Glucoamylase is an extracellular enzyme secreted by microorganisms and has exonuclease activity.
The main function of glucoamylase is to hydrolyze the α-1, 4-glucoside bond from the non-reducing end of the carbon chain such as starch, dextrin and glycogen, cut off glucose units, and change the configuration of glucose like β-amylase to form β-D-glucose.
When the substrate of grape amylase is amylopectin, it can hydrolyze the branch points formed by α-1,6 glucoside bonds, and degrade all amylopectin into glucose.
Glucoamylase can also weakly hydrolyze the carbon chains formed by α-1,3 glucoside bonds, but the rate of hydrolysis of α-1,4 glucoside bonds in starch molecules is faster than that of other types of glucoside bonds in starch molecules.
Glucoamylase usually hydrolyzes 100 percent of starch molecules to produce glucose.
Glucoamylase is widely distributed in microorganisms. The glucoamylase used in industrial production mainly comes from filamentous fungi and yeast such as Aspergillus Niger, Aspergillus oryzae and rhizopus.
Thermally stable glucoamylase can also be isolated from bacteria, and it is present in human saliva and animal pancreases.
The structure and function of glucoamylase vary at certain degree with its source.
Among many kinds of microorganisms, glucoamylase produced by fungi has the strong ability to hydrolyze raw starch.
Glucoamylase is one kind of glycoprotein with a molecular weight of 6×104 ~ 106, containing mannose, glucose, uronic acid and galactose.
In general, the carbohydrate content of glucoamylase is 4% to 18%, but there are exceptions, such as glucoamylase produced by saccharifying yeast, which contains up to 80% carbohydrates, and these carbohydrates are mainly composed of glucose, galactose, mannose and glucosamine.
It has been reported that the molecular weight of A. Saritoa glucoamylase GAM1 is 9×104, its glycoprotein contains 18% neutral sugar and 0.77% glucosamine, and the n-terminal amino acid is alanine.
Through the separation and purification research of glucoamylase, it was found that glucoamylase can be divided into 3 classification: G, GI, GII or GAI, GAI', GAII.
The substrate of different kinds of glucoamylase is also different, such as GAI', GAII(GI, GII) can only hydrolyze gelatinized starch, can not hydrolyze raw starch or hydrolysis is very weak, while GAI(GIII) type glucoamylase can hydrolyze raw starch.
It was also found that GAI', GAII can be obtained by the action of subtilisin on GAI.
For example, the glucoamylase from Aspergillus awamori var.Kawachi has molecular weight of GAI MW90,000, GAI'MW73000, GAII MW57000.
Glucoamylase GAI hydrolyzes starch because it not only contains the catalytic site GAI', but also has the affinity site Cp region and GP-1 (MW 13200) that binds to raw starch.
Substrate specificity of glucoamylase The hydrolysis rate of glucoamylase is affected by the molecular structure of glucoamylase itself, the structure of the substrate and the size of the substrate.
The results showed that the affinity of glucoamylase between dexlase and substrate increased with the increase of the carbon chain length of substrate, and showed a linear change.
Glucoamylase can hydrolyze α-1,4 glucoside bonds and α-1,6 and α-1,3 glucoside bonds of starch molecules, but the effect of α-1,4 glucoside bonds is the most important and strongest, and the hydrolysis ability of the latter two glucoside bonds is weak.
Wang Yangsheng ,etc. reported that monascus saccharifying enzyme can completely hydrolyze corn starch, potato starch, soluble starch, amylose, glycogen and maltose to produce glucose, but the decomposition limit of other substrates with smaller carbon chains decreases with the reduction of carbon chains.
Guan Hancheng ,etc studied the substrate specificity of glycosylase of Aspergillus Niger variant strains, and the results showed that the glycosylase GAI only hydrolyzed starch and malt oligosaccharides, but did not hydrolyze dextrose, xylan, lichen polysaccharide, mannose and α-, β-, γ-cyclodextrin, indicating that GAI had strong specificity to the sugar components and glucoside bonds in polysaccharides. It hydrolyzes the alpha-1,6 bond less than one hundredth of the rate at which it hydrolyzes the alpha-1,4 bond.
The same glucoamylase, because of its structure and composition has the variety, resulting in different types of glucoamylase to the same substrate hydrolysis rate is also different.
Fang Shankang ,etc obtained the three components of Aspergillus Niger S4 saccharase by isolating and purifying it, namely GI, GII and GIII. The study showed that the three components had different adsorption capacities for the same substrate, namely GI had the strongest adsorption capacity for the same substrate, while GII and GIII had much weaker adsorption capacity for the same substrate.
Fang Shankang ,etc studied the properties of purified components of A. niger S4 saccharifying enzyme, and found that GI, GII and GIII could all act on sweet potato starch, potato starch and raw corn starch, and the hydrolyzing capacity of these three kinds of saccharifying enzyme components for raw starch was lower than that for soluble starch.
It is estimated that the hydrolysis ability of each glucoamylase component to substrate decreases with the increase of Km value.
According to the kinetic parameters of the hydrolysis of maltose by glucoamylase, although only one glucose unit was increased, the Km value was greatly decreased and the Vmax was increased, that is, the affinity hydrolysis ability of each glycosylase component to maltose is higher than that of maltose disaccharide, indicating that maltose disaccharide is the smallest substrate of each glycosylase component. The structure of the enzyme and the length of the oligosaccharide chain affect the affinity of the glycosylase to the substrate.