what type of macromolecule is an enzyme？
As we all know, living organisms are composed of cells. Enzymes are the catalysts of metabolism in the human body, and only when enzymes are present can chemical reactions take place in the human body, metabolism in the body can proceed, and each cell can show all kinds of life activities. The more enzymes in the body, the more complete, the healthier the life. Most human diseases are related to enzyme deficiency or synthesis disorder.
As a matter of fact, enzymes are often encountered in our lives, such as enzyme-enriched laundry detergent, creatine kinase and other types of “enzyme” indicators in the blood, and so on, which are everywhere “enzymes”. Today, let’s take a look at enzymes and their functions.
What is an enzyme?
Enzymes are proteins with high efficiency and specific catalytic effects. Almost all metabolic reactions in the body require the participation of enzymes, and the control of material metabolism is mostly realized through the regulation of enzyme activity. It is now clear that many human diseases are due to the mutation, reduction or absence of certain enzymes, and therefore enzyme deficiency or mutation can cause metabolic disorders and lead to disease. A catalyst only accelerates a chemical reaction to an equilibrium point, it does not change the equilibrium point.
The same is true for enzymes, although they are extremely efficient compared to non-enzymatic catalysts; moreover, enzymes catalyze only certain chemical reactions of specific substances (called effectors), producing certain products without by-products, i.e., enzymes have a very high degree of specificity. The catalytic ability of an enzyme is called enzyme activity, which can be measured, and the amount of enzyme is often expressed in terms of its activity. Measurement of the activity of certain enzymes often helps in the diagnosis of diseases, so enzymology is very close to the etiology, diagnosis and treatment of diseases.
The nature of enzymes
During the Shang and Zhou Dynasties in China, there are records of the production activities of enzymes in microorganisms, such as brewing, making sauce, and making syrup. However, it was not until the beginning of the 20th century that a conclusion was reached about the nature of enzymes; in the middle of the 19th century, it was still believed that enzymes had to work in living organisms; the original Greek meaning of the word “enzyme” was “in yeast”, and it was only when it was discovered in 1897 that cell-free yeast extracts could be fermented that it was realized that enzymes could still work outside the cell.
In 1926, J.B. Sumner, an American biochemist, purified urease and crystallized it, proving that it was a protein, which was the first to propose the concept that enzymes are proteins. However, the academic authority at the time more objection, do not think that the enzyme has been crystallized out, on the contrary, think that the crystallization of the protein is not functioning, while the role of the pollutants attached to the nature of the unknown. Later, other scientists also purified and crystallized such as pepsin and trypsin and other protein hydrolases, and also proved that they are proteins, the essence of the enzyme is a protein this conclusion is recognized by the scientific community.
Thousands of enzymes have been discovered, hundreds of enzymes have been purified and crystallized, as well as analyzed and determined the chemical structure of the first level of the enzyme, have been proved to be proteins. The concept that enzymes are proteins is so solid that it would be inappropriate to call them enzymes if macromolecules with catalytic properties other than proteins were discovered. Therefore, several newly discovered ribonucleic acids with catalytic activity have been called enzyme-like.
Enzyme specificity
One of the most striking features of an enzyme is the specificity (or specificity) of the reaction it catalyzes. This refers both to the enzyme’s choice of effectors and to the specificity of the reaction it catalyzes. The degree of specificity varies from enzyme to enzyme.
For example, urease only catalyzes the hydrolysis of urea to CO2 and NH3; succinate dehydrogenase only succinic acid as the effector, their specificity is extremely strict, which can be referred to as absolute specificity, more enzymes have a common group or chemical bond selectivity; such as phosphatase can catalyze the hydrolysis of many kinds of phosphoric acid-containing compounds to remove phosphoric acid, and esterases catalyze the hydrolysis of the ester bond of many different compounds, the selection of the less stringent, the. This can be called relative specificity.
It can be seen that the specificity of different enzymes for the action of substances varies greatly, even the same class of enzymes, due to different sources, the degree of specificity of the strict degree of inconsistency.
Importance of enzymes
The human body and other organisms undergo thousands of different chemical reactions. All activities such as digestion, absorption, transportation, synthesis, secretion, locomotion, and reproduction (commonly referred to as substance metabolism) are based on chemical reactions. Most of these reactions are slow, but enzymes accelerate them so that the various activities on which life depends can be carried out in a timely manner. The vast majority of these reactions take place in the cell; each reaction is catalyzed by a different enzyme; the cell contains thousands of enzymes, segregated in various organelles, which methodically catalyze life-critical reactions.
Take the starch that we eat every day as an example, starch is digested in the digestive tract and hydrolyzed to glucose by amylase and other catalytic enzymes, while glucose enters the cell, which is also catalyzed by enzymes, and the various metabolisms of glucose in the cell are even more a series of enzyme-catalyzed reactions, which oxidize the glucose into carbon dioxide and water and supply energy, and can also be converted into other substances such as fat. Compared to the oxidation of glucose in the body and its combustion outside the body, although the products are carbon dioxide and water, and both release energy, but the oxidation of glucose in the body is catalyzed by enzymes, and is carried out under mild conditions, such as room temperature, and goes through a number of steps and gradually releases energy that can be easily utilized, which is extremely different from the combustion outside the body.
I. In the food fermentation industry
The brewing of soy sauce involves the use of proteases secreted by Aspergillus oryzae to break down the proteins in the raw materials and degrade them into peptides, amino acids, etc., so as to produce soy sauce with color, aroma and taste. For example, acidic protease used in soy sauce production can make up for the lack of enzyme in the currant, promote the decomposition of protein in raw materials of soy sauce and vinegar, strengthen the production process, and facilitate the large-scale production. Moreover, the hydrolysis of protease can increase the content of amino nitrogen in soy sauce spirits, thus promoting the fermentation process and the formation of aroma and flavor substances. At the same time it also helps to improve the utilization rate of raw materials, save food, reduce costs, and contribute to the improvement of production and the stability of product quality.
II. In beer brewing
When the amount of malt is reduced and auxiliary materials are increased, supplemental protease is often needed to fully degrade proteins, and microbial acid protease is also an effective beer clarifier.
III. In tanning production
Tanning raw material skin fibrous protein is a useful component of leather, but there are also many non-fibrous protein exists in the fiber gap and epidermis, these proteins content is small, if not removed, the finished leather will become stiff and brittle. Thus we have to rely on protease, protease is used in leather processing decomposition of interstitial proteins, domestic production of neutral and alkaline protease preparation can be used for enzyme dehairing.
Four. Used in the manufacture of gelatin and soluble collagen fiber:
Industry with lime water leaching skin, bone and other raw materials in the oil and grease and miscellaneous proteins, etc., this process time-consuming up to several months, labor-intensive, low rate of gelatin and energy consumption, with protease purification of collagen, gelatin purity, quality, relative molecular mass uniformity, molecular arrangement of the whole, the production cycle is short, gelatin yield is high.
V. Used for pretreatment of wool low temperature dyeing:
Wool with high-temperature dyeing, will make the strength of wool damage, and easy to cause fiber felting contraction and hair body erection, with protease treatment of wool, dyeing at the boiling point, the color rate of 2min almost up to 100%, the finished product color and lustre is bright, feel plump, and the waste water in the fuel content is greatly reduced.
For silk degumming:
Raw silk fabrics must be degumming, silk glue is a protein, our country has traditionally used alkali soap method of high temperature refining for degumming, many shortcomings, alkali invasion of silk pigment, easy to affect the luster, and with protease degumming, the finished product is lubricated and soft to the touch, glossy and bright, and degumming time is short, the operating temperature is low, and productivity is increased.
Enzyme genetic engineering and protein engineering industrial biocatalysis in the 1990s, the rise of protein directed evolution, genomics and proteomics technology development. The core of industrial biocatalysis is the application of enzymes. Compared with traditional chemical catalysis, biocatalysis has the advantages of site-specificity and stereospecificity, which allows people to carry out “re-evolution” according to human wishes without the need to understand the structure of the enzyme, and can be used for gene cloning, random mutation or hybridization, targeted mutation, and the construction of mutation libraries by error-prone PCR methods. Mutation libraries can be constructed by cloning, random mutation or hybridization, targeted mutation, and error-prone PCR, which can be combined with high-throughput screening strategies to improve enzyme activity, stability, stereoselectivity, and non-aqueous reaction properties.
Xylanase is a key enzyme for hemicellulose degradation, and in China, we used the gene of Streptomyces olivaceus to transfer it to the Pichia piscine yeast Pichiapastoris to obtain efficient expression. The enzyme activity was 1,200 U/mL and specific activity was 2,869 U/mg. It has very good resistance to protease degradation [3]. The four genes of the acidophilic fungus Biospora sp. MEY-1 were successfully cloned into Saccharomyces cerevisiae for heterologous expression, and the specific activity of the recombinant yeast XYL11 enzyme was 1,8831 U/mg, and the enzyme retained more than 87% of its viability at 90 ℃ for 10 min. Degradation of oat xylan mainly produces xylose and xylan-disaccharide, which has good resistance to protease degradation [8]. The xylan-producing gene of black currant IME-216 was cloned and expressed in Saccharomyces cerevisiae, and the vigor was increased to 90 000 U/mL [8], and the rest of the more than 30 xylanase genes were expressed in Escherichia coli and other papers will not be mentioned in detail.
Enzymes for feed have become the fastest growing and strongest enzyme industry in the world industrial enzyme industry. Phytase is a feed additive for degradation of phytic acid into inorganic phosphate and inositol in feed. The Institute of Feed Research, Chinese Academy of Agricultural Sciences, recombined the phytase gene of Aspergillus niger963 into Saccharomyces cerevisiae to obtain highly efficient expression, and the enzyme activity reached 8×105IU/mL, which was more than 3,000 times higher than that of the original Aspergillus niger, and was much higher than that of the engineered fungi reported abroad [4, 11]. 11], and several production enterprises have been established in China.
Cellulase is a multi-enzyme complex enzyme system, irrational design is the current method of cellulase directed evolution, Aspergillus xylosus cellulose exonuclease and cellulose endonuclease has been in phage to demonstrate the function. At present, a number of genes for medium alkaline cellulase have been cloned and expressed, which can be used in textiles and detergents, and the optimized cultivation of neutral endocellulase engineering bacteria for paper industry use, with enzyme activity up to 32,529 U/mL [10], which increased 7.8 times of the original strain. The multifunctional cellulase gene of Fusarium ampullaia crossean was expressed in E. coli, and a high specific activity cellulase line was obtained, with good hydrolytic activity against xylan, p-nitrophenol fiber disaccharide glycosides, and carboxymethyl cellulose [5]. Cloning of Mycobacterium anthropophilum S38 Swollenin gene may be disrupting hydrogen bonds, which is a component of the fungal degradation cellulase system [6]. In addition, research on the lignocellulose degrading enzyme system of the yellow-winged giant termite has been carried out in China
Lipase is an important enzyme class in biosynthesis. At present, China has established a technical platform for the modification, production and application of three lipase genes matured through rational design. The enzyme activity of Penicillium expansum FS8486 was increased by 317% using gene reorganization technology [7]. Lipase “cap” structure for the targeted mutation, to obtain open cap lipase, enzyme specific activity increased by 5.7 times, two-phase catalytic efficiency increased by 1.8 times [8].
China also constructed the surface display engineering bacteria, E. coli engineering bacteria, Picrosporum engineering bacteria, high-density culture technology platform, Pseudohyphae Candida sp. 99-125 lipase activity reached more than 15 000 U/mL [9]. The cloned Rhizopus miehei lipase was successfully expressed in two Pichia yeasts, and the highest enzyme activity reached 18 000 U/mL. The enzyme activity did not decrease at 4 ℃ for 6 months, and the biodiesel yield was more than 95% at 12 h [9]. Rhizopuschinensis lipase gene was successfully cloned into Picrosylla, and the two co-expressed chaperone proteins could increase the enzyme activity by 30%, and the enzyme activity reached 16 200 U/mL [10-11]. At the same time, the study of cell-bound lipase with good tolerance to organic solvents and thermal stability was also carried out.
Amylase is a very important industrial enzyme, accounting for 25% of the enzyme market. At present, the industry is mainly high-temperature enzyme, deep-sea liquid mouth thermophilic anaerobic archaea Thermococcus genus Thermococcus produced extracellular heat-resistant high-temperature enzyme, the optimal temperature of 95 ℃, 100 ℃ still has 60% of the viability of the enzyme gene was cloned by PCR, and was expressed in E. coli [7]. Two strains of heat-resistant starch-producing Geobacillus bacteria were also isolated from Tengchong, Yunnan, and the specific viability was 1,320 and 890 U/mg after purification [7]. The gene of Bacillus alcalophilus was cloned by PCR, in which Bacillus subtilis was heterologously expressed with an activity of 450 U/mL [9]. Mesophilic acidic α-amylase, which is energy-saving and energy-reducing for starch processing, Bacillus sp. α-amylase gene has 98% homology with B. amyliquefaciens α-amylase gene [7].
Mannanase belongs to hemicellulase and is suitable for mannan oligosaccharide preparation. Zhaodong City, Richeng enzyme preparation company with Aspergillus niger engineering bacteria acidic β-mannanase expression activity of 20 000 U/mL, for the current level of production strains of 25 times, in the leading position of fungal genetic engineering bacteria [10]. A. tabescens MAN 47 β-mannanase mutant with high temperature and acid resistance was obtained by DNA shuffling targeted mutation, and the enzyme activity at high temperature of 80 ℃ and pH 4.0 was 10 times that of the wild type. The targeted introduction of N-glycosylation sites enabled the expression of both wild-type and mutant in A. tabescens, and the heat stability, acid stability, and protease resistance were improved. Three mutants with 3-5 times higher mannanase activity than the wild type were also obtained [10-11]. A heat-stable β-1,4-mannanase was cloned from Thermoanaerobacter thermophilus, which had the highest activity at 80 ℃ in high-temperature, low-permeability oil wells and against hydroxyguar gum. The half-life of this enzyme is 46 h [10].
Laccase is a copper-containing polyphenol oxidase, ligninolytic enzyme, can also catalyze the synthesis of phenols, aromatic amines, oligomers. The expression activity of laccase gene cloned from Tanya ruderalis into Saccharomyces cerevisiae was 9.03 U/mL, which was 3 times higher than that of the original strain [5]. Wild Gramineae Panus rudis laccase was transformed into Picros yeast for secretion and expression, and the specific activity of the enzyme was 16.17 U/mg, which was 4.4-fold increased by targeted mutation and random mutation [7]. The novel marine bacterial laccase was subjected to amino acid targeted mutagenesis, and the half-life of the mutant was extended by 3-fold, and the soluble protein was increased by 244% compared with that before optimization. The fermentation yield was 7.9 times higher than that of the wild type [10]. The enzyme activity of laccase from a tropical white-rot fungal strain reached 11,400 U/L (guaiacol method) [7].
Pullulanase, also known as thaumatin polysaccharidase, is a debranching enzyme that breaks down α-1,6-glucosidic bonds. Thermococcus sp. HJ21 produces extracellular high-temperature Pullulanase with an optimal temperature of 95 ℃, and the enzyme activity is still more than 50% at 100 ℃ for 2 h [7]. The gene for this enzyme was cloned by PCR. Anoxy bacillus sp. p28 was cloned into the purulanase gene sequence and recombinant plasmid was constructed. Transformed into E. coli, the product was a single maltotriose, a type I Pullulanase. The purulanase gene of heat-resistant anaerobic Bacillus sp. isolated from Tengchong hot spring was introduced into Bacillus subtilis and expressed. 60 ℃ and 48 h still retained more than 50% viability, and the extracellular enzyme activity was 42 U/mL, which was a 40-fold increase in expression viability [10]. Through gene knockout and recombination technology, Nanjing Bestjie Bioengineering Company modified the gene of wild Bacillus Pullulanase, and made a composite enzyme with glucoamylase, which can prepare glucose with DE value over 96.5, and the trade name is High DEX series, which can satisfy the production of glucose, and it reaches the international level [10].
Penicillin acylase is the key enzyme in β-lactam antibiotic industry. China has successfully entered the enzyme synthesis antibiotic industry, penicillin acylase by mutagenesis to obtain a mutant strain viability of 820 U/mL [2]. Cloning of Bacillus megaterium penicillin acylase gene was expressed in Bacillus subtilis with a viability of 30 U/mL [4]. Penicillin acylase was secreted and expressed in Bacillus subtilis at 864 U/L, which was 144 times higher than the yield of wild-type Bacillus-like producer A. faecalis [5]. The penicillin acylase of B. faecalis was secreted and expressed in E. coli, and the enzyme activity of the improved culture of the engineered bacterium was >2,000 U/L. 7-amino cephalosporanic acid acylase (GL-7-ACA acylase) could transform cephalosporin C, and was expressed in E. coli, with the highest enzyme activity up to 266 U/L [5], and the penicillin acylase from Bacillus megaterium immobilized in Eupergitc vector produced 30 batches of successive transformations. The enzyme was immobilized with Eupergitc vector and produced in 30 consecutive batches with no decrease in activity [6].
D-amino acid oxidase can catalyze the production of D-amino acids to produce the corresponding keto acids and ammonia, this enzyme and 7-aminocephalosporanic acid acylase (7-ACA acylase) two-step production of cephalosporins important raw material 7-aminocephalosporanic acid (7-ACA). A highly expressed recombinant Picrosporum spp. was constructed using D-amino acid oxidase expressed by methanolic yeast, and the fermentation viability reached 8 000-1 2000 U/L in 14 L tanks [5]. Picot yeast fusion-expressed D-amino acid oxidase with a viability of 1 700 U/L was constructed [5], and two types of recombinant GL-7-ACA acylases were also constructed, with constitutive strains up to 1 500 U/L and inducible strains up to 900 U/L, and the conversion rate of the immobilized enzymes reached 97% [5]. The D-amino acid oxidase gene of Saccharomyces cerevisiae was transferred to E. coli with an expression vigor of 23.3 U/mL[5] .
The D-amino acid oxidase was immobilized and transformed on Amberzyme epoxy resin for 14 batches with no decrease in viability [6]. P-hydroxyphenylglycine is an important intermediate in the semi-synthesis of β-lactam antibiotics, which can be prepared by two-step catalytic preparation of Amberzyme and D-carbamoyl hydrolase, and the solubility was increased by 6-fold in E. coli by random mutation of D-carbamoyl hydrolase[6], and recombinant expression of E. coli with poor solubility of D-arbamoyl hydrolase, and co-expression of molecular chaperones increased the solubility of the expression by about 3-fold[7].
Carbonyl reductase asymmetric reduction of carbonyl compounds is widely used for the preparation of chiral alcohols. Ethyl (S)-4-chloro-3-hydroxybutyrate was prepared by Streptomyces azureus carbonyl reductase. Its recombinant bacterium, E. coli BL21, prepared ethyl (S)-4-chloro-3-4-phenylbutyrate and methyl (S)-o-chloromandelate with conversion and ee values of more than 99% The enantiomeric ee values and yields of the products of homologous expression of the baker’s yeast carbonyl reductase gene were increased. A novel (S)-type carbonyl reductase was cloned from the genome of near-smooth pseudohyphae, and the recombinant bacterium E. coliBL 21 was able to prepare (S)-phenylglycol with an optical purity of 99.1% and a yield of 89.6% [8,11].
β-Glucosidase is an important component of cellulase system, which can hydrolyze cellobiose and soluble cellobiose into glucose and corresponding ligands, hydrolyze β-glycosidic bonds and synthesize new sugar derivatives. The β-glucosidase from Aspergillus suisse, through gene knockout and amino acid mutation, the enzyme activity of the mutant was 143 times higher than that of the wild type [9]. Sphingobacterium neoformans β-glucosidase gene was successfully expressed in E. coli, which can convert isoflavone glycosides to produce the corresponding glycosides [10]. Heterologous expression of β-glucosidase in the intestinal tract of the Taiwan milk termite E. coli using a prokaryotic expression system resulted in a 2.6-fold increase in the activity of the mutant enzyme compared to the wild-type enzyme. It also improved glucose tolerance and thermal stability [10]. Penicillium obliquum β-glucosidase was genetically modified, and three expression strains were obtained by transformation, and the enzyme activity was increased by 3.4-3.7 times compared with that of the original strain [10], and the heat-resistant β-glucosidase gene was isolated from a non-decompetent prion bacterium and cloned in E. coli, and the enzyme activity was increased by 17 times [4]. A glucose-tolerant β-glucosidase was screened from a marine macrogenome and cloned into E. coli for efficient expression, and glucose concentrations below 400 mmol/L promoted the enzyme, and up to 1,000 mmol/L glucose concentration, the enzyme activity was 50% [8,11]. DNA reorganization and targeted mutagenesis were used to improve the stability of β-glucosidase, and the heat inactivation half-life at 61 ℃ of the four mutants was increased by 14.16-, 68- and 44-fold compared with that of the wild type. The heat resistance was significantly improved [8].
Galactosidase, also known as lactase, breaks down lactose into glucose and galactosyl groups and can synthesize oligosaccharides. β-Galactosidase, an efficient transglycosylating enzyme, was obtained from seabed mud by macro-genomic method, and was solubilized and expressed in E. coli, tolerated organic solvents, and synthesized oligo-galactose in yields of up to 51.6% [9]. Aspergillus α-galactosidase is solidly fermented with an enzyme activity of 305 IU/g [6]. The β-galactosidase of Sulfolobus solfataricus P2 was molecularly modified to construct a mutant, and the mutant enzyme, F441Y, produced oligogalactose with a yield of 61%, which was about 10% higher than the wild type [9].
Nitrile hydratase has important applications in the synthesis of amides, carboxylic acids and their derivatives, catalyzing the production of acrylamide from acrylonitrile, with a fermentation viability of up to 6,000 international units in Nocardia sp. YS-2002, which is expressed in E. coli and Picrosporum [5].
Inulinase is a hydrolytic enzyme that hydrolyzes the β-2,1-D-fructofructose fructoside bond of inulin to produce oligofructose, and is divided into two types: endonuclease and exonuclease. Inulin inulin endonuclease gene of Aspergillus niger was cloned into Bichiria yeast to get efficient expression, and the recombinant enzyme activity reached 128 U/mL, which reached the international level [9].
Alginate synthase, alginate is widely used in the field of food, medicine, light industry, can be used to produce alginate from maltose by single enzyme method, through the rational design of enzyme molecules and DNAshuffling technology from two GRAS strains and two thermophilic bacteria cloned to alginate synthase genes and successfully carried out the expression of high efficiency, and has been realized in industrial production [5].
Neutral protease is widely used in industrial production, the recombinant plasmid of Bacillus subtilis AS1398 gene Npr was transferred into B. subtilisAS 1398 to obtain multi-copy recombinant bacterium, and the recombinant bacterium plasmid stability was maintained at 100% for 30 consecutive generations, and the expression level of protease was increased by more than 1-fold to reach 24 000-28 000 U/ [10]. mL [10]. The strain of B. subtilis with efficient nematicidal virulence was optimized for culture to obtain protease activity up to 12 379 U/mL, which was 5 times higher than that before optimization, and the expression system of B. subtilis WB600 was applied to construct a random mutation library to obtain a heat-stable mutant, which was used as a basis for biocide [8]. Human matrix metalloproteinase is closely related to tumor metastasis, and the enzyme was successfully cloned and expressed in E. coli, which lays the foundation for the study of the mechanism of the enzyme and tumor metastasis and the search for specific inhibitors of the enzyme [5].
Glucanase is a hydrolase, divided into endonuclease and exonuclease, which is used in beer production and feed addition. Penicillium thermophilum β-1,3-1,4-glucanase gene was cloned into a prokaryotic expression vector and transfected into E. coli BL 21 induced expression with an enzyme activity of 240 U/mL [8], and Streptomyces sp. S27 endonuclease β-1,3-glucanase gene was efficiently expressed in E. coli, which hydrolyzes kombucha polysaccharides, etc., and can inhibit pathogenic and toxin producing fungi [8]. The endoglucanase gene of Aspergillus longum was introduced into Picrosporum to express, and the enzyme activity of recombinant bacterium III reached 110 U/mL, which was optimized to increase by 50% [8]. Point mutation extremely heat-resistant Thermotogamaritima endoglucanase Cell-12B, the enzyme optimal temperature of 95 ℃, 90 ℃ still retained 70% live.
N-acetylneuraminic acid is one of the most important salivary acids in living organisms, and the salivary acid sugar chain is involved in many life processes, CMP-salivary acid promotes the regeneration of neuronal cells, and base modification of CMP-salivary acid synthetase gene resulted in highly efficient expression in E. coli, with the enzyme expression accounting for 26.5% of the total protein, and the enzyme activity of 100 U/L, which was 850 times that of the departure strain [4]. Aflatoxin detoxification enzyme is an oxygenase, and the gene was constructed by recombinant technology to be expressed in high-density fermentation in Saccharomyces cerevisiae, and the enzyme accounted for 56% of the total protein, and the expression amount reached 814.5 mg/L [6]. The recombinant plasmid of the enzyme mutation gene was introduced into E. coli to amplify and transfer into Saccharomyces cerevisiae, and a mutation library was established, and the enzyme activity of the mutant A1773 was increased by 5 times. Mutant A1242 showed a 3.5-fold increase in high temperature resistance. Mutant DS1474, with a specific enzyme activity of 56 U/mg, and mutant DS896, with a specific enzyme activity of 44 U/mg [9], have been approved as enzyme products for detoxification in feed.
Aspergillus fumigatus fungal infection is a difficult problem for human health, the systematic study of Aspergillus fumigatus genome more than 50 glycosylation pathway related genes, has been cloned from Aspergillus fumigatus, chitinase, phosphomannan isomerase, O-mannosyltransferase, α-1,4-N-acetylglucosaminyltransferase, α-glucosidase I and CMP-salivaryl synthetase genes, the mechanism of fungal glycosylation to understand, development of genetically engineered drugs and antifungal infections [6].
L-asparaginase has a clear therapeutic effect on leukemia, through the DNA recombination technology, the L-asparaginase specific single-chain antibody fragment and the enzyme to construct a fusion protein to improve the stability of the enzyme in vivo, recombinant E. coli AS 1357 engineering enzyme activity increased up to 228 U/mL, which is comparable to the wild bacterium more than 50 times. The esterase gene was transferred into E. coli by macrogenomics method, and the constructed engineering bacterium improved the vigor, with the expression amount of 200 mg/L, the enzyme vigor up to 180 U/mg, and the thermal stability at 60 ℃ was improved by 144-fold and 196-fold comparing with that of the wild type, and a novel polyester pesticide-degrading mutant esterase that could degrade chlorpyrifos, deltamethrin, deltamethrin, and flucythrins was obtained [9].
Alkaline pectinase was produced by recombinant Picher yeast fermentation, and the highest enzyme production was 1,315 U/mL from 1 t fermenter [8]. The pectinase activity of Aspergillus niger EIM-6 culture was optimized to 30 231 U/mL [8,11]. Pseudomonas salicylate hydroxylase gene cloned into E. coli expressed two naphthalene-degrading enzymes that degrade salicylic acid, acetylsalicylic acid, sulfosalicylic acid, salicylaldehyde, m-nitrobenzaldehyde, 5-chlorosalicylic acid, octanal and o-nitrobenzaldehyde [5].
Organophosphate esters are a class of highly toxic compounds used as insecticides, herbicides or as nerve agents. The organophosphate ester hydrolase of Pseudomonas aeruginosa was cloned and recombinantly expressed in E. coli by amino acid site mutation, which increased the hydrolysis capacity of organophosphate esters by about 7,000-fold [10].
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| Compound Glucoamylase | 9032-08-0 |
| Pullulanase | 9075-68-7 |
| Xylanase | 37278-89-0 |
| Cellulase | 9012-54-8 |
| Naringinase | 9068-31-9 |
| β-Amylase | 9000-91-3 |
| Glucose oxidase | 9001-37-0 |
| alpha-Amylase | 9000-90-2 |
| Pectinase | 9032-75-1 |
| Peroxidase | 9003-99-0 |
| Lipase | 9001-62-1 |
| Catalase | 9001-05-2 |
| TANNASE | 9025-71-2 |
| Elastase | 39445-21-1 |
| Urease | 9002-13-5 |
| DEXTRANASE | 9025-70-1 |
| L-Lactic dehydrogenase | 9001-60-9 |
| Dehydrogenase malate | 9001-64-3 |
| Cholesterol oxidase | 9028-76-6 |