What is the EC4 enzyme?
EC4 enzyme, known as Lyases, catalyze a unique reaction that causes the cleavage of C-C, C-O, C-N, and various other chemical bonds, primarily by means other than hydrolysis and oxidation.
In living organisms, enzymes act as highly efficient catalysts to drive various chemical reactions.
In order to facilitate research and differentiation, a systematic enzyme classification system has been established internationally, which divides enzymes into six categories according to the type of catalytic reaction, namely REDOX enzyme (EC1), transferase (EC2), hydrolase (EC3), lyase (EC4), isomerase (EC5) and ligase (EC6).
Figuratively speaking, it is like a skilled craftsman, precisely removing specific groups from the substrate, while cleverly leaving double bonds;
In the reverse reaction, it can precisely add a group to the double bond like the splicing master.
Significantly different from other enzymes, it requires only one substrate in the forward reaction and two substrates in the reverse reaction.
In the EC4 enzyme family, there are many familiar members, such as decarboxylase, aldolase and dehydrase.
In the case of decarboxylase, it can precisely remove carboxyl groups from the substrate molecules when catalyzing the reaction, allowing them to be released in the form of carbon dioxide, while cleverly forming double bonds in the remaining molecular structure.
Aldolase plays a key role in the process of sugar metabolism, which can efficiently cleave aldolase and lay the foundation for the subsequent metabolic reaction.
Dehydrases focus on catalyzing the removal of water molecules from the substrate, prompting the rearrangement and formation of chemical bonds.
Classification and analysis of EC4 enzymes
1. Seven sub-categories are introduced in detail
The EC4 enzyme family is huge, in order to study and use them more carefully, scientists have further divided it into seven unique subclasses according to the type of chemical bonds in its catalytic cleavage.
(1) EC4.1 subclass is mainly responsible for the cleavage of carbon-carbon bonds
In this subclass, pyruvate decarboxylase is a typical representative.
It plays a key role in the microbial fermentation process, which can cleverly remove carboxyl groups from pyruvate molecules, convert them into carbon dioxide and release them, while forming acetaldehyde.
This process is crucial in alcohol fermentation, laying the foundation for subsequent alcohol generation.
For example, in the fermentation process of cerevisiae cerevisiae, pyruvate decarboxylase plays an indispensable role in promoting the gradual conversion of sugars into alcohol and carbon dioxide.
(2) EC4.2 subclass focuses on the cleavage of carbon-oxygen bonds
Fumarate hydratase, for example, plays an important role in the tricarboxylic acid cycle.
This enzyme can catalyze fumaric acid to react with water and precisely split the carbon-oxygen bond to produce malic acid.
This reaction is like an important link in the metabolic chain of substances in the body, ensuring the continuous supply of energy and the orderly transformation of substances.
(3) EC4.3 subclass is responsible for the cleavage of carbon-nitrogen bonds
Aspartic acid deaminase is one of them, which can precisely catalyze aspartic acid molecules to break carbon-nitrogen bonds, and then produce fumaric acid and ammonia.
This reaction plays an important role in amino acid metabolism and nitrogen cycle.
(4) EC4.4 subclass is responsible for the cleavage of carbon-sulfur bonds
Desulphurases, for example, can efficiently catalyze the break of carbon-sulfur bonds in the substrate, releasing sulfur elements from the compound.
This plays a key role in the metabolism of sulfur-containing compounds as well as some special biosynthetic pathways.
(5) EC4.5 subclass is mainly involved in the splitting of halogen bonds
Like halogenated alkane dehalogenase, it can specifically act on halogenated alkane to break the carbon-halogenated bond, so as to achieve the degradation of halogenated alkane.
It has a broad application prospect in the field of environmental remediation, especially in the treatment of soil and water polluted by organic halides.
(6) EC4.6 subclass focuses on the cleavage of phosphorus oxygen bonds
For example, adenylate cyclase, which catalyzes the reaction of ATP, splits the phospho-oxygen bond, and generates cyclic adenosine phosphate (cAMP).
cAMP, as an important second messenger in the cell, plays a central role in the process of cell signal transduction and regulates various physiological functions of cells.
(7) EC4.99 subclass is special
Contains other lyases that are not specifically classified.
Although relatively few in number, the reactions they catalyze are often unique, adding color to the diversity of biochemical reactions.
2. Classification basis and significance
This classification has a rigorous scientific basis, mainly based on the substrate specificity and action mechanism of enzyme-catalyzed reactions.
Through the in-depth study of the substrates and reaction processes of different subclasses of enzymes, scientists can clearly define their boundaries, thus building a systematic and orderly classification system.
Classification is of great significance to scientific research. It is like a precise “navigator”, giving scientists clear directions for screening enzymes for specific functions.
When studying a specific chemical reaction, researchers can quickly locate the subclasses of enzymes that may be involved according to the classification, which greatly improves the research efficiency. Classification also provides a solid foundation for enzyme modification and optimization.
In industrial applications, the importance of classification is self-evident.
Understanding the relationship between the structure and function of enzymes can help scientists to carry out targeted transformation of enzymes through genetic engineering and other means to improve the catalytic activity, stability and specificity of enzymes, so that they can better meet the needs of scientific research.
In the food processing industry, if a specific conversion of a sugar substance is required, the appropriate enzyme can be selected according to the classification of EC4 enzymes to catalyze the reaction, thereby improving the quality and yield of the product.
In the pharmaceutical field, the precise selection of enzymes that can catalyze specific reactions helps to synthesize target drug molecules, reduce production costs, and improve the success rate of drug development.
Core function of EC4 enzyme
1. Catalytic specific reaction
The EC4 enzyme’s core function is to catalyze a series of specific chemical reactions.
Pyruvate decarboxylase is a model in the cleavage of carbon-carbon bonds.
In the key stage of alcohol fermentation, it precisely acts on pyruvate, breaking the carbon-carbon bond, releasing carbon dioxide, and successfully forming acetaldehyde, laying the foundation stone for the final production of alcohol.
Aldolase plays a key role in the process of sugar metabolism in vivo. It skillfully cleats fructose-1, 6-diphosphate to produce glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, which provide important intermediates for subsequent energy generation and substance conversion.
Fumarate hydratase performed well in the cleavage reaction of carbon-oxygen bond.
In the tricarboxylic acid cycle, it causes fumaric acid to react with water, precisely breaking the carbon-oxygen bond and producing malic acid.
This reaction acts as a key cog in a precise clock, ensuring the stable operation of the tricarboxylic acid cycle and providing a constant stream of energy to the cell.
Aspartate deaminase plays an important role in the cleavage of carbon and nitrogen bonds.
It acts on aspartic acid to break the carbon-nitrogen bond and generate fumaric acid and ammonia, which is of great significance for the metabolism of amino acids and the circulation of nitrogen in organisms.
2. Functional differences with other enzymes
Compared with hydrolase, hydrolase mainly breaks chemical bonds by adding water, for example, amylase hydrolyzes starch to glucose, and its reaction process cannot be separated from the participation of water molecules.
EC4 enzymes, on the other hand, split the bonds in a unique way other than hydrolysis and oxidation, without the direct involvement of water molecules in the reaction process.
In terms of substrate specificity, EC4 enzymes exhibit a high degree of specificity.
Pyruvate decarboxylase has catalytic activity only for a specific substrate of pyruvate, and can accurately identify and act on pyruvate molecules for the cleavage of carbon-carbon bonds.
Other enzymes, such as some transferases, although they also have a certain substrate specificity, they mainly catalyze the transfer reaction of the group, and the chemical bond cleavage or addition reaction catalyzed by EC4 enzymes is quite different.
From the perspective of reaction conditions, most EC4 enzymes can function under relatively mild conditions.
At normal temperature, atmospheric pressure and near neutral pH in living organisms, they can catalyze reactions efficiently.
This characteristic of EC4 enzymes is particularly valuable compared to some chemical reactions that require high temperatures, high pressures, or special pH environments.
This makes the biochemical reaction using EC4 enzymes more energy efficient and environmentally friendly, and also provides convenient conditions for its normal operation in industrial production and in organisms.
Application of EC4 enzyme in synthetic biology
1. Preparation of low phenylalanine casein
Phenylketonuria (PKU) is an autosomal recessive genetic disorder in which patients are unable to metabolize phenylalanine properly due to a lack of phenylalanine hydroxylase, resulting in the accumulation of phenylalanine and its ketoacids, which are excreted in large quantities from the urine.
For PKU patients, lifelong consumption of low phenylalanine foods is the key to controlling the condition.
In this context, a novel saccharomyces rhodocerevisiae phenylalanine ammoniase (EC4.3.1.24; PAL played an important role.
According to the research report “Preparation of low phenylalanine casein using a new type of Saccharomyces rhodosus phenylalanine lyase” published in the “Biotechnology Bulletin”, the research team successfully cloned RmPAL and RdPAL genes from saccharomyces colloides and saccharomyces biobovate, and achieved heteroexpression and purification in Escherichia coli.
The results showed that these two enzymes showed good activity and high thermal stability at 50℃ and pH8.9.
The results of the experiment were surprising. They were able to efficiently remove L-phenylalanine from commercially available casein hydrolysates, and the conversion rate of substrate L-phenylalanine was as high as 89% and 93%, respectively, after 24 hours of reaction.
This result is significant and offers new hope for patients with PKU.
The preparation of low phenylalanine casein by this enzymatic method can meet the needs of patients for low phenylalanine food, effectively control the level of phenylalanine in patients, thereby slowing or preventing nervous system damage and improving the quality of life of patients.
Moreover, compared with the traditional preparation methods, the enzymatic method has the advantages of green environmental protection and strong specificity, and has injected new vitality into the technical innovation and production application in the field of low phenylalanine protein.
2. Synthesis of p-sulfoxylphenylserine
P-methylsulfoxylphenylserine is a key chiral block for the synthesis of β-aminoalcohol-based broad-spectrum antibiotics sulfoxamycin and flufenicol, which plays an important role in the field of medicine.
The traditional chemical synthesis method has many disadvantages in the preparation of p-methylsulfoxylphenylserine.
For example, in the process of synthesizing p-methylsulfone phenylserine copper salt, a large amount of copper sulfate wastewater will be produced, causing serious pollution to the environment;
In the process of racemic separation, not only the steps are complicated, but also a lot of waste water is generated, and the theoretical yield of the target product can only reach 50%.
The emergence of L-threonine aldolase (EC4.1.2.5) provides an effective solution to this problem. The patent invention of Fujian Changsheng Biotechnology Development Co., Ltd. describes its application in detail.
The researchers mined and screened the L-threonine aldolase gene from Actinocorallia herbida, and successfully expressed the enzyme in recombinant Escherichia coli with the help of genetic engineering technology.
(2S,3R) -sulfoxylphenylserine can be synthesized asymmetrically from p-sulfoxylbenzaldehyde and glycine by whole cell catalysis under mild reaction conditions, that is, 4-12 hours at 25-35 ℃.
This biocatalysis method has a number of significant advantages.
The reaction conditions are mild, avoiding the requirements of high temperature, high pressure and other harsh conditions on the equipment and energy consumption;
It is friendly to the environment and greatly reduces the discharge of pollutants such as wastewater, which is in line with the development concept of green chemistry;
With strong stereoselectivity, p-methylsulfoxylphenylserine with specific configuration can be synthesized efficiently, and the purity and quality of the product are improved.
This application promotes the innovation of p-sulfoxylphenylserine synthesis process, provides a more efficient and environmentally friendly way for the production of related antibiotics, and also makes a positive contribution to the development of the pharmaceutical field.
EC4 enzyme market industry status
1. Market size and growth trend
With the vigorous development of biotechnology, the enzyme preparation market has shown a strong growth momentum.
According to the data of China Research Industry Research Institute, the global industrial enzyme market size is about 6.3 billion US dollars in 2020, and is expected to reach nearly 9 billion US dollars by 2026, with a compound annual growth rate of 6% from 2021 to 2026.
Although the separate market size data of EC4 enzymes have not obtained, as an important part of the field of enzyme preparations, EC4 enzymes have also taken the east wind of this development and ushered in a broad market space.
The rising demand for EC4 enzymes in the food and beverage industry is strongly driving the market growth.
In the process of juice processing, the use of specific EC4 enzymes can effectively improve the juice yield, improve the clarity and taste of the juice, so as to improve the quality of the product and market competitiveness.
In baked goods preparation, certain EC4 enzymes can optimize the properties of dough, increase the volume and softness of bread, and extend the shelf life of products.
With the increasing attention of consumers to healthy food, the demand for natural and efficient enzyme preparations continues to grow, which provides a broad space for the application of EC4 enzymes in the food and beverage industry.
In the pharmaceutical field, the application of EC4 enzymes also brings new opportunities for drug development and production.
It can used to catalyze specific chemical reactions, synthesize drug molecules with specific structures and functions, improve the purity and activity of drugs, and reduce production costs.
With the development of precision medicine and personalized medicine, the demand for highly efficient and highly specific enzyme preparations will continue to increase, which will further promote the market growth of EC4 enzymes in the pharmaceutical field.
The enhancement of environmental awareness has prompted people to seek more environmentally friendly industrial production methods. EC4 enzyme has broad application prospects in bioreactor, sewage treatment and other fields because of its high efficiency and environmental protection characteristics.
In bioremediation, the use of EC4 enzymes that can degrade specific pollutants can transform harmful substances in the environment into harmless substances to achieve the restoration and treatment of polluted environments.
In sewage treatment, by adding specific EC4 enzymes, the decomposition of organic matter in sewage can accelerated, the efficiency of sewage treatment can improved, and the treatment cost can reduced.
With the increasingly strict environmental regulations and people’s increasing attention to environmental protection, the market demand for EC4 enzymes in the field of environmental protection expected to continue to grow.
2. Major firms and competitive landscape
In the global enzyme preparations market, Dubongenko, Royal DSM, Novozymes, BASF, Germany AB enzyme preparations and other enterprises occupy a dominant position, they rely on strong research and development strength, advanced production technology and extensive market channels, in the high-end market occupy a large share.
These companies are also industry leaders in the development and production of EC4 enzymes, constantly introducing new products and application solutions to meet the needs of different industries.
In China, the enzyme preparation industry has developed rapidly in recent years, and a number of competitive enterprises such as E-Doli, Xinhua Yang and Azure Biology have emerged.
They have reached the international leading level in the field of feed enzymes and other segments, and have also achieved certain results in the development and application of EC4 enzymes.
Etori focuses on the research and development, production and sales of biological enzyme preparations, and establishes a good brand image in the market through continuous technological innovation and product optimization.
In the development and production process of enzyme preparations, Xinhua Yang pays attention to cooperation with universities and scientific research institutions, strengthens technological innovation, and improves product quality and performance.
Azure Biological committed to providing customers with high-quality enzyme products and professional technical services, and increasing market share through continuous expansion of market channels.
Domestic enterprises in the low-end market competition is fierce, product homogenization phenomenon is more serious.
In order to stand out in the market competition, enterprises need to continuously increase investment in research and development, improve the technical level, optimize product structure, improve product quality and performance, and strengthen brand building and market promotion to enhance brand awareness and reputation.
Through continuous innovation and improving their competitiveness, domestic enterprises expected to occupy a larger share in the EC4 enzyme market and promote the development of the industry.
Future development prospects of EC4 enzymes
Looking ahead, EC4 enzymes show great potential in emerging fields.
In the field of gene editing, with the deepening of the research on gene regulation mechanism, it may found that some EC4 enzymes involved in the process of specific gene cutting and repair, providing new tools and ideas for the development of gene editing technology.
Just as the CRISPR-Cas system revolutionized the field of gene editing, the EC4 enzyme of the future could become another bright star in the field of gene editing.
In terms of biosensors, the specific catalytic reaction of EC4 enzymes can combined with the signal transduction system to develop highly sensitive and highly selective biosensors for detecting harmful substances in the environment, disease markers in organisms, etc.
Imagine that in the future, we may be able to quickly and accurately detect potential disease risks in the body through a small biosensor, which will greatly promote the progress of medical diagnosis technology.
With the in-depth research and continuous innovation of enzyme technology, its believed that more new EC4 enzymes will discovered and developed, and their catalytic activity, stability and specificity will continue to improve.
They will play a key role in more areas, providing strong support for solving many problems facing individuals, such as improving environmental quality, promoting the development of green energy, overcoming difficult diseases, and making important contributions to the sustainable development of individuals.
