Obtaining Of An Efficient Glucoamylase And Its Properties

Glucoamylase (GA,1,4-α-D-glucan glucohydrolase, EC3.2.1.3) fromAspergillus niger is an exo-acting enzyme that could catalyze the α-1,4-andα-1,6-glucosidic linkages from the non-reducing ends of starch and otherpolysaccharides to β-D-glucose. There are two kinds of glucoamylase produced byfungi: type I and type II. Three domains have been identified in type I. Type I GA canhydrolyze raw starch directly and the saccharification process was thoroughly.Whereas the type II don`t have the starch binding domain, so type II lacks the abilityto combining raw starch and it could hydrolyzed raw starch hardly.This paper obtained the following two outcomes by protoplast fusion breedingtechnology and compounding technique of GA:1. The methods of preparation of protoplasts and fusants screening for A. niger.2. A new type GA that can hydrolys substrates efficiently.In industry, GA was produced mainly by A. niger, Asperillus awamori andRhizopus oryzae. In our lab, there are two A. niger strains and they all have high yieldof GA: A. niger A and A. niger B. The major ingredient produced by A. niger A is typeI GA, it was called GAA. And the major ingredient produced by A. niger B is type IIGA, it was called GAB.The main work of this paper is divided into the following three aspects:1. The mathermatical model for A. niger glucoamylase fermentationThe processes of A. niger fermentation in5L fermentation tank was been studied.The data of biomass, enzyme activity of GA and reducing sugar in different time weremeasured by timing sampling during the fermentation period. And then the models ofbiomass growth, product synthesis and nutrient consumption were built by usingLogistic Equation and Leudeking-Piret Equation. According to the models, thelogarithmic phase and stable phase has been cleared. The fungus in the logarithmic phase are suitable for the preparation of protoplast. And only in the end of the stablephase we can obtain most GA from the fermentation liquor. So the model is the basicof all the work in this paper.2. The preliminary studied of protoplast fusion for A. nigerProtoplast fusion breeding had been done to obtain the newly and highlyeffective glucoamylase. The stabilizer was0.6M NaCl. The mixture of snailase,cellulose and lywallzyme had been used for breaking cell wall. After the vibrant anddense protoplasts were obtained, the protoplasts inactivated them by Iodoacetamide(IOA) and Amphotericin B (AmpB) respectively. And then protoplast fusion had beendone mediated by30%PEG-3350. As evidence, the fusionts observed under opticalmicroscope. Because of the complementary of metabolic pathways, the colonies grewin hypertonic culture dish were fusants. A. niger protoplast preparation and fusantsscreening methods had been established.3. The obtain of newly and highly effective glucoamylase by compoundingtechnique of GA and its enzymatic propertiesThe compound of GAA and GAB has been researched. At first, pure GAA andGAB was obtained by using hydrophobic interaction chromatography anddiethylaminoethlcellulose anion exchange chromatography, respectively. According toa series of enzyme activity ratios, GAA and GAB has been mixed. Then the enzymeactivities of these mixed enzymes have been measured. The data analyzed by T-testindicated that the experimental groups: GAB content of40%,50%,60%and70%were significantly improved. Among all four groups, GAB content of50%improvedmost, approximately8%. After that, mixed GAA and GAB in an enzyme activityratio of1:1. It was called GAA&GAB. And then the optimum temperatures and pH,thermostability and pH tolerance of these enzymes (GAA, GAB and GAA&GAB)had been evaluated. The optimum temperatures (60-65℃) and pH (3.9-4.9) of GAA,GAB and GAA&GAB were similarly. The thermostability and pH tolerance of themwere very similar. The experiments of dextrin hydrolyzed for mixed enzyme, GAAand GAB have been executed. The concentration of glucose, disaccharide andtrisaccharide in the process of the reaction was measured by HPLC. The results indicated that glucose was always existed and rising up all the time, the disaccharideand trisaccharide offered upgrade firstly than descending latter tendency. The abilitiesto hydrolyzed dextrin of GAA, GAB and GAA&GAB were similarly. At last, theability of hydrolysis of corn starch has been studied by starch hydrolized experiment.The abilities to hydrolyzed starch of GAA and GAA&GAB were similarly, but therewere some differences. The ability of GAB was weakest. The above experimentsshowed that The GAA&GAB exhibited a higher conversion efficiency and efficiencyboth in hydrolyzed dextrin and corn starch.This paper had established a mathematical model of Aspergillus nigerfermentation to produce glucoamylase, and simulated by Simulink. It offered a certainfoundation for pratical production. Set up the method of filtering Aspergillus nigerfusion by using two-parents inactivation. The method is simple and effective. Bymixed the two glucoamylases, a newly glucoamylase has been obtained. Theenzymatic activity of has been improved8%. And it showed up some newcharacteristics in hydrolysis experiments of dextrin and starch. This research has asignificance guding for industrial applications of glucoamylase, and provides a newway for efficient glucoamylase production strains screening.