Amylase is a kind of hydrolase and is a biocatalyst for starch hydrolysis. According to the degradation mode, it is divided into α-amylase, β-amylase, glucoamylase, isoamylase and cyclodextrin-producing enzyme.
When α-amylase hydrolyzes the long-chain starch molecules into short-chain molecules, it cuts the α-1,4 glycoside bonds in the starch macromolecules in a random manner to make the starch produce dextrins, oligosaccharides, etc., due to the end of the product In the glucose residue, C, the carbon atom is in the α-configuration, so it is called α-amylase. Most α-amylases (except for individual varieties) cannot cut the α-1,6 glycoside bond, nor the α-1,4 glycoside bond near the branch point. Currently, α-amylase has a relatively large output in my country and is widely used in food, brewing, pharmaceutical and textile fields. When using α-amylase to hydrolyze starch, pay attention to the hydrolysis conditions. The main factors that affect the activity of α-amylase: pH, temperature and metal ions.
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Generally, the activity of α-amylase is relatively stable when the pH value is 5.5-8.0, and when the pH value is less than 4, the enzyme is easy to lose its activity. However, some α-amylases are slightly acidic or alkaline. For example, the most suitable pH value of α-amylase produced by Aspergillus niger is 4. When the pH value is 2.5 and the temperature is 40°C, it still remains after 30 minutes of treatment. Certain activity, but at a pH of 7 and a temperature of 55°C for 15 minutes, the α-amylase is almost inactivated. The opposite is true for the α-amylase produced by rice koji enzyme. When the pH value is 7 and the temperature is 55°C, the enzyme activity is not lost after treatment for 15 minutes, but when the pH value is 2.5, the enzyme activity completely disappears.
The α-amylases currently used mainly have resistance to medium temperature (60-70℃) and high temperature (90-95℃). According to the analysis of enzyme reaction kinetics, every time the temperature increases by 10°C, the reaction speed increases by 1-2 times, so it is resistant to high temperature.
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Alpha-amylase has the advantages of fast reaction speed, high efficiency and low cost. Alpha-amylase is a kind of metalloenzyme. Ca2+ keeps alpha-amylase in a proper conformation, thereby maintaining its maximum activity and stability. In addition to Ca2+, other divalent alkali metal ions Ba2+, Mg2+, etc. also have the effect of maintaining α-amylase activity. In order to adapt to the high temperature conditions of the production process, the medium temperature-resistant alpha-amylase sometimes needs to add stabilizers such as Ca2+, but the high-temperature resistant alpha-amylase has good stability when the Ca2+ concentration is very low. In actual use, there is no need to add stabilizers such as Ca2+.
The current production problems of the bidding project are: 1. BF7658α-amylase production: In the case of continuous rain, the activity of fermentation enzymes fluctuates up and down, the high can reach 450μ/ml, and the low is only 300μ/ml. It is urgent to solve the fermentation enzyme. The problem of ups and downs. 2. High conversion rate saccharification enzyme production: the fermentation cycle (time) can be delayed up to 40 hours, and the fermentation enzyme activity is only 28,000 μ/ml, and the extraction yield averages 60%. It is urgent to solve the long fermentation cycle, low enzyme activity, The problem of low yield.
Glucoamylase (EC 3.2.1.3), also known as glucoamylase, is a highly efficient biocatalyst produced by fermentation of mutant strains of Aspergillus niger. Glucoamylase can cleave the α-1,4 and 1,6 glycosidic bonds of starch molecules under normal temperature conditions to convert starch into glucose. Any production process that uses starch as a raw material and requires saccharification can use glucoamylase to increase the saccharification yield of starch. The temperature is below 40℃, the pH is stable from 4.0 to 5.5, and the temperature is higher than 60℃, and the pH is lower than 2.5 or higher than 6.0. It is easy to inactivate.
Enzyme activity definition 1 ml of enzyme solution at 40°C, pH 4.6, hydrolyze soluble starch for 1 hour to produce 1 mg of glucose as 1 unit of enzyme activity. Use conditions When saccharification enzyme is saccharifying starch, the optimum temperature is 55～60℃, and the optimum pH is 4～5. In the normal temperature or low temperature fermentation process required by wine, glucoamylase can continue to function in the presence of yeast to gently saccharify starch materials and carry out alcohol fermentation.
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The application range of amylase is very wide, and there are many kinds of enzymes. Different kinds of enzymes can be selected according to different needs. Alpha-amylase is mainly used to produce maltodextrin. Glucoamylase catalyzes dextrin to get glucose, β-amylase can get maltose, and glucose isomerase can convert glucose into fructose. The general amylase catalysis is carried out under high temperature conditions.
The application of protein engineering can improve the thermal stability of enzymes. Someone got the double mutant A209V/H133T of B. licheniformmis α-amylase. The half-life of the enzyme at 90°C was extended by 9 times. Mitchinson et al. used site-directed mutagenesis and high-throughput screening methods to obtain a mutant whose optimal pH increased by 0.5 to 1.0. Gloria et al. [13] used Phe or Tyr to replace Ala at position 289 of B. stearothermophilus α-amylase, which has the ability to catalyze alcoholization reactions. Sierks et al. [14] reported that by changing the three amino acid residues of the active site of glucoamylase, the Kcat/Km ratio of 1,4-glycosidic bonds to 1,6-glycosidic bonds was increased by 300 times.
Concept and characteristics of enzymes
(1) The concept of enzymes: organic substances with biocatalytic activity produced by living cells.
(2) Characteristics of enzymes:
- ATP and energy
(1) ATP is a direct energy source for life activities, but its content in cells is not high, so the formation and decomposition of ATP in cells are quite frequent.
(2) Energy is involved in the formation and decomposition of ATP, but the energy properties of the two are different.
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