Feed enzyme preparation refers to the extracellular enzyme produced by using microbiological technology and is usually used as a feed additive. It plays an important role in eliminating anti-nutritional factors, increasing resource utilization, opening up new feed sources, and solving environmental pollution in animal husbandry. In recent years, feed enzyme preparations have been widely used in agricultural production, research is very active, and has broad application prospects.
At present, there seems to be controversy regarding the effectiveness of enzymes and the ratio of output to output. In the future, apart from focusing on the application conditions of enzymes in feeds and designing enzyme preparations based on specific feed components, the focus is to improve the quality and efficacy of the produced enzyme preparations, and to use microorganisms, plant seeds, and animals with DNA recombination technologies. Produce enzymes on its own, establish standard procedures for evaluating various enzyme products, and increase research efforts in predicting enzymes for various animals and various feed effects models. In particular, the application of genetic engineering technology in the study of feed enzyme preparations has become the main method for enzyme preparations research and production in countries all over the world, especially in developed countries.
The application of genetic engineering technology in feed enzymes mainly includes two aspects: (1) The use of recombinant microbial reactors to efficiently express the target enzyme and reduce production costs. (2) Using genetic engineering technology to improve feed enzyme preparations to improve the quality and efficiency of enzymes. With the development of genetic engineering technology, through the genetic modification of some microorganisms, for example, by means of genetic engineering, the basic structure of enzyme proteins has been changed, and this method of enhancing the functional properties of enzymes in certain aspects has become a model of commercial success. However, this practice poses a safety hazard to the application of enzyme preparations. Therefore, the enzyme preparations that have been transformed, especially genetically engineered, must undergo a reasonable and necessary safety evaluation before they can be industrialized and applied.
Progress in protease research
Proteases are enzymes that catalyze the hydrolysis of proteins and are one of the most widely used enzyme preparations in agriculture. There are many kinds of protease preparations, important are pepsin, trypsin, papain, and so on. Proteases are widely present in the digestive tracts of humans and animals and are abundant in plants and microorganisms. Due to limited animal and plant resources, protease preparations in agriculture are mainly produced by microbial fermentation. Therefore, feed proteases are mainly derived from microorganisms. There are many types of microorganisms that produce proteases, including bacteria and fungi, such as Bacillus, Aspergillus ( Aspergillus), Penicillum, and the like.
The addition of protease in monogastric animal feed mainly has two major functions. One is to supplement the deficiency of animal endogenous proteases. The digestive system of young pigs and young birds is imperfectly developed, with insufficient enzyme production and low microbial activity. The addition of protease to feed can help young animals digest the protein. The second is to eliminate the role of anti-nutritional factors. Botanical ingredients in feeds, especially legumes, are widely available with protease inhibitors and lectins. They have a great influence on the nutritional value of feed, but for poultry, further research is needed. Experiments have shown that many microbial proteases can degrade protease inhibitors and lectins that inhibit proteases in the digestive tract of animals, eliminate their effects, and reduce the anti-nutritional effects of these factors.
Ruminants can use both plant and animal proteins. Most plant proteins and all non-protein nitrogens are broken down by microorganisms in the rumen to synthesize bacterial proteins. The bacterial proteins are digested and absorbed by proteases secreted by animals.
At present, many advances have been made in improving the thermal stability of proteases through gene mutations. Imanaka et al. proposed a strategy for increasing the thermal stability of proteases through site-directed mutagenesis. He compared the resistance to Bacillusstearothermophilus and Bacillustherproteolyticus. The amino acid sequence and higher structure of the hot protease determine the amino acid residues that may further improve the heat resistance of Bacillus stearothermophilus. Gly144 is located in the a-helix and replacing it with Ala (M1 mutation) is expected to increase the hydrophobicity of the α-helix. And stability, thereby improving its heat resistance, on the contrary, replaced by Ser (M3 mutation) after Thr66 is expected to reduce its heat resistance, the test results fully confirmed this idea. Yamashita et al. used the Asp protease MPR obtained from Mucorus illus as a material, replacing Ala101 with Thr, Asp replacing Gly186, and then expressing the mutated gene in S. cerevisiae Saccharomyces cerevisiae. The results showed that these two mutations, especially those at Gly186, caused a significant decrease in the enzyme's thermostability. If these two mutations occur on the same molecule, the thermal stability of the enzyme is further reduced. Although these above studies are still in the laboratory research stage, two examples, one positive and one negative, fully illustrate the theoretical feasibility of using this method to improve the heat resistance of feed enzymes.
Many extracellular enzymes produced by microorganisms are glycosylases, oligosaccharides and enzymes are covalently linked in N-glycosylation or O-glycosylation, and some are connected in non-covalent forms through various physics. Method to separate it. For these extracellular protease-linked oligosaccharides, it is generally believed that the enzyme is protected from the hydrolysis of the immune protease and enhances the reversibility of the enzyme during extracellular secretion. Tsujita isolated an acidic protease A1 from A. oryzea, A1 It can also be separated into two components, A1a and A1b, and the glycosylation ratio of each component is 50%. Tests have shown that glycosylated enzymes are resistant to the degeneration of 50% citric acid. According to this feature, the properties of the protease can also be improved by selecting different expression systems to change the degree and type of glycosylation of the enzyme.
Progress in Amylase Research
Starch is generally composed of amylose and amylopectin. Amylases mainly used in the feed industry include a-amylase, pullulanase, and glucoamylase. Pullulanase is characterized by the ability to specifically cut the a1-6 glycosidic linkages at branch points such as starch and glycogen to form amylose. Therefore, the use of this enzyme in combination with other amylases can completely saccharify the starch. The strains that produce pullulanase in microorganisms are very widespread. Yeasts were first discovered, in addition to bacteria such as Pseudomonas, Bacillus, Azotobacter and some actinomycetes. In recent years, pullulanase has been used as a new type of amylases in feed industry and food industry. At present, the main component of feed is corn, of which amylopectin is difficult to use, resulting in wastage of feed. Such as adding a certain pullulanase, and then under the action of animal endogenous and exogenous amylase, can be a good digestion and absorption, improve feed utilization, reduce costs.
Since the high temperature stage in feed processing and the main role of amylase in feeds are in the acidic gastrointestinal tract of animals, the research on genetic engineering of amylase for feeds is mainly focused on enzyme resistance as other feed enzymes. Thermal and acid stability. Through years of efforts, this research has made some progress.
Since the 1980s, many amylase research efforts have focused on the mechanism of heat inactivation of enzymes. There are two main causes of thermal inactivation of a-amylase. The first is that the high temperature causes the conformation of the protein molecule to change and the ordered structure of the molecule becomes disorganized. At pH 5.0, 6.0, and 8.0, the α-amylase in Bacillus amyloliquefaciens is irreversibly inactivated when the temperature is increased to 90°C, but when the pH is 8.0, the presence of starch inhibits the loss of enzyme. live. The second and more important reason is that at high temperatures, deamidation of Glu or Asp residues occurs, resulting in the loss of enzyme activity. The main reason for heat inactivation of a-amylase in Geobacillus is mainly Glu or Asp. Deamidation. Compared with mesophilic enzymes, high-temperature enzymes have an additional salt bridge consisting of 2 to 3 specific Lys residues. DNA recombination technology provides new ideas for obtaining thermostable amylase. Substitution of the 88th and 253th amino acid residues in the Bacillus stearothermophilus a-amylase molecule by Lys and/or Arg can increase the half-life of the enzyme at high temperatures. Similarly, when the 88th, 253rd, and 385th amino acid residues in the B. amyloliquefacis α-amylase molecule are replaced by Lys or Arg, their thermal stability is also improved.
In 1999, Mitchinson et al. issued a patent to improve the temperature resistance and acid resistance of a-amylase. The gene of interest for the study was derived from the a-amylase encoding gene of Bacillus licheniformis NCIB8061. Using genetic mutation techniques, they changed the nature of the enzyme. The mutations mainly included three types: one site-directed mutation of 188 Asn; the second 188 Asn and 15 Met were replaced by other amino acids at the same time; the third mutation included not only the first two amino acid substitutions but also other positions. Point amino acid substitutions. The target gene was transformed into the recipient strain Bacillus subtilis after site-directed mutagenesis. It was found that both the recombinant a-amylase secreted by the recipient strain and the a-amylase expressed by the original strain were improved in temperature resistance, acid resistance and effectiveness. For example, the M15T/N188S mutant a-amylase activity was increased 2.1-fold (soluble substrate) and 1.7-fold (starch substrate), respectively, compared to the original enzyme. M15T/H133Y/N188S three-point mutation of the a-amylase specific activity increased by 2.8 times; compared with the original a-amylase, M15T/N188S mutant a-amylase has higher enzyme activity under acidic conditions, when When the pH value drops to 5.2, the original enzyme has no activity, but the mutant a-amylase is still active; the mutant enzyme reduces the dependence of the enzyme on Ca2+. These properties of mutant a-amylase improve the effectiveness of the application of a-amylase in feed and provide new ideas for the genetic engineering of feed amylase.
The construction of hybrid genes is also an effective method for improving amylase properties. For example, Shibuya hybridizes the a-amylase gene derived from A. shirousamii and the glucoamylase gene to express a large 145 kDa protein in Saccharomyces cerevisiae. The bifunctional fusion protein, which has both a-amylase activity and glucoamylase activity, greatly increased the efficiency of the enzyme's degradation of starch, but the expression level was only 2.3 mg/L. In the same year, Shibuya et al. expressed the heterozygous gene in Aspergillus oryzae to increase the expression level to 500 mg/L, 200 times more than that in Saccharomyces cerevisiae.
Although most of the work on amylase modification by genetic engineering is still in the laboratory stage, its potential has been recognized by scientists.
With the improvement of people's living standards and the enhancement of environmental awareness, feed enzyme preparations will be further promoted for their advantages of non-residue, non-resistance, and environmental pollution. Feed enzyme preparation can not only improve the digestibility and utilization rate of feed, increase the production performance of livestock and fish, but also reduce the excretion of nitrogen and phosphorus in livestock excrement and protect water and soil from pollution. Of course, the universal application of feed enzyme preparations in research and production is still faced with many problems. The Chinese government is also actively protecting feed and safety by prohibiting the use of antibiotics and hormones in feeds to maintain ecological balance. However, with the improvement of biotechnology, especially genetic engineering technology and production technology, it will be gradually resolved. Therefore, the feed enzyme preparation as a high-efficiency, non-toxic side-effect and environment-friendly "green" feed additive will have a very broad application prospect in the 21st century.
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