WO2019091366A1

WO2019091366A1 - Method for preparing optically pure l-tertiary leucine by using active inclusion body

Info

Publication number: WO2019091366A1
Application number: PCT/CN2018/114105
Authority: WO
Inventors: 方柏山; 张永辉
Original assignee: 厦门大学
Priority date: 2017-11-08
Filing date: 2018-11-06
Publication date: 2019-05-16
Also published as: CN107858384A; CN107858384B

Abstract

Disclosed is a method for preparing an optically pure L-tertiary leucine by using an active inclusion body, the method comprising the following steps: (1) preparing a bifunctional enzyme active inclusion body, wherein the active ingredient of the bifunctional enzyme active inclusion body is a fusion bifunctional enzyme which comprises a LeuDH part and a polymerase part, linked by a linker peptide, for coenzyme NAD⁺ regeneration; and (2) adding the above-mentioned bifunctional enzyme active inclusion body to a mixed reaction liquid of pH 6.0 to 10.0 for resuspension, and then reacting the mixture at 20ºC to 40ºC, with the pH being controlled to be 6.0 to 10.0 during the reaction, wherein the mixed reaction liquid contains 50 to 1000 mM of trimethylpyruvate, 50 to 1000 mM of ammonium formate, and 0.05 to 5 mM of the coenzyme NAD⁺.

Description

Method for preparing optically pure L-tert-leucine by using active inclusion body

Technical field

The present application belongs to the field of bioengineering technology, and particularly relates to a method for preparing optically pure L-tert-leucine by using active inclusion bodies.

Background technique

The tert-butyl group in the structure of L-tertiary bright amino acid (L-Tle) facilitates the reaction from the back side due to its large steric hindrance, so L-Tle and its derivatives are often used as catalysts for inducing asymmetric reactions. The products are highly selective, and are commonly used as templates for inducing asymmetric synthesis reactions, and are widely used in asymmetric synthesis. In addition, the tert-butyl structure of L-Tle is highly hydrophobic and can effectively control the molecular configuration; in the polypeptide component, L-Tle is gradually replacing Val, Leu and Ile, because it can enhance the hydrophobicity of the polypeptide and Stability to prevent degradation by enzymes.

L-Tle has a wide range of applications in feed additives and nutritional supplements. In addition, L-Tle and its derivatives are often used as metal chiral ligands or ligands for chemical enzyme catalysts to provide a more efficient catalytic mode for asymmetric amination reduction reactions. Another important use of L-Tle is as a pharmaceutical intermediate, which is widely used in the synthesis of anti-AIDS drugs and biological inhibitors.

The methods for producing L-tert-leucine have been reported mainly as chemical reagent resolution, chiral source synthesis, chemical synthesis and biological enzymatic methods. Among them, the splitting method is limited by the yield, the chiral source method is limited by the natural product productivity, and the chemical synthesis method is costly. Therefore, these synthetic methods have not been successfully industrialized. The bioenzymatic method is currently implementing L-tert-leucine. The main method of industrial production.

The bioenzymatic synthesis of tert-leucine has been reported to be mainly divided into two classes, using free enzymes and whole cells as catalysts. Kragl and Dreisbach use repeated leucine dehydrogenase and formate dehydrogenase in repeated batch reactions in tons of enzyme biofilm reactors, which can be recovered by ultrafiltration (Angewandte Chemie International Edition 1996, 6, 684) Codexis uses a free leucine dehydrogenase to react with keto reductase for L-Tle, keto reductase for NAD ⁺ reduction (US Patent 9080192B2); Hoechst, Germany uses transaminase (Free enzyme or microbial whole cell form) catalyzes the transamination of tert-butyl keto acid and an amino donor compound to synthesize tert-leucine (Danmark Patent DK175472B1). Inclusion bodies are often formed during the expression of heterologous proteins in prokaryotic expression systems and are generally considered to be adverse by-products, severely reducing the expression of soluble recombinant proteins. However, studies on inclusion bodies in recent years have shown that they are mainly composed of recombinant proteins of interest, and that aggregation of recombinant proteins to form inclusion bodies does not mean loss of biological activity. According to reports in the literature and patents, it can be confirmed that inclusion bodies are still quite Biological activity of soluble recombinant proteins (Trends in Biotechnology 2012, 30: 65-7; Trends in Biochemical Sciences, 2017, 42(9): 726-737).

The target enzyme can be self-immobilized by fusion with an appropriate label to form a stable, reusable biocatalyst. Nahlka et al. fused maltodextrin phosphorylase to the cellulose binding site of Clostridium cellulovorans to construct active inclusion bodies. 83% of maltodextrin phosphorylase was found in inclusion bodies and could be used for D-glucose-1. - Repeated batch catalysis of phosphoric acid (Journal of Industrial Microbiology and Biotechnology 2008, 35: 219-223); Diener et al. used the coiled-coil region (53 amino acids) of the cell surface protein Tetrabrachion from Staphylothermus marinus as a fusion tag, respectively The lipase, the hydroxynitrile lyase and the 2-succinyl-5-enolpyruvate-6-hydroxy-3-cyclohexene-1-carboxylic acid synthase are fused to obtain corresponding active inclusion bodies, and Increased stability and recyclability of enzymes (Chemcatchem 2016, 8: 142-152); Li and Zhang et al. fused elastin polypeptides with xylanases to induce expression of active inclusion bodies, the specific activity of which was wood 92% of the glycanase, and pH stability, thermal stability and storage stability were greatly improved (Journal of biotechnology, 2014, 177: 60-66.).

The currently reported labels for inducing inclusion body formation include a cellulose binding site, a tetramerization site of the cell surface protein Tetrabrachion, a foot-and-mouth disease virus VP1 capsid protein, a green fluorescent protein, an elastin polypeptide, and the like. However, there is no precedent for the preparation of L-Tle or other products by preparing bifunctional enzymes for the preparation of active inclusion bodies. Therefore, genetic engineering methods are used to construct bifunctional enzyme active inclusion bodies, and they are used as high-efficiency and economical biocatalysts to prepare optically pure L- Tle has an important meaning.

Summary of the invention

The purpose of the present application is to overcome the deficiencies of the prior art and to provide a process for the preparation of optically pure L-tert-leucine using active inclusion bodies.

The technical solution of the present application is as follows:

A method for preparing optically pure L-tert-leucine using active inclusion bodies, comprising the following steps:

(1) preparing a bifunctional enzyme activity inclusion body, wherein the active component of the bifunctional enzyme activity inclusion body is a fusion bifunctional enzyme comprising a LeuDH moiety linked by a linker peptide and a regeneration enzyme for NAD ⁺ The polymerase portion, wherein the LeuDH portion comprises a sequence as shown in SEQ ID NO: 01, wherein the linker peptide is a rigid linker peptide or a flexible linker peptide, and the rigid linker peptide is capable of forming an alpha helix to effectively isolate the LeuDH portion and the polymerase portion. The flexible linker peptide described above does not have the ability to form a particular secondary structure, and is generally present in the form of a random coil to provide the flexibility required for the protein in the catalytic process;

(2) The bifunctional enzyme activity inclusion body is resuspended in a reaction mixture having a pH of 6.0 to 10.0, and then reacted at 20 to 40 ° C, and the pH is controlled to be 6.0 to 10.0 during the reaction; the reaction mixture contains 50 to 1000 mM. Methylpyruvate, 50-1000 mM ammonium formate and 0.05-5 mM coenzyme NAD ⁺ .

In a preferred embodiment of the present application, the rigid linker peptide comprises a plurality of amino acid sequences as shown in SEQ ID NO 02, which are contiguously linked.

In a preferred embodiment of the present application, the flexible linker peptide comprises a plurality of amino acid sequences as shown in SEQ ID NO: 03.

In a preferred embodiment of the present application, the polymerase moiety is an FDH moiety, a glucose dehydrogenase moiety, a glycerol dehydrogenase moiety, an alcohol dehydrogenase moiety, a glucose-6-phosphate dehydrogenase moiety, and a lactate dehydrogenase. Department or hydrogenase part.

Further preferably, the polymerase moiety is an FDH moiety, and the FDH moiety comprises the amino acid sequence set forth in SEQ ID NO 04.

In a preferred embodiment of the present application, the pH of the reaction mixture in the step (2) is 8.5 to 9, the temperature is 30 ° C, and the pH is controlled to be 8.5 to 9 during the reaction.

In a preferred embodiment of the present application, the reaction mixture in the step (2) comprises 50 to 710 mM of trimethylpyruvate, 50 to 780 mM of ammonium formate, and 0.05 to 0.5 mM of the coenzyme NAD ⁺ .

The beneficial effects of the application are:

1. The present application can greatly reduce the cost of catalyst preparation in a double enzyme system by constructing a fusion enzyme.

2. The method for constructing the bifunctional enzyme activity inclusion body of the present application is low in cost and easy for industrial application.

3. The bifunctional enzyme activity inclusion body in the present application belongs to the self-assembly immobilization without carrier, avoids the cost of enzyme immobilization, and facilitates separation and purification of downstream products. The bifunctional enzyme activity inclusion body, typically, the FDH-LeuDH bifunctional enzyme activity inclusion body has high optical selectivity, thermal stability is improved compared with the soluble bifunctional enzyme, and can be reused as an immobilized enzyme. The cost-effective preparation of optically pure tert-leucine has a good industrial application prospect.

4. The present invention prepares a bifunctional enzyme active inclusion body by a genetic engineering bacteria containing a bifunctional enzyme expression vector, and can optimize the overall structure of the bifunctional enzyme activity inclusion body by adjusting the ligation peptide configuration, thereby improving the coupling efficiency of the double enzyme.

5. The application process is simple, no special requirements for equipment, suitable for industrial production.

DRAWINGS

Figure 1 is a diagram showing the agarose gel electrophoresis of the PCR product of the FDH-LeuDH bifunctional enzyme gene in Example 1 of the present application.

2 is a whole cell SDS-PAGE diagram of FDH-LeuDH bifunctional enzyme genetically engineered bacteria mediated by different linker peptides in Example 2 of the present application.

Figure 3 is a SEM image of the bifunctional enzyme active inclusion body in Example 3 of the present application, wherein A is FDH-R1-LeuDH, B is FDH-R2-LeuDH, C is FDH-S1-LeuDH, and D is FDH-S2- LeuDH.

4 is a recombinant protein distribution (A), an FDH enzyme activity distribution (B), and a LeuDH enzyme activity distribution (C) of the bifunctional enzyme activity inclusion body in Example 3 of the present application.

5 is a comparison of the enzyme activities of the bifunctional enzyme activity inclusion body and the free enzyme in Example 3 of the present application, wherein A is a LeuDH moiety and B is an FDH moiety, and the relative enzyme activity is calculated by using the enzyme activity of the free single enzyme as 100%.

Figure 6 is a comparison of the catalytic ability of the FDH-R3-LeuDH soluble fraction and the active inclusion body in Example 4 of the present application.

Figure 7 is a graph showing the results of liquid chromatography in Example 4 of the present application, wherein A is a liquid chromatogram of standard L-Tle and standard D-Tle, and B is a liquid phase of a catalytic product of FDH-R3-LeuDH active inclusion body. Chromatogram, asterisk indicates D-Tle peak time.

Figure 8 is a SDS-PAGE diagram of FDH-R3-LeuDH active inclusion bodies of different IPTG concentrations in Example 5 of the present application.

Figure 9 is a comparison of the catalytic capabilities of FDH-R3-LeuDH active inclusion bodies of different IPTG concentrations in Example 5 of the present application.

Figure 10 is a comparison of the thermal stability of FDH-R3-LeuDH active inclusion bodies and soluble fractions in Example 6 of the present application, wherein A is FDH enzyme activity and B is LeuDH enzyme activity.

Figure 11 is a continuous recovery catalysis of FDH-R3-LeuDH active inclusion bodies in Example 7 of the present application. The relative yield of the recovered catalysis was calculated as the first catalyzed yield of 100%.

Detailed ways

The technical solutions of the present application are further described and described in the following with reference to the accompanying drawings.

In the following examples, the active ingredient of the bifunctional enzyme activity inclusion body of the present application is a fusion bifunctional enzyme comprising a LeuDH moiety linked by a linker peptide and a polymerase moiety for coenzyme NAD ⁺ regeneration. The above-mentioned linker peptide is a rigid linker peptide or a flexible linker peptide capable of forming an α-helix to effectively isolate the above-mentioned LeuDH moiety and the polymerase moiety, and the flexible linker peptide has no ability to form a specific secondary structure, generally The form of the coil is present to provide the flexibility required for the protein in the catalytic process. The polymerase part is the FDH part, the glucose dehydrogenase part, the glycerol dehydrogenase part, the alcohol dehydrogenase part, the glucose-6-phosphate dehydrogenase part, The lactate dehydrogenase moiety or the hydrogenase moiety, the polymerized form of the above polymerase moiety, and the referenced PDB structure ID are shown in the following table. The enzyme capable of forming a multimer and capable of being used for coenzyme regeneration is preferably FDH, and the polymerase for coenzyme regeneration is preferably a polymer of FDH, and in this case, the bifunctional enzyme activity inclusion body is preferably FDH-LeuDH bifunctional enzyme activity. Inclusion body, that is, FDH is responsible for the regeneration of coenzyme.

Multimeric morphology information of the enzyme commonly used for coenzyme regeneration and the referenced PDB ID

In the following examples, if no specific operating conditions are indicated, they can usually be carried out according to conventional experimental conditions, such as the Guide to Molecular Cloning, prepared by J. Sambrook, and the Manual of Protein Technology, written by Wang Jiazheng. The conditions described in the article, or the conditions recommended by the manufacturer.

Example 1

Construction of recombinant strain of FDH-LeuDH bifunctional enzyme

Overlap extension polymerase chain reaction (OE-PCR) was used to construct FDH-LeuDH fusion enzyme mediated by different ligation peptides. The construction of FDH-R1-LeuDH fusion enzyme gene was used as an example to describe the construction process. First, primers were designed based on LeuDH, FDH, the linker sequence and the restriction sites on the pET28a plasmid:

P1: 5'-GGAATTC CATATG AAAATTGTCCTGGTCCTGT-3' (SEQ ID NO 05), underlined for the NdeI restriction site sequence.

Ligation peptide primer: 5'-GCCTATGGCAAACACGATAAAAAG XXX ATGACATTGG AAATCTTCGA-3', XXX refers to the linker peptide sequence, as shown in Table 1.

P3: 5'-ATGACATTGGAAATCTTCGAATAT-3' (SEQ ID NO 06).

P4: 5'-CCG CTCGAG TTACCGGCGACTAATGATGT-3' (SEQ ID NO 07), underlined for the XhoI restriction site sequence.

Using FDH and LeuDH genes as templates, the FDH gene was amplified by P1 and ligation peptide primers, and the LeuDH gene was amplified by P3 and P4. The PCR amplification system: template 2uL, primers 1.5uL, PCR Mix 25uL, ddH2O 20uL. PCR conditions: 94 ° C pre-denaturation, 5 min; 94 ° C denaturation, 1 min, 56 ° C annealing, 1 min, 72 ° C extension, 15 s, 30 cycles; 72 ° C extension, 10 min. The FDH and LeuDH genes were recovered using a gel recovery kit, and then PCR amplification was carried out using primers 1 and primers 4 using equimolar two enzyme genes as templates, and the fusion enzyme gene inserted into the linker peptide was obtained under the same conditions as above. , as shown in Figure 1, where M represents a DNA marker, and bands 1-7 are FDH-DL-LeuDH, FDH-S1-LeuDH, FDH-S2-LeuDH, FDH-S3-LeuDH, FDH-R1-LeuDH, The FDH-R2-LeuDH and FDH-R3-LeuDH genes were recovered from the fusion enzyme gene using a gel recovery kit. The obtained fusion enzyme gene and pET-28a plasmid were digested with NdeI/XhoI, and the fusion enzyme gene and plasmid backbone were recovered by gel recovery kit, and the ligated plasmid was transformed into E. coli BL21 (DE3). Positive clones were screened using kanamycin resistant plates. The obtained positive clones were cultured at 37 ° C overnight, and the plasmid was extracted, and after double enzyme digestion verification, the strains were stored in a -80 ° C refrigerator.

The amino acid sequence of the above FDH is shown in SEQ ID NO 04, the nucleotide sequence is shown in SEQ ID NO: 08, the amino acid sequence of the above LeuDH is shown in SEQ ID NO 01, and the nucleotide sequence is shown in SEQ ID NO 09.

Table 1 shows the amino acid sequences of different fusion enzyme linker peptides and the primer sequences used for insertion of the linker peptide.

^a DL represents a direct linkage; R1-R3 represents an EAAAK linking peptide of 1 to 3 repeating units; and S1-S3 represents a GGGGS linking peptide of 1 to 3 repeating units.

^The underlined portion of ^b is a primer for the corresponding fusion enzyme construction.

Example 2

Preparation of bifunctional enzyme activity inclusion bodies

The bifunctional enzyme recombinant strain was inoculated into LB medium, activated at 37 ° C, 200 rpm overnight, transferred to Lb medium, inoculated in an amount of 1%, cultured at 37 ° C, 200 rpm until the OD600 was about 0.5, and the final concentration was 0.2. Expression was induced for 24 h at mM IPTG, 16 ° C, 200 rpm. After the completion of the culture, the cells were collected, and the cells were washed twice with PBS buffer (pH = 7.2) and stored at -80 ° C until use. The whole cell SDS-PAGE was used to verify whether the recombinant bifunctional enzyme was successfully expressed. The results are shown in Figure 2, where M represents the protein marker, and bands 1-9 are FDH single enzyme, LeuDH single enzyme, FDH-DL-LeuDH, FDH-, respectively. S1-LeuDH, FDH-S2-LeuDH, FDH-S3-LeuDH, FDH-R1-LeuDH, FDH-R2-LeuDH and FDH-R3-LeuDH, it can be seen that all of the seven linker-mediated bifunctional enzymes have been successfully obtained. expression.

100 mg of the cells were suspended in 5 mL of ddH ₂ O, and the bacterial cells were disrupted by an ultrasonic cell disrupter, centrifuged at 12,000 × g for 20 min, and the supernatant was temporarily stored at 4 ° C. The precipitate obtained by centrifugation was first dissolved in PBS buffer supplemented with 1% by volume of ethylphenyl polyethylene glycol (NP-40), placed at 4 ° C for 45 min, and then 25 μL of DNAse and MgSO ₄ (final concentration 10 mM) were added. After shaking at 37 ° C, 100 rpm for 45 min, centrifugation at 12000 × g for 20 min at 4 ° C, the precipitate was washed once with PBS buffer (containing 1% Trition X-100), and washed with PBS buffer twice to obtain purified inclusion bodies. .

Example 3

Characterization of bifunctional enzyme activity inclusion bodies

The morphology of the inclusion bodies was observed directly by scanning electron microscopy. The sample preparation method was as follows: 5 μL of the inclusion body sample was dropped on a single crystal silicon wafer, air-dried overnight, and then plated with about 2 nm thick platinum in a JFC-1600 (JEOL, Tokyo, Japan) sputtering apparatus (sputtering conditions) : 10 mA, 30 s), and the coated samples were placed in a field emission Sigma-type scanning electron microscope (Carl-Zeiss AG, Germany) for observation. Figure 3 is a SEM structural diagram of a partial inclusion body, wherein A is FDH-R1-LeuDH, B is FDH-R2-LeuDH, C is FDH-S1-LeuDH, and D is FDH-S2-LeuDH. The mediated bifunctional enzyme activity inclusion bodies exhibit a lamellar structure, while the flexible linker-mediated bifunctional enzyme activity inclusion bodies exhibit an irregular globular aggregate structure.

The bispecific enzyme activity inclusion body distribution and enzyme activity distribution were studied. Figure 4A shows the distribution of recombinant protein. It can be seen that more than 80% of the recombinant protein is present in the active inclusion body, and the recombinant protein distribution in the supernatant is higher. Less, Figure 4B and Figure 4C show the distribution of enzyme activity. It can be seen that more than 90% of FDH activity and LeuDH activity are distributed in the inclusion body part, and the FDH and LeuDH activity distribution in some bifunctional enzyme inclusion bodies exceeds 95. %, the experimental results indicate that most of the FDH-LeuDH bifunctional enzyme is expressed as an active inclusion body.

The enzyme activity of the bifunctional enzyme activity inclusion body was studied. Figure 5A shows the comparison of the LeuDH activity of the bifunctional enzyme activity inclusion body and the free enzyme, and Fig. 5B shows the comparison of the FDH activity of the bifunctional enzyme activity inclusion body and the free enzyme. The relative enzymatic activity was calculated by using the enzyme activity of LeuDH and FDH single enzymes as 100%. It can be seen that the activity of the FDH in the active inclusion body was significantly higher than that of the single enzyme (24.7%-146.6%), while the free enzyme fraction The enzyme activity of FDH showed a significant decrease. The enzymatic activity of LeuDH is lower than that of free enzyme, but since the FDH is the rate-limiting enzyme in the L-Tle double enzyme catalytic system, the enzyme activity of LeuDH is much higher than that of FDH, so the decrease of LeuDH activity does not decrease. Overall catalytic efficiency.

Example 4

Bifunctional enzyme activity inclusion body preparation L-Tle

On the basis of the above Example 2, the precipitate was added to the reaction mixture after the purification treatment, and 10 mL of the reaction mixture was suspended to start the reaction. The supernatant was added with 5 mL of the 2× reaction mixture to keep the concentrations of the two experiments equal, and the two groups were placed at 30 ° C, 200 rpm. Reaction for 48 h. The reaction mixture contained 50 mM trimethylpyruvate, 50 mM ammonium formate, 0.04 mM NAD ⁺ , adjusted to pH 8.5 with aqueous ammonia, and the solvent was H ₂ O.

The soluble fraction of FDH-R3-LeuDH and the active inclusion body partially catalyze the catalytic ability of TMA to form L-Tle. The results are shown in Fig. 6. It can be seen that the conversion of the soluble fraction under the same conditions is only 14.6%, and the activity includes The conversion rate of the bulk fraction was 93.5%, which was about 6.4 times that of the soluble fraction, and the L-Tle ee value obtained by the active inclusion body catalysis was more than 99%. The results are shown in Fig. 4, wherein Fig. 7A is the standard L-Tle and D. The HPLC spectrum of -Tle, Figure 7B is the spectrum of the catalytic product of FDH-R3-LeuDH active inclusion body. The flow rate is reduced by 0.8 mL/min due to the increase of column pressure, and the asterisk indicates the theoretical D-Tle peak time. The above results indicate that the vast majority of fusion enzymes are expressed as active inclusion bodies and have a higher utilization potential in the biocatalytic conversion of L-Tle.

Example 5

Effect of Inducer Concentration on Catalytic Ability of Bifunctional Enzyme Activity Inclusion Body

FDH-R3-LeuDH was induced to culture using different IPTG concentrations, and then the distribution of recombinant protein was analyzed by SDS-PAGE, as shown in Figure 8, where M represents the protein standard, 1 is the supernatant (0 mM); (0 mM); 3 is the supernatant (0.01 mM); 4 is the inclusion body (0.01 mM); 5 is the supernatant (0.2 mM); 6 is the inclusion body (0.2 mM); 7 is the supernatant (1 mM); It is an inclusion body (1 mM). It can be seen that the content of recombinant protein in the inclusion body increases with the increase of IPTG concentration, while the soluble part of the recombinant protein shows an opposite trend. The FDH-R3-LeuDH activity induced by 50 mg of different IPTG concentrations was suspended in 10 mL of the reaction mixture, and placed at 30 ° C, and reacted at 200 rpm for 16 h. The reaction mixture contained 50 mM trimethylpyruvate, 50 mM ammonium formate, 0.01 mM NAD ⁺ , adjusted to pH 8.5 with aqueous ammonia, and the solvent was H ₂ O. Catalytic activity of the inclusion bodies As shown in Figure 9, it can be seen that the activity of inclusion bodies increased with the increase of IPTG concentration at the beginning, but when the concentration of IPTG increased to 1 mM, the catalytic efficiency decreased, at 0.2 mM. At the concentration of IPTG, the recombinant protein was expressed in the inclusion body at a high level and maintained a good active conformation.

Example 6

Thermal stability of bifunctional enzyme activity inclusion bodies

The FDH-R3-LeuDH soluble fraction and the active inclusion body were placed in a water bath at 20-50 ° C for 1 h, respectively, and the residual enzyme activity was measured. The results are shown in Figure 10. The FDH partial enzyme activity was maintained at 70-112% after the water bath, LeuDH Part of the enzyme activity was maintained at 87-108%, while the FDH activity of the soluble fraction was maintained at 47-94%, and the LeuDH enzyme activity was maintained at 74-94%. It can be seen that the thermal stability of the active inclusion bodies FDH and LeuDH were better than The fusion enzyme, the increase in thermal stability may be due to the aggregation of the fusion enzyme in the cells to form aggregates to prevent the subunits of the enzyme from dissociating at high temperatures. The enzyme activity of the active fusion enzyme inclusions increased slightly after incubation for 1 h at low temperature. The reason for this phenomenon may be that the misfolded recombinant protein in the inclusion body refolds under heat shock to form an active conformation.

Example 7

Repeated catalysis of bifunctional enzyme activity inclusion bodies

100 mg (wet weight) of the active inclusion body was added with 10 mL of the reaction mixture (containing 50 mM trimethylpyruvate, 50 mM ammonium formate, 0.04 mM NAD ⁺ , pH=8.5), suspended, and reacted at 30 ° C, 200 rpm for 24 h, and the reaction was completed. After taking 200 μL of the sample, it was stored at -80 ° C for testing. The active inclusion bodies were centrifuged at 5000 x g for 10 min at 4 ° C, and the precipitate was washed twice with ddH ₂ O to remove the reaction residue, followed by the addition of 10 mL of the reaction mixture, suspended, and the next reaction was started, and the batch catalysis was repeated for six rounds. The yield of the first batch of catalysis is recorded as 100%, and the relative yield of the latter batch is calculated. The results are shown in Fig. 11. It can be seen that the yield of L-Tle decreases with the increase of the number of times of recovery, without additional immobilization. In the case of medium and other modifications, after 2, 4, and 6 consecutive recovery catalysis, the yields were maintained at 86.0%, 72.0%, and 54.3%, respectively, of the first catalytic yield. The experimental results show that the FDH-R3-LeuDH active inclusion body has good reusability, and the multi-enzyme active inclusion body can simultaneously realize the construction of the multi-enzyme catalytic system and the immobilization of the enzyme.

Example 8

Batch catalysis of bifunctional enzyme activity inclusion bodies

10 g (wet weight) of the active inclusion body was added with 200 mL of the reaction mixture (containing 710 mM trimethylpyruvate, 780 mM ammonium formate, 0.5 mM NAD ⁺ , pH=9), suspended, and reacted at 30 ° C, 200 rpm for 16 h, every The sample was stored at -20 °C for a period of time. The conversion rate was 31.8% after 2 hours, the conversion rate was 48.7% after 4 hours, and the maximum conversion rate was 87.4% at 16h. The experimental results showed that the FDH-R3-LDH activity inclusion body energy was compared. Good catalysis, with good application potential.

The above is only the preferred embodiment of the present application, and thus the scope of the application is not limited thereto, that is, equivalent changes and modifications made in accordance with the scope of the patent application and the contents of the specification should still be covered by the present application. In the range.

Claims

A method for preparing optically pure L-tert-leucine using active inclusion bodies, comprising the steps of:

(1) preparing a bifunctional enzyme activity inclusion body by genetically engineered bacteria containing a bifunctional enzyme expression vector, wherein the active ingredient of the bifunctional enzyme activity inclusion body is a fusion bifunctional enzyme comprising a linked peptide a LeuDH portion and a polymerase portion for coenzyme NAD + regeneration, wherein the LeuDH portion comprises an amino acid sequence as shown in SEQ ID NO: 01, wherein the linker peptide is a rigid linker peptide or a flexible linker peptide, and the rigid linker peptide is capable of Forming an alpha helix to effectively isolate the LeuDH moiety and the polymerase moiety described above, the flexible linker peptide having no ability to form a particular secondary structure, typically in the form of a random coil to provide the flexibility required for the protein in the catalytic process;

(2) The bifunctional enzyme activity inclusion body is resuspended in a reaction mixture having a pH of 6.0 to 10.0, and then reacted at 20 to 40 ° C, and the pH is controlled to be 6.0 to 10.0 during the reaction; the reaction mixture contains 50 to 1000 mM. Methylpyruvate, 50-1000 mM ammonium formate and 0.05-5 mM coenzyme NAD + .
The method of claim 1, wherein the rigid linker peptide comprises a plurality of amino acid sequences as shown in SEQ ID NO 02, which are sequentially linked.
The method of claim 1, wherein the flexible linker peptide comprises a plurality of amino acid sequences as shown in SEQ ID NO: 03 in sequence.
The method according to claim 1, wherein the polymerase moiety is an FDH moiety, a glucose dehydrogenase moiety, a glycerol dehydrogenase moiety, an alcohol dehydrogenase moiety, a glucose-6-phosphate dehydrogenase moiety, and a dehydrogenation of lactate. Enzyme or hydrogenase part.
The method according to claim 4, wherein the polymerase moiety is an FDH moiety, and the FDH moiety comprises the amino acid sequence set forth in SEQ ID NO: 04.
The method according to claim 1, wherein the pH of the reaction mixture in the step (2) is 8.5 to 9, the temperature is 30 ° C, and the pH is controlled to be 8.5 to 9 during the reaction.
The method according to claim 1, wherein the reaction mixture in the step (2) comprises 50 to 710 mM of trimethylpyruvate, 50 to 780 mM of ammonium formate, and 0.05 to 0.5 mM of coenzyme NAD + .