Fungal α-amylase has been widely used in modern starch syrup, baking products, beer brewing, raw alcohol industries,etc.With the development of modern sugar industry and fermentation industry and the usage requirement for fungal α-amylase, fungal α-amylase plays an important role in modern industrial enzyme preparations.
Similar with most α-amylase, fungal α-amylase usually contain three structural domains, they are called A, B and C.
Structural domain A is the central zone of the catalytic reaction of enzymes, and its typical structure is (a/b) 8TIM-barrel structure.
Structural domain B and structural domain C are basically located at opposite ends of domain A.Among them, the conserved binding site of Ca2+ is located in the surface region between structural domain A and structural domain B, and the presence of
Ca2+ in most cases is necessary for the α-amylase family to maintain its enzymatic activity and stability.
Structural domain B is located between the third β-fold and the third α-helix of the Tim-barrel domain. This region is rich in irregular
β-lamellar structure, which varies greatly in size and structure among different amylases and is regarded to be related to the fifth specificity of α-amylase.At the same time, site-specific mutation or random mutation results showed that this site
was relatively weak in amylase, and was closely related to the overall stability of α-amylase, in which the change of some amino acids had a significant effect on the pH stability and thermal stability of the enzyme.
Structural domain C forms
the carboxyl terminal of α-amylase proteins and contains the Greek-key β-sandwich structure characteristic of theα-amylase family, which is generally thought to stabilize the catalytic region or TIM barrel structure by isolating the hydrophobic region
of structural domain A from the solvent phase.
By means of X-ray crystal structure, chemical modification and site-specific mutation, it is shown that Asp206, Glu230 and Asp2973 amino acids may be the core catalytic sites of α-amylase family.
In the process of α-amylase catalysis, the enzyme first
immobilizes the heterotopic (α-conformation) and then catalyzes it through a double substitution reaction.
In the first replacement process, the acidic group of the enzyme (Glu230) protonates the oxygen atom in the glycoside and breaks the link
between carbon and oxygen, forming a onium salt transition state. Then in the second replacement process, the protophile acidic group of the protein attacks the heterotope center of the sugar, forming a temporary state of β-glycobase and enzyme complex.
The glycosylligand of the substrate then leaves the active site.
In the reported literature, α-amylases of various fungal sources can be roughly divided into three types according to enzymatic properties or action conditions:
①Neutral fungal α-amylase
Unlike bacterial α-amylase, the sources of fungal α-amylase
are relatively few, and the action temperature and pH of most fungal α-amylase are relatively mild, such as the optimal pH is between 5.0 and 5.5, and the optimal action temperature is about 50 ℃ to 55℃. When the temperature exceeds 60℃, the enzyme
begins to be inactivated.At present, the most commercially produced and widely used α-amylase from Aspergillus oryzae (variety) belongs to this enzyme.
②Heat or acid resistant fungal α-amylase
These enzymes are between pH2.5 and pH4.5, and have
good thermal stability when the operating temperature exceeds 60℃.Compared with neutral fungal α-amylase, heat or acid resistant fungal α-amylase can simplify the liquefaction and saccharification process, reduce the probability of bacterial contamination
in the deep processing of starch such as sugar production and reduce the corresponding production cost. At present, these enzymes have begun to be produced and used in industry, and have great potential for development and utilization.
③Fungal
α-amylase with bioamylase activity
Due to the unique properties of fungal α-amylase, such as mild reaction temperature, neutral pH, etc., fungal α-amylase is mainly used in the actual application of starch saccharification. Compared with bacterial α-amylase, the application of fungal α-amylase
is mainly concentrated in the field of food application.
①Production of high malt syrup
Malt syrup is a maltose based syrup hydrolyzed by enzyme or acidase. According to the different content of maltose, it can be roughly divided into malt sugar
(Maltose content is 20%-30%), high maltose syrup (Maltose content is 40%-60%), ultra-high maltose syrup (Maltose content is greater than 70%) ,crystal maltose and so on.
The sweetness of high malt syrup is low and mild, and the taste is strong.
It is stable under high temperature and acidic conditions, and has the advantages of high boiling temperature and less Maillard reaction,etc.
In the food industry, it has the effect of preventing starch aging, moisturizing and extending shelf life
in the processing of bread, pastries and baked goods.In preserves, convenience food, soy sauce, candy, oral liquid, health drinks, malted milk and frozen food and other food industry as a nutritional sweetener, filler agent has been widely used.
In
the pharmaceutical industry, it can be added to a variety of traditional Chinese medicine for use, with functions such as moistening the lung, tonifying deficiency, relieving cough and treating lumbago,etc.
In addition, the ultra high malt syrup
can be hydrohydrogenated to produce maltitol. Maltitol is a sweetener with a sweetness equivalent to sucrose but a low caloric value. It is also a raw material for preparing another functional food raw material, maltitoketose and isomaltooligosaccharide.
②Baked
goods
In 1955 and 1963, the United States and the United Kingdom, respectively, identified fungal α-amylase as a generally safe (GRASstatus) additive and permitted its use in the bread baking process.
At present, it has been used in different
extent all over the world. Because the neutral fungus α-amylase is not heat-resistant and other characteristics, it is easy to inactivate it by heating during dough fermentation, which is conducive to controlling the fermentation speed and degree
of dough, so as to avoid producing a large amount of dextrin for too long.
The fungal α-amylase can hydrolyze the damaged starch in flour to produce small molecules of dextrin, which are further fermented by yeast to produce alcohols and carbon
dioxide, thereby increasing the volume of bread.
The reducing sugar produced in this process can participate in the Maillard reaction during bread baking, helping to improve the appearance of the bread color.
Therefore, the addition of fungal
α-amylase can not only accelerate the fermentation rate of dough, improve the structure and volume of bread, but also promote the taste, color , quality of bread,etc.
In addition, fungal α-amylase has been used in beer brewing industry (improving
the fermentability of wort), rice wine brewing industry (improving wine quality and increasing liquor yield) and raw alcohol industry to liquefy starch in the mash at low temperature (50-60℃) and isomaltose oligosaccharide production.
In the research and development process of fungal α-amylase, the research on further increasing the unit yield of amylase is still an effective way to improve production efficiency and promote the development of the industry.
However, the development
of new products with new properties is also essential to improve product performance, deepen the industrial structure, broaden the application of products,etc.
For example, the development and use of the fungal α-amylase with the hydrolyzing ability
of raw amylase for synchronous fermentation to produce bioalcohol can save energy and increase the alcohol yield at the same time, which has a certain promoting effect on improving industrial benefits and promoting the development of bioalcohol industry.
The use of fungal α-amylase, a heat-resistant and acid-resistant type of fungal α-amylase, can reduce the problem of bacterial contamination and the corresponding cost in the production of high maltose.
The traditional fungal α-amylase has poor efficiency in the action of β-1, 6 bonds in starch, and the limit value of maltose in the product is only 60%. Other enzymes must be added in the production process of high malt syrup.
Therefore, the research and development of α-amylase with high maltose forming ability have good application potential to change the traditional maltose production mode, save the use of other enzyme preparations and improve the production efficiency of maltose.
In conclusion, improving the performance of fungal α-amylase or researching and developing enzymes with special application value is of great significance to meet the domestic market demand, adjust the industrial structure of enzyme preparations in China, expand the market, drive the development of related food or fermentation industries,etc.