CN118421577B - Tyrosinase mutant and use thereof - Google Patents

Abstract

The present embodiment proposes a tyrosinase mutant and its use. The tyrosinase mutant undergoes an amino acid mutation on the amino acid sequence shown in SEQ ID NO.2; wherein, the amino acid mutation site is one or a combination of F197A, P201A, R209A, V218A, F197D, F197E, F197M, F197N, F197Q, F197R, F197S, F197V, N205D, N205I, N205K, N205L, N205Q, N205R, N205S, N205T, N205V, R209D, R209G, R209N, R209S, R209V, V218G, V218I, V218M, V218Q, V218S. The tyrosinase mutant provided in the embodiments of the application can better catalyze the production of 5,6-dihydroxyindole from levodopa and has a higher conversion rate, indicating that this tyrosinase mutant has good application prospects in the biotransformation of levodopa to prepare 5,6-dihydroxyindole.

Description

Tyrosinase mutant and its use

Technical Field

The present application relates to the field of biological enzyme technology, and in particular to a tyrosinase mutant and its use.

Background Art

Tyrosinase is widely present in animals, plants and microorganisms in nature, and has monophenolase activity and diphenolase activity. In organisms, tyrosinase is the key rate-limiting enzyme in melanin synthesis.

5,6-dihydroxyindole (DHI) is an important intermediate and component structure of melanin synthesis. Due to the activity of 5,6-dihydroxyindole intermediate, it is easy to oxidize and self-polymerize under air conditions without the stimulation of initiators, and is less irritating to the human body. In addition, since 5,6-dihydroxyindole is naturally present in the human body as a natural component in the human body, 5,6-dihydroxyindole melanin hair dye is safer for the human body and is gradually replacing p-phenylenediamine compounds as a better choice for hair dyes.

In the prior art, the synthesis methods of 5,6-dihydroxyindole are mainly divided into phenylethylamine method, benzaldehyde method and benzyl cyanide method according to different raw materials. However, the above synthesis methods require the use of highly toxic and carcinogenic materials, and have the disadvantages of long preparation time and complicated steps.

At present, some biological hair dyes use tyrosinase to catalyze the conversion of L-DOPA to dopaquinone, which is then reduced to 5,6-dihydroxyindole. However, the current conversion rate of tyrosinase to 5,6-dihydroxyindole from L-DOPA is low.

In view of this, discovering more tyrosinases and modifying their structures to achieve efficient synthesis of 5,6-dihydroxyindole is of great significance to promoting the industrial production of 5,6-dihydroxyindole.

Summary of the invention

The purpose of the present application is to provide a tyrosinase mutant and its use.

To achieve the above objectives, this application proposes the following technical solutions:

In a first aspect, an embodiment of the present application provides a tyrosinase mutant, wherein the tyrosinase mutant has an amino acid mutation in the amino acid sequence shown in SEQ ID NO. 2;

Wherein, the site of the amino acid mutation is one or a combination of two of F197A, P201A, R209A, V218A, F197D, F197E, F197M, F197N, F197Q, F197R, F197S, F197V, N205D, N205I, N205K, N205L, N205Q, N205R, N205S, N205T, N205V, R209D, R209G, R209K, R209N, R209S, R209V, V218G, V218I, V218M, V218Q, and V218S.

As an embodiment, the tyrosinase mutant undergoes any one of the following amino acid mutations:

F197A, P201A, R209A, V218A, F197D, F197E, F197M, F197N, F197Q, F197R, F197S, F197V, N205D, N205I, N205K, N205L, N205Q, N205R, N205S, N205T, N2 05V, R209D, R209G, R209K, R209N, R209S, R209V, V218G, V218I, V218M, V218Q, V218S, N205D+F197A, N205D+F197D, N205D+F197E, N205D+R209D, N205D+R209N, N205T+F197A, N205T+R209D, N205T+R209N, R209D+F197A, R2 09D+F197D, R209D+F197E, R209N+ F197A, R209N+F197D, R209N+F197E.

A second aspect of an embodiment of the present application provides a DNA molecule, wherein the DNA molecule comprises a nucleotide sequence encoding the tyrosinase mutant as described in the first aspect or its complementary sequence.

The third aspect of the embodiment of the present application provides a recombinant plasmid, which contains the DNA molecule as described in the second aspect.

The fourth aspect of the embodiments of the present application provides a recombinant strain, wherein the recombinant strain contains the recombinant plasmid as described in the third aspect.

As an embodiment, the host cell of the recombinant strain is a prokaryotic cell or a eukaryotic cell, and the eukaryotic cell is a yeast cell.

As an embodiment, the host cell of the recombinant strain is a competent cell.

As an embodiment, the competent cell is Escherichia coli BL21 (DE3).

The fifth aspect of the embodiments of the present application provides the use of the tyrosinase mutant described in the first aspect, the DNA molecule described in the second aspect, the recombinant plasmid described in the third aspect, and the recombinant strain described in the fourth aspect in catalyzing the production of 5,6-dihydroxyindole from L-dopa.

As an embodiment, the preparation method of 5,6-dihydroxyindole comprises:

preparing a recombinant plasmid containing a tyrosinase mutant;

Transforming the recombinant plasmid into a host cell to obtain a recombinant strain;

Using L-DOPA as substrate, the recombinant strain catalyzes L-DOPA to produce 5,6-dihydroxyindole.

The embodiments of the present application have at least the following beneficial effects:

The present application example uses the amino acid sequence shown in SEQ ID NO. 2 as a template (basis), selects key amino acid sites in the active center for mutation modification according to the binding conformation of L-DOPA and the tyrosinase active center, constructs a mutant library, and screens to obtain a series of mutants with significantly improved catalytic activity; the tyrosinase mutant provided in the present application example can better catalyze L-DOPA to produce 5,6-dihydroxyindole, and has a higher conversion rate, indicating that the tyrosinase mutant has good application prospects in the biotransformation of L-DOPA to prepare 5,6-dihydroxyindole.

Additional aspects and advantages of the present application will be partially given in the following description, which will become apparent from the following description, or will be understood through the practice of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a reaction scheme of tyrosinase mutants catalyzing L-DOPA to produce DHI;

FIG2 is a HPLC detection diagram of the template pET-30a-TYR catalyzing L-DOPA to obtain DHI;

FIG. 3 is a HPLC detection chart of the tyrosinase mutant N205T+R209D mutant catalyzing L-DOPA to produce DHI.

DETAILED DESCRIPTION

The technical solutions in the embodiments will be described clearly and completely below in combination with the embodiments and drawings of the present application. Obviously, the embodiments described below are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.

It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and/or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and/or combinations thereof.

It should also be understood that the terms used in this specification of the embodiments of the present application are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present application. As used in the specification of the embodiments of the present application and the appended claims, unless the context clearly indicates otherwise, the singular forms of "a", "an" and "the" are intended to include plural forms.

Some of the terms and materials involved in this embodiment are explained below to facilitate understanding by those skilled in the art.

DHI: 5,6-dihydroxyindole, the English name is 5,6-Dihydroxyindole; the structural formula of DHI is: .

L-DOPA: Levodopa or DOPA, English name Levodopa. The structural formula of L-DOPA is: .

DQ: Dopaquinone; the structural formula of dopaquinone is: .

DC: Dopachrome; the structural formula of dopachrome is: .

The culture medium used in this example is as follows:

LB liquid medium: Take a 1 L beaker, add 5 g yeast powder, 10 g sodium chloride, 10 g peptone, add 900 mL distilled water and mix well, adjust the pH to 7.0, and then make up to 1000 mL. Finally, dispense into 250 mL conical flasks, 100 mL per bottle, and sterilize in an autoclave at 121°C for 20 min before use;

TB medium: Accurately weigh 7.2 g of yeast extract and 3.6 g of peptone, add 200 mL of sterile water and 1.2 mL of glycerol into a 1 L beaker, make up to 270 mL, and dispense into 150 mL conical flasks, 22.5 mL per bottle; accurately weigh 16.43 g (0.72 M) of dipotassium hydrogen phosphate trihydrate and 2.31 g (0.17 M) of anhydrous potassium dihydrogen phosphate, dissolve into a 100 mL beaker, and make up to 100 mL to prepare potassium phosphate buffer; after each of them is dispensed, sterilize them at 121°C in an autoclave for 20 min. When using, add 2.5 mL of potassium phosphate buffer to 22.5 mL of culture medium and mix them.

The tyrosinase mutants and their uses in the examples of the present application are described in detail below.

Tyrosinase mutants

In the prior art, traditional hair dyes such as p-phenylenediamine hair dyes are allergenic and carcinogenic. However, since 5,6-dihydroxyindole is easily oxidized and self-polymerized under air conditions and does not require the stimulation of an initiator, 5,6-dihydroxyindole melanin hair dyes are gradually replacing traditional aniline compounds as a better choice for synthetic hair dyes.

According to existing literature reports, the synthesis methods of 5,6-dihydroxyindole are mainly divided into phenylethylamine method, benzaldehyde method and benzyl cyanide method according to the different raw materials. The benzyl cyanide method will be used as an example below.

The preparation method of 5,6-dihydroxyindole by the benzyl cyanide method comprises:

Demethylating 3,4-dimethoxybenzeneacetonitrile to obtain the intermediate product 3,4-dihydroxybenzeneacetonitrile (demethylation reaction);

3,4-dihydroxybenzyl acetonitrile is reacted with benzyl chloride to obtain 3,4-dibenzyloxybenzyl acetonitrile to protect the hydroxyl group (hydroxyl protection);

The protected intermediate is nitrated to obtain 2-nitro-3,4-dibenzyloxyphenylacetonitrile (nitration reaction);

3,4-dibenzyloxyindole is obtained by reduction and cyclization reaction, and finally the benzyloxy protection is removed to obtain the product 5,6-dihydroxyindole (reductive cyclization reaction).

It can be seen from the above records that the above preparation method takes a long time and has complicated steps, and the materials used, such as pyridine used in the demethylation reaction, are highly toxic and carcinogenic.

Based on the above problems, there is currently a synthesis method for obtaining 5,6-dihydroxyindole by using tyrosinase to catalyze the substrate (levodopa). However, the current conversion rate of tyrosinase to obtain 5,6-dihydroxyindole by catalyzing l-dopa is low.

In view of this, the present embodiment provides a tyrosinase mutant, which has an amino acid mutation in the amino acid sequence shown in SEQ ID NO. 2; wherein the site of the amino acid mutation is one or a combination of two of F197A, P201A, R209A, V218A, F197D, F197E, F197M, F197N, F197Q, F197R, F197S, F197V, N205D, N205I, N205K, N205L, N205Q, N205R, N205S, N205T, N205V, R209D, R209G, R209K, R209N, R209S, R209V, V218G, V218I, V218M, V218Q, and V218S.

The tyrosinase in this example is derived from Bacillus megaterium . The nucleotide sequence of the tyrosinase is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.

In this example, the amino acid sequence shown in SEQ ID NO. 2 was used as a template (basis), and according to the binding conformation of L-DOPA with the active center of tyrosinase, key amino acid sites in the active center were selected for mutation modification, a mutant library was constructed, and a series of mutants with significantly improved catalytic activity were screened.

It can be understood that the catalytic activity described in this example refers to the ability of the mutant to better catalyze the conversion of L-dopa to 5,6-dihydroxyindole per unit time (having a higher conversion rate).

Therefore, the mutant obtained in this example has a higher conversion rate in the reaction (enzymatic conversion reaction) of levodopa to 5,6-dihydroxyindole.

Wherein, the nucleotide sequence of the above tyrosinase (SEQ ID NO. 1) is:

ATGAGTAACAAGTATAGAGTTAGAAAAAACGTATTACATCTTACCGACACGGAAAAAAGAGATTTTGTTCGTACCGTGCTAATACTAAAGGAAAAAGGGATATATGACCGCTATATAGCCTGGCATGGTGCAGCAGGTAAATTCATACTCCTCCGGGCAGCGATCGAAATGCAGCACATATGAGTTCTGCTTTTTTACCGTGGCATCGTGAATACCTTTACGATTCGAACGTGACCTTCAGTCAATCCAGAAGTAACCCT TCCTTATTGGGAATGGGAAACGGACGCACAGATGCAGGATCCCTCACAATCACAAATTTGGAGTGCAGATTTTATGGGAGGAAACGGAAATCCCATAAAAGATTTTATCGTCGATACCGGGCCATTTGCAGCTGGGCGCTGGACGACGATCGATGAACAAGGAAATCCTTCCGGAGGGCTA AAACGTAATTTTGGAGCAACGAAAGAGGCACCTACACTCCCTACTCGAGATGATGTCCTCAATGCTTTAAAAATAACTCAGTATGATACGCCGCCTTGGGATATGACCAGCCAAAACAGCTTTCGTAATCAGCTTGAAGGATTTATTAACGGGCCACAGCTTCACAATCGCGTACACCGTTGGGTTGGCGGACAGATGGGCGTTGTGCCTACTGCTCCGAATGATCCTGTCTTCTTTTTACACCACGCAAATGTGGATCGT ATTTGGGCTGTATGGCAAATTATTCATCGTAATCAAAACTATCAGCCGATGAAAAACGGGCCATTTGGTCAAAACTTTAGAGATCCGATGTACCCTTGGAATACAACCCCTGAAGACGTTATGAACCATCGAAAGCTTGGGTACGTATACGATATAGAATTAAGAAAATCAAAACGTTCCTCATAA.

The amino acid sequence of the above tyrosinase (SEQ ID NO. 2) is:

MSNKYRVRKNVLHLTDTEKRDFVRTVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQMQDPSQSQIWSADFMGGNGNPIKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRNFGATKEAPTLPTRDDVLNALKITQYDTPPWDMTSQNSFRNQLEGFINGPQLHNR VHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIIHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS.

In this embodiment, the site of the above-mentioned amino acid mutation can be a single site mutation, that is, a mutation at the site F197A, P201A, R209A, V218A, F197D, F197E, F197M, F197N, F197Q, F197R, F197S, F197V, N205D, N205I, N205K, N205L, N205Q, N205R, N205S, N205T, N205V, R209D, R209G, R209K, R209N, R209S, R209V, V218G, V218I, V218M, V218Q, V218S (single mutants) ); it may also be a combined mutation of two sites, that is, mutations occur at any two sites among sites F197A, P201A, R209A, V218A, F197D, F197E, F197M, F197N, F197Q, F197R, F197S, F197V, N205D, N205I, N205K, N205L, N205Q, N205R, N205S, N205T, N205V, R209D, R209G, R209K, R209N, R209S, R209V, V218G, V218I, V218M, V218Q, and V218S (mutants with multiple mutation points).

Illustratively, the above-mentioned tyrosinase mutant uses the amino acid sequence shown in SEQ ID NO. 2 as a template (based on), and any one of the following amino acid mutations occurs:

F197A, P201A, R209A, V218A, F197D, F197E, F197M, F197N, F197Q, F197R, F197S, F197V, N205D, N205I, N205K, N205L, N205Q, N205R, N205S, N205T, N2 05V, R209D, R209G, R209K, R209N, R209S, R209V, V218G, V218I, V218M, V218Q, V218S, N205D+F197A, N205D+F197D, N205D+F197E, N205D+R209D, N205D+R209N, N205T+F197A, N205T+R209D, N205T+R209N, R209D+F197A, R2 09D+F197D, R209D+F197E, R209N+ F197A, R209N+F197D, R209N+F197E.

As a preferred embodiment of this embodiment, the mutant of this embodiment undergoes any one of the following amino acid mutations:

F197A, F197D, F197E, N205T, R209D, R209N, V218G, N205D+F197A, N205D+R209D, N205D+R209N, N205T+R209D, N205T+R209N, R209D+F197A, R209D+F1 97D, R209D+F197E, R209N+F197A, R209N+F197D, R209N+F197E.

It is understandable that the tyrosinase mutant of this embodiment can be mutated at the corresponding site by existing site-directed mutagenesis technology to obtain the target mutant. Among them, the mutant with multiple mutation points can be superimposed on the basis of a single mutant, that is, on the basis of a single mutant, a mutation is performed at another amino acid mutation site to obtain a mutant with multiple mutation points.

FIG1 is a reaction scheme of the tyrosinase mutant prepared in this example catalyzing DOPA to generate 5,6-dihydroxyindole, and TYR in FIG1 is the tyrosinase mutant. Referring to FIG1 , the tyrosinase mutant provided in this example can catalyze L-DOPA to generate dopaquinone, and the generated dopaquinone will spontaneously cyclize and decarboxylate to convert into 5,6-dihydroxyindole.

Compared with the prior art, the tyrosinase mutant provided in this example can not only catalyze L-dopa to produce 5,6-dihydroxyindole, but also has a higher conversion rate, indicating that the tyrosinase mutant has good application prospects in the biotransformation of L-dopa to prepare 5,6-dihydroxyindole.

Recombinant plasmid

The recombinant plasmid provided in this embodiment is a DNA molecule containing a nucleotide sequence encoding any of the above-mentioned tyrosinase mutants or its complementary sequence.

Illustratively, the recombinant plasmid of this embodiment can be selected from any one of the following: pET-21b (+), pET-22b (+), pET-3a (+), pET-3d (+), pET-11a (+), pET-12a (+), pET-14b (+), pET-15b (+), pET-16b (+), pET-17b (+), pET-19b (+), pET-20b (+), pET-21a (+), pET-23a (+), pET-23b (+), pGEX-6p-1, pET-24a (+), pET- ET-25b(+), pET-26b(+), pET-27b(+), pUC-19, pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b (+), pET-41a (+), pET-41b (+), pET-42a (+), pET-43a (+), pET-43b ( +), pET-44a (+), pET-49b (+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pB V221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pUC-18, pUC-19.

The recombinant plasmid of this embodiment can be prepared by a known preparation method, for example:

Using the expression vector containing the amino acid sequence shown in SEQ ID NO. 2 as a template, mutations were performed at the corresponding sites to obtain a recombinant plasmid containing the encoded tyrosinase mutant.

Among them, the above-mentioned expression vector includes but is not limited to pET-30a; the above-mentioned expression vector containing the amino acid sequence shown in SEQ ID NO. 2 can be synthesized into the pET-30a vector by a gene synthesis company (such as Tianyi Huiyuan Gene Technology Co., Ltd.) to obtain the corresponding template (such as the template pET-30a-TYR in Example 1).

Recombinant strains

It is understandable that the recombinant strain provided in this embodiment contains the above-mentioned recombinant plasmid.

In this embodiment, the host cell of the recombinant strain is a prokaryotic cell or a eukaryotic cell, and preferably the eukaryotic cell is a yeast cell.

The host cell may be a competent cell, and preferably the competent cell is Escherichia coli BL21 (DE3).

The recombinant strain of this embodiment can be prepared by the following preparation method, for example:

The prepared recombinant plasmid was transferred into Escherichia coli BL21 (DE3) for cultivation to obtain a recombinant strain.

Usually, after obtaining the recombinant plasmid, it will be treated with restriction nuclease (such as Dpn I enzyme) and then transformed into Escherichia coli BL21 (DE3) for culture.

It is understandable that the prepared recombinant strain needs to be stored in a refrigerator (-80°C).

Uses of tyrosinase mutants

As mentioned above, the tyrosinase mutant of this example can be used to catalyze the production of 5,6-dihydroxyindole from L-dopa.

In fact, based on the use of tyrosinase mutants, the DNA molecules, recombinant plasmids, and recombinant strains of this embodiment can also be used to catalyze L-DOPA to produce 5,6-dihydroxyindole.

The preparation method of 5,6-dihydroxyindole will be introduced in detail below.

Illustratively, the method for preparing 5,6-dihydroxyindole in this embodiment comprises:

(I) preparing a recombinant plasmid containing a tyrosinase mutant;

(II) transforming the recombinant plasmid into a host cell to obtain a recombinant strain;

(III) Using L-DOPA as a substrate, the recombinant strain catalyzes L-DOPA to produce 5,6-dihydroxyindole.

The steps of the above preparation method will be further described below.

In step (I), preparing a recombinant plasmid containing a tyrosinase mutant, comprising:

Using the expression vector (pET-30a) containing the amino acid sequence shown in SEQ ID NO. 2 as a template, mutations (site-directed mutagenesis) were performed at the corresponding sites to obtain a recombinant plasmid containing the encoded tyrosinase mutant.

Among them, site-directed mutagenesis can adopt existing mutagenesis techniques, such as polymerase chain reaction (PCR) method.

In step (II), the recombinant plasmid is transformed into a host cell to obtain a recombinant strain, comprising:

The recombinant plasmid obtained in step (I) was digested with Dpn I enzyme and then transferred into Escherichia coli BL21 (DE3) for culture to obtain a recombinant strain.

In step (III), levodopa is used as a substrate, and the recombinant strain is used to catalyze levodopa to produce 5,6-dihydroxyindole, comprising:

The recombinant strain obtained in step (II) is added to a solution containing levodopa, and the recombinant strain is used to catalyze levodopa to generate 5,6-dihydroxyindole.

It can be understood that, referring to FIG. 1 , the generation of DHI from L-DOPA in step (III) is mainly divided into two reactions; in the first reaction, L-DOPA generates DQ and is quickly converted into DC; in the second reaction, the generated DC is converted into DHI.

Among them, in the first part of the reaction, oxygen is required to participate in the reaction so that L-DOPA generates DQ and converts into DC, so the first part of the reaction can be carried out in the air; in the second part of the reaction, nitrogen is usually introduced or sealed to protect DHI, which can reduce the oxidative polymerization of DHI.

Typically, the reaction temperature of the first reaction is 10-40°C, preferably 25-38°C (e.g., 37°C); the reaction time is 3-20 min, preferably 5-15 min (e.g., 5 min). During the first reaction, stirring is usually performed to increase the reaction rate. Typically, the stirring rate is 100-500 r/min, preferably 200-400 r/min (e.g., 220 rpm).

Usually, in the second reaction, that is, after the first reaction reaches a preset time (e.g., 5 minutes), the reaction liquid is taken out, and anhydrous ethanol and concentrated acid (e.g., 37% concentrated hydrochloric acid) are added to the reaction liquid to accelerate the conversion of DC to DHI. For example, anhydrous ethanol and concentrated acid are added to the reaction liquid, and the reaction is carried out at 20°C and 220 rpm for 30 minutes in a sealed state to obtain DHI.

The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate/explain the present application and are not used to limit the scope of the present application.

In the following examples, the materials, reagents and instruments used can be purchased from commercial sources unless otherwise specified.

Example 1

Synthesis and recombinant expression of template pET-30a-TYR

In this example, tyrosinase TYR from Bacillus megaterium was used as a template, and its optimized nucleotide sequence is shown in SEQ ID NO. 1, and its amino acid sequence is shown in SEQ ID NO. 2.

Among them, tyrosinase TYR can be obtained from the NCBI database, and its NCBI accession number is EU627691.1.

Based on this, in this example, tyrosinase TYR was used as a parent template to synthesize and recombinantly express the template pET-30a-TYR.

Specifically:

The gene sequence of tyrosinase TYR was synthesized into the pET-30a vector to obtain pET-30a-TYR;

pET-30a-TYR was transferred into Escherichia coli BL21 (DE3), and placed in a shaker at 37°C and 220 rpm for 60 min of recovery. After recovery, the bacterial solution was spread on an LB plate containing 50 μg/mL kanamycin and incubated at 37°C overnight.

A single colony on the plate was picked for activation and inoculated into 25 mL of TB medium containing 50 μg/mL kanamycin. The culture was cultured at 37°C and 220 rpm until OD ₆₀₀ was 0.6-0.8, and then induced. The induction conditions were: adding 0.6 mM inducer IPTG, shaking and culturing at 25°C and 180 rpm for 12 h.

The cells were collected at 6000 rpm, 4°C, and 10 min, and the collected cells were washed twice with 1×PBS buffer solution. The washed cells (wet cells) were stored in a -80°C refrigerator for later use.

Example 2

Using pET30a-TYR plasmid as template, design mutation primers and place the base to be mutated in the middle of the primer;

The PCR system was as follows: template 1 ng, upstream and downstream mutation primers 1.5 μL each, 2×PCR Buffer for KOD FXNeo 25 μL, KOD FX Neo enzyme 1 μL, and finally sterile distilled water was added to make up the volume to 50 μL.

The sequences of the upstream and downstream mutation primers used in this embodiment are shown in Table 1 below;

Table 1: Sequences of upstream and downstream mutation primers

Primer name Primer sequence (5'-3') F197A-F GTAATCAGTTAGAAGGCGCAATTAACGGCCCGCAACTGC F197A-R GTTGCGGGCCGTTAATTGCGCCTTCTAACTGATTACGAAAG P201A-F GCTTTATTAACGGCGCACAACTGCATAATCGTGTTCATCG P201A-R CGATTATGCAGTTGTGCCGCCGTTAATAAAGCCTTCTAACTG R209A-F CATAATCGTGTTCATGCATGGGTAGGTGGCCAAATGGG R209A-R GGCCACCTACCCATGCATGAACACGATTATGCAGTTG V218A-F GTGGCCAAATGGGTGTTGCACCTACCGCGCCGAAC V218A-R CGGCGCGGTAGGTGCAACACCCATTTGGCCACCTAC F197D-F GTAATCAGTTAGAAGGCGATATTAACGGCCCGCAACTGC F197D-R GTTGCGGGCCGTTAATATCGCCTTCTAACTGATTACGAAAG F197E-F G GTAATCAGTTAGAAGGCGAAATTAACGGCCCGCAACTGC F197E-R GTTGCGGGCCGTTAATTTCGCCTTCTAACTGATTACGAAAG F197M-F GTAATCAGTTAGAAGGCATGATTAACGGCCCGCAACTGC F197M-R GTTGCGGGCCGTTAATCATGCCTTCTAACTGATTACGAAAG F197N-F GTAATCAGTTAGAAGGCAATATTAACGGCCCGCAACTGC F197N-R GTTGCGGGCCGTTAATATTGCCTTCTAACTGATTACGAAAG F197Q-F GTAATCAGTTAGAAGGCCAGATTAACGGCCCGCAACTGC F197Q-R GTTGCGGGCCGTTAATCTGGCCTTCTAACTGATTACGAAAG F197R-F GTAATCAGTTAGAAGGCCGTATTAACGGCCCGCAACTGC F197R-R GTTGCGGGCCGTTAATACGGCCTTCTAACTGATTACGAAAG F197S-F GTAATCAGTTAGAAGGCAGCATTAACGGCCCGCAACTGC F197S-R GTTGCGGGCCGTTAATGCTGCCTTCTAACTGATTACGAAAG F197V-F GTAATCAGTTAGAAGGCGTTATTAACGGCCCGCAACTGC F197V-R GTTGCGGGCCGTTAATAACGCCTTCTAACTGATTACGAAAG N205D-F GGCCCGCAACTGCATGACCGTGTTCATCGCTGGGTAG N205D-R CCCAGCGATGAACACGGTCATGCAGTTGCGGCCGTTAATAAAG N205I-F GGCCCGCAACTGCATATTCGTGTTCATCGCTGGGTAG N205I-R CCCAGCGATGAACACGAATATGCAGTTGCGGGCCGTTAATAAAG N205K-F GGCCCGCAACTGCATAAACGTGTTCATCGCTGGGTAG N205K-R CCCAGCGATGAACACGTTTATGCAGTTGCGGGCCGTTAATAAAG N205L-F GGCCCGCAACTGCATCTGCGTGTTCATCGCTGGGTAG N205L-R CCCAGCGATGAACACGCAGATGCAGTTGCGGGCCGTTAATAAAG N205Q-F GGCCCGCAACTGCATCAGCGTGTTCATCGCTGGGTAG N205Q-R CCCAGCGATGAACACGCTGATGCAGTTGCGGGCCGTTAATAAAG N205R-F GGCCCGCAACTGCATCGTCGTGTTCATCGCTGGGTAG N205R-R CCCAGCGATGAACACGACGATGCAGTTGCGGGCCGTTAATAAAG N205S-F GGCCCGCAACTGCATAGCCGTGTTCATCGCTGGGTAG N205S-R CCCAGCGATGAACACGGCTATGCAGTTGCGGGCCGTTAATAAAG N205T-F GGCCCGCAACTGCATACCCGTGTTCATCGCTGGGTAG N205T-R CCCAGCGATGAACACGGGTATGCAGTTGCGGGCCGTTAATAAAG N205V-F GGCCCGCAACTGCATGTTCGTGTTCATCGCTGGGTAG N205V-R CCCAGCGATGAACACGAACATGCAGTTGCGGCCGTTAATAAAG R209D-F CATAATCGTGTTCATGATTGGGTAGGTGGCCAAATGGG R209D-R GGCCACCTACCCAATCATGAACACGATTATGCAGTTG R209G-F CATAATCGTGTTCATGGCTGGGTAGGTGGCCAAATGGG R209G-R GGCCACCTACCCAGCCATGAACACGATTATGCAGTTG R209K-F CATAATCGTGTTCATAAATGGGTAGGTGGCCAAATGGG R209K-R GGCCACCTACCCATTTATGAACACGATTATGCAGTTG R209N-F CATAATCGTGTTCATAATTGGGTAGGTGGCCAAATGGG R209N-R GGCCACCTACCCAATTATGAACACGATTATGCAGTTG R209S-F CATAATCGTGTTCATAGCTGGGTAGGTGGCCAAATGGG R209S-R GGCCACCTACCCAGCTATGAACACGATTATGCAGTTG R209V-F CATAATCGTGTTCATGTTTGGGTAGGTGGCCAAATGGG R209V-R GGCCACCTACCCAAACATGAACACGATTATGCAGTTG V218G-F GTGGCCAAATGGGTGTTGGTCCTACCGCGCCGAAC V218G-R CGGCGCGGTAGGACCAACACCCATTTGGCCACCTAC V218I-F GTGGCCAAATGGGTGTTATTCCTACCGCGCCGAAC V218I-R CGGCCGCGTAGGAATAACACCCATTTGGCCACCTAC V218M-F GTGGCCAAATGGGTGTTATGCCTACCGCGCCGAAC V218M-R CGGCGCGGTAGGCATAACACCCATTTGGCCACCTAC V218Q-F GTGGCCAAATGGGTGTTCAGCCTACCGCGCCGAAC V218Q-R CGGCGCGGTAGGCTGAACACCCATTTGGCCACCTAC V218S-F GTGGCCAAATGGGTGTTAGCCCTACCGCGCCGAAC V218S-R CGGCGCGGTAGGGCTAACACCCATTTGGCCACCTAC

The PCR reaction procedure was as follows: (1) pre-denaturation at 94°C for 2 min; (2) denaturation at 98°C for 10 s; (3) annealing temperature was set according to the mutant primers, and the annealing time was 15 s; (4) extension at 68°C for 60 s, steps (2) to (4) were repeated for 30 cycles, and finally extension was performed at 68°C for 10 min. The PCR product was stored at 12°C.

The PCR product was digested with Dpn I enzyme, and after removing the template, it was transferred into Escherichia coli DH5α competent cells. The bacterial solution after recovery culture was spread on LB medium plates containing 50 μg/mL kanamycin and inverted cultured at 37°C overnight.

Pick monoclonal colonies for single colony verification, select 1-3 single colonies for gene sequencing, and extract the strains with correct sequencing.

Among them, the mutant with multiple mutation points is obtained by superimposing mutations on a single mutant using the same method as above, thereby obtaining a mutant with multiple mutation points.

The inducible expression of the mutants was consistent with that of the above templates.

Example 3

Activity screening of tyrosinase mutants

In this example, L-DOPA was used as a substrate to determine the catalytic activity of the tyrosinase mutant.

The specific method is:

The obtained wet bacteria were resuspended in 7.5 mL (1×PBS buffer solution) at a ratio of 1 g (wet bacteria), and 600 μL was used for ultrasonic disruption, with 1 second on and 2 seconds off for a total of 10 minutes. The disrupted liquid was centrifuged at 12,000 rpm for 15 minutes. The supernatant was collected, 500 μL of the supernatant was taken, and 500 μL of the substrate L-DOPA (prepared with DDW to 8 mM stock solution) was added to a final volume of 1 mL;

In an air environment, place it at 37°C and 220 rpm for 5 min, take out the reaction solution, add 2 μL concentrated hydrochloric acid (37%) and 1 mL anhydrous ethanol, and react at 20°C and 220 rpm for 30 min in a sealed state; after the reaction is completed, centrifuge at 12000 rpm for 10 min, filter with a 0.22 μm organic filter membrane, and use HPLC to detect the content. The activity test results of the tyrosinase mutants are shown in Table 2 below;

Table 2: Activity test results of tyrosinase mutants

Template/Mutant Yield (%) Template (pET-30a-TYR) 19.89±0.4 F197A 35.62±2.07 P201A 22.03±0.95 R209A 25.15±0.22 V218A 27.29±1.01 F197D 38.15±2.49 F197E 39.61±2.25 F197M 25.78±0.11 F197N 23.61±0.13 F197Q 23.8±0.02 F197R 21.02±0.13 F197S 24.55±0.16 F197V 22.21±0.02 N205D 29.63±1.35 N205I 23.73±0.16 N205K 24.18±0.66 N205L 21.83±0.16 N205Q 22.31±0.08 N205R 27.67±0.21 N205S 26.22±0.16 N205T 37.99±1.55 N205V 22.9±0.17 R209D 42.42±1.17 R209G 21.1±0.15 R209K 23.4±0.17 R209N 36.61±0.9 R209S 24.69±0.14 R209V 28.16±0.67 V218G 31.16±0.33 V218I 23.79±0.85 V218M 24.24±0.94 V218Q 20.36±0.78 V218S 26.84±0.99 N205D+F197A 44.86±0.18 N205D+F197D 28.12±0.12 N205D+F197E 24.8±17.54 N205D+R209D 42.83±1.46 N205D+R209N 40.43±1.21 N205T+F197A 24.65±0.02 N205T+R209D 63.77±1.67 N205T+R209N 44.62±1.31 R209D+F197A 43.21±1.19 R209D+F197D 37.34±5.14 R209D+F197E 46.78±1.47 R209N+F197A 32.66±0.6 R209N+F197D 47.57±0.86 R209N+F197E 53.88±1.5

Note: The test results in Table 2 were obtained after three parallel reactions under the same reaction conditions. The yield is defined as: molar concentration of product at the end of the reaction/molar concentration of the initial substrate × 100%.

According to the test results in Table 2, in this example, the key active sites were mutated based on the template pET-30a-TYR to obtain a mutant with significantly improved activity in catalyzing levodopa (L-DOPA) to generate 5,6-dihydroxyindole.

Therefore, compared with the template pET-30a-TYR, the above mutants have improved yields of L-DOPA to DHI.

Specifically, FIG. 2 is a HPLC detection diagram of the template pET-30a-TYR catalyzing L-DOPA to obtain DHI, and FIG. 3 is a HPLC detection diagram of the tyrosinase mutant N205T+R209D mutant catalyzing L-DOPA to obtain DHI.

Further combined with Table 2, Figure 2 and Figure 3, it can be seen that the mutant N205T+R209D showed higher catalytic activity than the template pET-30a-TYR, and the yield of DHI increased from 19.89% to 63.77%.

It can be understood that the mutant N205T+R209D is considered as a preferred embodiment in this example.

Example 4

Based on the test results of Example 3, this example uses mutants N205T, R209D and N205T+R209D as exemplary mutants to further verify the enzyme activity of tyrosinase mutants;

The induction expression method of the mutant in this example is consistent with that in Example 1;

Take 20 μL of mutant N205T, R209D, and N205T+R209D recombinant strains, add them to 5 mL of LB medium containing 50 μg/mL, and culture them overnight at 37°C and 220 rpm. Take 2.5 mL of bacterial solution and add it to 250 mL of TB medium containing 50 μg/mL kanamycin, culture it at 37°C and 220 rpm until OD ₆₀₀ is 0.6-0.8, and then induce it; the induction conditions are: add 0.6 mM IPTG, and induce it at 25°C and 180 rpm for 12 h. The culture solution was collected by centrifugation at 6000 rpm and 4°C for 5 minutes, washed twice with 1× PBS buffer solution, and the obtained wet bacteria were stored at -80°C for use;

After induction, the cells were resuspended in 15 mL of equilibration buffer (500 mM NaCl, 20 mM imidazole, 20 mM Tris-HCL buffer, pH = 7.2) according to 1 g (wet cells). Ultrasonic disruption was performed in an ice bath, with the cell disruption solution on for 1 second and off for 2 seconds, for a total of 60 minutes. The cell disruption solution was centrifuged at 12,000 rpm and 4°C for 15 minutes, and the supernatant was filtered with a 0.22 μm filter membrane for subsequent purification.

The purification method is nickel column affinity chromatography: the purification column is a 5 mL pre-packed column, and the entire purification system is first balanced with an equilibrium buffer (500 mM NaCl, 20 mM imidazole, 20 mM Tris-HCL buffer, pH=7.2), and then the sample is loaded at a flow rate of 1 mL/min, and the impurities that are not attached to the column are further removed with an equilibrium buffer (500 mM NaCl, 20 mM imidazole, 20 mM Tris-HCL buffer, pH=7.2), and then gradient elution is performed with an elution buffer (500 mM NaCl, 500 mM imidazole, 20 mM Tris-HCL buffer, pH=7.2);

The target protein was eluted at an imidazole concentration of about 300 mM, and the target protein was collected, concentrated and desalted using an ultrafiltration tube (cutoff 30 KDa) to a final volume of 1 mL, and the protein solution was aliquoted and stored at -80°C for later use;

Because L-DOPA is oxidized to dopaquinone (DQ) under the catalysis of tyrosinase, and DQ has extremely high reactivity and will spontaneously cyclize to dopachrome (DC) at a high rate. DC will then further decarboxylate to form DHI;

Since the intermediate product DC has a maximum absorption peak at 475 nm (ε=3700 M ^-1 cm ^-1 ), the colorimetric method can be used to detect the absorbance at 475 nm to explore the enzyme activity and catalytic efficiency of the mutant.

The specific steps are:

Add 130 μL of 100 mM sodium phosphate buffer (pH = 7.0) and 50 μL of L-DOPA solution of different concentrations to a 96-well plate, shake for 30 seconds, place in a 37°C constant temperature incubator for 5 minutes, and then add 20 μL of pure enzyme solution (use 1×PBS buffer solution to prepare the protein solution into a 1 μM concentration mother solution), the total volume is 200 μL; react in a 37°C constant temperature incubator for 5 minutes, use a microplate reader to measure the absorbance at 475 nm to determine the generation of DC (ε=3700 M ^-1 cm ^-1 ), the optical path length l=0.6 cm, and set three parallels for each test. The test results are shown in Table 3 below. Among them, K _cat /K _M (S ^-1 mM ^-1 ) is the catalytic efficiency;

Table 3: Enzyme activity test results of mutants N205T, R209D and N205T+R209D:

Template/Mutant K _M (μM) V _max (μM ^-1 min ^-1 ) K _cat (S ^-1 ) K _cat /K _M (S ^-1 mM ^-1 ) Template pET-30a-TYR 0.41±0.06 26.86±0.75 4.48±0.13 10.92±2.08 N205T 0.28±0.15 28.92±0.64 4.82±0.11 17.21±0.71 R209D 1.02±0.43 125.05±2.6 20.84±0.43 20.23±1.01 N205T/R209D 1.06±0.11 139.27±1.06 23.21±0.18 21.9±1.61

Combined with the test results in Table 3, it can be seen that the mutants N205T, R209D and N205T/R209D in this example have better catalytic efficiency than the template pET-30a-TYR. Therefore, the tyrosinase mutant provided in this example can better catalyze L-DOPA to generate 5,6-dihydroxyindole with a higher yield.

In summary, this example uses the amino acid sequence shown in SEQ ID NO. 2 as a template (basis), selects key amino acid sites in the active center for mutation modification according to the binding conformation of L-DOPA and the tyrosinase active center, constructs a mutant library, and screens a series of mutants with significantly improved catalytic activity; the tyrosinase mutant provided in this example can better catalyze L-DOPA to produce 5,6-dihydroxyindole, and has a higher conversion rate, indicating that the tyrosinase mutant has good application prospects in the biotransformation of L-DOPA to prepare 5,6-dihydroxyindole.

The technical solutions provided in the embodiments of the present application are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present application. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present application. At the same time, for those skilled in the art, according to the embodiments of the present application, there will be changes in the specific implementation methods and application scopes. In summary, the contents of this specification should not be understood as limiting the present application.

Claims (9)

1. A tyrosinase mutant, characterized in that the tyrosinase mutant has the following amino acid mutations in the amino acid sequence shown in SEQ ID NO. 2: N205T+R209D. 2. A DNA molecule, characterized in that the DNA molecule is a nucleotide sequence encoding the tyrosinase mutant according to claim 1 or its complementary sequence. 3. A recombinant plasmid, characterized in that the recombinant plasmid contains the DNA molecule according to claim 2. 4. A recombinant strain, characterized in that the recombinant strain contains the recombinant plasmid according to claim 3. 5. The recombinant strain according to claim 4, characterized in that the host cell of the recombinant strain is a prokaryotic cell or a eukaryotic cell, and the eukaryotic cell is a yeast cell. The recombinant strain according to claim 4 , characterized in that the host cell of the recombinant strain is a competent cell. 7. The recombinant strain according to claim 6, characterized in that the competent cell is Escherichia coli BL21 (DE3). 8. Use of the tyrosinase mutant according to claim 1, the DNA molecule according to claim 2, the recombinant plasmid according to claim 3, or the recombinant strain according to any one of claims 4 to 7 in catalyzing the production of 5,6-dihydroxyindole from L-dopa. 9. The use according to claim 8, characterized in that the preparation method of 5,6-dihydroxyindole comprises: preparing a recombinant plasmid containing a tyrosinase mutant; Transforming the recombinant plasmid into a host cell to obtain a recombinant strain; Using L-DOPA as substrate, the recombinant strain catalyzes L-DOPA to produce 5,6-dihydroxyindole.