Arginase mutant, encoding gene, plasmid, genetically engineered bacteria and application

Abstract

The application belongs to the technical field of bioengineering, and discloses an arginase mutant, a coding gene, a plasmid, a genetically engineered bacterium and application. The arginase mutant is obtained by mutating methionine at the 276th position of arginase into threonine, serine or cysteine, the corresponding coding gene is obtained, and then the corresponding plasmid and genetically engineered bacterium are obtained. The arginase mutant has high enzyme activity and high stability after high-temperature treatment. After being prepared into immobilized enzyme, the repeated batches can reach more than 50 batches, and the production price of L-ornithine is greatly reduced. The application is suitable for constructing the arginase mutant, and the constructed arginase mutant is further applied to the production of L-ornithine.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to an arginase mutant, specifically an arginase mutant, its encoding gene, plasmid, genetically engineered bacteria, and its applications. Background Technology

[0002] L-Ornithine, also known as 2,5-diaminovalerate, is an unconventional amino acid that forms proteins and is therefore relatively rare in nature. It is commonly found in the formation of antimicrobial peptides, such as the short bacterial peptide S. L-Ornithine has many important physiological functions, and its main product form is L-ornithine hydrochloride. L-Ornithine helps the liver clear toxic ammonia and can alleviate the effects of cirrhosis associated with liver dysfunction. Studies have also found that L-ornithine helps promote recovery and tissue repair, thus its application in the pharmaceutical and food industries is becoming increasingly widespread.

[0003] The main methods for synthesizing L-ornithine include: 1. chemical synthesis; 2. bio-fermentation; and 3. enzymatic conversion. In recent years, the chemical synthesis of amino acids has been gradually replaced by bio-fermentation due to problems such as severe environmental pollution, complex processes, and lack of chirality. The production of L-ornithine is no exception.

[0004] One biosynthetic route starts from glucose metabolism and produces L-ornithine through fermentation. The fermentation strains are generally *Corynebacterium glutamicum* or *Escherichia coli*. Chinese patent CN101955901 B discloses a *Corynebacterium glutamicum* strain and a method for preparing L-ornithine and its salts using this strain, and discloses that the accumulation of L-ornithine using this strain can reach 35-45 g / L. Chinese patent CN109161507B discloses a high-yield *Corynebacterium glutamicum* strain for L-ornithine production and its application. This strain uses ammonium sulfate as the sole nitrogen source for fermentation to produce L-ornithine, with a fermentation cycle of 65 hours, and the L-ornithine yield can reach 45 g / L after fermentation. Chinese patent CN113604390A discloses *Corynebacterium glutamicum* ACOrn202, a high-yield strain of L-ornithine obtained through multiple mutagenesis. In a 10L fermenter, the L-ornithine yield can reach 86.5 g / L within 72 hours, with a sugar-acid conversion rate of 37.72%. This route involves fermentation metabolism control. The reported yield of this project is currently low, generally below 90 g / L. In addition to the low yield, this route also has low purification efficiency because various nutrients need to be separated from L-ornithine during the fermentation process, making the subsequent separation and purification process quite complicated.

[0005] Another biological route involves the application of enzyme engineering, utilizing arginase from the urea cycle metabolism to convert L-arginine into ornithine. This process can be accomplished using bacterial cells expressing the enzyme, crude enzyme solution, or immobilized enzyme. This route can achieve conversion concentrations as high as 200–300 g / L, or even higher, and the production process is short; by controlling the enzyme dosage, L-ornithine can be completely converted within 0.5–4 hours. Depending on the choice of enzyme preparation, especially immobilized enzymes, product quality can be further improved. Chinese patent CN104830922B discloses a method for enzymatic conversion of L-arginine solution to produce L-ornithine using Enterococcus faecalis, with an L-ornithine mass concentration of 112.7 g / L and a molar conversion rate of 99%.

[0006] In enzyme engineering applications, immobilization is highly beneficial for enzymatic reactions. On the one hand, it increases the number of enzyme batches and reduces enzyme costs; on the other hand, immobilization reduces enzyme protein detachment, thus facilitating subsequent product purification and quality improvement. However, not all enzymes can be successfully immobilized for industrial production. Many factors determine successful enzyme immobilization, including the enzyme's higher-order structure, stability, the need for coenzymes, prosthetic groups, substrate properties, immobilization materials, and screening conditions; among these, the stability of the enzyme's own structure plays a particularly important role. Chinese patent CN103014086B discloses a method for the continuous production of L-ornithine complex salts by enzyme immobilization, which uses a macroporous adsorption resin with an amino matrix for covalent linkage. However, the reproducibility of this method is low, reaching only about 30 times. Chinese patent CN114196712 A discloses a method for producing L-ornithine by immobilization of enzymes, which uses a nickel column affinity histidine tagging method to immobilize the enzyme. This design requires the vector to be modified with 6 or more consecutive histidine residues (L-His) on the gene, and immobilized by affinity interaction between L-His and metallic nickel. This immobilization vector is relatively expensive and has a small particle size, and is generally used in higher value-added fusion proteins and therapeutic protein drugs. This method does not disclose reproducibility. Summary of the Invention

[0007] The purpose of this invention is to construct an arginase mutant using genetic engineering techniques and obtain its encoding gene, so as to improve the enzyme catalytic activity of the arginase mutant and enable it to be used for the efficient fermentation production of L-ornithine.

[0008] Another object of the present invention is to provide the application of strains carrying the above-mentioned arginase mutant encoding gene in the fermentation production of L-ornithine.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] An arginase mutant, wherein the arginase mutant is a mutation at position 276 of the wild-type arginase protein sequence shown in SEQ ID NO.2, in which methionine is replaced by other amino acids.

[0011] Specifically, the sequence of SEQ ID NO.2 is:

[0012] 1MetSerSerLysProLysSerLeuG lu I l eI l eG lyAl aProPheSerLysG lyG lnPro21ArgG l yG lyVa l G l uLysG lyProAl aAl aLeuArgLysAl aG lyLeuLeuG luLysLeu41LysG l uThrG l uTyrAspVa lArgAspH i sG lyAspLeuAl aPheVa lAspVa lProAsn61AspSerSerPheG ln I l eVa l LysAsnProArgSerVa l G lyLysAl aAsnG l uGl uLeu81Al aG l yVa lVa lAl aG l uVa l G l nLysAsnG lyArgVa l SerVa lVa lLeuG lyG lyAsp101H i sSerLeuAl aVa lG lySer I l eSerG lyHi sAl aArgVa l HisProAspLeuCysVa l121I l eTrpVa lAspAl aH i sThrAspI leAsnThrProLeuThrThrSerSerG lyAsnLeu141H i sG l yG l nProVa l SerPheLeuLeuLysGl uLeuLysG lyLysPheProAspVa l Pro161G lyPheSerTrpVa l ThrProCys I l ​​eSerAlaLysAspI l eVa l Tyr I l eG lyLeuArg181AspVa lAspProG lyG l uH i sTyr I l eIl eLysThrLeuG lyI l eLysTyrPheSerMet201ThrG l uVa lAspLysLeuG lyI l eGlyLysVa l MetG l uG l uThrPheSerTyrLeuLeu221G lyArgLysLysArgPro I l eH isLeuSerPheAspVa lAspG lyLeuAspProAl aPhe241ThrProAl aThrG lyThrProVa l LeuGlyG lyLeuSerTyrArgG l uG lyLeuTyr I l e261ThrG l uG lu I li sLysProG lyThrAspTyrLeuLys321ProProLys

[0013] The wild-type nucleotide sequence encoding the wild-type arginase protein shown in SEQ ID NO.2 is SEQ ID NO.1:

[0014]

[0015]

[0016] As a limitation, the methionine at position 276 is mutated to threonine, serine, or cysteine.

[0017] The present invention also provides a coding gene that encodes the above-mentioned arginase mutant.

[0018] The present invention also provides a plasmid in which the above-mentioned encoding gene is expressed in a free overexpression vector.

[0019] As a limitation, the free overexpression vector is pET29a or other vectors that can be expressed in Escherichia coli.

[0020] The present invention also provides a genetically engineered bacterium, wherein the genetically engineered bacterium uses Escherichia coli as a host and expresses the above-mentioned plasmid.

[0021] The present invention also provides the application of the above-mentioned genetically engineered bacteria, which produce L-ornithine through fermentation.

[0022] As a limitation, the genetically engineered bacteria, enzyme solution, or immobilized enzyme produce L-ornithine through fermentation.

[0023] As a further limitation, the immobilized enzyme is prepared by immobilizing a bacterial cell-derived enzyme solution with epoxy resin.

[0024] The epoxy resin fixation was performed for 24 hours at a salt ion concentration of 500 mM PBNa, a pH value of 8.3–8.7, a final protein concentration of 10–20 g / L, and a temperature of 25°C.

[0025] As another limitation, the concentration of L-arginine substrate in the fermentation broth is 100–300 g / L, the pH value is 6–9, and the temperature is 28–37 °C.

[0026] By adopting the above technical solution, the technical progress achieved by this invention compared with the prior art is as follows:

[0027] ①The arginase mutant provided by this invention increases the enzyme activity to twice that of the wild type, and the mutant has high stability after high temperature treatment;

[0028] ②The arginase mutant provided by this invention, after being prepared into an immobilized enzyme, can be replicated in more than 50 batches, which greatly reduces the production cost of L-arginine to L-ornithine by enzymatic method. Detailed Implementation

[0029] The present invention will be further described in detail below through specific embodiments. It should be understood that the described embodiments are only for explaining the present invention and do not limit the present invention.

[0030] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available. Experimental methods not specifically described in the embodiments are generally performed under standard conditions or as recommended by the manufacturer.

[0031] Example 1: Obtaining and expressing wild-type and mutant arginase

[0032] This embodiment includes the following steps performed sequentially:

[0033] S1. Construction of wild-type expression strain

[0034] Arginase sequences (Sequence ID: NP_031508.1) encoding the CDS region were selected from bacteria, fungi, yeasts, and eukaryotes in the Musmusculus species. A full-gene DNA template was synthesized through codon optimization for PCR amplification of the target gene fragment. The target gene sequence SEQ ID NO.1 is:

[0035]

[0036]

[0037] The primers for PCR amplification are:

[0038] SEQ ID NO.3(F1):5'AATTCCATATGAGTTCAAAACCCAAGTC

[0039] SEQ ID NO.4(R1):5'AAGCTTTTATTTAGGCGGCTTCAGGTAG

[0040] The PCR system consisted of 50 μL of the following: 10× Reaction Buffer, 5 μL; Sample plasmid (10 ng / μL, total 20 ng), 2 μL; F1 primer (10 pmol / μL), 1 μL; R1 primer (10 pmol / μL), 1 μL; dNTP primer (each 2.5 mM), 2 μL; dH2O, 38 μL; and Enzyme Pyrobest DNA Polymerase (5 U / μL) (Takara), 1 μL.

[0041] The specific PCR reaction procedure is as follows: Initially, perform one cycle at 95℃ for 30 seconds; then perform 30 cycles at 95℃ for 60 seconds, 55℃ for 15 seconds, 72℃ for 1 minute; finally, extend at 72℃ for 5 minutes. The target gene fragments were separated by agarose gel electrophoresis, and the PCR products were purified using a gel extraction kit (Tiangen Biotech (Beijing) Co., Ltd.).

[0042] The target gene fragment was directionally cloned into the Pet29a vector via NdeI and HindII restriction sites. The recombinant expression plasmid was transformed into competent E. coli Top10 (purchased from TransGen Biotech). Positive clones were then screened on LB agar plates containing kanamycin (50 μg / mL). The selected positive clones (transformants) were verified by colony PCR using appropriate primers, and the positive clones were sequenced. The correctly sequenced recombinant plasmid was transformed into E. coli BL21(DE3) (purchased from TransGen Biotech) to obtain the wild-type expression strain.

[0043] S2. Obtaining mutant arginase using error-prone PCR

[0044] Using a mutation kit (BTN101005), wild-type expression strains were mutated according to the instructions. Mutated strains were selected and cultured in deep-well plates. High-throughput screening was conducted to identify strains with increased enzyme activity (chromogenic method). The obtained cells with increased activity were sequenced to determine the mutation sites. The results are shown in Table 1. After multiple rounds of testing, a positional change (Mut1-17) was obtained, resulting in the Mut1-17 mutant, which showed a certain promoting effect on arginase activity. This position is M276T. The amino acid sequence of the Mut1-17 mutant is shown in SEQ ID NO. 5.

[0045] SEQ ID NO.5: (m1-17 amino acid sequence)

[0046] 1MetSerSerLysProLysSerLeuG lu I l eI l eG lyAl aProPheSerLysG lyG lnPro21ArgG l yG lyVa l G l uLysG lyProAl aAl aLeuArgLysAl aG lyLeuLeuG luLysLeu41LysG l uThrG l uTyrAspVa lArgAspH i sG lyAspLeuAl aPheVa lAspVa lProAsn61AspSerSerPheG ln I l eVa l LysAsnProArgSerVa l G lyLysAl aAsnG l uGl uLeu81Al aG l yVa lVa lAl aG l uVa l G l nLysAsnG lyArgVa l SerVa lVa lLeuG lyG lyAsp101H i sSerLeuAl aVa lG lySer I l eSerG lyHi sAl aArgVa l HisProAspLeuCysVa l121I l eTrpVa lAspAl aH i sThrAspI leAsnThrProLeuThrThrSerSerG lyAsnLeu141H i sG l yG l nProVa l SerPheLeuLeuLysGl uLeuLysG lyLysPheProAspVa l Pro161G lyPheSerTrpVa l ThrProCys I l ​​eSerAlaLysAspI l eVa l Tyr I l eG lyLeuArg181AspVa lAspProG lyG l uH i sTyr I l eIl eLysThrLeuG lyI l eLysTyrPheSerMet201ThrG l uVa lAspLysLeuG lyI l eGlyLysVa l MetG l uG l uThrPheSerTyrLeuLeu221G lyArgLysLysArgPro I l eH isLeuSerPheAspVa lAspG lyLeuAspProAl aPhe241ThrProAl aThrG lyThrProVa l LeuGlyG lyLeuSerTyrArgG l uG lyLeuTyr I l e261ThrG l uG lu I li sLysProG lyThrAspTyrLeuLys321ProProLys

[0047] Subsequently, a rational design approach was adopted to perform saturation mutations at this position, including the original amino acid and the mutant amino acid, to obtain two mutants with increased activity, namely Mut2-2 (M276S) and Mut2-6 (M276C).

[0048] Table 1. Mutant sequences and methods of obtaining them

[0049] 1 Mut1-17 M276T Irrational design 2 Mut2-2 M276S Rational design; saturation mutation 3 Mut2-6 M276C Rational design; saturation mutation

[0050] The amino acid sequence of the Mut2-2 mutant is shown in SEQ ID NO. 6:

[0051] SEQ ID NO.6: (m2-2 amino acid sequence)

[0052] 1MetSerSerLysProLysSerLeuG lu I le I l eG l yAl aProPheSerLysG l yGl nPro21ArgG l yG l yVa l G l uLysG l yProA l aA l aLeuArgLysA l aG lyLeuLeuG l uLysLeu41LysG l uThrG l uTyrAspVa lArgAspH i sG l yAspLeuA laPheVa lAspVa l ProAsn61AspSerSerPheG ln I l eVa l LysAsnProArgSerVa l G lyLysA l aAsnG l uG l uLeu81A l aG l yVa lVa lA l aG l uVa l G l nLysAsnG lyArgVa l SerVa lVa l LeuG l yG l yAsp101H i sSerLeuA l aVa l G l ySer I leSerG l yH i sA l aArgVa l H i ​​sProAspLeuCysVa l121I l eTrpVa lAspA l aH isThrAsp I l eAsnThrProLeuThrThrSerSerG l yAsnLeu141H i sG l yG l nProVa lSerPheLeuLeuLysG l uLeuLysG l yLysPheProAspVa l Pro161G l yPheSerTrpVa lThrProCys I l ​​eSerAl aLysAsp I l eVa l Tyr I l eG l yLeuArg181AspVa l AspProGl yG l uH i sTyr I le I l eLysThrLeuG ly I l eLysTyrPheSerMet201ThrG l uValAspLysLeuG ly I l eG l yLysVa l MetG l uG l uThrPheSerTyrLeuLeu221G lyArgLysLysArgPro I l eH i sLeuSerPheAspVa lAspG l yLeuAspProAl aPhe241ThrProAl aThrG l yThrProVa l LeuG l yG l yLeuSerTyrArgGl uG l yLeuTyr I l e261ThrGl uG lu I l eTyrLysThrG l yLeuLeuSerG l yLeuAsp I l eSerG l uValAsnPro281ThrLeuG l yLysThrA l aG l uG l uVa l LysSerThrVa lAsnThrA l aVa lAl aLeuThr301LeuA l aCysPheG l yThrG l nArgG l uG l yAsnH i sLysProG lyThrAspTyrLeuLys321ProProLys

[0053] The amino acid sequence of the Mut2-6 mutant is shown in SEQ ID NO.7:

[0054] SEQ ID NO.7: (m2-6 amino acid sequence)

[0055] 1MetSerSerLysProLysSerLeuG lu I le I l eG l yAl aProPheSerLysG l yGl nPro21ArgG l yG l yVa l G l uLysG l yProA l aA l aLeuArgLysA l aG lyLeuLeuG l uLysLeu41LysG l uThrG l uTyrAspVa lArgAspH i sG l yAspLeuA laPheVa lAspVa l ProAsn61AspSerSerPheG ln I l eVa l LysAsnProArgSerVa l G lyLysA l aAsnG l uG l uLeu81A l aG l yVa lVa lA l aG l uVa l G l nLysAsnG lyArgVa l SerVa lVa l LeuG l yG l yAsp101H i sSerLeuA l aVa l G l ySer I leSerG l yH i sA l aArgVa l H i ​​sProAspLeuCysVa l121I l eTrpVa lAspA l aH isThrAsp I l eAsnThrProLeuThrThrSerSerG l yAsnLeu141H i sG l yG l nProVa lSerPheLeuLeuLysG l uLeuLysG l yLysPheProAspVa l Pro161G l yPheSerTrpVa lThrProCys I l ​​eSerAl aLysAsp I l eVa l Tyr I l eG l yLeuArg181AspVa l AspProGl yG l uH i sTyr I le I l eLysThrLeuG ly I l eLysTyrPheSerMet201ThrG l uValAspLysLeuG ly I l eG l yLysVa l MetG l uG l uThrPheSerTyrLeuLeu221G lyArgLysLysArgPro I l eH i sLeuSerPheAspVa lAspG l yLeuAspProAl aPhe241ThrProAl aThrG l yThrProVa l LeuG l yG l yLeuSerTyrArgGl uG l yLeuTyr I l e261ThrGl uG lu I l eTyrLysThrG l yLeuLeuSerG l yLeuAsp I l eCysG l uValAsnPro281ThrLeuG l yLysThrA l aG l uG l uVa l LysSerThrVa lAsnThrA l aVa lAl aLeuThr301LeuA l aCysPheG l yThrG l nArgG l uG l yAsnH i sLysProG lyThrAspTyrLeuLys321ProProLys

[0056] Construction of S3. Arginase mutant strain

[0057] Referring to the method for constructing the wild-type expression strain in step S1, Mut1-17, Mut2-2 and Mut2-6 were directionally cloned to obtain the Mut1-17 expression strain, the Mut2-2 expression strain and the Mut2-6 expression strain.

[0058] Example 2: Evaluation Method for Arginase Activity and Stability

[0059] This example measures the enzyme activity and stability of wild-type expression strains, Mut1-17 expression strain, Mut2-2 expression strain, and Mut2-6 expression strain. The specific procedure is as follows:

[0060] Wild-type, Mut1-17, Mut2-2, and Mut2-6 expression strains were cultured in LB broth at 37°C with shaking at 200 rpm. When the cell density reached approximately OD600 of 1.2, 0.1 mM IPTG (isopropyl-β-D-thiogalactoside) was added, and the culture was induced at 20°C for 12 h to express arginase. Cells were collected after centrifugation at 6000 rpm for 5 min, and sonicated in 50 mM PBNa buffer (pH 6.0). After centrifugation at 12000 rpm for 10 min, the supernatant (enzyme solution) was collected for activity assessment.

[0061] Both wild-type and mutant expression strains possess catalytic activity; direct introduction into the bacterial cells can also complete the catalytic reaction, with reaction parameters similar to those of enzyme solutions. For ease of later evaluation, both activity and stability assessments were performed using cell lysate.

[0062] The enzyme activity reaction system consists of 1 mL of 400 μL of 1M sodium acetate buffer (pH 6.0), 200 μL of 100 g / L arginine solution, 300 μL of purified water, and 100 μL of enzyme solution. After preheating all components except the enzyme solution to 37°C, the enzyme solution is added and reacted for 5 min. Then, 0.1 mL of the reaction solution is serially diluted 10–500 times. The optimal dilution factor varies depending on the enzyme activity. The solution is then used for spectrophotometer or HPLC detection.

[0063] Definition of enzyme activity unit: One international unit of activity refers to the amount of enzyme required to hydrolyze 1 micromole of arginine per minute under specified conditions. HPLC conditions: C18 column, UV detector 205 nm, flow rate 1 mL / min, injection 10 μL, column temperature 25℃, mobile phase: 5% acetonitrile, 1% sodium heptanesulfonate, and 13 g / L potassium dihydrogen phosphate.

[0064] Stability test method: Place the enzyme solution in a 50℃ constant temperature water bath for 30 minutes, then remove it and determine the enzyme activity according to the above method. See Table 2 for specific activity results.

[0065] Table 2. Vitality evaluation results of different strains

[0066]

[0067] Table 2 shows that the error-prone PCR screening revealed that the T mutation at position 276 had high activity and exhibited some resistance to higher temperature treatment. Subsequent rational mutations at this site yielded better M276S (Mut2-2 expressing strain) and M276C (Mut2-6 expressing strain) mutations, increasing activity by 58% and 35%, respectively.

[0068] During stability testing, the wild-type strain showed rapid inactivation. However, the first round of screening mutations at this position had a relatively small impact on the improvement effect, only 30%. In the second round of rational mutations, the M276S and M276C mutations not only significantly improved activity but also had a greater impact on stability, especially M276S, which improved stability by more than 2 times, and M276C, which also improved stability by nearly 2 times. Enzyme activity residue data at 50℃ showed that, compared to the wild-type strain, the enzyme activity residue rates of the Mut1-17, Mut2-2, and Mut2-6 expression strains were effectively improved, with the Mut2-2 and Mut2-6 expression strains showing particularly significant improvements. Comparison of enzyme activity and enzyme activity residue rates at 50℃ showed that the Mut1-17, Mut2-2, and Mut2-6 expression strains all exhibited high stability at 50℃.

[0069] Example 3: Small-volume catalytic experiment and verification of mutant transformation results

[0070] This embodiment is a small-volume catalysis experiment and a mutant transformation result verification experiment.

[0071] (1) The small-volume catalytic experiment was a 100 mL reaction, designed in parallel. For the specific experimental design, please refer to Table 3.

[0072] Specifically, the following steps are included:

[0073] Weigh 20g of arginine substrate, dissolve it in water, adjust the pH to 7 with hydrochloric acid, bring the volume to 100mL, transfer the reaction solution to a 250mL shake flask, maintain the temperature at 30℃, add enzyme at a final concentration of 1g / L, stir at 100r / min for 30 minutes, and then take samples for detection.

[0074] The sampling time for both wild-type and mutant was 30 min, and small-volume evaluation was conducted under the same reaction conditions. The results are shown in Table 3.

[0075] Table 3. Small-volume validation results

[0076] 1 wild type 200 7 1 31 2 Mut1-17 200 7 1 38 3 Mut2-2 200 7 1 56 4 Mut2-6 200 7 1 44

[0077] As shown in Table 3, under the same screening conditions, Mut1-17, Mut2-2, and Mut2-6 all showed higher conversion rates than the wild type (31%), at 30 min, which were 38%, 56%, and 44%, respectively.

[0078] (2) Mut2-2 mutant was selected for conversion determination at different concentrations and pH values. The specific experimental design is shown in Table 4.

[0079] Specifically, the following steps are included:

[0080] According to the detection requirements in Table 4, weigh different weights of arginine substrate, dissolve in water, adjust to the corresponding pH with hydrochloric acid, bring the volume to 100 mL, transfer the reaction solution to a 250 mL shake flask, maintain the temperature at 30℃, add different final concentrations of enzyme, stir at 100 r / min for 30 minutes, and then take samples for detection. The results are shown in Table 4.

[0081] Table 4. Conversion results of Mut2-2 under different conditions

[0082]

[0083] As shown in Table 4, Mut2-2, which has a high conversion rate, was selected for transformation exploration under different conditions. It was found that, with a conversion rate >98% as the reaction endpoint, Mut2-2 could complete the catalytic reaction well under conditions of L-arginine substrate concentration of 100–300 g / L and pH 6–8.5. The reaction completion time varied slightly depending on the enzyme dosage and specific conditions. Mut2-2 was selected for subsequent immobilization studies. However, other mutants at this position disclosed in this invention can also complete the reaction well under suitable substrate concentrations and pH conditions, and are also within the scope of protection of this invention.

[0084] Example 4: Scale-up Fermentation Culture Conditions and Control

[0085] This embodiment is a scaled-up fermentation culture experiment, including the following steps:

[0086] S1. Seed activation

[0087] Mut2-2 expression strain was taken from a -80℃ freezer and activated in LB medium with a final kanamycin concentration of 50 mg / L. The seed activation volume was 5 mL, the inoculum size was 0.5-1%, and the culture was carried out at 37℃ and 200-220 rpm for 8-12 h.

[0088] S2. Primary Seed Preparation

[0089] After seed activation, LB medium was still used, with kanamycin at 50 mg / L. The activated seeds were inoculated into 150–500 mL of medium at an inoculation rate of 1–5%, and cultured at 37°C and 200–220 rpm for 8–12 h.

[0090] S3. Fermentation culture

[0091] After the primary seed preparation was completed, the strain was fermented using a fermentation medium. The fermentation medium consisted of: 13.8 g / L KH2PO4, 1.82 g / L (NH4)2SO4, 3.63 g / L MgSO4·7H2O, 2.35 g / L KOH, 1 g / L yeast extract, 20 g / L glucose, and 0.5 g / L antifoaming agent.

[0092] The prepared culture medium was poured into the fermenter and sterilized at 121℃ for 30 min. After cooling, the temperature was controlled at 37℃, the air-to-gas ratio was 1:1, the rotation speed was 200 rpm, and the dissolved oxygen was calibrated to 100% after stabilizing for 20 min. The pH of the culture medium was measured to be 6.0±0.1. During fermentation, glucose and ammonia were added to maintain the pH at 6.5±0.2. OD600 was induced between 15 and 20, 0.2 M I PTG was added, and the temperature was adjusted to 25±1℃. The fermentation cycle was 36–48 h, and the fermenter was discharged until the OD600 reached 60 or above.

[0093] Process control: Initial speed 400 rpm; pH adjusted to 6.5±0.2 using ammonia; Oxygen control: DO controlled at 25-35%, speed increased when dissolved oxygen ≤25%.

[0094] Under these conditions, the cells in the lower tank are homogenized and broken down. The breaking conditions are: 50-60 Hz, 800 bar pressure, and broken down 5-8 times. The cell lysate is collected and is enzyme solution X.

[0095] Example 5: Preparation of Immobilized Enzymes

[0096] The concentrated enzyme solution X was mixed with epoxy resin (LX-109S) (1g resin for every 50-200mg of protein) and immobilized in an immobilization reactor for 24 hours. The salt ion concentration in the immobilization reactor was 500mM PBNa, the pH was 8.5±0.2, the final protein concentration was controlled at 10-20g / L, and the temperature was 25℃. After immobilization, the enzyme was washed 2-3 times with PBNa buffer (pH 8.5±0.2), filtered, and stored at 4℃ for later use.

[0097] When detecting immobilized enzyme activity, the enzyme activity assay system needs to be modified as follows: increase the reaction volume to 50 mL, containing 200 mM sodium acetate buffer, pH 7; the final concentration of L-arginine is 100 g / L, add 0.1 g of immobilized enzyme, react at 37 °C for 10 min, then boil to terminate the reaction, and measure after 10-100 times dilution.

[0098] The activity of the successfully immobilized enzymes was measured to be 0.6–1.1 million U / g.

[0099] Example 6: Application Method of Immobilized Enzyme

[0100] Immobilized enzyme prepared with 200–300 g / L Mut2-2 enzyme solution was added to a 1 L reaction system. The pH was maintained at 6–9, and the arginine conversion was measured. When the conversion rate was >98%, the resin was collected for the next batch assay. The reproducibility of the batches was recorded. Samples were taken before the reaction and at the 10th batch to measure the activity. The results are shown in Table 6.

[0101] Table 6. Reproducibility Tests of Immobilized Enzymes

[0102] 1 200 5 37 6 1.5-2.5h 16 2 200 10 30 7 3-4h 10 3 300 8 37 7 4-5h 26 4 300 10 37 8 4.5-5.5h 15 5 300 10 28 8.5 6-8h 12 6 300 15 37 9 5-7h 8

[0103] Generally speaking, increasing substrate concentration and decreasing temperature will reduce batch reaction time. Lower temperatures also result in a smaller decay rate of immobilized enzymes. The specific results are also affected by the amount of immobilized enzyme added.

[0104] As shown in Table 6, the Mut2-2 immobilized enzyme can complete batch reactions under the following conditions: L-arginine substrate concentration of 200–300 g / L, pH 6–9, and temperature 28–37 °C. When re-verifying activity in 10 batches, varying degrees of attenuation were observed, with attenuation rates ranging from 8% to 26%, and a maximum activity retention of 92% was achieved, with only an 8% loss. The reaction time can be reduced and the number of usable batches extended by supplementing with a certain amount of enzyme. For industrial scale-up validation, a substrate concentration of 300 g / L, pH 6–9, and more than 50 replicate batches are possible.

[0105] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may use the above technical content as inspiration to make changes or modifications to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the claims of the present invention.

Claims

1. An arginase mutant, characterized in that, The arginase mutant is a mutation of methionine at position 276 of the sequence shown in SEQ ID NO. 2 to threonine, serine, or cysteine.

2. A gene encoding a gene, characterized in that, The encoding gene encodes the arginase mutant as described in claim 1.

3. A plasmid, characterized in that, The plasmid is used to express the encoding gene as described in claim 2 in a free overexpression vector.

4. The plasmid according to claim 3, characterized in that, The free overexpression vector is pET29a or other vectors that can be expressed in Escherichia coli.

5. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria use Escherichia coli as a host to express the plasmid as described in claim 4.

6. The application of the genetically engineered bacteria according to claim 5, characterized in that, The genetically engineered bacteria produce L-ornithine through fermentation.

7. The application of the genetically engineered bacteria according to claim 6, characterized in that, The genetically engineered bacteria, enzyme solution, or immobilized enzyme produce L-ornithine through fermentation.

8. The application of the genetically engineered bacteria according to claim 7, characterized in that, The immobilized enzyme is prepared by immobilizing a bacterial cell-derived enzyme solution with epoxy resin. The epoxy resin fixation was performed for 24 hours at a salt ion concentration of 500 mM PBNa, a pH value of 8.3–8.7, a final protein concentration of 10–20 g / L, and a temperature of 25°C.

9. The application of the genetically engineered bacteria according to any one of claims 7 to 8, characterized in that, During fermentation, the concentration of L-arginine substrate in the fermentation broth was 100–300 g / L, the pH value was 6–9, and the temperature was 28–37℃.

Images