Application of high-activity nitrile hydratase mutant in preparation of nicotinamide
Through the design and genetic engineering expression of nitrile hydratase mutants and combined with immobilization technology, the problem of inactivation of nitrile hydratase catalyst in hydration reaction is solved, efficient and stable production of niacinamide is achieved, industrialization process is simplified and reduced cost.
Patent Information
- Application Number
- CN202411260100.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-10
- Publication Date
- 2024-12-13
AI Technical Summary
The nitrile hydratase catalyst is inactivated due to sudden temperature rise in hydration reaction, and the high product concentration is toxic to cells, resulting in a decrease in economic benefits. It is difficult for the prior art to build a catalyst with high stability and high product tolerance.
A nitrile hydratase mutant was designed, and a highly efficient nitrile hydratase mutant protein was constructed by replacing valine at the amino acid position 53, and expressed in E. coli through genetic engineering, combining Ni The IDA resin is immobilized to form an immobilized enzyme, which is used to catalyze 3-cyanopyridine to form nicotinamide.
It significantly improves the catalytic vitality and temperature stability of nitrile hydratase, simplifies the industrial production process, reduces costs, and achieves efficient and low-pollution production of niacinamide.
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Figure CN119120436A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of bioengineering and enzyme engineering, in particular to the application of a highly active nitrile hydratase mutant in the preparation of nicotinamide. Background Art
[0002] Nitrile hydratase (EC 4.2.1.84, nitrile hydratase, abbreviated as NHase) is a metalloenzyme that can hydrate nitrile substances into more valuable amide compounds. The enzyme from Rhodococcus rhodochrous J1 (Arthrobacter) is the first nitrile hydratase discovered and reported. At present, nitrile hydratase is gradually replacing traditional chemical methods with its advantages of being green and environmentally friendly, mild reaction conditions, and high safety factor, making the production of amides in line with the concept of sustainable development and green production.
[0003] Nicotinamide, commonly known as vitamin B3, has a molecular formula of C6H6N2O and a molecular weight of 22. It is widely found in nature and belongs to the B vitamins. As a coenzyme, nicotinamide participates in a variety of redox reactions in the body and directly participates in the metabolism of carbohydrates, proteins and fats. As a drug, nicotinamide can treat skin diseases, neurological diseases, diabetes, liver diseases and solar dermatitis. In the field of additives, it can be added to food as a supplement of essential vitamins for the human body. It can also be used in animal husbandry as an animal feed additive. In addition, nicotinamide is also widely used in medicine and can be used as a pharmaceutical intermediate to treat various diseases.
[0004] The synthesis method of nicotinamide has always attracted attention. Its production process mainly includes chemical synthesis and biological synthesis. The chemical synthesis method of nicotinamide uses pyridine as raw material, first obtains 3-cyanopyridine through oxidation reaction, and then 3-cyanopyridine is subjected to alkaline hydrolysis reaction under certain conditions to obtain nicotinamide. The traditional chemical synthesis method has disadvantages such as low process yield and purity, harsh reaction conditions, and certain pollution to the environment. The biological method is to use the known nitrile hydratase to catalyze 3-cyanopyridine to produce nicotinamide. With the development of DNA recombinant technology and the development and maturity of the current Escherichia coli expression system, the heterologous expression of nitrile hydratase has been realized, and the use of nitrile hydratase to catalyze nicotinonitrile to synthesize nicotinamide has become the most common method. However, nitrile hydratase catalysts often face the problem of temperature rise caused by the exothermic effect of the hydration reaction, resulting in enzyme inactivation and cell rupture, resulting in a decrease in economic benefits. In addition, the toxic effect on cells is greater at high product concentrations, which will increase the inactivation of the catalyst. Therefore, the construction of a new generation of catalysts with high stability and high product tolerance is an important demand for the industrial production of amide compounds. Summary of the invention
[0005] The technical problem to be solved by the present invention is to provide a nitrile hydratase mutant, gene, engineering bacteria, immobilized enzyme and application to improve the catalytic activity of nitrile hydratase.
[0006] The embodiment of the present invention provides a nitrile hydratase mutant, wherein the amino acid sequence of the nitrile hydratase α subunit is SEQ ID NO.1.
[0007] SEQ ID NO.1 sequence is: MSAHEASTSTPGERARALLQVLKQKDLIPDGYIEHMTQLMAHQWSPENGARVVAKAWVDPQFRELLLKDGTAACAQFGYTGPQGEYIVALPDTPSVKNVIVCSLCSCTNWPVLGLPPEWYKGFEFRARLVREGRTVLRELGTELPADVIVKVWDTSAETRYLVLPVRPKGSEAMDEEALRKLITKDVLIGVALPYVA SEQ ID NO.3 is a nitrile hydratase mutant obtained by mutating the amino acid at position 53 based on SEQ ID NO.2. The specific mutation method is: the valine (V) at position 53 is mutated to tyrosine (Y).
[0008] The original SEQ ID NO.2 sequence is: MDGFHDLGGFQGFGKVPHTINSLSYQPVFKQDWEHLAYSLMFVGADHLKKFSVDEVRHAVERLDVRQHVGTQYYERYVIATATLLVETGVITQAELDQALGSHFKLANP AHASGRPAITGRAPFEVGDRVVVRDEYVAGHTRMPAYVRGKEGVVVHRTTQKWPFPDAIGHGDLSAAHQPTYHVEFRVKELWGDAADEGFVVVDLFESYLDHADSTVNA The present invention provides a gene, which encodes the nitrile hydratase mutant. The sequence of the gene is SEQ ID NO.3 ATGGATGGCTTTCACGATCTCGGTGGGTTT CAAGGCTTTGGCAAAGTCCC 50 TCACACCATC AACAGCCTGA GCTACCAACC CGTGTTCAAG CAGGACTGGG 100 AGCACCTGGC CTACAGCCTG ATGTTTGTCG GCGCCGATCA CTTGAAAAAG 150 TTCAGCTATG ACGAAGTGCG CCATGCCGTG GAGCGCCTGG ATGTGCGCCA 200 ACATGTCGGC ACCCAATACT ACGAACGCTA TGTCATCGCG ACCGCCACCC 250 TGCTGGTCGA AACCGGTGTA ATCACCCAGG CGGAACTCGA TCAGGCCCTG 300 GGCTCGCACT TCAAGCTGGC GAACCCCGCG CATGCCAGTG GGCGCCCAGC 350 AATCACCGGCCGCGCGCCCTTTGAGGTGGGCGACCGGGTGGTGGTGCGAG 400 ACGAGTATGT GGCCGGGCAT ACCCGCATGC CGGCCTATGT GCGCGGCAAA 450 GAAGGCGTGG TTGTGCACCG CACCACGCAA AAGTGGCCCT TCCCCGACGC 500 GATCGGCCAC GGTGATCTGA GCGCGGCGCA TCAGCCGACC TACCACGTCG 550 AGTTTCGCGT GAAAGAGCTC TGGGGCGACG CAGCAGATGA GGGGTTCGTG 600 GTGGTCGACC TGTTCGAAAG CTACCTGGAC CACGCCGATA CACGGTGAA 650 CGCATGA 657 The invention provides an engineering bacterium, wherein the engineering bacterium contains the gene.
[0009] The invention provides an immobilized enzyme, which is obtained by the following preparation method: mixing an enzyme solution containing the nitrile hydratase mutant with an IDA resin containing Ni to obtain the immobilized enzyme.
[0010] Preferably, the method for preparing the enzyme solution containing the nitrile hydratase mutant is to culture an engineered bacterium containing a gene encoding the nitrile hydratase mutant, resuspend the engineered bacterium with a buffer solution and break it to obtain an enzyme solution containing the nitrile hydratase mutant.
[0011] Preferably, the engineering bacteria is Escherichia coli, more preferably Escherichia coli BL21 (DE3), and the expression vector is a pET series vector, more preferably pET 24 (+).
[0012] Preferably, the engineered bacteria are cultured under the conditions of induction culture at a final concentration of 0.4-0.6 mM (preferably 0.5 mM) of IPTG at 22-28° C. (preferably 25° C.) (preferably at a stirring speed of 180 rpm), preferably for 12-16 h.
[0013] Preferably, the engineered bacteria are washed with physiological saline (0.9% NaCl solution) before being resuspended with a buffer and disrupted, and the buffer is a sodium phosphate buffer (20 mM PBS, pH 7.2) with a pH of 7.0 to 7.5.
[0014] The present invention provides an application of the immobilized enzyme, wherein the immobilized enzyme, substrate 3-cyanopyridine, buffer and cobalt ion solution (cobalt-dependent nitrile hydratase, the enzyme active center needs to bind cobalt ions to exhibit complete activity) are mixed to catalyze hydration reaction to obtain corresponding products.
[0015] Preferably, the buffer is sodium phosphate buffer (20 mM PBS, pH 7.2) with a pH of 7.0 to 7.5.
[0016] Preferably, the cobalt ion solution is a divalent cobalt ion emulsion, preferably a cobalt chloride aqueous solution.
[0017] The product obtained in the immobilized enzyme catalyzed reaction is nicotinamide, that is, an amide compound of nicotinic acid. The enzyme has strong stereoselectivity and regioselectivity, so no other conformational impurities are generated.
[0018] The beneficial effects of the present invention are The invention constructs a thermostable Escherichia coli engineering strain which can efficiently react 3-cyanopyridine to obtain nicotinamide, and obtains the relevant immobilized enzyme by a simple method, so that the industrial production of nicotinamide becomes simple, low-cost, efficient and environmentally friendly.
[0019] The invention constructs and mutates a nitrile hydratase from Pseudomonas, obtains a mutant protein with significantly improved enzyme activity and reaction temperature, and provides the mutant gene, a recombinant plasmid containing the gene, and a host cell containing the recombinant plasmid.
[0020] The mutant nitrile hydratase protein constructed by the invention can be heterologously expressed and effectively transform 3-cyanopyridine, and the preparation process of the immobilized enzyme is simple and the property is stable.
[0021] The optimal reaction temperature of the nitrile hydratase mutant protein constructed by the invention is 25° C., and it has great industrial application potential. The invention has simple operation, mild reaction conditions, is green and pollution-free, has high specificity for 3-cyanopyridine, and has high yield. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 This is a comparison chart of the enzyme activities of the wild-type PsNHase and the mutant V53Y of nitrile hydratase.
[0023] FIG2 is a liquid chromatogram showing the formation of nicotinamide by nicotinonitrile catalyzed by wild-type PsNHase and mutant V53Y of nicotinamide. Figure 2A For wild type, Figure 2B For mutants. DETAILED DESCRIPTION
[0024] The present invention is further described in detail below in conjunction with specific examples. The following examples are explanations of the present invention, and it should be understood that the protection scope of the present invention is not limited by the specific implementation methods. The materials used in the examples are all commercially available unless otherwise specified.
[0025] Example 1 Construction of nitrile hydratase engineered bacteria E. coli BL21(DE3) / pET28a Ps NH, the operation steps are as follows: The present invention Pseudoxanthomonas sp The nitrile hydratase NHase of AAP-7 was used as the parent. The amplification primers with restriction sites BamHI and NotI were designed as shown in Table 1. The amplification system was shown in Table 2. The PCR amplification reaction conditions were 98°C pre-denaturation for 30s, 98°C denaturation for 10s, 63°C annealing for 5s, 72°C extension for 30s, and 72°C final extension for 1min, for a total of 30 cycles.
[0026] The complete gene sequence was obtained by PCR, including the gene sequence of the β subunit as SEQ ID NO. 4. The recombinant plasmid pET28a was obtained by enzyme ligation and ligation. Ps NH. The constructed recombinant plasmid pET28a PsNH was transformed into competent cells E. coli BL21 (DE3) prepared by the calcium chloride method, and then spread on LB medium (containing 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, and a final kanamycin concentration of 50 ug / mL) containing 50 mg / L kanamycin, and cultured overnight at 37°C. Positive clones were selected and sent to a sequencing company for verification. Recombinant E. coli BL21 (DE3) / pET28a expressing wild-type nitrile hydratase was obtained. Ps NH.
[0027] SEQ ID NO.4 atggatggct ttcacgatct cggtgggttt caaggctttg gcaaagtccc 50 tcacaccatc aacagcctga gctaccaacc cgtgttcaag caggactggg 100 agcacctggc ctacagcctg atgtttgtcg gcgccgatca cttgaaaaag 150 ttcagcgtgg acgaagtgcg ccatgccgtg gagcgcctgg atgtgcgcca 200 acatgtcggc acccaatact acgaacgcta tgtcatcgcg accgccaccc 250 tgctggtcga aaccggtgta atcacccagg cggaactcga tcaggccctg 300 ggctcgcact tcaagctggc gaaccccgcg catgccagtg ggcgcccagc 350 aatcaccggc cgcgcgccct ttgaggtggg cgaccgggtg gtggtgcgag 400 acgagtatgt ggccgggcat acccgcatgc cggcctatgt gcgcggcaaa 450 gaaggcgtgg ttgtgcaccg caccacgcaa aagtggccct tccccgacgc 500 gatcggccac ggtgatctga gcgcggcgca tcagccgacc taccacgtcg 550 agtttcgcgt gaaagagctc tggggcgacg cagcagatga ggggttcgtg 600 gtggtcgacc tgttcgaaag ctacctggac cacgccgata gcacggtgaa 650 cgcatga 657 Table 1 Primer sequences Primer name Primer sequence (5'-3') NHase-F CGGATCCagcgcacacgaagcaa NHase-R TTGCGGCCGCgtggttcggctgaaaaagagcat Table 2 PCR amplification system Element Volume (μL) <![CDATA[ddH2O]]> 20 2×Phanta Flash Master(Dye Plus) 25 NHase-F 2 NHase-R 2 template 1 Example 2 Construction of nitrile hydratase mutant engineered bacteria E. coli BL21(DE3) / pET28a Ps NHV53Y, the operation steps are as follows: In this example, the amino acid sequence of nitrile hydratase SEQ ID NO.2 was used for multi-template modeling on the protein three-dimensional structure online prediction website SWISS-MODEL (https: / / swissmodel.expasy.org / ), and the PyMOL software was used to simulate the docking of the enzyme protein and the substrate 3-cyanopyridine. According to the docking results, the 53rd amino acid was selected for site-directed saturation mutation to obtain the mutant E. coli BL21 (DE3) / pET28a Ps NHV53Y, it was found that the enzyme activity and reaction temperature were significantly improved, and the amino acid sequence after mutation was SEQ ID NO.4.
[0028] Mutant E. coli BL21(DE3) / pET28a Ps The NHV53Y construction process is as follows: Based on the sequence of SEQ ID NO.4, bases 157 to 159 were mutated. Site-directed mutagenesis primers were designed as shown in Table 3, and the amplification system was shown in Table 4. The PCR amplification reaction conditions were 98°C pre-denaturation for 30s, 98°C denaturation for 10s, 56°C annealing for 5s, 72°C extension for 1min20s, and 72°C extension for 1min, for a total of 30 cycles. The recombinant plasmid pET28a in Example 1 was mutated by inverse PCR. Ps The inverse PCR product was treated with DpnI enzyme (purchased from Takara) and purified, and then transformed into competent cells E. coli BL21 (DE3). The positive clones were screened and sequenced to obtain the mutant strain E. coli BL21 (DE3) / pET28a. Ps NHV53Y.
[0029] Table 3 Primer sequences Primer name Primer sequence (5'-3') V53Y-F aagttcagcTATgacgaagtgcgccat V53Y-R gcacttcgtcATAgctgaacttt Table 4 PCR amplification system Element Volume (μL) <![CDATA[ddH2O]]> 20 2×Phanta Flash Master(Dye Plus) 25 NHase-F 2 NHase-R 2 template 1 Example 3 Preparation of immobilized nitrile hydratase mutant (1) Inducing and culturing the nitrile hydratase mutant engineered bacteria E. coli BL21 (DE3) / pET28a obtained in Example 2 with IPTG at a final concentration of 0.5 mM and CoCl2·6H2O at a final concentration of 0.4 mM at 25°C Ps NHV53Y, after 1216h of induction fermentation, the culture solution was centrifuged at 6000rpm for 10min, the supernatant was discarded, the bacterial cells were collected, the wet weight of the bacterial cells was weighed, and the cells were stored in a -80℃ ultra-low temperature refrigerator for later use; (2) According to the wet weight of the bacteria, the fermented bacteria were made into a 300 g / L bacterial suspension, and an appropriate amount of sodium phosphate buffer (20 mM PBS, pH 7.2) was added during the preparation process to control the pH between 7.0 and 7.5; (3) Using a high-pressure homogenizer to break up the bacterial suspension, and the cell wall breaking rate of E. coli under a microscope is >90%, thus obtaining the nitrile hydratase mutant engineered bacteria E. coli BL21(DE3) / pET28a Ps Crude enzyme solution of NHV53Y; (4) The crude enzyme solution of the mutant nitrile hydratase engineered bacteria was mixed with the activated and Ni-bound IDA resin, and immobilized at 35° C. and 110 rpm for 47 h to obtain the immobilized enzyme of the mutant nitrile hydratase engineered bacteria.
[0030] Preparation of Nicotinamide Using Immobilized Enzyme The immobilized enzyme, substrate tricyanopyridine, buffer and cobalt ion solution are mixed to catalyze hydration reaction to obtain the corresponding product nicotinamide, and the cobalt ion solution is a cobalt chloride aqueous solution.
[0031] The immobilized enzyme, substrate 3 cyanopyridine, buffer and cobalt ion solution (cobalt-dependent nitrile hydratase, the enzyme active center needs to bind cobalt ions to exhibit complete activity) are mixed to catalyze the hydration reaction to obtain the corresponding product nicotinamide.
[0032] Example 4 Ps Activity test of wild type and mutant NHase Enzyme activity assay method: The concentrations of the wild-type and mutant nitrile hydratase pure enzymes were diluted to 1 mg / mL with 20 mM KPB solution at pH 7.2, and 10 uL was taken to a 1.5 mL centrifuge tube and placed in a 25°C constant temperature water bath; 400 uL of 50 mM nicotinamide solution and 90 ul of 10 mM phosphate buffer were added to the centrifuge tube, fully vortexed and mixed, reacted at 25°C for 10 minutes, and then 500 uL of acetonitrile solution was added to terminate the reaction, and the solution in the centrifuge tube was filtered through a 0.22 um filter membrane; the solution after the filter membrane was subjected to liquid chromatography detection, and the detection method was as follows: the mobile phase composition was: acetonitrile: water = 1:2 (v / v), the flow rate was 0.6 mL / min, the detection wavelength was 215 nm, the column temperature was 40°C, and the amount of product nicotinamide generated in the reaction system was determined.
[0033] Enzyme activity of nitrile hydratase (U): Unit enzyme activity is defined as the amount of enzyme required to catalyze nicotinonitrile to produce 1 umol nicotinamide per minute at 25°C.
[0034] Specific enzyme activity of nitrile hydratase (U / mg): the enzyme activity per milligram of nitrile hydratase.
[0035] The wild type purified in Example 3 was determined by the above method. Ps The specific enzyme activity of pure enzyme protein of NHase and mutant V53Y is shown in the following figure. Figure 1 As shown, the liquid chromatography results of the mutants are shown in Figure 2.
[0036] from Figure 1 It can be seen that the wild type Ps The enzyme activity of NHase was 122±1.4U / ml, and the specific enzyme activity of mutant V53Y was 1064±10.3U / ml. This indicates that the specific enzyme activity of the mutant was significantly improved compared with the wild type, by more than 7 times. That is, when the 53rd amino acid residue on the β subunit of the wild type nitrile hydratase mutated, the specific enzyme activity of the nitrile hydratase increased, indicating that the wild type Ps The 53rd amino acid residue of NHase may be in the key structural domain of nitrile hydratase and play an important role in the catalytic activity of nitrile hydratase.
[0037] Figure 2A This is the liquid chromatogram of 0.06g / L nicotinamide standard. The peak at 5.003min is nicotinamide.
[0038] Figure 2B This is the liquid chromatogram of nicotinonitrile to nicotinamide catalyzed by nitrile hydratase mutant V53Y. The peak at 4.990 min is nicotinamide.
[0039] One or more embodiments of the present application are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the present application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of the present application should be included in the protection scope of the present application.
Claims
1. A nitrile hydratase mutant, characterized in that The mutant is derived from Pseudoxanthomonas sp The nitrile hydratase NHase of AAP-7 is used as the parent; the amino acid sequence of the α subunit of the nitrile hydratase parent is shown in SEQ ID NO.1, and the amino acid sequence of the β subunit is shown in SEQ ID NO.2; the mutant is a substitution at position 53 of the β subunit.
2. The mutant according to claim 1, characterized in that The mutant is a mutant in which position 53 of the parent is substituted with tyrosine, and its gene sequence is shown in SEQ ID NO.
3.
3. A gene characterized by: The gene encodes the nitrile hydratase mutant according to claim 1.
4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the gene according to claim 3.
5. A host cell, characterized in that Comprising the gene of claim 3 or the expression vector of claim 4; the host cell includes Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Mycobacterium, and Pichia pastoris.
6. An immobilized enzyme, characterized in that: The method comprises the following steps: mixing an enzyme solution containing the nitrile hydratase mutant according to claim 1 with an IDA resin containing Ni to obtain an immobilized enzyme.
7. The immobilized enzyme according to claim 6, characterized in that: The preparation method of the enzyme solution containing the nitrile hydratase mutant comprises the following steps: culturing an engineered bacterium containing a gene encoding the nitrile hydratase mutant, resuspending the engineered bacterium with a buffer solution and breaking the engineered bacterium to obtain the enzyme solution containing the nitrile hydratase mutant.
8. The immobilized enzyme according to claim 7, wherein The culture conditions of the engineered bacteria are as follows: IPTG with a final concentration of 0.5 mM and CoCl2·6H2O with a final concentration of 0.4 mM at 25°C for induction culture.
9. The immobilized enzyme according to claim 7, wherein Before resuspending the engineered bacteria with a buffer solution and breaking them, the engineered bacteria were washed with physiological saline. The buffer solution was pH 7.07.5 sodium phosphate buffer (20 mM PBS, pH 7.2).
10. Use of the immobilized enzyme according to any one of claims 6 to 9, characterized in that: The method of preparing nicotinamide by using the immobilized enzyme comprises the following steps: mixing the immobilized enzyme, substrate 3-cyanopyridine, buffer solution and cobalt ion solution, and catalyzing a hydration reaction to obtain the corresponding product nicotinamide.