Lysine decarboxylase mutant and application thereof in synthesis of 1, 5-pentamethylene diamine

Abstract

The invention discloses a lysine decarboxylase mutant, a coding gene of the lysine decarboxylase mutant, a recombinant vector constructed by the coding gene, recombinant genetically engineered bacteria obtained by transforming the recombinant vector, and an immobilized enzyme prepared from the lysine decarboxylase mutant. The invention also discloses an application of the lysine decarboxylase mutant and the immobilized enzyme prepared from the lysine decarboxylase mutant in synthesis of 1, 5-pentamethylene diamine. The lysine decarboxylase mutant AfLDCT88S, which is high in catalytic activity, wider in pH tolerance range and good in thermal stability, is screened out by performing site-directed mutagenesis on lysine decarboxylase from different sources, and is successfully used for catalyzing L-lysine to synthesize 1, 5-pentamethylene diamine, so that the lysine decarboxylase mutant AfLDCT88S shows a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of enzymatic catalytic synthesis technology, specifically to a lysine decarboxylase mutant and its application in the synthesis of 1,5-pentanediamine. Background Technology

[0002] 1,5-Pentanediamine, also known as cadaverine, can be used as a precursor for polymer synthesis to replace traditional petroleum-based hexamethylenediamine. It can be used to prepare novel bio-based polyamides by polycondensation with various diacids. The novel bio-based polyamide materials have excellent chemical corrosion resistance, heat resistance and mechanical strength, and show great application potential in industrial fields such as packaging, medical devices and home appliances.

[0003] Currently, the biosynthetic pathways of 1,5-pentanediamine are mainly divided into two categories: microbial fermentation and enzymatic catalysis. While microbial fermentation can achieve de novo synthesis, the product 1,5-pentanediamine is toxic to host cells. Enzymatic catalysis, on the other hand, constructs an in vitro reaction system to efficiently convert the substrate L-lysine to 1,5-pentanediamine under specific catalytic conditions. This method is not only simple and easy to control, but also avoids the toxicity of the product to cells.

[0004] Lysine decarboxylase (LDC) is a specific enzyme that catalyzes the decarboxylation of L-lysine to 1,5-pentanediamine. Current research on this enzyme largely focuses on sources such as *Escherichia coli*, *Klebsiella pneumoniae*, and *Haffniella vesicae*. However, these lysine decarboxylases generally suffer from low catalytic activity and poor structural stability in practical applications. Therefore, it is necessary to further explore novel lysine decarboxylase resources from different bacterial strains and combine them with enzyme engineering techniques to improve their catalytic activity and structural stability, thereby promoting their industrial application. Summary of the Invention

[0005] The purpose of this invention is to provide a lysine decarboxylase mutant and its application in the synthesis of 1,5-pentanediamine, in order to overcome the shortcomings of the prior art.

[0006] The present invention adopts the following technical solution: The first aspect of the present invention provides a lysine decarboxylase mutant, which is a mutant of lysine decarboxylase AfLDC. The amino acid sequence of lysine decarboxylase AfLDC is shown in SEQ ID NO: 5. The lysine decarboxylase mutant is obtained by mutating threonine at position 88 of lysine decarboxylase AfLDC to serine, i.e., T88S.

[0007] A second aspect of the present invention provides the encoding gene for the above-mentioned lysine decarboxylase mutant.

[0008] A third aspect of the present invention provides a recombinant vector for constructing the encoding gene of the above-mentioned lysine decarboxylase mutant.

[0009] The fourth aspect of the present invention provides recombinant genetically engineered bacteria obtained by transformation of the above-mentioned recombinant vector.

[0010] The fifth aspect of the present invention provides an immobilized enzyme prepared from the above-mentioned lysine decarboxylase mutant.

[0011] Further, the immobilized enzyme is prepared by the following steps: using 50-200mM phosphate buffer, pH 6.0-8.0 as a solvent, the lysine decarboxylase mutant and amino resin are added, the mass ratio of the lysine decarboxylase mutant to the amino resin is 20-200mg:1g, the amino resin includes LX-1000NH amino resin or ESR-1 amino resin, and 1v / v%-5v / v% glutaraldehyde and 1v / v%-5v / v% polyethyleneimine are added. The immobilization temperature is 4-10℃, the stirring speed is 20-100rpm, the immobilization time is 2-5h, and the enzyme is filtered and washed to obtain the immobilized enzyme.

[0012] The sixth aspect of the present invention provides the application of the above-mentioned lysine decarboxylase mutant in the synthesis of 1,5-pentanediamine. In the application, the lysine decarboxylase mutant is used as a catalyst, L-lysine is used as a substrate, pyridoxal 5-phosphate is used as a coenzyme, and the enzyme catalysis to generate 1,5-pentanediamine is carried out in a reaction medium under controlled temperature and stirring speed conditions.

[0013] Furthermore, the reaction medium is an aqueous solution with pH 5.0-9.0, the amount of lysine decarboxylase mutant is 20-200 mg / L, the concentration of L-lysine is 0.5-2.0 M, the concentration of pyridoxal 5-phosphate is 0.05-0.5 mM, the temperature is controlled at 40-60℃, the stirring speed is 70-300 rpm, and the reaction time is 1-4 h.

[0014] The seventh aspect of the present invention provides the application of the above-mentioned immobilized enzyme in the synthesis of 1,5-pentanediamine. In the application, the immobilized enzyme is used as a catalyst, L-lysine is used as a substrate, pyridoxal 5-phosphate is used as a coenzyme, and the enzyme catalysis to generate 1,5-pentanediamine is carried out in a reaction medium under controlled temperature and stirring speed conditions.

[0015] Furthermore, the reaction medium is an aqueous solution with pH 5.0-9.0, the amount of immobilized enzyme is 5-20 g / L, the concentration of L-lysine is 0.5-2.0 M, the concentration of pyridoxal 5-phosphate is 0.05-0.5 mM, the temperature is controlled at 40-60℃, the stirring speed is 70-300 rpm, and the reaction time is 1-4 h.

[0016] The beneficial effects of this invention are: This invention screened out a lysine decarboxylase mutant, AfLDC_T88S, by site-directed mutagenesis of lysine decarboxylases from different sources. The mutant exhibited high catalytic activity (enzyme activity reaching 2513.7 U / mg, approximately 3 times higher than that of wild-type lysine decarboxylase AfLDC), a wider pH tolerance range (relative enzyme activity maintained above 75% in pH 5.0-9.0), and good thermal stability (relative enzyme activity maintained at 80% after treatment at 60℃ for 150 h). This mutant was successfully used to catalyze the synthesis of 1,5-pentanediamine from L-lysine, demonstrating promising prospects for industrial application.

[0017] This invention utilizes the immobilized lysine decarboxylase mutant AfLDC_T88S as a catalyst and L-lysine as a substrate to synthesize 1,5-pentanediamine via enzymatic catalysis under appropriate conditions and reaction medium, achieving a conversion rate of 99.4%. The immobilized lysine decarboxylase mutant AfLDC_T88S retains 77% of its relative enzyme activity even after 50 cycles, enabling enzyme reuse and reducing industrial production costs. Attached Figure Description

[0018] Figure 1 Results of pH stability determination for lysine decarboxylase VpLDC and its mutants.

[0019] Figure 2 Results of pH stability determination for lysine decarboxylase RtLDC and its mutants.

[0020] Figure 3 Results of pH stability determination for lysine decarboxylase AfLDC and its mutants.

[0021] Figure 4 Results of thermostability determination of lysine decarboxylase VpLDC and its mutants.

[0022] Figure 5 Results of thermostability determination of lysine decarboxylase RtLDC and its mutants.

[0023] Figure 6 Results of thermostability determination of lysine decarboxylase AfLDC and its mutants.

[0024] Figure 7 The results of 50 relative enzyme activity assays were performed on each immobilized enzyme. Detailed Implementation

[0025] The present invention will be further explained below with reference to embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0026] Unless otherwise specified, the following examples are typically conducted under standard testing conditions or as recommended by the reagent company. Unless otherwise stated, all materials and reagents used are commercially available.

[0027] The culture medium formulations involved in the following examples are as follows: SOC medium (1L): 20g tryptone, 5g yeast extract, 0.5g sodium chloride, 10mL 250mmol / L potassium chloride, 10mL 1M magnesium chloride, 10mL 1M magnesium sulfate, 5mL 1M D-glucose (added separately before use after sterilization by filtration through a 0.22μm filter membrane); pH 7.2.

[0028] LB liquid medium (1L): 10g tryptone, 5g yeast extract, 10g sodium chloride; pH 7.0.

[0029] LB solid medium is made by adding 20g of agar powder to 1L of LB liquid medium.

[0030] All of the above culture media need to be autoclaved at 121°C for 20 minutes.

[0031] Example 1: Construction of recombinant Escherichia coli with lysine decarboxylase I. Construction of Recombinant Plasmids Vibrio paracholerae ( Vibrio paracholerae The gene fragment encoding the wild-type lysine decarboxylase VpLDC in the genome (gene sequence shown in SEQ ID NO.2, amino acid sequence shown in SEQ ID NO.1), *Laurella terreus* ( Raoultella terrigena The gene fragment encoding the wild-type lysine decarboxylase RtLDC in the genome of *Streptococcus finisteri* (gene sequence shown in SEQ ID NO.4, amino acid sequence shown in SEQ ID NO.3) is from *Streptococcus finisteri*. Aliivibrio finisterrensis The gene fragment encoding the wild-type lysine decarboxylase AfLDC in the genome (gene sequence as shown in SEQ ID NO. 6, amino acid sequence as shown in SEQ ID NO. 5) was synthesized by Sangon Biotech (Shanghai) Co., Ltd. after codon optimization of E. coli. The optimized gene fragment was then constructed into the vector pET-28a(+) to obtain the recombinant plasmid.

[0032] II. Transformation of Escherichia coli with recombinant plasmids E. coli BL21(DE3) The recombinant plasmid obtained in step one of Example 1 was transformed into the strain using a heat shock method. E. coli In BL21(DE3). The specific steps are as follows: 100 μL in each tube E. coliAdd 10 μL of 1 ng / μL recombinant plasmid to a suspension of BL21(DE3) competent cells (OD approx. 0.4-0.6), mix gently, and incubate on ice for 30 min. Transfer to a 42°C water bath and heat shock for 90 s. Quickly transfer to an ice bath and cool for 3 min. Add 700 μL of antibiotic-free SOC liquid medium to each tube and incubate at 37°C and 100 rpm on a shaker for 40 min. After incubation, centrifuge the bacterial culture at 4°C and 12000 rpm for 10 min, discard 600 μL of supernatant, and spread the remaining bacterial culture onto LB agar plates containing 50 μg / mL kanamycin sulfate. Incubate overnight at 37°C with the plates inverted.

[0033] Selection of positive clones: Four clones were selected and transferred to 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate. The cultures were incubated at 37°C and 200 rpm for 8 h. Plasmids were extracted using the Mini-Plasmid Rapid Isolation Kit (Beijing Bodatech Biotechnology Co., Ltd.). 20 μL of plasmid was taken and sent to Sangon Biotech (Shanghai) Co., Ltd. for DNA sequencing to confirm successful construction.

[0034] Example 2 Construction of recombinant Escherichia coli with lysine decarboxylase mutant I. Construction of Recombinant Plasmids The sequences of each wild-type lysine decarboxylase in step one of Example 1 were analyzed, and multiple single-point or multi-point mutants were designed using bioinformatics methods, as shown in Table 1.

[0035] Table 1

[0036] VpLDC_R69D is a mutation of arginine at position 69 of the lysine decarboxylase VpLDC into aspartic acid.

[0037] VpLDC_K116C / E141C is a lysine decarboxylase VpLDC where lysine at position 116 is mutated to cysteine ​​and glutamate at position 141 is mutated to cysteine.

[0038] RtLDC_V258T is a lysine decarboxylase RtLDC in which valine is mutated to threonine at position 258.

[0039] RtLDC_R116C / Q141C is a lysine decarboxylase RtLDC where arginine at position 116 is mutated to cysteine ​​and glutamine at position 141 is mutated to cysteine.

[0040] AfLDC_T88S is a mutation where the threonine at position 88 of the lysine decarboxylase AfLDC is replaced with serine.

[0041] AfLDC_T88Y is a mutation where the threonine at position 88 of the lysine decarboxylase AfLDC is changed to tyrosine.

[0042] AfLDC_A175T is a mutation where the alanine at position 175 of the lysine decarboxylase AfLDC is replaced with threonine.

[0043] All the above lysine decarboxylase mutants were obtained by optimizing the E. coli codons using Sangon Biotech (Shanghai) Co., Ltd., synthesizing the optimized gene fragments, and constructing them into the vector pET-28a(+) to obtain recombinant plasmids.

[0044] II. Transformation of recombinant plasmid into Escherichia coli BL21(DE3) The recombinant plasmid obtained in step one of Example 2 was transformed into the strain using a heat shock method. E. coli In BL21(DE3). The specific steps are as follows: 100 μL in each tube E. coli Add 10 μL of 1 ng / μL recombinant plasmid to a suspension of BL21(DE3) competent cells (OD approx. 0.4-0.6), mix gently, and incubate on ice for 30 min. Transfer to a 42°C water bath and heat shock for 90 s. Quickly transfer to an ice bath and cool for 3 min. Add 700 μL of antibiotic-free SOC liquid medium to each tube and incubate at 37°C and 100 rpm on a shaker for 40 min. After incubation, centrifuge the bacterial culture at 4°C and 12000 rpm for 10 min, discard 600 μL of supernatant, and spread the remaining bacterial culture onto LB agar plates containing 50 μg / mL kanamycin sulfate. Incubate overnight at 37°C with the plates inverted.

[0045] Selection of positive clones: Four clones were selected and transferred to 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate. The cultures were incubated at 37°C and 200 rpm for 8 h. Plasmids were extracted using the Mini-Plasmid Rapid Isolation Kit (Beijing Bodatech Biotechnology Co., Ltd.). 20 μL of plasmid was taken and sent to Sangon Biotech (Shanghai) Co., Ltd. for DNA sequencing to confirm successful construction.

[0046] Example 3: Induced expression culture of recombinant Escherichia coli The recombinant *E. coli* strains constructed in Examples 1 and 2, as well as the recombinant *E. coli* strain from the control group (pET-28a(+) empty vector transformant, as a negative control), were inoculated into 10 mL of LB broth containing 50 μg / mL kanamycin sulfate and cultured overnight at 37°C with shaking at 200 rpm. 10 mL of the culture was then transferred to 1 L of LB broth containing 50 μg / mL kanamycin sulfate. -1 In LB broth containing kanamycin sulfate, the culture was carried out at 37°C with shaking at 200 rpm until OD reached.600 The concentration was approximately 0.6-0.8. Isopropyl-β-D-thiogalactoside (IPTG) was added to the culture to a final concentration of 0.5 mM, and the culture was induced at 28°C and 200 rpm for 12 h. The culture medium was centrifuged at 4°C and 12000 rpm for 10 min, the supernatant was discarded, and the precipitate (bacterial cells) was collected. The bacterial cells were dispersed in 14 mL of 50 mM phosphate buffer (pH 7.2) and sonicated in an ice bath. The sonication parameters were as follows: the ultrasonic power of the ultrasonic cell disruptor (purchased from Ningbo Xinzhi Biotechnology Co., Ltd., model JY92-IIN) was set to 10%, and the sonication time was 30 min (2 s working time, 3 s interval). A portion of the sonicated bacterial cells (i.e., whole cell lysate) was directly used for SDS-PAGE analysis, and the other portion was centrifuged at 4°C and 12000 rpm for 10 min. The supernatant and precipitate were then used for SDS-PAGE analysis separately. SDS-PAGE results showed that the target protein was expressed at a high soluble level and had the correct molecular weight.

[0047] Example 4 Purification of recombinant protein The recombinant *E. coli* strains constructed in Examples 1 and 2 were inoculated into 100 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate and cultured overnight at 37°C with shaking at 200 rpm. 100 mL of the culture was then transferred to 10 L of LB liquid medium containing 50 μg / mL kanamycin sulfate and cultured at 37°C with shaking at 200 rpm until OD reached [value missing]. 600 Approximately 0.6–0.8. Add IPTG to the culture to a final concentration of 0.5 mM and induce culture at 28°C and 200 rpm for 12 h. Centrifuge the culture medium at 4°C and 12000 rpm for 10 min, discard the supernatant, collect the precipitate (bacterial cells), and wash the bacterial cells three times with physiological saline to obtain wet bacterial cells.

[0048] Weigh 40g of wet bacterial cells and suspend them in 140mL of 50mM phosphate buffer (pH 7.2). Sonicate the cells in an ice bath. The sonication parameters were: ultrasonic power of the ultrasonic cell disruptor (Ningbo Xinzhi Biotechnology Co., Ltd., model JY92-IIN) set to 10%, and sonication time to 30min (2s working time, 3s interval). Centrifuge the disrupted cells at 4℃ and 12000rpm for 10min, and collect the supernatant as the crude enzyme solution. Purify the crude enzyme solution using a His-Trap HPaffinity column. After ultrafiltration and desalting, obtain pure enzyme solution. Concentrate the pure enzyme solution to 5mg / mL using a 30KD ultrafiltration tube at 4℃ and 6000rpm for 30min. Store the obtained lysine decarboxylase solutions (5mg / mL) and lysine decarboxylase mutant solutions (5mg / mL) at 4℃ for later use.

[0049] Example 5: HPLC determination of 1,5-pentanediamine content Sample preparation method: Dilute the sample with ultrapure water to a 1,5-pentanediamine content of 1-20 mg / mL, filter it through a 0.22 μm aqueous filter membrane, and then inject the sample for detection.

[0050] HPLC detection method: An Agilent 1260 Infinity HPLC system was used with an evaporative light scattering detector (ELSD). The chromatographic column was a Phenomenex Titank-plus C18 column (250 × 4.6 mm, 5 μm). The mobile phase was 0.4% v / v heptafluorobutyric acid aqueous solution (v: acetonitrile, v = 80: 20). The column temperature was 35℃, the flow rate was 0.8 mL / min, and the detector parameters were: evaporator temperature 80℃, nebulizer temperature 70℃, carrier gas flow rate 1.0 SLM, and injection volume 5 μL.

[0051] Example 6 Enzyme activity assay of lysine decarboxylase and its mutants Enzyme activity assay conditions: Total reaction volume 10 mL, solvent was pure water, including 1 M L-lysine, 0.1 mM pyridoxal 5-phosphate, 40 μL of 5 mg / mL lysine decarboxylase solution or lysine decarboxylase mutant solution obtained in Example 4, pH controlled at 7.2 (adjusted with 1 M hydrochloric acid), temperature 50 °C, stirring speed 70 rpm, reaction time 10 min (the reaction time of 10 min is for determining enzyme activity; for higher conversion rates, the reaction time can be 1-4 h). At the end of the reaction, 3 mL of 37 wt% concentrated hydrochloric acid was added to quench the reaction. The amount of 1,5-pentanediamine generated was detected by HPLC according to the method described in Example 5. Enzyme activity was defined as: 1 U is the amount of enzyme required to generate 1 μmol of 1,5-pentanediamine per minute.

[0052] The enzyme activity test results of lysine decarboxylase and its mutants are shown in Table 2. Under the condition of pH 7.2, the lysine decarboxylase mutants constructed in Example 2 have higher enzyme activities than wild-type lysine decarboxylase. Among them, the enzyme activity of lysine decarboxylase mutant AfLDC_T88S reached 2513.7 U / mg, which is about 3 times higher than the enzyme activity of wild-type lysine decarboxylase AfLDC.

[0053] Table 2

[0054] Example 7 pH stability determination of lysine decarboxylase and its mutants Take 8 mL of each of the 5 mg / mL lysine decarboxylase enzyme solution or the lysine decarboxylase mutant enzyme solution obtained in Example 4, and adjust the pH to 5.0, 6.0, 7.0, 8.0, and 9.0 respectively using 1M hydrochloric acid or 1M sodium hydroxide. After standing at 4°C for 10 h, measure the enzyme activity under the same reaction conditions according to the enzyme activity determination method in Example 6, and calculate the relative enzyme activity of lysine decarboxylase and its mutant after different pH treatments (with the enzyme activity of each enzyme and each mutant measured in Example 6 at pH 7.2 as 100%).

[0055] The results are as follows Figures 1-3 As shown, the pH stability of the various lysine decarboxylase mutants was significantly improved compared to the wild-type lysine decarboxylase. Among them, the lysine decarboxylase mutants AfLDC_T88S and RtLDC_R116C / Q141C maintained a relative enzyme activity of over 75% in the pH range of 5.0-9.0.

[0056] Example 8: Determination of the thermal stability of lysine decarboxylase and its mutants Two mL of either the 5 mg / mL lysine decarboxylase enzyme solution or the lysine decarboxylase mutant enzyme solution obtained in Example 4 were placed in a 60°C constant temperature water bath and incubated for 25 h, 50 h, 75 h, 100 h, 125 h, and 150 h, respectively. After incubation, the heat-treated samples were immediately transferred to an ice-water mixture to terminate the heat effect. Following the enzyme activity assay method in Example 6, enzyme activity was measured under the same reaction conditions, and the relative enzyme activities of lysine decarboxylase and its mutants after heat treatment at different times were calculated (with the enzyme activity of each enzyme and mutant at 0 h as 100%).

[0057] The results are as follows Figures 4-6 As shown, the thermostability of the various lysine decarboxylase mutants was significantly improved compared to the wild-type lysine decarboxylase. After treatment at 60℃ for 150 h, the lysine decarboxylase mutants VpLDC_K116C / E141C, RtLDC_R116C / Q141C, and AfLDC_T88S still maintained more than 70% of their relative enzyme activity. Among them, the relative enzyme activity of AfLDC_T88S remained at 80%.

[0058] Example 9 Determination of enzyme kinetic parameters of lysine decarboxylase and its mutants The enzyme kinetic parameters of wild-type lysine decarboxylases VpLDC, RtLDC, AfLDC, and the more stable mutants VpLDC_K116C / E141C, RtLDC_R116C / Q141C, and AfLDC_T88S were determined under the following conditions: the substrate lysine concentration in the reaction system was set to 0.25-50 mM (gradients of 0.25, 0.5, 1, 2, 4, 6, 8, 10, 15, 25, and 50 mM), and the other conditions were the same as in Example 6; the enzyme kinetic parameters were calculated by fitting the Michaelis-Menten equation by monitoring the reaction rate at different substrate concentrations.

[0059] The results are shown in Table 3. Significant differences in the catalytic efficiency of wild-type lysine decarboxylases from different sources were observed; the catalytic efficiency of the obtained lysine decarboxylase mutants was improved. Among them, the lysine decarboxylase mutant AfLDC_T88S exhibited excellent catalytic efficiency and has significant application value.

[0060] Table 3

[0061] Example 10 Preparation of immobilized enzyme and its application in the synthesis of 1,5-pentanediamine Preparation of immobilized enzyme: The total volume of the reaction system was 20 mL, and the solvent was 50 mM phosphate buffer (pH 7.2), including 12 mL of VpLDC enzyme solution (5 mg / mL), VpLDC_K116C / E141C enzyme solution (5 mg / mL), RtLDC enzyme solution (5 mg / mL), RtLDC_R116C / Q141C enzyme solution (5 mg / mL), AfLDC enzyme solution (5 mg / mL) or AfLDC_T88S enzyme solution (5 mg / mL) obtained in Example 4, 1 g LX-1000NH amino resin (Xi'an Lanxiao Technology New Material Co., Ltd., the amino resin is not included in the total volume of the system), 0.5 mL glutaraldehyde, and 0.2 mL polyethyleneimine. The mixture was stirred at 10 °C and 50 rpm for 5 h, and then filtered to obtain a solid. The solid was washed with 100 mL of deionized water and filtered again to obtain the immobilized enzyme.

[0062] Immobilized enzyme-catalyzed reaction: The total volume of the reaction system was 10 mL, the solvent was pure water, and it included 1 M L-lysine, 0.1 mM pyridoxal 5-phosphate, and 10 g / L immobilized enzyme (the immobilized enzyme is not included in the total volume of the reaction system). The pH was controlled at 7.2 (adjusted with 1 M hydrochloric acid), the temperature was 50 °C, the stirring speed was 70 rpm, and the reaction time was 3 h. The immobilized enzyme in the reaction solution was filtered off, and then 3 mL of 37 wt% concentrated hydrochloric acid was added to quench the reaction. The amount of 1,5-pentanediamine generated in the reaction was detected by HPLC according to the method described in Example 5. The conversion rate was calculated using the following formula: Conversion rate (%) = (mass concentration of 1,5-pentanediamine / molar mass of 1,5-pentanediamine / molar concentration of lysine in feed) × 100% In the conversion formula, the mass concentration of 1,5-pentanediamine was obtained by HPLC detection according to the method described in Example 5.

[0063] The results are shown in Table 4. AfLDC_T88S had the highest conversion rate, reaching 99.4%.

[0064] Table 4

[0065] The immobilized enzyme was washed with 10 mL of deionized water and filtered, then reused in the reaction cycle for a total of 50 cycles. Enzyme activity was defined as the amount of enzyme required to generate 1 μmol of 1,5-pentanediamine per minute (1 U). The relative enzyme activity of each immobilized enzyme was calculated at different cycles (with the enzyme activity measured at 0 cycles being 100%).

[0066] The results are as follows Figure 7 As shown, the three immobilized enzymes, VpLDC_K116C / E141C, RtLDC_R116C / Q141C, and AfLDC_T88S, maintained a relative enzyme activity of >60% after 50 consecutive applications. Among them, the immobilized enzyme AfLDC_T88S had the highest relative enzyme activity, retaining 77% of its relative enzyme activity.

Claims

1. A lysine decarboxylase mutant, characterized in that, The lysine decarboxylase mutant is a mutant of lysine decarboxylase AfLDC. The amino acid sequence of lysine decarboxylase AfLDC is shown in SEQ ID NO:

5. The lysine decarboxylase mutant is obtained by mutating threonine at position 88 of lysine decarboxylase AfLDC to serine, i.e., T88S.

2. The encoding gene of the lysine decarboxylase mutant according to claim 1.

3. A recombinant vector constructed from the encoding gene of the lysine decarboxylase mutant of claim 2.

4. The recombinant genetically engineered bacteria obtained by transformation of the recombinant vector according to claim 3.

5. The immobilized enzyme prepared from the lysine decarboxylase mutant of claim 1.

6. The immobilized enzyme according to claim 5, characterized in that, The immobilized enzyme is prepared by the following steps: using 50-200mM phosphate buffer (pH 6.0-8.0) as a solvent, the lysine decarboxylase mutant and amino resin are added, with a mass ratio of 20-200mg:1g. The amino resin includes LX-1000NH amino resin or ESR-1 amino resin. 1v / v%-5v / v% glutaraldehyde and 1v / v%-5v / v% polyethyleneimine are added. The immobilization temperature is 4-10℃, the stirring speed is 20-100rpm, and the immobilization time is 2-5h. The enzyme is then filtered and washed to obtain the immobilized enzyme.

7. The application of the lysine decarboxylase mutant of claim 1 in the synthesis of 1,5-pentanediamine, characterized in that, In application, the lysine decarboxylase mutant is used as a catalyst, L-lysine is used as a substrate, and pyridoxal 5-phosphate is used as a coenzyme. Under controlled temperature and stirring speed conditions, the enzyme catalyzes the production of 1,5-pentanediamine in the reaction medium.

8. The application according to claim 7, characterized in that, The reaction medium is an aqueous solution with pH 5.0-9.

0. The amount of lysine decarboxylase mutant is 20-200 mg / L, the concentration of L-lysine is 0.5-2.0 M, the concentration of pyridoxal 5-phosphate is 0.05-0.5 mM, the temperature is controlled at 40-60℃, the stirring speed is 70-300 rpm, and the reaction time is 1-4 h.

9. The use of the immobilized enzyme according to claim 5 or 6 in the synthesis of 1,5-pentanediamine, characterized in that, In application, the immobilized enzyme is used as a catalyst, L-lysine as a substrate, and pyridoxal 5-phosphate as a coenzyme. Under controlled temperature and stirring speed conditions, enzyme catalysis is carried out in the reaction medium to generate 1,5-pentanediamine.

10. The application according to claim 9, characterized in that, The reaction medium is an aqueous solution with pH 5.0-9.0, the amount of immobilized enzyme is 5-20 g / L, the concentration of L-lysine is 0.5-2.0 M, the concentration of pyridoxal 5-phosphate is 0.05-0.5 mM, the temperature is controlled at 40-60℃, the stirring speed is 70-300 rpm, and the reaction time is 1-4 h.

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