312 results about "Cofactor" patented technology

The invention belongs to the technical field of genetic engineering, and discloses a Yarrowia lipolytica engineering strain capable of efficiently producing eicosapentaenoic acid through a cofactor strategy, Yarrowia lipolytica Po1f is used as an original strain, and genes of delta 9 elongase, delta 8 desaturase, delta 5 desaturase and delta 17 desaturase are over-expressed, so that the eicosapentaenoic acid can be efficiently produced. And simultaneously overexpressing glucose-6-phosphate dehydrogenase (G6PDH) and malic enzyme (ME) to obtain the gene. The construction method is suitable for constructing a genetic engineering strain for producing eicosapentaenoic acid, the aim of simply, conveniently, effectively and accurately constructing the strain is achieved, the constructed engineering strain is further applied to production of eicosapentaenoic acid, and an effective way is provided for commercially producing eicosapentaenoic acid by applying yarrowia lipolytica.

The invention discloses a P450 mutant and application thereof in synthesis of calcifediol, and belongs to the technical field of bioengineering. According to the present invention, the P450 enzyme mutant enzyme is constructed, and the single plasmid three-enzyme co-expression system of the redox partner and the cofactor cycle enzyme is constructed, such that the sufficient electron transport chain is provided for the P450 enzyme, and the calcifediol can be efficiently synthesized under the mild condition by using the vitamin D3 as the substrate; the conversion rate of calcifediol can reach 63.1%, and the conversion rate of the reaction is greatly improved. By adopting the method disclosed by the invention, the catalytic efficiency of the catalyst is improved, and the reaction cost is reduced.

The invention discloses a biological catalytic synthesis method of norambrox ether, which comprises the following steps: step 1, starting from trans-nerolidol, carrying out carroll rearrangement reaction to synthesize farnesyl acetone; 2, in the presence of a cofactor, converting farnesyl acetone into high farnesol acetate under catalysis of Bayer-Villiger monooxygenase; step 3, catalyzing the deacetylation of the high-farnesol acetate by using esterase to obtain EE-high-farnesol; and step 4, carrying out enzymatic conversion on farnesol by using the squalene hopaene cyclase mutant to obtain norambrox ether. The transformation process is safe and non-toxic, Bayer-Villiger monooxygenase which is new with a key enzyme, natural co-expressed esterase, alcohol dehydrogenase and squalene and hopaene cyclase mutants matched with the system are used for synergistic transformation, so that not only is the coenzyme for oxidation reaction promoted to be recycled, but also the conversion efficiency is improved; conversion from farnesyl acetone to norambrox ether can be efficiently and specifically catalyzed, and a final product can be directly obtained from a reaction system without separation and purification of an intermediate.

The invention discloses a Sarcabomyces lipolytica strain for synthesizing (2S)-naringenin and application of the Sarcabomyces lipolytica strain, and belongs to the technical field of engineering and bioengineering. According to the invention, a key enzyme CHS is subjected to semi-rational transformation, so that the yield of the strain (2S)-naringenin expressing the mutant V196A / T132C is increased by 60%. According to the method, the modified CHS is further integrated into a yeast genome, conversion of p-coumaric acid to (2S)-naringenin is promoted by strengthening a shikimic acid pathway and enhancing supply of cofactors, so that the yield of the (2S)-naringenin reaches 776mg / L after the constructed strain is fermented in a 24-pore plate for 96 hours, and the yield of the constructed strain can reach 8.65 g / L after the strain is fermented in a 5L fermentation tank; the method lays a foundation for the subsequent biosynthesis of flavonoid compounds, and has potential value and significance for the development of synthetic biology.

The invention belongs to the technical field of biology, and discloses an exogenous cofactor enhanced yarrowia lipolytica strain, a method and application in efficient synthesis of EPA (eicosapentaenoic acid) and lipids.The chassis strain of the yarrowia lipolytica strain is yarrowia lipolytica Po1f, desaturase is partially prolonged in an overexpression fatty acid synthesis route in the strain, and the EPA and the lipids.The yield of the EPA and the lipids.The yield of the EPA and the lipids.The yield of the EPA and the lipids. The desaturase gene mainly comprises a delta 9 elongase gene, a delta 8 desaturase gene, a delta 5 desaturase gene and a delta 17 desaturase gene, and G6PDH and malic enzyme are overexpressed at the same time on the basis. The strain provided by the invention improves the EPA and lipid yield of yarrowia lipolytica; the method is suitable for constructing a genetic engineering strain with high yield of EPA and lipid, provides a basis for producing grease rich in EPA from yarrowia lipolytica, and solves the problems of non-sustainability, high cost and the like caused by obtaining EPA from fish.

The invention provides an engineering strain for producing mannitol from methanol and / or formaldehyde and fructose and a method for producing mannitol by using the engineering strain. According to the method, a reducing agent is methanol or formaldehyde, a catalyst is dehydrogenase capable of utilizing the methanol or the formaldehyde, a reduction-state cofactor NADH is provided while the methanol or the formaldehyde is oxidized by the dehydrogenase, and D-fructose is converted into D-mannitol by coupling mannitol dehydrogenase. According to the method and the technology, methanol dehydrogenase and / or formaldehyde dehydrogenase, formate dehydrogenase and mannitol dehydrogenase are over-expressed in recombinant escherichia coli, so that a novel method and a novel technology for synthesizing D-mannitol through multi-enzyme coupling catalysis are constructed. The invention establishes a preparation method for co-producing D-mannitol, methanol (or formaldehyde) and reduced NADH from cheap raw materials, and the preparation method has important significance for green and efficient production of D-mannitol.

The present invention discloses a genetically engineered bacterium producing furfural amine, and a preparation method and application thereof, and the present invention belongs to the technical field of fermentation engineering. The present invention is to solve the problems of large amount of amino donor, high price, metal ion cofactor, large amount of co-substrate, etc. in the existing preparation of furfural amine by bio-transamination catalysis. The method of the present invention comprises the following steps: constructing a recombinant Escherichia coli cell; adding the whole cell of the recombinant engineered bacillus induced for expression into the reaction system; recovering the whole cell catalyst to prepare the product. Furfural amine is prepared by using a cheap inorganic amino donor and glucose as a co-substrate; the amount and cost of the amino donor are effectively reduced, and the amount of glucose is greatly reduced, thereby realizing the green and efficient synthesis of bio-based furfural amine.

The invention provides a 7beta-hydroxysteroid dehydrogenase mutant and application thereof in synthesis of ursodesoxycholic acid by a biological enzyme method, through a genetic engineering technology and computer-aided design, phosphoric acid coordination residues of protein are modified, the 7beta-hydroxysteroid dehydrogenase (7beta-HSDH) mutant with higher activity under an NADH (Nicotinamide Adenine Dinucleotide Hydroxide) system is constructed, and the 7beta-hydroxysteroid dehydrogenase (7beta-HSDH) mutant can be used for synthesizing ursodesoxycholic acid. The modification strategy is suitable for other proteins for modifying cofactor preference and improving enzyme activity, and has remarkable industrial value when being used for efficiently catalyzing CDCA to be converted into UDCA through a one-pot one-step method.

The invention discloses a reagent for detecting the activity of isocitrate dehydrogenase. The reagent consists of a first reagent and a second reagent, the first reagent comprises an electron carrier, isocitric acid or isocitrate, divalent metal ions, a first reagent buffer solution, a first reagent additive, a first reagent preservative and a first reagent surfactant; the divalent metal ions comprise metal ions; the second reagent comprises a dye which comprises tetrazolium salt and has an oxidation state and a reduction state, a cofactor, a second reagent buffer solution, a second reagent additive, a coenzyme stabilizer, a second reagent surfactant and a second reagent preservative; the pH is buffered to 7-9.5 and 5-9 by the first buffer solution and the second buffer solution respectively; the first reagent additive and the second reagent additive comprise divalent metal salt, sugar, sugar alcohol, protein, chelating agent and basic amino acid; the first and second reagent surfactants comprise nonionic and anionic surfactants; the first reagent preservative and the second reagent preservative comprise sodium azide or isothiazolinones. The invention further discloses a corresponding detection method.

The present invention discloses a method for synthesizing rasagiline in a microfluidic field. The method is based on a double enzyme hydrogenation cascade reaction, which efficiently converts racemic alcohol into enantiomeric amines, realizes the direct synthesis of chiral amines, and has high atom economy and reaction efficiency. Racemic 1-indanol is completely oxidized to generate 1-indanone by alcohol dehydrogenase catalysis, and the cofactor NADP+ is reduced to NADPH to provide the required reducing power for subsequent reactions; using ( R )-selective reductive amino enzyme catalyzes the reductive amination reaction of 1-indanone to generate the target product rasagiline. The present invention adopts a dual enzyme synergistic catalytic strategy to successfully achieve the efficient conversion from racemic alcohol to chiral amine, has the advantages of mild reaction conditions, green sustainability, high product yield and optical purity, and provides a new efficient, economical and environmentally friendly way for the synthesis of rasagiline.

The invention discloses a recombinant bacterium and application of the recombinant bacterium in production of benzophenanthridine alkaloid. The synthetic route of the benzophenanthridine alkaloid is divided into a plurality of modules for research, and de novo synthesis of the benzophenanthridine alkaloid is realized through a plurality of strategies, for example, in order to improve the activity of BBE, a new endoplasmic reticulum engineering strategy is developed, so that the yield of a key intermediate S-Soulerine is increased by 3 times; the McDBOX2 is identified as the only reported DBOX gene which plays a role in saccharomyces cerevisiae at present, and the reconstruction of the whole biosynthetic pathway of the benzophenanthridine alkaloid is completed; by carrying out N-terminal truncation operation on DBOX, cytoplasmic expression and endoplasmic reticulum amplification are improved, and supply of a cofactor FAD is increased, so that the yield of benzophenanthridine alkaloid is increased. Experiments prove that the recombinant strain constructed by the invention can be used for efficiently producing benzophenanthridine alkaloid. The method has an important application value.

A method of synthesising a 1N-alkyl derivative of pseudouridine comprises contacting a substrate with a methyltransferase in the presence of a cofactor to produce the 1N-alkyl derivative of pseudouridine. The substrate has a structure of: Formula (I). The 1N-methyl derivative of pseudouridine has a structure of: Formula (II). R1 is a hydroxyl group, a monophosphate group, a diphosphate group, or a triphosphate group. R2 is H or a hydroxyl group. R3 is a methyl group or an ethyl group. The cofactor is a source of a methyl group or an ethyl group. The methyltransferase is configured to transfer the methyl group or ethyl group from the cofactor to the substrate. Enzymatic synthesis of 1N-methyl derivatives of pseudouridine may be more efficient and produce less waste than chemical syntheses. Also provided are methyltransferases and buffers useful in the method; and polynucleotides, expression vectors, and microorganisms useful for producing the methyltransferases.

Heterologous synthesis of a nitrogen-fixing system in a non-diazotrophic organism is a long-sought-after goal because of the significance of nitrogenase for areas related to agronomy, energy and environment. Two simplified nitrogenase analogs from Azotobacter vinelandii in Escherichia coli were successfully heterologously synthesized. One analog comprise the reductase component (NifH) and, in place of the catalytic component (NifDK), a cofactor maturase (NifEN); whereas the other analog comprised only NifEN. Metal, EPR and activity analyses verified the cluster composition and functional competence of the heterologously expressed NifH and NifEN. Moreover, NMR, nanoSIMS and growth experiments illustrated the abilities of both systems to mimic the nitrogenase in reducing N2 and incorporating the reduced N into the cellular mass. These results firmly established NifEN / NifH or NifEN as a simplified nitrogenase analog that could be engineered or expanded to facilitate future transgenic expression of nitrogenase and development of nitrogenase-based biotechnological applications.

This invention relates to a combined module for increasing the yield of polyketide natural products from actinomycetes, and the construction and application of recombinant bacteria containing this module, belonging to the field of genetic engineering technology. To improve the yield of natural products such as polyketides and nonribosomal peptides, this invention utilizes a metabolic engineering strategy to combine and modify the CoASH synthetic pathway and the PPTase post-modification pathway, providing a combined module that can increase the yield of natural products from actinomycetes. This module consists of the CoASH cofactor synthesis module element SCoaA. R106A Composed of SCoaD and PPTase post-modification module element HPC3, recombinant vectors and recombinant bacteria containing this module were constructed. It was found that overexpression of this module can effectively increase the yield of natural products such as actinomycete polyketides, polyethers, and non-ribosomal peptides.

The invention discloses construction and application of a recombinant escherichia coli engineering strain with high yield of D-pantothenic acid, and belongs to the technical field of genetic engineering and biological engineering. According to the invention, acetic acid branch pathway genes are knocked out, so that accumulation of precursor pyruvic acid is improved; carrying out free expression and integrated expression on panBC respectively; by weakening a cofactor 5, 10-CH2-THF degradation pathway and strengthening an de novo synthesis pathway of the cofactor 5, 10-CH2-THF, supply of 5, 10-CH2-THF is promoted, and the yield of the constructed engineering strain D-pantothenic acid can reach 4.83 g / L. The Escherichia coli recombinant strain with high yield of D-pantothenic acid, constructed by the invention, lays a foundation for subsequent biosynthesis of D-pantothenic acid and derivatives, and has potential value and significance for development of synthetic biology.

The invention belongs to the technical field of biochemical engineering, and provides a method for increasing the yield of L-histidine by strengthening a metabolic pathway of escherichia coli C1. The content of a cofactor fTHF in cells is increased by increasing supply of C1 in escherichia coli, namely up-regulating expression of FolD, so that the yield and the yield of L-histidine are remarkably increased. The invention provides a new strategy for improving the fermentation level of L-histidine.

The invention relates to the technical field of synthetic biology, in particular to a squalene high-yield saccharomyces cerevisiae engineering bacterium and a construction method and application thereof.Saccharomyces cerevisiae CEN.PK.2-1D is used as an original strain, pentose phosphate pathway genes ZWF1, GND1, TAL1 and TKL1 and IDP1 genes of TCA circulation are integrated in a genome of the original strain through homologous recombination, and the squalene high-yield saccharomyces cerevisiae engineering bacterium is obtained. Constructing a dual-channel cofactor supply system to improve the intracellular NADPH level and the NADPH / NADP + ratio; according to the saccharomyces cerevisiae genetic engineering strain Sc-Sq04 constructed by the method disclosed by the invention, the yield of squalene obtained by shake flask fermentation reaches 741.08 + / -28.9 mg / L by further integrating all genes of an MVA way, ERG20 and ERG9 genes, and a new thought is provided for transforming saccharomyces cerevisiae to synthesize terpene compounds and promoting industrial application.

The invention provides a method for improving tetrahydrofolic acid biosynthesis based on NADPH cofactor circulation, and aims to catalyze folic acid into tetrahydrofolic acid through an in-vitro enzyme catalysis technology. According to the invention, escherichia coli BL21 (DE3) is used as a host cell, and dihydrofolate reductase (SmDHFR) derived from serratia marcescens and formate dehydrogenase (pseudoFDH) derived from pseudomonas, which are obtained by screening, are expressed. A TB culture medium is used for fermentation, ultrasonic cell disruption and purification to obtain pure enzyme liquid of SmDHFR and pseFDH. SmDHFR is responsible for converting folic acid into tetrahydrofolic acid, and NADPH consumed in the process is converted into NADP < + >. And the pseudoFDH is used for reducing the generated NADP < + > into NADPH so as to realize the cyclic utilization of the NADPH cofactor. By exploring the optimal ratio of the addition concentrations of the two enzymes, the optimal reaction pH and the optimization of the substrate concentration, the final yield of tetrahydrofolic acid is 3661.14 mu M.

The invention discloses a flavin monooxygenase FMO mutant and application thereof in indigo production, and belongs to the technical field of microbial engineering. Through rational design, directional mutation or random mutation, a mutant with catalytic efficiency several times higher than that of wild-type flavin monooxygenase is obtained, then the mutant is integrated into escherichia coli to obtain a recombinant strain capable of expressing the mutant, and the obtained recombinant strain not only can realize cyclic regeneration of cofactors, but also can be used for preparing the flavin monooxygenase. An expensive cofactor NADPH does not need to be additionally added, so that the effects of saving materials and reducing the production cost are achieved; the fermentation period can be shortened, and the yield of indigo blue is increased and reaches the g-level level. Therefore, the flavin monooxygenase mutant and the constructed recombinant bacteria have good application prospects in the field of indigo production, and large-scale industrial production of indigo is promoted.

The invention relates to the technical field of synthetic biology, in particular to a microbial cell factory for producing catechol. Pseudomonas putida without a pcaHG gene, a catA gene and a catA2 gene is used as a chassis bacterium, and exogenous AroY and / or kpdB genes are / is introduced into the chassis bacterium, or the expression of endogenous genes VanAB and PobA is improved. The lignin-derived monomer is efficiently converted into catechol through the strategy of cofactor regulation and key enzyme overexpression, the conversion rate reaches 98.5%, the highest yield is 14.1 mM, and compared with the previous 64% conversion rate, the conversion rate is greatly improved.

The invention provides a method for improving fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan, alpha-ketoglutaric acid ferrous salt, alpha-ketoglutaric acid zinc salt, alpha-ketoglutaric acid cobalt salt and alpha-ketoglutaric acid manganese salt are added into a fermentation culture medium according to a specific proportion, alpha-ketoglutaric acid directly participates in TCA circulation, circulation operation can be accelerated, generation of NADH and FADH2 is increased, the yield of 5-hydroxytryptophan is increased, and the yield of 5-hydroxytryptophan is increased. The ATP synthesis efficiency is improved, and more energy is provided for cell growth; the alpha-ketoglutaric acid can balance amino acids required in the growth process of escherichia coli, promote the generation of proteins and nucleic acids and accelerate the growth of thalli; the alpha-ketoglutaric acid metal salt can improve the utilization efficiency of microelements by thalli; ferrous ions are cofactors of 5-hydroxytryptophan hydroxylase and can accelerate the hydroxylation process of tryptophan. Through the synergistic effect of the added components, the yield of the 5-hydroxytryptophan and the sugar-acid conversion rate are improved.

A method for contamination control when growing yeasts is provided. Bacterial contamination is controlled by using urea as the primary nitrogen source while simultaneously limiting the amount of nickel available to contaminating bacteria. Bacteria require nickel as a cofactor for urease enzymes in order to use urea for growth while yeasts do not require nickel as a cofactor for any enzymes. Nickel is limited by using metals in heat exchangers that do not leach nickel. Ethyl carbamate is limited by using a carbon / nitrogen ratio that consumes all urea during fermentation. After fermentation completes, yeast is recycled using centrifugation, enabling use of high concentrations of yeast to reduce fermentation time from 48 hours to 12 hours and to eliminate bacterial contamination from growth on free amino nitrogen.

The present disclosure relates to multi-enzyme pathways for the in vitro cell-free biosynthesis of the cofactor, coenzyme A ("CoA") from lower-cost substrates, such as D-pantothenate, L-cysteine, and ATP, and the use of these pathways in CoA-dependent cell-free systems for the biobased production of chemicals, such as acetyl-CoA, hexanoyl-CoA, malonyl-CoA, and cannabigerolic acid (CBGA).

A method for bacterial contamination control of heterotrophic growth of green algae is provided. Bacterial contamination is controlled by using urea as the primary nitrogen source while simultaneously limiting the amount of nickel available to contaminating bacteria. Bacteria require nickel as a cofactor for urease enzymes in order to use urea for growth while green algae do not require nickel as a cofactor for any enzymes. Green algae use biotin as a cofactor to use urea and are biotin prototrophic. Nickel is limited by using titanium in plate heat exchangers instead of stainless steel. The preferred green algae are Chlorella sorokiniana, Chlorella vulgaris, and Auxenochlorella protothecoides.

The invention provides a preparation method and application of a preparation for RNA cell-free synthesis and belongs to the technical field of molecular biology, the preparation method of the preparation for RNA cell-free synthesis mainly comprises the steps that escherichia coli serves as a chassis strain, a pET-28a carrier is utilized, a recombinant strain for expressing T7RNA polymerase is constructed, and the T7RNA polymerase has enzyme activity at the temperature of 50-60 DEG C; inducing, culturing and collecting thalli, carrying out ultrasonic lysis, and treating a lysis solution to prepare a preparation for RNA cell-free synthesis; the preparation provided by the invention naturally contains various cofactors, and RNA polymerase does not need to be extracted and purified, so that the production cost is reduced.

The invention discloses a method for producing sterols and sulfated derivatives thereof through fermentation and improving sulfation degree, and belongs to the field of bioengineering. The yeast engineering bacteria for fermentation production of sterols and sulfated derivatives thereof are constructed by taking yeast as an original strain and knocking out and introducing genes. The engineered yeast can efficiently produce 7-dehydrocholesterol and cholesterol, especially cholesterol sulfate, the shake flask fermentation yield of the engineered yeast is close to 249.0 mg / L, and the fermentation yield of a 5L fermentation tank can reach 544.7 mg / L. By highly expressing key rate-limiting enzyme in a metabolic pathway, enhancing precursor supply and enhancing cofactor PAPS supply, the yield and sulfation degree of the cholesterol sulfate are improved, and the pichia pastoris is utilized to efficiently generate the cholesterol sulfate, so that the method has the characteristics of short period, environmental protection and resource conservation. The yeast engineering bacterium for high-yield production of cholesterol sulfate has a wide application prospect, and provides important reference for production of other sulfated products.

Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and / or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties can include enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased thermostability, increased accuracy, increased speed, increased readlength, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.

This invention discloses an enzyme preparation for catalytic synthesis of epiinositol and a method for synthesizing epiinositol, belonging to the field of genetic engineering technology. The enzyme preparation includes squalinositol dehydrogenase, NADH oxidase, formate dehydrogenase, and muscle inositol dehydrogenase; the amino acid sequence of squalinositol dehydrogenase is shown in SEQ ID NO. 6, and the amino acid sequence of muscle inositol dehydrogenase is shown in SEQ ID NO. 8. The enzyme activities of the squalinositol dehydrogenase mutant and the muscle inositol dehydrogenase mutant obtained by mutation in this invention are significantly increased, thus further improving the yield and conversion rate of epiinositol during catalytic synthesis. Furthermore, the enzyme preparation of this invention also contains NADH oxidase and formate dehydrogenase, achieving in-situ regeneration of NADH, thereby effectively reducing the amount of cofactors and NADH used in the catalytic synthesis of epiinositol, and thus reducing production costs.

The invention discloses a difunctional alcohol dehydrogenase derived from Geobacillus thermodenitrifis, the difunctional alcohol dehydrogenase takes NAD < + > as a cofactor, can specifically catalyze a continuous oxidation reaction of omega-hydroxy fatty acid, and catalyzes the omega-hydroxy fatty acid to synthesize alpha, omega-binary fatty acid through single enzyme catalysis without an aldehyde intermediate. The alcohol dehydrogenase has dual catalytic functions of alcohol oxidation and aldehyde oxidation, the oxidation process from hydroxymethylene to carboxyl can be completed in one step, the reaction steps are simplified, accumulation and separation of intermediate aldehyde are avoided, the conversion efficiency is improved, and the alcohol dehydrogenase is alpha, omega-dibasic fatty acid and is a novel alcohol dehydrogenase. The invention provides a simple and efficient biocatalysis way for biosynthesis of dodecanedioic acid, especially dodecanedioic acid, and has important industrial application value.

The invention provides a slow-release organic-inorganic compound fertilizer based on synergistic interaction of a carrier immobilized enzyme and a cofactor and a preparation method of the slow-release organic-inorganic compound fertilizer, and belongs to the technical field of organic fertilizers. According to the application, heat-insulation enzyme-loaded particles are added during pile building, a semi-coke-perlite low-heat-conduction framework forms a cold island in a reactor core, and a starch-polypeptide-sodium tripolyphosphate cross-linked membrane is used for firmly immobilizing enzyme, so that heat inactivation is prevented, fibrin is rapidly cracked, free ammonia is adsorbed, and the pile temperature is promoted to rapidly rise; then, dynamic humidity regulating particles are put, reversible water release-water absorption of montmorillonite is combined with glycerin, and a sorbitol hydrophilic film automatically and stably controls water content and supplements easily-degradable carbon, so that over-wet anaerobic or over-dry enzyme loss is avoided; the enzyme cofactor powder added in the later period slowly releases manganese, calcium and copper to serve as enzyme complement, alanyl glutamine serves as a molecular chaperone to repair damaged conformation, and copper and zinc are chelated to passivate heavy metal; the three agents sequentially realize'keep-alive, stable-alive ', prolong the high activity period of enzyme, accelerate lignin degradation, and reduce nitrogen volatilization, odor and heavy metal risks at the same time.