Organisms in nature generally produce α-amylase.
α-amylase is one kind of endoglucosidase. According to the standard of Enzyme Committee which is EC3.2.1.1, α-amylase refers to one kind of hydrolase which can incise the α-1, 4-glycosidic bond in starch molecules to form dextrin and reducing sugar. The terminal glucose residues C1 of the product are α-configurated. Hence it is named as α-amylase.
α-amylase plays an important role in the production of MSG, equal sugar, alcohol, glucose, fructose syrup, beer, citric acid, lactic acid,etc.
Fungal α-amylase was firstly used in industry, but some of the high temperature resistant α-amylase produced by bacteria, especially Bacillus ( for instance, Bacillus licheniae and Bacillus amylolyticus), are more suitable for the extreme conditions of high temperature, acid,alkali and so on in industrial production.
The main function of high temperature resistant α-amylase in the production process of high fructose syrup is to hydrolyze the α-1,4 glucoside bond of starch at high temperature (105 ~ 109℃), to degrade the molecular weight of starch, and to change the starch from large molecules to small molecules of dextrin.
Molecular structure of high temperature resistant α-amylase The molecular weight of high temperature resistant α-amylase is estimated to be between 40kD and 60kD by X-ray diffraction or gene sequence, and it exists in monomer or dimer form.
The amino acid sequences of α-amylase from bacteria with known structure are compared and can be found that it contains four conserved regions.
Divalent calcium ions are active catalysts for most bacterial α-amylases, but thermophilic α-amylase (PFA) from Pyrococcus furiosus does not require additional divalent calcium ions.
Their secondary structure is the common α-helix, β-fold and β-corner.
The tertiary structure of high-temperature resistant α-amylase is formed by the arrangement and accumulation of these secondary structures, and the tertiary structure has catalytic activity.
Although the primary structure of various alpha-amylases from different sources is different, their tertiary structure is very similar, which indicates that the tertiary structure is a key factor in the catalytic activity of high temperature resistant α-amylases.
The tertiary structure consists of three domains, which is also a common feature of the tertiary structure of α-amylase from different sources. Take Bacillus licheniformisαamylase (BLA) as an example, the α/β barrel structure composed of 8 α-helical and 8 β-folds alternately is domain A. Domain A contains about 280 amino acid residues to form the middle of α-amylase, which is A region with strong rigidity. Domain A maintains the basic conformation of the enzyme.
Between the third α-helix and the third β-fold of domain A is domain B, which consists of one or several β-layers as its secondary structure. This secondary structure is characterized by greater flexibility. It is speculated that domain B may be related to substrate specific binding, and the number of residues contained in domain B varies with the source of α-amylase, such as 58 PFA . There are about 100 BLAs, but their tertiary structure consists of β layers and relaxed random curls.
There are the pockets of catalytic activity of α-amylase between domains A and B, at the bottom of the α/β barrel structure, it is similar to the active centers of other α-amylases with this domain.
There are one or several calcium ion and other metal ion binding sites between the domain A and B of α-amylase, and it is speculated that the metal ion binding sites may be related to the structural stability of the enzyme.
The antiparallel β layer makes up domain C, which forms the carbon end of α-amylase, and domain C which contains a small number of amino acids, it is lack of flexibility due to its distance from the active site, and its function is unclear.
By studying the primary and tertiary structure characteristics of α-amylase, it is found that the four conserved regions displayed in the primary structure of α-amylase are located between the 3rd, 4th and 5th β-folds and the 7th β-folds and the 7th α-helix of domain A, respectively. The catalytic activity center of the enzyme is composed of these conserved regions, and the activity of the enzyme is controlled by these conserved regions.
The carbon end of the third β-fold is conserved region I, which contains three conserved amino acids Asp100, Asn104 and His105.
The fourth β-folded carbon end is conserved region II, which contains two conserved amino acids Asp231 and Arg229. The fifth β-folded carbon end is conserved region III, containing a fully conserved amino acid G1u261.
The joint between the 7th β-fold joins the 7th α-helix is conserved region IV, containing the fully conserved amino acid Asp328.
The bottom of the α/β barrel structure is the catalytic center of α-amylase, and the two amino acids Asp231 and Glu261 are important residues that play the catalytic role.
The whole catalytic process can be divided into three steps in sequence:
In the first step, the proton donor Glu261 protonates the glycoside oxygen in the starch chain.
In the second step, the C1 glycosidic bond of the glucose residue is broken by the nucleophilic group Asp231 and forms an ester bond with Asp231. At the same time, H+ in a water molecule is stripped by the deprotonated proton donor Glu261, then it produces an OH-.
In the third step, the ester bond is broken as C1 of the glucose residue is attacked by OH-, while Glu261 and Asp231 return to their original state. It contains two residues, Glu and Asp which is located at the bottom of the α/β barrel structure in the α-amylase with known structure, they have similar relative positions.
Since all α-amylases have the very similar overall two-dimensional structure, they should have the same or similar catalytic reaction mechanism.