α-glucosidase, also known as α-D-glucosidase, is a generic term for a family of enzymes that catalyze the hydrolysis of α-glucose from the non-reducing end of substrates containing α-glycosidic bonds. It cleaves α-1,4 glucosidic bonds from the non-reducing ends of oligosaccharide substrates, releases glucose, or transfers free glucose residues to another carbohydrate substrate to form α-1,6 glycosidic bond, resulting in non-fermentable isomaltooligosaccharide, glycolipid, glycopeptide or the like. It is widely present in all living organisms in nature and has a wide variety and diverse properties. Since the screening of α-glucosidase production strains, and industrial production of enzyme preparation, it has been used in the production of isomaltooligosaccharide and other fields, and has obtained much of the domestic and foreign food industry attention.
Sources
α-glucosidase is widely distributed in nature and exists in almost all organisms. The vast majority α-glucosidase already found, in addition to a small number of α-glucosidase from plants and animals, are from microorganisms. Some strains such as bacteria, mold and yeast can secrete α-glucosidase. Among them, Aspergillus niger yield higher, and most of α-glucosidase products on the market are produced by Aspergillus niger fermentation.
Properties
The relative molecular mass of α-glucosidase is generally between 40,000 and 150,000. The relative molecular mass of α-glucosidase from different sources varies greatly. It is also significantly different in different strains of the same genus or in the same plant tissue. The isoelectric point of all α-glucosidase enzymes that have been found are in the acidic range with little change, generally between 3.0 and 5.0, but the optimum pH can exceed 7.0. Most α-glucosidases have high thermal stability and optimum temperature, which is beneficial to their industrial application. Its thermal stability may be related to its amino acid composition, and with the increase of hydrophobic amino acids in the enzyme molecule. The increase of the number of hydrophobic amino acids such as glycine, alanine, proline and leucine in the enzyme molecule helps to improve its thermal stability, which may be due to the stronger three-dimensional structure of the enzyme molecule due to the strengthening of the hydrophobic interaction. α-glucosidase belongs to the bond-specific enzyme, which can specifically cut α-1,4 glucosidic bond in the molecule of carbohydrate substrate. Some kinds of α-glucosidase can also act on the α-1,2 glucosidic bond. Therefore, in general, α-glucosidase is less stringent on substrates and has a wide range of substrate specificity.
Catalytic Mechanism
α-glucosidase hydrolyzes terminal non-reducing (1→4)-linked α-glucose residues to release a single α-glucose molecule. α-glucosidase is a carbohydrate-hydrolase that releases α-glucose as opposed to β-glucose. β-glucose residues can be released by glucoamylase, a functionally similar enzyme. The substrate selectivity of α-glucosidase is due to subsite affinities of the enzyme’s active site. Two proposed mechanisms include a nucleophilic displacement and an oxocarbenium ion intermediate.
Metabolic Mechanism
α-glucosidase mainly includes enzymes such as maltase, sucrase, isomerized maltase, and lactase, which are mainly distributed along the brush border of the intestinal epithelium and play an important role in the catabolism of sugar. The process is that polysaccharides is digested by oral saliva, pancreatic amylase into oligosaccharides containing a small number of glucose molecules, α-glucosidase then cut α-1,4 glycosidic bond in these non-reducing end of the oligosaccharide, releasing glucose. Glucose is absorbed by the small intestine into the blood circulation, it becomes blood sugar. In the physiological state, the upper, middle and lower sections of small intestine all contain α-glucosidase, but the upper section is the main part of absorption.
Purification Method
Ammonium sulfate precipitation is a frequently used method for the initial purification of α-glucosidase. This is because ammonium sulfate precipitant produce less damage to enzyme activity, the precipitate can be stored for a long time. At the same time, due to a variety of protein structures, the initial concentration and complete precipitation concentration of ammonium sulfate precipitation is different, which can be used to coarsely extract the enzyme for pre-purification and remove most of the hybrid protein. Ammonium sulfate precipitation method is simple and convenient, but the resolution is poor, the purification multiple is not high, and a large amount of salt is mixed in the enzyme, further desalination is required when purifying. Acetone precipitation is characterized by high resolution and is another common method for the initial purification of α-glucosidase. Using 1.0 to 1.5 times the volume ratio of cold acetone direct precipitation of crude enzyme is more ideal, and remove a large number of impurities in protein and pigment material. Especially when acetone and enzyme volume ratio is 1: 2, purification fold and yield are compared high. Therefore, the ammonium sulfate (40% ~ 60%) by salt precipitation method combined with the acetone method is more conducive for α-glucosidase purification.
