis were appliedto achieve soluble production and secretion of recombinant pullulanase, respectively. Inaddition, the Tfu₁891gene of Thermobifida fusca was cloned and overexpressed, and anovel synchronous bioconversion strategy for the production of cyclodextrins by both CGTaseand isoamylase was developed. The main results are listed below:（1） Heterologous expression and characterization of pullulanase: The pullulanaseencoding gene of B. deramificans was expressed in E.coli, and the recombinant protein waspurified to homogeneity and characterized. The optimum temperature and pH of the enzymewas55°C and5.5, and the half life of the enzyme was20h at60°C, pH4.5. Kinetic studiesshowed that the Vmax, Kcat, Kmand Kcat/Kmof the enzyme was469.7U mg-1,2109.0s-1,0.7mg mL-1and3012.8s-1mg-1mL, respectively. When the enzyme was used in conjunctionwith glucoamylase to transform corn starch （30%, W/V） into dextrose, the total yield ofdextrose reached92.7%（W/W） after52h, which was1.3%higher than that utilizeglucoamylase alone.（2） Improving the thermostability and catalytic efficiency of pullulanase by site-directedmutagenesis: To improve the thermstability of pullulanase, four mutants were generated basedon a structure-guided consensus approach and characterized in detail. The results showed thatthe double mutant D437H/D503Y displayed a larger shift in thermostability, with an optimaltemperature of60°C and a half-life at60°C of more than4.3-fold that of the wild-typeenzyme. Kinetic studies showed that the Kmvalues of D503Y and D437H/D503Y was58.6%and54.3%of that of the wild-type enzyme, and the Kcat/Kmvalues increased by140%and 100%, respectively. Moreover, when used in conjunction with the glucoamylase, D503Y andD437H/D503Y exhibited an improved reaction rate and glucose yield during starch hydrolysis,confirming the enhanced properties of the mutants.（3） Enhance the soluble production of pullulanase in E. coli by regulation of processconditions and supplement with natural osmolytes：Shake flasks fermentation results revealedthat the activity of insoluble cell fraction was2.2-fold of that the the soluble fraction. Inaddition, SDS-PAGE, transmission electron microscope image of the cells and softwareanalysis clearly confirmed the in vivo formationed “IBs” were active protein aggregation. Itshowd that the yield of soluble pullulanase was found to be enhanced with lower temperature,lower IPTG concentration, and suppled with betaine in shake flasks. In addition, a modifiedapproach was applied in a3L fermentor supplied with20mmol mL-1betaine at25°C, thetotal soluble activity of pullulanase reached963.9U mL-1, which was30.5-fold higher thanthat observed without addition of betaine at37°C, and the yield of pullulanase represents thehighest yield reported so far.（4） Effects of the N-terminal domains truncation on the secretion efficiency and thermalstability of pullulanase: Four N-terminal truncated pululanase mutants were constructed. Theresults showed that the secretion efficiency of the Puld1′, Puld1and Puld2were3.1,3.4and3.8folds of that of the wild type enzyme. While the substance affinity of Puld1and Puld2wasonly62.5%and22.5%of that of the parent, respectively. In order to address this issuse, twocombined mutants were constructed. It showed that the extracellular pullulanase secrationratio of D437H/D503Y/d1and D437H/D503Y/d2was5.3and5.6folds of that of theD437H/D503Y, respectively. The half-life of D437H/D503Y/d1and D437H/D503Y/d2was203h and160h, respectively. Furthermore, the starch saccharification results showed that DXvalue of D437H/D503Y/d1and D437H/D503Y/d2was95.0%and94.1%.（5） Expression of a recombinant T. fusca isoamylase for facilitating α-cyclodextrinsynchronous production with α-CGTase: The Tfu₁891gene of T. fusca was cloned andoverexpressed in E. coli BL21（DE3）. The optimum temperature and pH of the recombinantenzyme was50°C and5.5. A two-stage feeding strategy was proposed and applied in a3Lfermentor, which achieved a dry cell weight of68.5g L-1, the activity of isoamylase was6876.9U mL-1. In addition, the recombinant isoamylase and α-cyclodextringlucanotransferase （α-CGTase） were used simultaneously to transform potato starch （15%,W/V） into cyclodextrins, and the conversation conditions were optimized. Under the optimalconditions, the total yield of cyclodextrins reached84.6%（W/W） after24h, and the ratio ofα-cyclodextrin was94.3%.