Mycobacterium hydrolase and application thereof in preparation of flavors and fragrances

Abstract

The invention relates to a mycobacterium hydrolase and application thereof in preparation of flavors and fragrances, and belongs to the fields of enzyme engineering and mining and application of new enzyme resources. The hydrolase comes from mycobacteria, and the amino acid sequence of the hydrolase is shown as SEQ ID No: 2; the hydrolase MdHDL disclosed by the invention has relatively high catalytic activity and substrate concentration tolerance, and can efficiently utilize benzyl alcohol or phenethyl alcohol to react with p-nitrophenyl methyl acetate pNPA, p-nitrophenyl methyl butyrate pNPB or p-nitrophenyl methyl caprylate pNPO to synthesize essence and perfume substances with different flavors; the method is suitable for preparing flower fragrance and fruit fragrance type flavors and fragrances such as jasmine flower fragrance, rose fragrance, pineapple fragrance, banana fragrance and apricot fragrance by an enzyme method, has obvious industrial production potential, and provides technical basis and method guidance for preparing the flower fragrance and fruit fragrance type flavors and fragrances by the enzyme method.

Description

TECHNICAL FIELD

[0001] The present application relates to a mycobacterial hydrolase and its application in preparing flavor and fragrance, belonging to the field of enzyme engineering, new enzyme resource mining and application. BACKGROUND

[0002] Ester flavor and fragrance is an important class of aromatic compounds, widely used in food, beverage, cosmetics, detergents and perfume industries. They are usually generated by esterification of alcohol and carboxylic acid, with a wide variety of aroma characteristics. Ester flavor and fragrance mainly includes aliphatic esters, aromatic esters, terpene esters, lactones.

[0003] The preparation methods of ester flavor and fragrance mainly include chemical synthesis (such as acid catalyzed esterification, phase transfer catalysis), enzyme catalysis (such as lipase catalyzed esterification or transesterification) and microbial fermentation (such as engineering bacteria synthesis). Chemical synthesis (such as sulfuric acid catalysis) has mature technology, low cost and high yield, but has strong corrosion, multiple side reactions, environmental pollution and other problems; enzyme catalysis (such as immobilized lipase) has mild conditions, good selectivity and environmental protection, but has high enzyme cost, slow reaction speed and substrate / product may inhibit enzyme activity; microbial fermentation is sustainable, with a wide range of substrates, but has complex metabolic regulation, difficult product separation and long production cycle. The enzymes that can be used for the preparation of ester flavor and fragrance reported so far mainly include Rhizopus arrhizus lipase, Candida antarctica lipase CALB, organic solvent-tolerant lipase SSL1970 from deep-sea Streptomyces, Rhizomucor miehei lipase RML and Lipozyme435. The reactions catalyzed by these enzymes are carried out in non-aqueous phase, which requires the use of a large amount of organic reagents, and has the problems of high cost and environmental pollution. At present, chemical method still dominates in industry, but enzyme method and fermentation method are gradually popularized in high-end fragrance field due to their environmental advantages.

[0004] Hydrolases are a class of biocatalysts with broad substrate scope and stereoselectivity and regioselectivity, which are very suitable for industrial applications. Many hydrolases, especially lipases and esterases, can remain active in organic solvents, which enables them to catalyze "reverse hydrolysis" or transesterification reactions. However, the use of organic solvents has disadvantages such as high cost, environmental damage, toxicity, etc., and cannot be applied to the food and cosmetic industries. Although some lipases can catalyze the selective acyl transfer of hydrophilic compounds such as sugars in organic solvents, the solubility of these hydrophilic compounds in pure organic solvents is very low, which greatly limits the efficiency of the selective acyl transfer reaction. In addition, since most enzymes have reduced activity in organic solvents, it is difficult to integrate lipase-catalyzed acyl transfer reactions into a cascade of biocatalytic reactions. Therefore, it is very necessary to find an enzyme that can perform selective acyl transfer reactions in water. Acyltransferases can perform acyl transfer reactions in water, but they can generally only catalyze one substrate and rely on structurally complex and expensive acyl donors such as acyl-CoA, sulfate esters, and pentafluorophenol esters. Therefore, it is very important to find an enzyme that can use simple and easily available acyl donors (such as methyl esters, ethyl esters, and vinyl esters) and catalyze acyl transfer reactions in water.

[0005] At present, a class of hydrolases with acyltransferase activity, Candida parapsilosis lipase CpLIP2, has been found to generate glycol oleate in catalytic reactions, which first reveals the acyltransferase activity of hydrolases. In recent years, by analyzing the hydrophobicity of the N-terminal domain of the bacterial hormone-sensitive lipase family, the acyltransferase activity of the carboxylic acid esterases of the VIII family (such as EstCE1 and EstA) has been successfully predicted and verified, and the application of this class of enzymes has also been expanded, such as EstXT1 and DLFae4 being used for the acetylation of anthocyanin-3-glucoside.

[0006] Hybrid hydrolase / acyltransferase is a class of hydrolases with acyltransferase activity, but it preferentially catalyzes acyl transfer reactions rather than hydrolysis reactions. Acyltransferases catalyze reactions using a concerted mechanism or a ping-pong mechanism, and a covalent acyl-enzyme intermediate is formed in the reaction, which can acylate organic nucleophiles such as alcohols, amines, and thiols. Hydrolases also catalyze reactions using a ping-pong mechanism, and an acyl-enzyme intermediate is also formed during the reaction process, so the hydrolysis process of hydrolases can be regarded as the process of acyl transfer to water. The mechanisms of hydrolysis and acyl transfer reactions are closely related to a covalent acyl-enzyme intermediate.

[0007] Hybrid hydrolyase / acyltransferases have a wide range of applications in the field of biocatalysis, especially in the synthesis process in aqueous solution and the combination with other enzymes in cascade reactions. For example, hybrid hydrolyase / acyltransferase PestE can efficiently catalyze the acyl transfer reaction of hydroxycinnamic acid ester and amine substrates in aqueous phase to synthesize hydroxycinnamic acid amide with antioxidant, anti-diabetic and neuroprotective activity, with a conversion rate of up to 97% within 2 hours, providing a green synthesis path for pharmaceutical intermediates, fragrances and cosmetic ingredients. In addition, the enzyme can also synthesize complex natural products through cascade reaction, such as coupling high-yield terpene modules with acyl transfer modules to achieve efficient conversion of drimenol to albicanol. In the field of biological materials and biofuels, hybrid hydrolyase / acyltransferase EstCE1 can catalyze the transesterification reaction of fatty acid esters and alcohols to generate biodiesel, significantly reducing production costs and improving environmental friendliness. Recently, there have been studies on the use of its acyl transfer activity to convert terephthalic acid (TPA) generated by depolymerization of waste PET plastics into degradable plastic PHA, with a concentration of up to 2.25 g / L, realizing the resource recycling of waste plastics. In the aspect of environmental remediation, this type of enzyme can degrade organic pollutants (such as pesticides) and plastic waste. For example, through artificial intelligence optimization of substrate binding groove flexibility, PET hydrolyase mutant TurboPETase can completely depolymerize high load PET plastics within 8 hours, solving the problem of 10% high crystallinity residue in traditional biological recycling.

[0008] In view of the problems of high cost and environmental pollution existing in the preparation process of ester flavor substances, a hydrolytic enzyme with acyltransferase activity is developed, which can efficiently catalyze acyl transfer reaction in aqueous phase, and use lower-cost acyl donors to synthesize fruit or floral essence with broad market prospects. SUMMARY

[0009] The purpose of the present application is to propose a mycobacterial hydrolytic enzyme and its application in preparing essence in view of some deficiencies in the prior art. The function and use of the enzyme are further verified through experiments, including the enzymatic properties, structural characteristics of the enzyme and the optimal reaction conditions for preparing essence, which makes up for the deficiency of enzymes for preparing ester essence aromatic compounds with independent intellectual property rights in China.

[0010] In order to achieve the above technical purpose, the present application provides the following technical scheme:

[0011] The present application first provides a hydrolytic enzyme MdHDL, which has amino transfer activity and is derived from Mycobacterium dioxanotrophicus. The amino acid sequence of the hydrolytic enzyme is shown in SEQ ID No: 2.

[0012] The application also provides a polynucleotide encoding the hydrolytic enzyme MdHDL, wherein the polynucleotide sequence is shown as SEQ ID No: 1.

[0013] The application also provides a recombinant expression vector comprising the polynucleotide.

[0014] The application also provides a recombinant engineering bacterium comprising the polynucleotide or the recombinant expression vector.

[0015] The application also provides an application of the hydrolytic enzyme MdHDL, or the polynucleotide, or the recombinant expression vector, or the recombinant engineering bacterium in preparing a fragrance or an ester flavoring substance.

[0016] Further, the fragrance or the ester flavoring substance comprises a fragrance or an ester flavoring substance with a flower or fruit fragrance; and the fragrance or the ester flavoring substance with a flower or fruit fragrance comprises a substance with any one of a jasmine fragrance, a rose fragrance, a pineapple fragrance, a banana fragrance, an apricot fragrance or a peach fragrance.

[0017] Further, the fragrance or the ester flavoring substance comprises one or more of benzyl acetate, benzyl butyrate, benzyl octanoate, phenethyl acetate, phenethyl butyrate and phenethyl octanoate.

[0018] The application also provides a method for preparing a fragrance or an ester flavoring substance, which comprises:

[0019] The method comprises: taking benzyl alcohol or phenethyl alcohol as an acyl acceptor, taking p-nitrophenyl acetate, p-nitrophenyl butyrate or p-nitrophenyl octanoate as an acyl donor, adding the hydrolytic enzyme MdHDL, and reacting to obtain a fragrance or an ester flavoring substance.

[0020] Further, the hydrolytic enzyme MdHDL is used in an amount of 0.05-50 μg, the reaction temperature is 4-80℃, and the reaction time is 30-360 min; further preferably, the concentration of the benzyl alcohol or the phenethyl alcohol is 10-200 mM, and the concentration of the p-nitrophenyl acetate, the p-nitrophenyl butyrate or the p-nitrophenyl octanoate is 1-20 mM.

[0021] The application also provides an application of the hydrolytic enzyme MdHDL, or the polynucleotide, or the recombinant expression vector, or the recombinant engineering bacterium, or the fragrance or the ester flavoring substance prepared by the above method in preparing a fragrance.

[0022] Further, the essence and flavor is an essence and flavor with any one of jasmine flower fragrance, rose flower fragrance, pineapple fruit fragrance, banana fruit fragrance, apricot fruit fragrance or peach fruit fragrance.

[0023] Compared with the prior art, the present application has the following advantages:

[0024] The Mycobacterium dioxanotrophicus hydrolytic enzyme MdHDL has a dual catalytic function of hydrolysis and acyl transfer.

[0025] The Mycobacterium dioxanotrophicus hydrolytic enzyme MdHDL prepared by the present application has good catalytic activity, can efficiently catalyze the reaction of benzyl alcohol or phenethyl alcohol with pNPA, pNPB or pNPO to produce essence and flavor substances with different fragrance types, and has high hydrolysis activity to pNPA and pNPB.

[0026] The Mycobacterium dioxanotrophicus hydrolytic enzyme MdHDL has a dual catalytic function of hydrolysis and acyl transfer, can efficiently catalyze the acyl transfer reaction in aqueous phase, and can be used to synthesize high-value-added products by using acyl donors with low cost, such as the essence and flavor substances prepared by the present application. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The figure is a schematic diagram of an expression plasmid constructed by the Mycobacterium dioxanotrophicus hydrolytic enzyme MdHDL gene.

[0028] Figure 2 The figure is a schematic diagram of double enzyme digestion verification results of an expression plasmid constructed by the Mycobacterium dioxanotrophicus hydrolytic enzyme MdHDL gene.

[0029] Figure 3The results show the effects of different temperatures, different induction times, and different IPTG concentrations on the expression concentration of the hydrolase MdHDL protein in Mycobacterium dioxanotrophicus.

[0030] in, Figure 3 SDS-PAGE images of crude enzyme supernatant and crude enzyme precipitate of MdHDL hydrolase obtained under IPTG conditions of 0.5 mM A at different temperatures and induction times. Figure 3 B uses ImageJ software to... Figure 3 A histogram of the target band obtained in A, analyzed for grayscale. Figure 3 C is an SDS-PAGE image of crude enzyme supernatant and crude enzyme precipitate of MdHDL obtained by induction with different IPTG concentrations at 37℃ for 4 h. Figure 3 D is using ImageJ software to... Figure 3 A bar chart of the target band obtained in C, analyzed for grayscale.

[0031] Figure 4 The image shows SDS-PAGE images of bovine serum albumin standard solutions of different concentrations (top image) and the standard curve of grayscale value versus protein concentration established after grayscale analysis of the target band using ImageJ software (bottom image).

[0032] Figure 5 SDS-PAGE images of different components during the purification process of the hydrolase MdHDL protein and the purified protein. Figure 5 A) and a bar chart of protein expression levels based on grayscale analysis (A) Figure 5 B).

[0033] Figure 6 This is a schematic diagram illustrating the principle of the reaction process involving mixed hydrolases / acyltransferases.

[0034] Figure 7 The figure shows the results of the analysis of the effect of different pH values ​​on the acyl transfer activity of the hydrolase MdHDL.

[0035] Figure 8 The figure shows the results of the analysis of the effect of different temperatures on the acyl transfer activity of the hydrolase MdHDL.

[0036] Figure 9 The figure shows the results of the analysis of the effects of different substrate types and concentrations on the acyl transfer activity of the hydrolase MdHDL. The final concentrations of pNPA, pNPB and pNPO were all 2 mM, and the concentrations of benzyl alcohol and phenylethanol were 0-100 mM.

[0037] Figure 10Figure 1 is the analysis result graph of the effect of different metal ions on the acyltransfer activity of hydrolytic enzyme MdHDL, in which the left graph is the change curve of absorbance value, and the right graph is the change curve of hydrolytic enzyme MdHDL activity.

[0038] Figure 11 Figure 2 is the gas chromatogram of benzyl alcohol and the standard curve established in the example, in which, Figure 11 Figure 3 is the gas chromatogram detection graph of different concentrations of benzyl alcohol (left graph) and the standard curve of benzyl alcohol concentration and gas chromatogram peak area (right graph), Figure 11 Figure 4 is the gas chromatogram detection graph of different concentrations of benzyl acetate (left graph) and the standard curve of benzyl acetate concentration and gas chromatogram peak area (right graph), Figure 11 Figure 5 is the gas chromatogram detection graph of benzyl alcohol of different concentrations reacting with 20 mM pNPA to generate benzyl acetate, and the enzyme kinetics curve fitted based on the Michaelis equation (right graph).

[0039] Figure 12 Figure 6 is the hydrolysis activity curve (A) and enzyme activity (B) graph of hydrolytic enzyme MdHDL on fatty acid esters of different chain lengths. Figure 12 A) and enzyme activity (B) graph of hydrolytic enzyme MdHDL on fatty acid esters of different chain lengths. Figure 12

[0040] Figure 13 Figure 7 is the content detection result of benzyl acetate prepared by hydrolytic enzyme MdHDL.

[0041] Figure 14 Figure 8 is the content detection result of benzyl butyrate prepared by hydrolytic enzyme MdHDL.

[0042] Figure 15 Figure 9 is the content detection result of benzyl octanoate prepared by hydrolytic enzyme MdHDL. DETAILED DESCRIPTION

[0043] The technical solutions of the present application are further described below through specific examples. It should be understood that the examples are preferred solutions of the present application, and are intended to assist in understanding the technical content of the present application, but not to limit the protection scope of the present application. Those skilled in the art can make various substitutions, equivalent improvements or combined applications without departing from the basic concept of the present application, which should be considered to fall within the protection scope of the present application.

[0044] In the following description, many specific details are set forth in order to provide a thorough understanding of the present application, but the present application can also be implemented in other different ways from those described herein, and those skilled in the art can make similar generalizations without departing from the concept of the present application, therefore the present application is not limited to the specific examples disclosed below.

[0045] ​Example 1: Heterologous expression and purification of Mycobacterium dioxanotrophicus hydrolase MdHDL in Escherichia coli

[0046] (1) Gene synthesis, PCR amplification and recombinant plasmid construction of Mycobacterium dioxanotrophicus hydrolase MdHDL

[0047] This example is based on the gene sequence of Mycobacterium dioxanotrophicus hydrolase published in NCBI database (Gene Accession No. WP_087073196.1), and the gene sequence of the optimized hydrolase MdHDL (see SEQ ID No: 1) obtained according to the codon bias of Escherichia coli. The gene sequence was submitted to Nanjing Kingsriver Biotechnology Co., Ltd. for gene synthesis. The synthesized hydrolase MdHDL gene was double-digested with restriction endonucleases Ndel and Xhol (purchased from Thermo Fisher Scientific), and then ligated with plasmid pET30a also double-digested with Ndel and Xhol. The ligation product was transformed into Escherichia coli DH5a, and positive transformants were screened. The plasmid was extracted after overnight culture at 37°C and 200 rpm, and the recombinant plasmid pET30a-MdHDL (plasmid map see Figure 1 ) was obtained. Double digestion verification was performed (verification results see Figure 2 , and DNA marker standard product was purchased from Shanghai Sangon Biotech Co., Ltd.).

[0048] The codon-optimized gene sequence of Mycobacterium dioxanotrophicus hydrolase MdHDL (SEQ ID No: 1) is as follows:

[0049] ATGGCAGCAAAACGCATTCTGTGCTTCGGCGACTCTCTGACGTGGGGCTGGGTGCCTGTTGCTGACGGTGCACCGACTGAGCGTTTCGCGCGTCACGTTCGTTGGACTGGTGTCCTGGCGGATCGCCTGGGCGCGGATTATGAAGTCATCGAGGAAGGCCTGAGCGCTCGTACGACCAACGTTGACGATCCGACCGACCCGCGTCTGAATGGCGCGGCATACCTGCCGTCTTGTCTGGCTGCTCACCTGCCGCTGGACCTGGTGGTGATCATGCTGGGCACCAACGATACCAAGGCATACTTCCGCCGTGAACCGCTGGATATCGCGCTGGGCATGTCTGTACTGGCTACCCAGGTTCTGACTTCTGCAGGCGGTGTAGGCACTGTCTACCCAGCTCCGCAGGTGCTGGTAGTAGCCCCGCCACCGCTGGCTCCGATGCCGCACCCGTGGTTCGAACTGATCTTCGAAGGCGGCCAGCGTAAAAGCGCGGAGCTGGCTCGTGTTTACTCTGCTCTGGCGTCTTTCATGAAGATCCCGTTCTTCGATGCTGGTAGCGTCCTGACTACTCAGGGCGTTGATGGCATCCACTTCACTGAGGCAAACAACCATGATCTGGGCGTTGCTCTGTCCGAGCAGGTGCGTGCCCTGGTACCAGCCCTCGAGCACCACCACCACCACCACTGA

[0050] (2) Construction of the hydrolytic enzyme MdHDL gene expression strain, induction of expression and purification

[0051] The recombinant plasmid pET30a-MdHDL was introduced into the chemically competent E. coli BL21 by heat shock method, and positive transformants were obtained by colony PCR verification, which were the protein expression strains of hydrolytic enzyme MdHDL. The E. coli into which the recombinant plasmid pET30a-MdHDL was introduced was inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37°C, 220 rpm until the OD value reached 0.6-0.8. The final concentration of IPTG was 0 mM, 0.05 mM, 0.1 mM and 0.5 mM, respectively, and the induction was carried out at 15°C, 30°C or 37°C for 4-16 h. The induced bacterial solution was centrifuged at 4°C, 8000 rpm for 12 min, and the supernatant was discarded. The precipitate was resuspended in 1 mL lysis buffer (0.9 g NaH2PO4·2H2O and 1.75 g NaCl were weighed, and the pH value was adjusted to 8.0 with NaOH, and the volume was made to 100 mL with ddH2O) and transferred to a 2 mL centrifuge tube for ultrasonic disruption. The power was set to 50%, and the ultrasonic treatment was 3 s, with an interval of 4 s. The total treatment time was 3 min. After treatment, the sample was centrifuged at 4°C, 15000 rpm for 12 min, and the crude enzyme supernatant and crude enzyme precipitate were separated and their protein concentrations were determined, respectively.

[0052] The experimental grouping is as follows:

[0053] Under the condition of 0.5 mM IPTG, the effects of 15°C induction for 16 h, 30°C induction for 4 h and 37°C induction for 4 h on protein expression were studied (results as shown in Figure 3 A, wherein Lane M: Protein maker; Lane 1: Cell lysate supernatant induced at 15°C for 16 h; Lane 2: Cell lysate supernatant induced at 30°C for 4 h; Lane 3: Cell lysate supernatant induced at 37°C for 4 h; Lane 4: Cell lysate precipitate induced at 15°C for 16 h; Lane 5: Cell lysate precipitate induced at 30°C for 4 h; Lane 6: Cell lysate precipitate induced at 37°C for 4 h; Lanes 7-9 are control groups without adding IPTG, Lane 7: Cell lysate supernatant cultured at 15°C for 4 h; Lane 8: Cell lysate supernatant cultured at 30°C for 4 h; Lane 9: Cell lysate supernatant cultured at 37°C for 16 h.)

[0054] Under the condition of 37°C induction for 4 h, the effects of 0 mM, 0.05 mM, 0.1 mM and 0.5 mM IPTG on protein expression were studied (results as shown in Figure 3C, wherein, Lane M: Protein maker; Lane 1: supernatant of cell lysate with IPTG concentration of 0.5 mM; Lane 2: supernatant of cell lysate with IPTG concentration of 0.1 mM; Lane 3: supernatant of cell lysate with IPTG concentration of 0.05 mM; Lane 4: supernatant of cell lysate without IPTG; Lane 5: precipitate of cell lysate without IPTG; Lane 6: precipitate of cell lysate with IPTG concentration of 0.05 mM; Lane 7: precipitate of cell lysate with IPTG concentration of 0.1 mM; Lane 8: precipitate of cell lysate with IPTG concentration of 0.5 mM.

[0055] The total protein concentration of each sample was determined by Nano-300, and each sample was diluted to 10 μg / μL with the lysis buffer to ensure that the total amount of protein in different samples was equal when loading on SDS-PAGE. Then, 40 μL of each diluted sample was taken and 10 μL of loading buffer (5x) was added, and the mixture was heated in a 100 °C water bath for 10 min, and then centrifuged at 15000 rpm for 5 min.

[0056] SDS-PAGE analysis was performed using FuturePAGETM precast gel (4-20%, 12 wells), and the sample loading amount was 15 μL, and the protein Marker loading amount was 6 μL, and the electrophoresis voltage was set to 170 V, and the electrophoresis time was about 1 h. After electrophoresis, the gel was stained and decolorized, and the target band was analyzed by ImageJ software, and the results are shown in Figure 3 B and 3D.

[0057] SDS-PAGE was performed using gradient concentrations of bovine serum albumin (BSA) standard solution (0.1-1.0 mg / mL), and ImageJ software was used to analyze the gray value of the target band (results are shown in Figure 4 , wherein, Lane M: Protein maker; Lane 1: bovine serum protein solution with a concentration of 1.0 mg / mL; Lane 2: bovine serum protein solution with a concentration of 0.75 mg / mL; Lane 3: bovine serum protein solution with a concentration of 0.5 mg / mL; Lane 4: bovine serum protein solution with a concentration of 0.25 mg / mL; Lane 5: bovine serum protein solution with a concentration of 0.1 mg / mL). The standard curve of gray value and protein concentration was established by five independent experimental data, and the intDen value of the target band was introduced to obtain the concentration of the target protein. By comparing the concentrations of the target protein under different induction conditions, the optimal induction temperature and time, IPTG concentration were found.

[0058] The recombinant MdHDL protein expressed heterologously carries a 6xHis tag (amino acid sequence shown as SEQ ID No: 2) and is purified using a 5 mL HyPur TNi-NTA 6FF (His-Tag) PrePacked Gravity Column (Ni-NTA affinity column). The purification steps are as follows:

[0059] (1) Column equilibration: The Ni-NTA affinity column is washed with 5-8 column volumes of deionized water, and then equilibrated with two column volumes of Binding Buffer (1 mL of 3M imidazole solution, 8.76 g of NaCl, 0.936 g of NaH2PO4·2H2O, 260 mL of ddH2O, mix well, adjust pH to 6.0 with HCl or NaOH, and dilute to 300 mL with ddH2O).

[0060] (2) Sample treatment: The protein extract (crude enzyme supernatant obtained by separation, induced under the condition of 0.5 mM IPTG concentration and 4 h of incubation at 37°C) is mixed with Binding Buffer at a ratio of 1:1 to form a sample solution (1 mL of supernatant plus 1 mL of Binding Buffer), so that the total volume of the sample is twice the column volume.

[0061] (3) Sample loading: The sample solution is added to the chromatography column, and the resin is suspended by gentle shaking. The column is placed on a 400 rpm shaker for 1 h to promote the adsorption of the target protein to the Ni-NTA affinity column.

[0062] (4) Elution of impurities: The chromatography column is vertically stationary for 5-10 min, and the resin is precipitated after the liquid is discharged. The flow-through is collected for analysis of protein binding. 3 mL of Binding Buffer is added, and the above operation is repeated for a total of 3 times to remove non-specifically bound impurities without affecting the binding of the target protein to the resin. The discharged liquid is collected to evaluate the cleaning effect.

[0063] (5) Elution: 2 mL of Elution Buffer (8.335 mL of 3M imidazole solution, 2.99 g of NaH2PO4·2H2O, 0.312 g of NaCl, 80 mL of ddH2O, mix well, adjust pH to 6.0 with HCl or NaOH, and dilute to 100 mL with ddH2O) is added, the resin is suspended by gentle shaking, and after 5-10 min of standing, the eluate is collected in 1.5 mL centrifuge tubes, about 1 mL per tube. Repeat this step until the absorbance of the eluate at 280 nm approaches the baseline.

[0064] (6) Column post-treatment: elute the column with 5 column volumes of Elution Buffer, then equilibrate the column with 5 column volumes of Binding Buffer, and finally wash the column with 5 column volumes of deionized water, add 20% ethanol protection solution, and store at 4°C. Purify three samples with the same crude enzyme supernatant, and mark them as purified protein 1, purified protein 2 and purified protein 3, respectively; use the flow-through, the crude enzyme supernatant and the crude enzyme precipitate obtained under the same induction conditions (IPTG concentration of 0.5 mM, 37°C incubation for 4 h) as controls.

[0065] The purity of the purified protein was detected by SDS-PAGE, and the concentration of the purified protein was further estimated by gray scale analysis. The results are shown in Figure 5 ( Figure 5 In Figure A, Lane M: Protein maker; Lane 1: purified protein 1; Lane 2: purified protein 2; Lane 3: purified protein 3; Lane 4: flow-through; Lane 5: washing liquid; Lane 6: crude enzyme precipitate; Lane 7: crude enzyme supernatant.

[0066] The results show that the optimal induction conditions for the recombinant Escherichia coli strain expressing MdHDL are an IPTG concentration of 0.5 mM Figure 3 C and Figure 3 D), 37°C incubation for 4 h Figure 3 A and Figure 3 B); the Ni-NTA affinity column is effective for purifying MdHDL, and SDS-PAGE analysis shows that the target band in the purified sample is obvious and almost free of other impurity bands Figure 5 A); gray scale analysis shows that the concentration of the purified MdHDL protein is 0.5-0.7 mg / mL Figure 5 B).

[0067] The amino acid sequence (SEQ ID No: 2) of the recombinant Mycobacterium dioxanotrophicus hydrolase MdHDL protein is as follows:

[0068] MAAKRILCFGDSLTWGWVPVADGAPTERFARHVRWTGVLADRLGADYEVIEEGLSARTTNVDDPTDPRLNGAAYLPSCLAAHLPLDLVVIMLGTNDTKAYFRREPLDIALGMSVLATQVLTSAGGVGTVYPAPQVLVVAPPPLAPMPHPWFELIFEGGQRKSAELARVYSALASFMKIPFFDAGSVLTTQGVDGIHFTEANNHDLGVALSEQVRALVPALEHHHHHH

[0069] The hydrolytic enzyme MdHDL obtained in the embodiment of the present application is further verified whether it has the mixed hydrolytic enzyme and acyltransferase activity:

[0070] Using p-nitrophenyl ester as the acyl donor, p-nitrophenyl (pNP) is released while the acyl-enzyme intermediate is formed, and pNP has light absorption at 405 nm, so the speed of pNP release can be determined by measuring the absorbance at 405 nm. In the absence of any other nucleophile except water, the half-life of the acyl-enzyme intermediate only depends on the hydrolysis rate. If the nucleophilicity of the organic nucleophile is higher than that of water, the half-life of the acyl-enzyme intermediate will be shorter, resulting in a faster overall release of pNP. To explore whether a hydrolytic enzyme has acyltransferase activity, the ratio of the absorbance of the experimental group with the addition of an organic nucleophile to the absorbance of the control group without the addition of an organic nucleophile under the same conditions can be compared.

[0071] Relative activity = OD 405 Experimental group / OD 405 If the ratio is greater than 1, it can be preliminarily judged that the enzyme has both hydrolytic enzyme and acyltransferase activity. Here, the same conditions refer to the same volume of the reaction system, the same content of acyl donor in the system, and the same content of enzyme.

[0072] The relevant experimental process is described in the following examples.

[0073] Example 2: Effect of different pH on the mixed hydrolytic enzyme and acyltransferase activity of MdHDL

[0074] The hydrolytic enzyme MdHDL purified in Example 1 was diluted with 0.2M potassium phosphate buffer (pH 7.5) to obtain a hydrolytic enzyme MdHDL solution of 100 ng / μL.

[0075] The reaction system was carried out according to Table 1, benzyl alcohol was selected as the acyl acceptor, pNPA was the acyl donor, and standard buffers with pH values of 2, 4, 6, 7, 8, 10, and 12 (glycine-HCl buffer with pH 2.0, citric acid-disodium hydrogen phosphate buffer with pH 4.0, phosphate buffer with pH 6.0, phosphate buffer with pH 7.0, Tris-HCl buffer with pH 8.0, carbonate-bicarbonate buffer with pH 10.0, and phosphate-NaOH buffer with pH 12.0) were used as the buffer of the reaction system. The reaction system was 200 μL, the enzyme was added in an amount of 100 ng, the final concentration of pNPA was 2 mM, the final concentration of benzyl alcohol in the experimental group was 100 mM, and no acyl acceptor benzyl alcohol was added in the control group. The reaction was carried out in a 96-well plate, and the OD 405 was measured in real time and dynamically by a microplate reader. The incubator temperature of the microplate reader was set to 20°C, the total reaction time was 20 min, a value was read once every 20 s, and after the reaction was completed, the reading results were exported in Excel format, the absorbance ratio of the experimental group to the control group without organic nucleophile was calculated, and a curve was drawn.

[0076] The results are shown in Table 1. Figure 7 The results show that the acyl transfer activity of MdHDL is highest at pH 8.0, and the ratio of the experimental group to the control group is close to 1 at pH 2.0, 4.0, 10.0, and 12.0, indicating that the acyl transfer activity of the enzyme in the system is almost 0. Therefore, both the over-acid and over-alkali environments can inhibit the acyl transfer activity of MdHDL, and the optimal pH for MdHDL catalytic reaction is 8.0.

[0077] Table 1. Reaction system at different pH values

[0078]

[0079] Example 3: Effect of different temperatures on the mixed hydrolytic enzyme and acyl transferase activity of MdHDL

[0080] The hydrolytic enzyme MdHDL purified in Example 1 was diluted with 0.2 M potassium phosphate buffer (pH 7.5) to obtain a hydrolytic enzyme MdHDL solution of 100 ng / μL.

[0081] The method was the same as in Example 2, and the reaction system was carried out according to Table 2, benzyl alcohol was selected as the acyl acceptor, pNPA was the acyl donor, and the pH was 7.0. The enzyme was added in an amount of 100 ng, the final concentration of pNPA was 2 mM, the final concentration of benzyl alcohol in the experimental group was 100 mM, and the reaction was carried out in a water bath at 4°C, 25°C, 37°C, 50°C, 70°C, and 100°C, respectively. After 20 min of reaction, the reaction solution was moved into a 96-well plate, and the absorbance at 405 nm was measured by a microplate reader.

[0082] Results are shown in Figure 8 Table 1. The results show that MdHDL has higher acyltransferase activity at temperatures of 4°C, 25°C and 37°C. As the temperature increases, the ratio of the experimental group to the control group decreases, and at 80°C, the ratio is close to 1, indicating that the acyltransferase activity of the enzyme in the system is almost 0. High temperature can inhibit the acyltransferase activity of MdHDL, and the optimum temperature for MdHDL catalytic reaction is 25°C.

[0083] Table 2. Reaction system at different temperatures

[0084]

[0085] Example 4: Effect of type and concentration of reaction substrate on the mixed hydrolytic enzyme and acyltransferase activity of MdHDL

[0086] (1) Dilute the hydrolytic enzyme MdHDL purified in Example 1 with 0.2M potassium phosphate buffer (pH 7.5) to obtain a 50μL, 50ng / μL hydrolytic enzyme MdHDL solution.

[0087] (2) The method is the same as Example 1, and different acyl acceptors are selected: benzyl alcohol (BeOH), phenethyl alcohol (PEA), acyl donors: 4-nitrophenyl acetate (pNPA), 4-nitrophenyl butyrate (pNPB) and 4-nitrophenyl octanoate (pNPO), according to the reaction system in Table 3. The OD 405 absorbance is measured by an enzyme marker, the incubator temperature is set to 20°C, the total reaction time is 30min, and a value is read every 20s, the data is analyzed, and the optimum acyl acceptor type and the best concentration ratio of acceptor and donor are determined.

[0088] Results are shown in Figure 9 The results show that MdHDL has higher catalytic activity and tolerance in the benzyl alcohol and pNPA system, and the relative activity is as high as 3.19. The optimum concentration ratio of benzyl alcohol and pNPA reaction is 25:1.

[0089] Table 3. Reaction system for the effect of type and concentration of reaction substrate on MdHDL activity

[0090]

[0091] Example 5: Effect of different metal ions on the mixed hydrolytic enzyme and acyltransferase activity of MdHDL

[0092] Dilute the hydrolytic enzyme MdHDL purified in Example 1 with 0.2M potassium phosphate buffer (pH 7.5) to prepare a 50μL, 50ng / μL hydrolytic enzyme MdHDL solution.

[0093] The method is the same as in Example 1, and the reaction system in Table 4 is used, with benzyl alcohol and 4-nitrophenyl acetate as the substrates, and the concentration of each metal ion being 1 mM. The OD is measured using an enzyme marker, the incubator temperature is set to 20°C, the total reaction time is 30 min, and a value is read once every 20 s, the data is analyzed, and the effects of different metal ions on the mixed hydrolytic enzyme and acyltransferase activity of MdHDL are determined. 405 The absorbance is measured, the incubator temperature is set to 20°C, the total reaction time is 30 min, and a value is read once every 20 s, the data is analyzed, and the effects of different metal ions on the mixed hydrolytic enzyme and acyltransferase activity of MdHDL are determined.

[0094] The results, as shown in Figure 10 The results show that: Ca 2+ has an activating effect on the acyltransferase activity of the enzyme at the beginning of the reaction, Fe 2+ , Co 2+ , Cu 2+ , and Mg 2+ have an inhibitory effect on the acyltransferase activity of the enzyme at the beginning of the reaction, Fe 3+ , Mn 2+ , and Ni 2+ have no significant inhibitory or activating effect on the acyltransferase activity of the enzyme.

[0095] Table 4. Reaction system for determining the effects of metal ions on the activity of MdHDL

[0096]

[0097] Example 6: Determining the enzyme kinetic properties of the acyltransferase activity of MdHDL

[0098] The hydrolytic enzyme MdHDL purified in Example 1 is diluted with 0.2 M potassium phosphate buffer (pH 7.5) to prepare a 500 ng / μL hydrolytic enzyme MdHDL solution.

[0099] Benzyl alcohol and pNPA are used as the reaction substrates, and the system in Table 5 is used for sample addition. After 30 min of reaction, 500 μL of ether is added to rapidly vortex quench the enzyme, and the reaction solution is fixed on a vortex for 2 h of continuous shaking at 14000 rpm to extract the product. After the extraction is complete, the layers are allowed to separate, and the upper organic phase is carefully transferred to a new centrifuge tube, an excess of magnesium sulfate powder is added to adsorb residual moisture, and after thorough mixing, the mixture is centrifuged at 15000 rpm for 5 min, and the lower layer containing the magnesium sulfate precipitate is discarded. The upper clear organic phase is taken, and the product concentration is determined using a gas chromatograph.

[0100] Gas chromatography parameters and method settings: the initial temperature of the chromatographic column was 70℃, maintained for 2 min; the temperature was increased to 90℃ at a rate of 15℃ / min, maintained for 2 min; then the temperature was increased to 100℃ at a rate of 15℃ / min, maintained for 2 min; then the temperature was increased to 115℃ at a rate of 10℃ / min, maintained for 2 min; then the temperature was increased to 125℃ at a rate of 10℃ / min, maintained for 1 min; finally, the temperature was increased to 300℃ at a rate of 30℃ / min; the temperatures of the injection port and the detector were 250℃ and 275℃, respectively.

[0101] According to the product yield and reaction time, the initial reaction rate V under each substrate concentration was calculated. The obtained data was imported into GraphPad Prism software, the Michaelis equation curve was fitted by nonlinear regression, the relationship graph of substrate concentration and reaction rate was obtained, and the Michaelis constant (Km), the maximum reaction rate (Vmax) and the catalytic constant (Kcat) were further calculated.

[0102] The results are shown in Figure 11 , Figure 11 A is the gas chromatogram of different concentrations of benzyl alcohol (left) and the standard curve of benzyl alcohol concentration and gas chromatogram peak area (right), Figure 11 B is the gas chromatogram of different concentrations of benzyl acetate (left) and the standard curve of benzyl acetate concentration and gas chromatogram peak area (right), Figure 11 C is the gas chromatogram of benzyl alcohol with different concentrations and 20mM pNPA to generate benzyl acetate (MdHDL), and the enzyme kinetics curve MdHDL of MdHDL based on Michaelis equation fitting (right).

[0103] The results show that the maximum reaction rate V max of the acyl transfer activity of MdHDL to the substrate benzyl alcohol is 409.1 μM / min, the Michaelis constant K m is 46.23 mM, the catalytic constant K cat is 8.71 s -1 , and the catalytic efficiency constant is 0.1884 mM -1 s -1 , and the specific enzyme activity is 54.55 U / mg; the above kinetic parameters for characterizing enzyme properties show that the hydrolytic enzyme of the application has acyl transfer activity and can catalyze the synthesis of ester compounds in water phase.

[0104] Table 5. Composition of enzyme reaction system under different substrate concentrations

[0105]

[0106] Example 7: verification of MdHDL hydrolytic activity

[0107] The enzyme activity releasing 1 μmoL pNP per unit time by hydrolyzing p-nitrophenyl acetate is defined as one enzyme activity unit U, and the calculation formula is: enzyme activity = 1000 * N * V * (Y - 0.0688) / 3.761T, wherein N is the enzyme liquid dilution multiple, T is the reaction time (unit: min), V is the total volume of the reaction system (unit: L), and 1000 is the ratio of mmoL to μmoL. The enzyme activity under each reaction condition can be obtained by substituting the absorbance Y of the reaction for 30 min, N = 1, and V = 200 μL, and the enzyme dosage in the system is 50 ng. The specific activity (unit: U / mg) can be obtained by measuring the enzyme activity and combining the enzyme dosage, and the specific activity = measured enzyme activity (U) / enzyme dosage (mg).

[0108] Reaction system: 50 ng of MdHDL was added to 4-nitrophenyl acetate (pNPA), 4-nitrophenyl butyrate (pNPB) and 4-nitrophenyl octanoate (pNPO) with a final concentration of 2 mM as substrates, respectively, under the conditions of pH 7.0 and 20°C, and the reaction was carried out for 30 min. The OD 405 The absorbance was measured by an enzyme marker every 20 s.

[0109] The results show that MdHDL has high hydrolysis activity to pNPA and pNPB, and the specific enzyme activities of MdHDL to pNPA, pNPB and pNPO are 103.65 U / mg, 37.14 U / mg and 4.65 U / mg, respectively (see Figure 12 ).

[0110] Example 8: Application of hydrolytic enzyme MdHDL in preparation of fragrances and flavors

[0111] In a 1 mL reaction system, 50 μg of MdHDL, 200 mM benzyl alcohol and 20 mM pNPA were added, and the rest was made up with a potassium phosphate buffer to 1 mL. The reaction was carried out at pH 8.0 and 25°C for 30 min, and the content of benzyl acetate in the product was determined. The results are shown in Figure 13 The results show that about 70% of pNPA can be converted into benzyl acetate (jasmine fragrance) after 30 min of reaction, and the yield reaches 6.9 mM.

[0112] The pNPA in the above reaction was replaced by pNPB for experimental verification:

[0113] In a 1 mL reaction system, 50 μg of MdHDL, 100 mM benzyl alcohol and 10 mM pNPB were added, and the rest was made up with a potassium phosphate buffer to 1 mL. The reaction was carried out at pH 8.0 and 25°C for 4 h, and the content of benzyl butyrate in the product was determined. The results are shown in Figure 14The results show that about 41% of pNPB can be converted into benzyl butyrate (apricot fruit flavor, jasmine flower fragrance) after 4h reaction, and the yield reaches 4.1mM.

[0114] The pNPA in the above reaction is replaced by pNPO to verify the experiment:

[0115] In 1mL of the reaction system, 50μg of MdHDL, 10mM of benzyl alcohol and 10mM of pNPO are added, and the volume is made up to 1mL with potassium phosphate buffer solution, and the reaction is carried out at 25℃ and pH 8.0 for 6h, and the content of benzyl octanoate in the product is determined. The results are shown in Table 2. Figure 15 The results show that about 22% of pNPO can be converted into benzyl octanoate (pineapple fruit flavor, banana fruit flavor) after 6h reaction, and the yield reaches 2.2mM.

[0116] In summary, the hydrolytic enzyme provided by the application is suitable for application in the preparation of aromatic compounds, and further application in the field of preparation of fragrances and flavors, and the shortcoming of needing to add an organic solvent in the traditional enzymatic hydrolysis method is improved, and the application is suitable for large-scale popularization and application.

[0117] Although the application has been described in detail in the foregoing with general description and specific embodiments, some modifications or improvements can be made on the basis of the application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the application, all belong to the scope of protection required by the application.

Claims

1. A hydrolytic enzyme, MdHDL, characterized in that, The hydrolytic enzyme has acyl transfer activity, and the amino acid sequence of the hydrolytic enzyme is shown in SEQ ID No:

2.

2. A polynucleotide encoding the hydrolytic enzyme MdHDL according to claim 1, characterized in that, The polynucleotide sequence is shown in SEQ ID No:

1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the polynucleotide of claim 2.

4. A recombinant engineered bacterium characterized in that, The recombinant engineering bacteria comprise the polynucleotide of claim 2 or the recombinant expression vector of claim 3.

5. Use of the hydrolytic enzyme MdHDL of claim 1, or the polynucleotide of claim 2, or the recombinant expression vector of claim 3, or the recombinant engineering bacteria of claim 4 in the preparation of a fragrance or an ester flavoring substance.

6. Use according to claim 5, characterized in that, The fragrance or the ester flavoring substance comprises any one of a fragrance with a floral or fruity fragrance, an ester flavoring substance.

7. Use according to claim 6, characterized in that, The fragrance with a floral or fruity fragrance comprises any one of a substance with a jasmine fragrance, a rose fragrance, a pineapple fragrance, a banana fragrance, an apricot fragrance or a peach fragrance. The ester flavoring substance comprises one or more of benzyl acetate, benzyl butyrate, benzyl octanoate, phenethyl acetate, phenethyl butyrate and phenethyl octanoate.

8. A process for the preparation of a flavouring substance of the class of the aroma chemicals or ester flavouring substances, characterized in that, The method comprises: The hydrolytic enzyme MdHDL of claim 1 is added to benzyl alcohol or phenethyl alcohol as an acyl acceptor, and p-nitrophenyl acetate, p-nitrophenyl butyrate or p-nitrophenyl octanoate as an acyl donor to obtain a fragrance or an ester flavoring substance by reaction.

9. The method of claim 8, wherein, The amount of the hydrolytic enzyme MdHDL is 0.05-50 μg, the reaction temperature is 4-80°C, and the reaction time is 30-360 min.

10. Use of the hydrolytic enzyme MdHDL of claim 1, or the polynucleotide of claim 2, or the recombinant expression vector of claim 3, or the recombinant engineering bacteria of claim 4, or the fragrance or the ester flavoring substance prepared by the method of any one of claims 8-9 in the preparation of a fragrance, wherein the fragrance comprises any one of a jasmine fragrance, a rose fragrance, a pineapple fragrance, a banana fragrance, an apricot fragrance or a peach fragrance.

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