A nucleoside hydrolase mutant, engineered bacteria and its application

Abstract

This invention discloses a nucleoside hydrolase mutant, engineered bacteria, and its applications. The mutant is obtained by single or double mutation of amino acids 12 and 190 in the amino acid sequence shown in SEQ ID NO.1. The nucleoside hydrolase mutants NH-S12P and NH-S190V of this invention achieve enzyme activities of 1030 U / mg and 1197 U / mg, respectively, representing increases of 18.8% and 38% in specific activity compared to the wild-type nucleoside hydrolase. Furthermore, the thermostability of the two mutants at 65℃ is increased by 8 times and 10 times, respectively, compared to the wild-type nucleoside hydrolase. This improved thermostability helps reduce the rate of enzyme thermal inactivation during production, transportation, application, and storage. This is of great significance for improving the industrial production efficiency of enzymatic hydrolysis of nucleosides to prepare purines.

Description

(I) Technical Field

[0001] This invention relates to a nucleoside hydrolase mutant, engineered bacteria, and its applications. (II) Background Technology

[0002] Nucleoside hydrolases are enzymes that break down nucleosides into nitrogenous bases and ribose. Nucleoside hydrolases can be used in the enzymatic hydrolysis of nucleosides to prepare purine bases. Adenine and guanine have important applications in the pharmaceutical industry. Adenine is a component of nucleic acids, participates in the synthesis of genetic material, promotes leukocyte proliferation, and increases the number of white blood cells. It is used to prevent and treat leukopenia caused by various reasons, especially leukopenia caused by chemotherapy for tumors, and is also used for acute granulocytopenia. It is an indispensable compound in the body. In addition, adenine is also an intermediate in the production of many drugs, including the plant hormone 6-BA, vitamin B4, and adefovir dipivoxil. Guanine is an important intermediate in the synthesis of the antiviral drugs acyclovir and ganciclovir. It is also used clinically to treat blind cytomegalovirus retinitis caused by immunodeficiency, and is used as an adjuvant therapy in organ transplantation, AIDS, and malignant tumors.

[0003] Currently, the main methods for producing adenine and guanine are natural product extraction and chemical synthesis. Due to the low efficiency and high cost of natural product extraction, industrial production primarily employs chemical methods. However, chemical methods generally suffer from drawbacks such as lengthy steps, demanding reaction conditions, and environmental unfriendliness. The preparation method disclosed in Chinese Patent CN101125854A utilizes high-temperature liquid water hydrolysis of adenosine to prepare adenine. The drawback of this method is that adenosine hydrolysis requires high temperatures, and the generated byproduct D-ribose is easily denatured at high temperatures and cannot be utilized, making it highly uneconomical. The preparation method disclosed in Chinese Patent CN103923083A uses adenosine as a substrate, obtaining acetylated adenine and tetraacetylribose through acetyl alcohol acetylation, followed by alkaline hydrolysis and neutralization to obtain adenine. Although this method yields both the main product adenine and the byproduct tetraacetylribose in high yields, the process is complex and generates a significant amount of waste. Chinese patent CN112143662A discloses a recombinant Pichia pastoris that produces adenosine hydrolase, but its adenosine hydrolase gene is derived from Escherichia coli, and the expression of prokaryotic genes in eukaryotes is subject to many limitations such as glycosylation. The two high-activity nucleoside hydrolase mutants provided by this invention have genes derived from fungi and exhibit better compatibility in the Pichia pastoris expression system. The enzyme activities of the shake-flask fermentation products reach 1030 U / mg and 1197 U / mg, respectively, representing a 18.8% and 38% increase in specific activity compared to wild-type nucleoside hydrolases, and a 10-fold and 8-fold increase in thermal stability, demonstrating greater industrialization potential.

[0004] The present invention aims to provide a recombinant strain of Pichia pastoris that produces nucleoside hydrolases with high enzyme activity, and secretes and expresses a nucleoside hydrolases mutant with a 6HIS purification tag. (III) Summary of the Invention

[0005] The purpose of this invention is to provide a nucleoside hydrolase mutant, engineered bacteria, and its application, which has application value in the field of enzymatic hydrolysis of nucleosides to prepare purines. It solves the problems of insufficient enzyme activity and low enzyme stability in the existing technology of microbially expressed nucleoside hydrolases, and is of great significance for the green biomanufacturing of pharmaceutical intermediates purines.

[0006] The technical solution adopted in this invention is:

[0007] This invention provides a nucleoside hydrolase mutant, which is obtained by single or double mutation of amino acids 12 and 190 in the amino acid sequence shown in SEQ ID NO.1.

[0008] Preferably, the nucleoside hydrolase mutant is obtained by mutating serine at position 12 of the amino acid sequence shown in SEQ ID NO.1 to proline (amino acid sequence shown in SEQ ID NO.2); or by mutating serine at position 190 to valine (amino acid sequence shown in SEQ ID NO.3).

[0009] The present invention also provides a coding gene for a nucleoside hydrolase mutant, the nucleotide sequence of which is shown in SEQ ID NO.4 and SEQ ID NO.5.

[0010] The present invention also relates to a recombinant vector containing the coding gene of the nucleoside hydrolase mutant, and a recombinant genetically engineered bacterium constructed with the recombinant vector, wherein the recombinant vector plasmid is pPIC9K and the recombinant genetically engineered bacterium host bacterium is Pichia pastoris GS115.

[0011] This invention also provides an application of the nucleoside hydrolase mutant in the catalytic preparation of purine from nucleosides. The application is carried out according to the following steps: the fermentation broth obtained by inducing culture of recombinant genetically engineered bacteria containing the coding gene of the nucleoside hydrolase mutant is centrifuged, and the supernatant is used as a catalyst. Using nucleosides as substrate, a transformation reaction is carried out under the conditions of 30-70℃, 50-200rpm, and pH 3-5 (preferably 50℃, pH 4.5, and 50rpm). The reaction solution is separated and purified to obtain the product purine.

[0012] Preferably, the final concentration of the substrate added is 50-150 g / L, more preferably 100 g / L; the amount of catalyst used is 100-130 mg / L, more preferably 130 mg / L, based on the protein content in the supernatant. Under the above conditions, the conversion rate of adenosine to adenine reaches 70% after 5 hours of catalysis, and 100% after 8 hours of catalysis.

[0013] Preferably, the catalyst is prepared by the following method: (1) the recombinant engineered bacteria are inoculated into BMGY medium and cultured at 30°C and 220 rpm for 16-18 h until the OD600 reaches 2-6 (preferably 5). The bacteria are then centrifuged at 4000 rpm-5000 rpm for 5 min at room temperature and the cells are collected; (2) the bacteria are resuspended in BMMY medium to make the OD600 1 and cultured at 30°C and 220 rpm for 3 days; methanol is added to the medium every 24 h until the final volume concentration is 1%; after fermentation, the bacteria are centrifuged and the supernatant is collected.

[0014] Compared with existing technologies, the beneficial effects of this invention are mainly reflected in the following aspects: Based on wild-type nucleoside hydrolase, this invention modifies nucleoside hydrolase NH through site-directed mutagenesis and heterologous expression biotechnology, ultimately obtaining two nucleoside hydrolase mutants with enhanced enzyme activity, NH-S12P and NH-S190V. Fermentation of these mutants NH-S12P and NH-S190V yields enzyme activities of 1030 U / mg and 1197 U / mg, respectively, representing increases of 18.8% and 38% in specific activity compared to the wild-type nucleoside hydrolase. Furthermore, the thermostability of the two mutants at 65℃ is increased by 8 times and 10 times, respectively, compared to the wild-type nucleoside hydrolase. This improved thermostability helps reduce the rate of enzyme thermal inactivation during production, transportation, application, and storage. This is of great significance for improving the industrial production efficiency of enzymatic hydrolysis of nucleosides to prepare purines. The nucleoside enzymes of this invention exhibit high conversion rates for the hydrolysis of adenosine, guanosine, or inosine. Taking adenosine as an example, the final concentration of the substrate added was 100 g / L, and the amount of catalyst used was 130 mg / L based on the protein content in the supernatant. After catalytic reaction at 50℃, pH 4.5, and 50 rpm for 5 h, the conversion rate of adenosine hydrolysis to adenine reached 70%, and after catalysis for 8 h, the conversion rate reached 100%. (iv) Description of the attached drawings

[0015] Figure 1 Map of the recombinant plasmid pPIC9K-NH of wild-type nucleoside hydrolase NH.

[0016] Figure 2 The image shows the liquid phase detection results of crude enzyme solutions of wild-type nucleoside hydrolase NH and its mutants NH-S12P and NH-S190V.

[0017] Figure 3 The results of SDS-PAGE analysis of fermentation products of wild-type nucleoside hydrolase NH and its mutants NH-S12P and NH-S190V are presented.

[0018] Figure 4 Bar graph showing the enzyme activity of wild-type nucleoside hydrolase NH and its mutants NH-S12P and NH-S190V.

[0019] Figure 5 The bar chart shows the optimal pH for wild-type nucleoside hydrolase NH and its mutants NH-S12P and NH-S190V.

[0020] Figure 6 The bar chart shows the optimal temperature for wild-type nucleoside hydrolase NH and its mutants NH-S12P and NH-S190V.

[0021] Figure 7 The graph shows the results of thermostability determination of wild-type nucleoside hydrolase NH and its mutants NH-S12P and NH-S190V.

[0022] Figure 8 The figure shows the liquid chromatography results at the start and 8 hours after the adenosine hydrolysis reaction.

[0023] Figure 9 This is a graph showing the liquid phase detection results during the hydrolysis reaction of guanosine and inosine. (V) Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0025] Culture medium formulations (all prepared with deionized water):

[0026] YPD medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, with 20 g / L agar powder added when preparing the solid medium. Autoclave at 115°C for 30 min.

[0027] MD medium: glucose 20 g / L, ammonium sulfate 10 g / L, YNB (yeast nitrogen base, without Amino Acids & Ammonium Sulfate) 3.4 g / L, with 20 g / L agar powder added when preparing the solid medium. Sterilize at 115℃ for 30 min, and add 0.2% 500× biotin after sterilization.

[0028] BMGY medium: yeast extract 10 g / L, peptone 20 g / L, glycerol 10 g / L, K₂HPO₄ 3.7 g / L, KH₂PO₄ 11.8 g / L, YNB (yeast nitrogen base, without Amino Acids & Ammonium Sulfate) 3.4 g / L, ammonium sulfate 10 g / L. Sterilize at 115℃ for 30 min, then add 0.2% 500× biotin.

[0029] BMMY medium: yeast extract 10 g / L, peptone 20 g / L, K₂HPO₄ 3 g / L, KH₂PO₄ 11.8 g / L, YNB (yeast nitrogen base, without Amino Acids & Ammonium Sulfate) 3.4 g / L, ammonium sulfate 10 g / L. Sterilize at 115℃ for 30 min. After sterilization, add 0.2% 500× biotin. Add methanol every 24 h until the final concentration reaches 1%.

[0030] 500×B (0.02% Biotin): Dissolve 2.0 mg of biotin in 10 mL of ddH2O, filter to sterilize, and store at 4°C protected from light.

[0031] LB medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, pH 7.0. Add 20 g / L agar powder when preparing solid medium. Autoclave at 121℃ for 20 min.

[0032] The room temperature described in this invention is 25-30℃.

[0033] Example 1: Cloning of wild-type nucleoside hydrolase gene, construction and expression of plasmid

[0034] 1. Wild-type Pichia pastoris recombinant engineered strain NH

[0035] The gene sequence (CN109563504A) encoding a nucleoside hydrolase from *Penicillium multicolor* was identified as the wild-type nucleoside hydrolase gene. Codon optimization of the wild-type nucleoside hydrolase gene was performed based on the codon preference pattern of *Pichia pastoris*, and a 6-his tag was added. The nucleoside hydrolase gene fragment was synthesized by Beijing Qingke Company and ligated between the EcoRI and NotI restriction sites on plasmid pPIC9K to obtain the recombinant plasmid pPIC9K-NH (see spectrum). Figure 1 The nucleoside hydrolase expression gene is 1161 bp in length and contains 387 amino acids. The amino acid sequence is shown in SEQ ID NO.1 and the nucleotide sequence is shown in SEQ ID NO.6.

[0036] The recombinant plasmid pPIC9K-NH was linearized by restriction endonuclease SacⅠ and then transformed into Pichia pastoris GS115 competent cells by electroporation. Positive recombinants were screened by histidine deficiency markers to obtain wild-type Pichia pastoris recombinant engineered strain NH.

[0037] SEQ ID NO.1

[0038] MHHHHHHHFPVSLPLLCGSLLPLITGTLAVPK

[0039] ASRADKHYAIMDNDWYTAGFVPYLIALDGDVE

[0040] VLGLASDTANTWQPQVALHAVATLEAGNLSCI

[0041] P V Y P G S T W P L I N T P N R F Q A W E M V H G K L P W E G A

[0042] F A P E N K T L E A E G N D P TS G N P N R I V K A A F K E G F

[0043] P K G K P E N R T S A A N F M VE M V H K Y P G Q V S I Y S A G

[0044] A L T N V A L A V R M D P Q F AS L A K E L V I M G G Y V D L N

[0045] M L Q A T G S V L L A D L Q S DI N L M I D P E A S K I A L T A

[0046] E F P N I T I A G N V A N Q V FP T K E F V D E I A S V P N P Y

[0047] S K L F H D Y Y D L S F P F W DE T A A A L M V D P T L A T N Q

[0048] T S V F L D V D T A Y G S P N YG N I H V Y Q K A L A P V G I R

[0049] E V N F V F Q V D G D R L K Q RI K H S L Q Y P K S C A D L R NE R。

[0050] SEQ ID NO.6

[0051]

[0052] 2. NH-induced expression in recombinant Pichia pastoris engineered strains

[0053] (1) Inoculate the recombinant engineered Pichia pastoris strain NH into BMGY medium and culture at 30℃ and 220rpm for 16-18h until OD. 600 Once the bacterial count reaches 5.0, centrifuge at 4000-5000 rpm for 5 minutes at room temperature and collect the bacterial cells.

[0054] (2) Resuspend the bacterial cells in BMMY medium to achieve OD 600 The culture medium was 1% and cultured at 30℃ and 220rpm for 3 days. Methanol was added to the culture medium every 24 hours until the final volume concentration was 1%. After fermentation, the supernatant of the fermentation broth (i.e., the crude enzyme solution of wild-type nucleoside hydrolase) was collected by centrifugation and the enzyme activity was detected. The results showed that the activity of nucleoside hydrolase produced by the recombinant engineered wild-type Pichia pastoris NH fermentation was 86.7U / ml.

[0055] 3. Enzyme activity detection

[0056] The enzyme activity detection reaction system is as follows: substrate adenosine concentration 100 g / L, fermentation supernatant 1 ml, and buffer solution is pH 4.5 acetate / sodium acetate buffer. The reaction was carried out at 50℃ and pH 4.5 for 30 min, followed by boiling in a water bath for 10 min to inactivate the enzyme. High-performance liquid chromatography (HPLC) was used to detect the peak area of ​​adenine. The adenine content in the reaction solution was obtained based on the standard curve of adenine concentration versus peak area (curve equation: adenosine y = 8307003.100x + 373096.500; adenine y = 741,826.200x - 42,780.400). The chromatogram for HPLC detection of adenine is shown below. Figure 2 As shown.

[0057] Enzyme activity unit (U) is defined as the amount of enzyme required to hydrolyze 1 μmol of adenosine per minute at 50°C and pH 4.5.

[0058] High performance liquid chromatography (HPLC) detection conditions: Column: C18 column; Mobile phase: 5% acetonitrile; Detection wavelength: 260 nm; Injection volume: 10 μL; Flow rate: 1 ml / min; Column temperature: 35 ℃; Detection was performed using a UV detector.

[0059] Example 2: Construction of nucleoside hydrolase mutants and expression plasmids

[0060] 1. Computer simulation screening of nucleoside hydrolase mutation sites

[0061] Based on the predicted thermal stability of point-mutated proteins using the ddg-monomer method in Rosetta software, this application uses wild-type nucleoside hydrolase crystal structure PDB data as input and simulates the structural model of the point mutant. ddg = mutant energy - wild-type energy, with a higher negative value indicating greater stability of the mutant protein. Ten high-resolution simulated mutation sites were obtained: S12P, P78W, D44L, D71V, S190V, P54A, D44I, P78L, Q77L, and E117L.

[0062] To ensure the mutation effect on enzyme activity, based on the molecular docking results, S12P and S190V, the two sites closest to the predicted enzyme activity center, were selected for actual mutation experiments.

[0063] 2. Nucleoside hydrolase mutant NH-S12P

[0064] (1) The recombinant plasmid pPIC9K-NH of wild-type Pichia pastoris recombinant engineered strain NH was extracted using a plasmid mini-extraction kit. Using pPIC9K-NH as a template, PCR amplification was performed using primer pair S12P-F / S12P-R to obtain the PCR product.

[0065] The primer pair S12P-F / S12P-R is as follows.

[0066] S12P-F:CCAGTT cca TTGCCATTGTTGTGTGGTTCCTTGTTGCCTT (Underlined bases indicate mutated bases)

[0067] S12P-R:AACAATGGCAA tgg AACTGGAAAGTGGTGGTGGTGGTGGT (Underlined bases indicate mutated bases)

[0068] The PCR amplification system is as follows: 1 μL S12P-F, 1 μL S12P-R, 1 μL plasmid template, 25 μL high-fidelity enzyme, and water to a final volume of 50 μL.

[0069] PCR reaction sequence: First, pre-denaturation at 95℃ for 3 min; then 25 cycles: denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 3 min; finally, extension at 72℃ for 10 min, and incubation at 4℃.

[0070] (2) The PCR product from step (1) was digested with DPNⅠ enzyme and incubated at 37℃ for 1 h to remove the original template interference.

[0071] The digestion system was as follows: 50 μL PCR stock solution, 1 μL DPNⅠ, and 5 μL 10×BUFFER.

[0072] (3) The PCR product digested in step (2) was purified using a purification kit (purchased from Shanghai Sangon Biotech Co., Ltd.). The specific operation was carried out according to the kit instructions.

[0073] (4) The purified product from step (3) was recombinantly ligated using a one-step cloning kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.). The specific operation was performed according to the kit instructions. The product was incubated at 37°C for 30 min to obtain the ligation product.

[0074] The reaction system is as follows: 1 μL recombinase, 2 μL buffer, 4 μL PCR purified product, and 13 μL water.

[0075] (5) Transform the ligation product from step (4) into commercial E. coli DH5α competent cells (purchased from Beijing Qingke Biotechnology Co., Ltd.). The specific operation is as follows: Take 100 μL of competent cells thawed on ice, add the target DNA (ligation product), mix gently, and let stand on ice for 5 min; heat shock in a 42℃ water bath for 45 s, and quickly put it back on ice for 2 min; add 900 μL of sterile LB liquid medium without antibiotics to a centrifuge tube, mix well, and revive at 37℃, 200 rpm for at least 20 min; take 100 μL of competent cells and add them to LB solid medium containing ampicillin and kanamycin at a final concentration of 1‰, spread evenly and blow dry the plate; invert the plate and incubate overnight in a 37℃ incubator. Single colonies from transformation plates were selected for colony PCR verification. The verification primers were α-F: TACTATTGCCAGCATTGCTGCT; AOX-R: GCAAATGGCATTCTGACATCC. The PCR product size was 1351 bp. The PCR product was sent to Qingke Biotechnology Co., Ltd. for sequencing to verify the correctness of the mutation site. Plasmids were extracted from positive transformants to obtain the nucleoside hydrolase mutant NH-S12P. The correct strain was stored at -80℃. The nucleoside hydrolase mutant NH-S12P is formed by mutating serine at position 12 of the NH amino acid sequence (shown in SEQ ID NO. 1) to proline. The full-length gene is 1161 bp, consisting of 387 amino acids, as shown in SEQ ID NO. 2 and the nucleotide sequence in SEQ ID NO. 4.

[0076] 3. Nucleoside hydrolase mutant NH-S190V

[0077] Using the method in step 2, PCR amplification was performed using primer pair S190V-F / S190V-R and pPIC9K-NH as a template. The amplified PCR product was purified and recombinantly ligated into a circular form using a one-step cloning kit. The ligation product was transformed into commercially available E. coli DH5α competent cells, positive transformants were screened, sequenced, and plasmids were extracted to obtain the nucleoside hydrolase mutant NH-S190V. The correct bacterial strain was stored at -80°C. The nucleoside hydrolase mutant NH-S12V is formed by mutating serine at position 190 of the NH amino acid sequence (shown in SEQ ID NO.1) to valine. The full-length gene is 1161 bp, consisting of 387 amino acids, as shown in SEQ ID NO.3 and the nucleotide sequence is shown in SEQ ID NO.5.

[0078] S190V-F:ATCTAC gt TGCTGGTGCTTTGACTAACGTTGCTTTGGCTG (underlined bases indicate mutated bases);

[0079] S190V-R:

[0080] TTAGTCAAAGCACCAGCA ac GTAGATTGAAACCTGACCTGGGTACT (underlined bases indicate mutations).

[0081] Example 3: Construction of recombinant Pichia pastoris engineered strains NH-S12P and NH-S190V

[0082] 1. Carrier linearization

[0083] In Example 2, the expression vectors pPIC9K-NH-S12P and pPIC9K-NH-S190V were digested with restriction endonuclease SacⅠ and linearized by incubation at 37°C for 3 hours.

[0084] The reaction system is as follows: 1 μg plasmid, 2 μL 10× buffer, 1 μL SacⅠ, and sterile water added to 20 μL.

[0085] 2. Preparation of Pichia pastoris GS115 competent cells

[0086] Pick a single colony of Pichia pastoris GS115 from a YPD plate and inoculate it into a test tube containing 3 mL of YPD liquid medium. Incubate overnight at 30°C with shaking at 220 rpm. Take 1000 μL of the overnight culture and inoculate it into a 250 mL Erlenmeyer flask containing 30 mL of fresh YPD liquid medium. Incubate at 30°C with shaking at 220 rpm for about 8 hours until the OD600 reaches 1.3. Transfer the above culture into a 50 mL sterile centrifuge tube and centrifuge at 4°C with 5000 rpm for 5 min. Remove the supernatant and place on ice. Resuspend the above cells in 20 mL of LiAc-DTT solution (100 mM LiAc, 10 mM DTT, 0.6 M sorbitol, 10 mM Tris-HCl, pH 7.5), incubate at 30°C with shaking for 30 min, centrifuge at 4°C with 5000 rpm for 5 min, and remove the supernatant. Repeat the above steps three times, or resuspend the bacterial cells in 1 mL of ice-chilled 1M sorbitol. Then transfer the resuspended cells to a 1.5 mL EP tube, centrifuge at 3000 rpm for 5 min, remove the supernatant, and repeat this step three times. Resuspend the collected bacterial cells in ice-chilled 1M sorbitol to a final volume of 0.5 mL; aliquot into 80 μL tubes and store at -80 °C for later use.

[0087] 3. Electroconversion of Pichia pastoris

[0088] Mix 1 μg of linearized plasmid with 80 μL of competent cells, transfer to a pre-chilled 0.2 cm electroporation cuvette, gently tap to ensure it is at the bottom, and place on ice for 5-10 min. Following the Bio-Rad electroporator's operating instructions, set the mode to Pic mode, wipe the outer wall of the cuvette dry and place it in the electroporation position, setting the voltage to 1.5 kV, capacitance to 25 μF, resistance to 200 Ω, and electroporation time to 5 msec. Immediately after electroporation, add 1 mL of pre-chilled 1 M sorbitol to the cuvette, gently pipette to mix, and quickly transfer to a 1.5 mL EP tube. Incubate at 30°C and 220 rpm for 1-2 h. Spread 100-200 μL of bacterial cells onto MD plates and incubate upside down at 30°C for 2-4 days until single colonies appear.

[0089] 4. PCR identification of recombinant transformants.

[0090] Select single colonies from step 3 for PCR identification. Using a sterile toothpick, pick an appropriate amount of single colony cells from a transformation plate and transfer them to a PCR tube containing 10 μL of 20 mM NaOH solution. Incubate at 98°C for 10 min, then at -80°C for 10 min. Repeat this process three times, followed by incubation at 98°C for 10 min. Use the above product as a template and perform PCR using verification primers (α-F, AOX-R). Agarose gel electrophoresis confirmed that the PCR product band size was 1351 bp. The positive recombinants were named pPIC9K-NH-S12P and pPIC9K-NH-S190V.

[0091] 5. Fermentation induced by recombinant Pichia pastoris NH-S12P and NH-S190V

[0092] The method is the same as described in step 2 of Example 1, to obtain crude enzyme solutions of nucleoside hydrolase mutants NH-S12P and NH-S190V.

[0093] 6. Enzyme activity detection

[0094] The method is the same as described in Example 1. The liquid phase detection results are as follows: Figure 2 The results showed that the enzyme activities of the fermentation supernatant of the recombinant Pichia pastoris strains expressing wild-type nucleoside hydrolases and their mutants were 86.7 U / ml for wild-type nucleoside hydrolases NH, 123.6 U / ml for mutant NH-S12P, and 155.61 U / ml for mutant NH-S190V.

[0095] 7. SDS-PAGE analysis of the fermentation broth yielded the following results: Figure 3 The results showed that the nucleoside hydrolase was approximately 50 kDa in size.

[0096] 8. Methods for determining protein content:

[0097] The Coomassie Brilliant Blue (Bradford) binding method for determining protein content in fermentation supernatant is a combined colorimetric and dye-based method. Coomassie Brilliant Blue G-250 is brownish-red in acidic solution, turning blue upon binding with protein. Within a certain protein concentration range, it follows Beer's Law and can be measured colorimetrically at 595 nm. It exhibits significant absorption within 3–5 minutes and remains stable for at least 1 hour. In the range of 10–1000 μg / mL, the absorbance is directly proportional to the protein concentration.

[0098] The enzyme solution and Coomassie Brilliant Blue solution were mixed at a volume ratio of 1:5, allowed to stand for 10 minutes, and then the protein content was determined according to the above method. The protein concentration standard curve was y = 3.4668x - 1.4271 (R²). 2=0.9972). The results showed that the protein content of the fermentation supernatant of the recombinant Pichia pastoris strains expressing wild-type nucleoside hydrolase and its mutants was as follows: wild-type nucleoside hydrolase: 0.1 mg / ml, mutant NH-S12P: 0.12 mg / ml, mutant NH-S190V: 0.13 mg / ml.

[0099] 9. Specific vitality calculation

[0100] Specific activity refers to the number of enzyme activity units per unit weight of protein, usually expressed as U / mg protein. The formula for calculating specific activity is: Specific activity (U / mg) = Enzyme activity (U / mL) / Protein content (mg / mL).

[0101] The specific activities of wild-type nucleoside hydrolases and their mutants NH-S12P and NH-S190V are shown in Table 1 and . Figure 4 .

[0102] Table 1 Comparison of specific activities of nucleoside hydrolases and their mutants

[0103] Wild-type nucleoside hydrolases and their mutants Specific activity (U / mg) Wild-type NH 867U / mg NH-S12P 1030U / mg NH-S190V 1197U / mg

[0104] Example 4: Determination of optimal pH and optimal temperature for enzyme

[0105] 1. Determination of optimal pH

[0106] The activity of the crude nucleoside hydrolase prepared in Examples 1 and 3 in catalyzing the hydrolysis of adenosine was determined under different pH conditions (3.2-6) at 50°C using the method described in Example 1. The relative enzyme activity under the optimal pH condition was defined as 100%.

[0107] Reaction system: 1 ml of crude enzyme solution (wild-type nucleoside hydrolase protein concentration 0.1 mg / ml, mutant NH-S12P protein concentration 0.12 mg / ml, mutant NH-S190V protein concentration 0.13 mg / ml) was placed in 1 ml of acetate / sodium acetate buffer at pH 3.2, 4, 4.5, 5, 5.5, and 6, respectively. Adenosine substrate was added at a concentration of 100 g / L. The reaction was carried out for 30 min, followed by inactivation in a boiling water bath for 10 min. Samples were taken for liquid chromatography analysis.

[0108] The optimal pH for both the wild-type nucleoside hydrolase and the two mutants is 4.5. Figure 5 The results showed that point mutation did not change its optimal pH, but slightly increased the residual enzyme activity at around pH 5.

[0109] 2. Determination of optimal temperature

[0110] The activity of the crude nucleoside hydrolase prepared in Examples 1 and 3 in catalyzing the hydrolysis of adenosine was determined under different temperature (40℃-65℃) reaction conditions at pH 4.5 using the method of Example 1. The relative enzyme activity under the optimal temperature condition was defined as 100%.

[0111] Reaction system: 1 ml of crude enzyme solution (wild-type nucleoside hydrolase protein concentration 0.1 mg / ml, mutant NH-S12P protein concentration 0.12 mg / ml, mutant NH-S190V protein concentration 0.13 mg / ml) was placed in 1 ml of pH 4.5 acetate / sodium acetate buffer (substrate adenosine concentration 100 g / L). The solutions were reacted at 40℃, 45℃, 50℃, 55℃, 60℃, and 65℃ for 30 min each, followed by inactivation in a boiling water bath for 10 min. Samples were then taken for liquid chromatography analysis.

[0112] The optimal pH temperature for both the wild-type nucleoside hydrolase and the two mutants is 50°C. Figure 6 The results showed that point mutations did not change its optimal temperature.

[0113] Example 5: Thermal Stability Determination

[0114] 1. Thermal stability

[0115] The crude enzyme solution of wild-type nucleoside hydrolase NH prepared in Example 1, and the crude enzyme solutions of nucleoside hydrolase mutants NH-S12P and NH-S190V prepared by the method in Example 4, were diluted with pH 4.5 acetate-sodium acetate buffer to the same protein concentration of 0.1 mg / ml. 1 ml of each solution was placed in a 1.5 mL EP tube and incubated at 65°C for different times (0, 5, 10, 20, 30, 60 min). The corresponding enzyme activities were then measured using the method in Example 1, with the activity at 0 min incubation as 100%, and the remaining enzyme activity at different incubation times was calculated. Figure 7 As shown, the residual enzyme activities of mutants NH-S12P and NH-S190V were 49% and 42% respectively after treatment at 65℃ for 60 min.

[0116] 2. Half-life

[0117] Half-life t 1 / 2 The half-life (t) refers to the time required for the initial activity to decrease by 50% at a given temperature. 1 / 2 The thermostability of enzymes is a commonly used parameter; a higher value indicates better thermostability. It is calculated using the following formula:

[0118] Where, k d The inactivation rate constant can be obtained through linear regression: In the formula, At refers to the residual activity, A0 is the initial activity, and t is the treatment time at the studied temperature.

[0119] according to Figure 7 Wild-type nucleoside hydrolase at 65℃ 1 / 2 The value was 5.6 min, while the mutant's t 1 / 2 The values ​​increased to varying degrees, with the mutant NH-S190V showing the highest t value at 65℃. 1 / 2 The t-value of the mutant NH-S12P at 65℃ was 44.8 min. 1 / 2 The highest value was 56.4 min, approximately 8 and 10 times that of the wild type, respectively, indicating a significant improvement in thermal stability. The improved thermal stability of nucleoside hydrolases helps reduce the rate of thermal inactivation during production, transportation, application, and storage.

[0120] Example 6: Application of the mutant NH-S190V in hydrolyzing adenosine, guanosine, and inosine to produce adenine, guanine, and hypoxanthine.

[0121] 1. Adenosine as substrate

[0122] The crude enzyme solution (0.13 mg / ml) of the nucleoside hydrolase mutant NH-S190V prepared by the method in Example 4 was used as a catalyst. Adenosine was used as the substrate, and a pH 4.5 acetate-sodium acetate buffer was used as the reaction medium. The protein concentration in the catalyst was 130 mg / L (based on buffer volume), and the substrate concentration was 100 g / L (based on buffer volume). The catalytic reaction was carried out at 50 °C, pH 4.5, and 50 rpm for 8 h. The enzyme activity was detected by the method in Example 1. The liquid chromatogram is shown in [Figure 1]. Figure 8 and Figure 9 As shown.

[0123] With adenosine as the catalytic catalyst, the conversion rate of adenosine to adenine reached 70% after 5 hours of hydrolysis, and 100% after 8 hours of catalysis. The initial and 8-hour liquid chromatography results are shown in [Figure number missing]. Figure 8 .

[0124] 2. Guanosine and inosine are substrates

[0125] In step 1, adenosine was replaced with guanosine and inosine, and enzyme activity was detected using the method described in Example 1.

[0126] Standard curve equation:

[0127] Guanosine y = 4,668,581.0000x + 10,012.9000 R 2 =1.0000;

[0128] Guanine y = 4,402,714.0000x - 13,159.6000 R 2 =0.9999;

[0129] Inosine y = 4,063,648.4000x + 289,589.2000 R 2 =0.9997;

[0130] Hypoxanthine y = 3,761,936.7000x + 1,092,177.1000 R 2 =0.9987.

[0131] The liquid chromatography results of the enzyme's hydrolysis of guanosine or inosine are shown in the figure. Figure 9 (Sampling during the intermediate stage of hydrolysis). The enzyme activities of the mutant NH-S190V in hydrolyzing guanosine and inosine reached 336.9 U / mg and 769 U / mg, respectively.

[0132] The nucleoside enzyme of this invention exhibits a high conversion rate for the hydrolysis of adenosine, guanosine, or inosine.

Claims

1. A nucleoside hydrolase mutant, characterized in that, The nucleoside hydrolase mutant is formed by mutating serine at position 12 of the amino acid sequence shown in SEQ ID NO.1 to proline; or by mutating serine at position 190 to valine.

2. The encoding gene of the nucleoside hydrolase mutant of claim 1.

3. A recombinant genetically engineered bacterium containing the coding gene of the nucleoside hydrolase mutant of claim 2.

4. The recombinant genetically engineered bacteria as described in claim 3, characterized in that, The host strain of the recombinant genetically engineered bacteria is Pichia pastoris GS115.

5. The use of the nucleoside hydrolase mutant of claim 1 in the catalytic preparation of adenine from adenosine.

6. The application as described in claim 5, characterized in that, The application is carried out according to the following steps: the fermentation broth obtained by inducing culture of recombinant genetically engineered bacteria containing the coding gene of nucleoside hydrolase mutant is centrifuged, the supernatant is used as a catalyst, nucleoside is used as a substrate, and a buffer solution with pH 3-5 is used as a reaction medium. The transformation reaction is carried out at 30-70℃ and 50-200rpm. The reaction solution is separated and purified to obtain the product purine.

7. The application as described in claim 6, characterized in that, The nucleosides include adenosine, guanosine, or inosine.

8. The application as described in claim 6, characterized in that, The substrate was added to a final concentration of 50-150 g / L buffer; the catalyst dosage was 100-130 mg / L buffer, based on the protein content in the supernatant.

9. The application as described in claim 6, characterized in that, The catalyst was prepared as follows: (1) Recombinant engineered bacteria were inoculated into BMGY medium and cultured at 30°C and 220 rpm for 16-18 h until OD. 600 Reach 2-6, centrifuge at room temperature, and collect the bacterial cells; (2) Resuspend the bacterial cells in BMMY medium to achieve OD200. 600 The culture medium was 1% and cultured at 30℃ and 220rpm for 3 days; methanol was added to the culture medium every 24 hours until the final volume concentration was 1%; after fermentation, the medium was centrifuged and the supernatant was collected.

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