High-temperature-resistant cellobiose epimerase mutant, engineering bacterium and application of high-temperature-resistant cellobiose epimerase mutant

Abstract

The invention discloses a high-temperature-resistant cellobiose epimerase mutant, an engineering bacterium and application of the high-temperature-resistant cellobiose epimerase mutant. According to the high-temperature-resistant cellobiose epimerase mutant, the half-life period of a mutant enzyme at the temperature of 80 DEG C is prolonged, the half-life period of the mutant DiCE / S184K / K7G / N238P at the temperature of 80 DEG C is 184.2 min and is 4.3 times that of an original enzyme, and the optimal temperature reaches 85 DEG C and is increased by 5 DEG C compared with that before mutation. According to the mutant enzyme disclosed by the invention, the substrate affinity of the enzyme to lactose serving as a substrate for synthesizing lactulose is improved, the conversion efficiency is improved, the yield of the product lactulose is obviously increased, the yield of a by-product epinose is obviously reduced, the yield of the product is increased by 11.8%, and the yield of the by-product is reduced by 1.4%. The method has the advantages of environmental protection, low toxicity, few byproducts and high product yield, overcomes the problem of three wastes easily generated by a chemical synthesis method, and has important industrial application prospects.

Description

(I) Technical Field

[0001] This invention relates to a thermoresistant cellobiose epimerase mutant, an engineered bacterium, and its application in the microbial catalytic isomerization of lactose to produce lactulose. (II) Background Technology

[0002] Lactulose is a reduced disaccharide composed of D-galactose and D-fructose groups linked by a β-1,4 glycosidic bond. It is used to treat chronic constipation and hepatic encephalopathy and is a common over-the-counter drug in over 100 countries, with huge demand. Lactulose can also act as a prebiotic to improve the gut microbiota. However, lactulose production relies on chemical methods, which use environmentally unfriendly catalysts and present significant challenges in downstream separation. In recent years, cellobiose epimerase has been discovered to efficiently catalyze the production of lactose into lactulose. This technology is environmentally friendly, simple, and has strong industrialization potential.

[0003] Biotransformation is a process that utilizes one or more specialized extracellular or intracellular enzymes produced by microorganisms as biocatalysts to convert substrates into products. It is characterized by mild reaction conditions and high raw material utilization, while also exhibiting excellent chemoselectivity, regioselectivity, and stereoselectivity, ensuring the efficient synthesis of target compounds. Currently, using isomerases or cells containing these enzymes as biocatalysts for isomerization reactions to prepare various carbohydrate compounds has become an important economic growth point for the sugar industry.

[0004] Cellobiose 2-epimerase (EC 5.1.1.11, abbreviated as CE) belongs to the N-acetyl-D-glucosamine 2-epimerase (AGE) family. It partially catalyzes the isomerization of lactose to lactulose, and some of these enzymes originate from thermophilic microorganisms. Caldicellulosiruptor saccharolyticus Cellobiose epimerase is the most widely used and has the most research reports; in addition, there are also epimerases derived from... Dictyoglomus turgidum , Caldicellulosiruptor obsidiansis , Dictyoglomus thermophilum CE can also be used to produce lactulose.

[0005] Although CE catalysis for lactulose production is highly efficient, several problems remain: First, temperature affects the lactulose yield during the isomerization reaction. According to the principle of thermodynamic reaction equilibrium, CE must maintain high activity at high temperatures to produce high concentrations of lactulose. Second, CE catalysis produces the byproduct ipilactose. Although ipilactose is a prebiotic, its content is strictly limited in pharmacopoeias of various countries. Therefore, the lower the ipilactose content in the lactulose synthesis reaction, the better.

[0006] Therefore, obtaining a highly efficient biocatalyst for lactulose preparation is of great significance for meeting the growing sugar intake needs of the people. (III) Summary of the Invention

[0007] The purpose of this invention is to provide a heat-resistant cellobiose epimerase mutant, its encoding gene, an engineered bacterium, and its application in the microbial catalytic lactose isomerization to prepare lactulose, thus providing a green, environmentally friendly, hygienic, and safe biological method for lactulose preparation.

[0008] The technical solution adopted in this invention is:

[0009] This invention provides a thermoresistant cellobiose epimerase mutant, which is obtained by performing a single point mutation or a combination mutation on the 184th, 7th, or 238th amino acid sequence shown in SEQ ID NO. 1.

[0010] Preferably, the mutant is obtained by mutating one, two, or three amino acids from the 184th, 7th, and 238th positions of the amino acid sequence shown in SEQ ID NO: 1 to lysine, glycine, or proline (in the case of multiple mutations, each position is mutated independently to one of the three).

[0011] Preferably, the mutant is an amino acid sequence shown in SEQ ID NO: 1 that is mutated to one of the following: (1) the serine at position 184 is mutated to lysine, proline, glycine, threonine, or tyrosine, respectively denoted as S184K, S184P, S184G, S184T, or S184Y; (2) the serine at position 184 (S) is mutated to lysine (K), and the lysine at position 7 is mutated to glycine, tyrosine, or alanine, respectively denoted as S184K / K7G, S184P / K7G, or S184Y. 184K / K7Y, S184K / K7A; (3) The 184th serine is mutated to lysine, the 7th lysine is mutated to glycine, and the 238th asparagine is mutated to proline, phenylalanine, isoleucine and arginine, respectively, and are recorded as S184K / K7G / N238P, S184K / K7G / N238F, S184K / K7G / N238I and S184K / K7G / N238R.

[0012] More preferably, the mutant amino acid sequence is shown in SEQ ID NO.6.

[0013] This invention also relates to a coding gene for the thermostable cellobiose epimerase mutant, a recombinant vector containing the coding gene, and a recombinant engineered bacterium containing the recombinant vector; the recombinant vector is based on pET28b, with the insertion site... Xba I and Xho I, the recombinant genetically engineered bacteria asE. coli BL21(DE3) is the host bacterium.

[0014] This invention also provides an application of the thermostable cellobiose epimerase mutant in the catalytic preparation of lactose from lactose, wherein the application is: using a recombinant genetically engineered bacterium (preferably) containing the gene encoding the cellobiose epimerase mutant. E. coli Using wet cell culture of BL21(DE3) / DiCE / S184K / K7G / N238P obtained by fermentation or pure enzyme solution extracted by ultrasonic disruption of wet cell culture as a biocatalyst, lactose as a substrate and buffer solution of pH 6-8 as a reaction medium constitute the reaction system. The reaction is carried out at 65-85℃ and 100-300 r / min. After the reaction is complete, the reaction solution is separated and purified to obtain lactulose.

[0015] Preferably, in the reaction system, the final concentration of lactose added is 300-400 g / L, preferably 350 g / L; the amount of catalyst added in wet cell form is 100-150 g / L, preferably 135 g / L; the amount of catalyst added in pure enzyme form, based on protein content, is 1.0-1.8 mg / mL, preferably 1.3 mg / mL. The preferred reaction medium is a 50 mM HEPES buffer solution at pH 7.5; the preferred reaction conditions are 85℃ and 200 r / min.

[0016] Preferably, the wet bacterial cells can be prepared as follows: Recombinant genetically engineered bacteria containing a cellobiose epimerase mutant encoding gene, stored at -80℃, are streaked onto LB solid medium containing a final concentration of 50 μg / mL kanamycin, and incubated upside down at 37℃ for 12 h. Single colonies are then picked and inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance, and cultured at 37℃ and 150-220 r / min until OD... 600 =0.6-0.8; culture medium at 2% ( v / v The transfer volume was transferred to LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance and cultured at 37°C and 150-220 r / min until OD. 600 =0.6-0.8, add isopropyl thiogalactoside (IPTG) to a final concentration of 0.1 mM to induce expression, and induce fermentation at 28℃ and 150-200 r / min for 8-10 h. Centrifuge, discard the supernatant, and collect the wet cells.

[0017] Preferably, the pure enzyme solution is prepared as follows: Wet cells of recombinant genetically engineered bacteria containing the cellobiose epimerase mutant encoding gene are resuspended at 1 g wet cells in 20 mL of 50 mM HEPES (pH 7.5) buffer. The mixture is ultrasonically disrupted at 200 W for 20 min, with 2 s intervals and 3 s intervals. The disrupted mixture is centrifuged at 8000 r / min for 10 min, and the supernatant is collected as the crude enzyme solution, used as the loading solution. Purification is performed using a Nickel-NTA affinity chromatography column (Bio-ScaleMini Profinity IMAC pre-packed column, 40 mm long × 12.6 mm inner diameter). The column is first equilibrated with equilibration buffer (20 mM phosphate buffer, 300 mM NaCl, 20 mM imidazole, pH 8.0). The loading solution is loaded at a rate of 1 mL / min for 4 column volumes. Then, elution buffer (50 mM phosphate buffer, 300 mM NaCl, 500 mM HEPES (pH 7.5)) is used. Elution was performed at a rate of 1 mL / min using mM imidazole (pH 8.0). The eluent was collected when the signals from the UV detector and the conductivity detector both increased simultaneously. Collection was stopped when the conductivity detector signal remained unchanged and the UV detector signal decreased. This eluent was the pure enzyme solution.

[0018] Preferably, the method for separating and purifying the reaction solution to prepare lactulose is as follows: after centrifuging the reaction solution at 8000 r / min for 20 min, the bacterial cells are removed, boric acid resin is added to the supernatant, and the solution is allowed to stand at 28℃ for 2 h before filtration. The obtained resin is washed twice with ultrapure water, and ultrapure water is added. The pH is adjusted to 2 with HCl, and the resin is removed again with a filter (the resin can be packed into a column, washed, and reused). The resulting acidic solution is adjusted to pH 7.5 with NaOH, and the solution is distilled under reduced pressure until no liquid flows out to obtain lactulose. The amount of boric acid resin added is 120-150 g / L based on the volume of the supernatant.

[0019] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0020] This invention utilizes genetic engineering to mutate wild-type enzymes, improving the short half-life of the original enzyme at high temperatures. A thermostable cellobiose epimerase mutant was obtained, enhancing the half-life of the mutant enzyme at 80°C. Specifically, the mutant DiCE / S184K / K7G / N238P exhibits a half-life of 184.2 min at 80°C, 4.3 times that of the original enzyme. The optimal temperature for this mutant reaches 85°C, an increase of 5°C compared to the original enzyme.

[0021] The mutant enzyme of this invention has a relatively long reaction time, which improves the substrate affinity of the enzyme for lactose, the substrate used to synthesize lactulose. Biotransformation using genetically engineered bacteria containing the mutant enzyme improves the transformation efficiency, significantly increases the yield of the product lactulose, and significantly reduces the yield of the byproduct ipilactose. The product yield is increased by 11.8%, and the byproduct yield is reduced by 1.4%.

[0022] The synthesis of lactulose using cellobiose epimerase mutants of this invention has the advantages of being green and environmentally friendly, having low toxicity, few by-products, and high product yield. It overcomes the three wastes problems that are easily generated by chemical synthesis methods and has important industrial application prospects. (iv) Description of the attached drawings

[0023] Figure 1 Electrophoresis diagrams of DiCE and DtCE expression; Lane 1: crude DiCE enzyme solution; Lane 2: crude DtCE enzyme solution.

[0024] Figure 2 This is a high-performance liquid chromatogram of a mixture of lactulose, lactose, and ipilactose standards.

[0025] Figure 3 The optimal reaction temperatures for DiCE and DiCE / S184K / K7G / N238P are given.

[0026] Figure 4 The half-life of DiCE and DiCE / S184K / K7G / N238P.

[0027] Figure 5 This is a diagram showing the conversion process of DiCE.

[0028] Figure 6 This is a conversion process diagram for DiCE / S184K / K7G / N238P. (V) Detailed Implementation Methods

[0029] 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:

[0030] Example 1: Screening and Activity Assay of Novel Cellobiose Epimerases

[0031] 1. Screening of potential CEs and construction of recombinant expression plasmids

[0032] Four potential CE strains were selected from the NCBI database, respectively from... Dictyoglomus sp. ob1-4 (GenBank ID WP_148806989.1) Dictyoglomus turgidum (GenBank ID YP_002352551.1) Dictyoglomaceaebacterium (GenBank accession number HOL39840.1) and Dictyoglomus (GenBank ID WP_424662166.1), named DiCE (amino acid sequence as shown in SEQ ID NO.1), DtCE, DbCE, and PsCE. Codon optimization was performed based on the codon preference of *E. coli*, and nucleotide sequences were synthesized using conventional genetic engineering methods. The nucleotide sequences of DiCE, DtCE, DbCE, and PsCE are shown in SEQ ID NO.2-5, respectively. A 6×his-tag was added to the end of the nucleic acid sequence, and restriction enzyme sites Xba I and Xho I were added to both ends. The above genes were cloned into the Xba I and Xho I sites corresponding to pET28b(+), obtaining recombinant expression plasmids pET28b / DiCE, pET28b / DtCE, pET28b / DbCE, and pET28b / PsCE.

[0033] SEQ ID NO.1

[0034] MDLKTLKNEVKNHLTEKIIPFWAKLMDKENGGYIGYVSFDLKKDPYAHKSLVLTTRILWFFSAVYNLTKEENLIPYMNHAYSFLVQKLWDHKNKGFYWMVDYKGEPIDKRKHIYGHAFSIYALSEFYKATKKDEALNIALETYNLLEEKCKDEYAYLEEFDEYWNPKENKAISEYGIITEKSMSSLLHILEAYTNLYTT WSHENLKKNIENLVKIFKDKIFNPETKHLGVFFDRKLNNIIDAISYGHDIEATWLLDESLKYINDANLKEEVNRITLEIADQVLEEAFENGSLINEKVRNILDKSRIWWVEAEALVGFLNAYQKSREEKFLNAVIELWKFIKNYMVDQRPDSEWFWKLDENYIPAPMPIVEPWKCPYHNGRMCIEAIKRINEDHHHHHHH

[0035] SEQ ID NO.2

[0036]

[0037] 2. Construction and Induction Expression of the Original Enzyme Recombinant Bacteria

[0038] The recombinant expression plasmids pET28b / DiCE, pET28b / DtCE, pET28b / DbCE, and pET28b / PsCE obtained in step 1 were transformed into [pET28b / DiCE, pET28b / DtCE, pET28b / DbCE, and pET28b / PsCE respectively]. E.coli BL21(DE3) recipient bacteria were plated on LB agar plates containing a final concentration of 50 μg / mL kanamycin. After incubation at 37°C for 12 h, colonies were randomly selected from the grown colonies, and plasmids were extracted for identification by agarose gel electrophoresis and nucleotide sequencing. Genetically engineered bacteria containing DiCE, DtCE, DbCE, and PsCE were obtained, which are the recipient bacteria. E.coli BL21(DE3) / pET28b / DiCE, E.coli BL21(DE3) / pET28b / DtCE, E.coli BL21(DE3) / pET28b / DbCE, E.coli BL21(DE3) / pET28b / PsCE.

[0039] The above-mentioned genetically engineered bacteria were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 150 r / min. OD 600 Seed culture was obtained by adjusting the growth rate to 0.6-0.8. The seed culture was then inoculated at a volume concentration of 2% into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and cultured at 37℃ and 150 r / min. OD 600 To a concentration of 0.6-0.8, IPTG was added to the culture medium to a final concentration of 1 mM. Expression was induced at 28℃ for 10 h, followed by centrifugation at 4℃ and 8000 r / min for 10 min. The supernatant was discarded, and the wet bacterial cells were washed twice with 0.85% physiological saline and collected for later use. The obtained wet bacterial cells were sonicated, and the supernatant was used for SDS-PAGE analysis of protein solubility. The results are shown below. Figure 1 Only DiCE and DtCE are soluble.

[0040] LB liquid medium composition: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, water as solvent, natural pH; LB solid medium is LB liquid medium with 20 g / L agar added; autoclave at 121℃ for 20 min; add kanamycin to a final concentration of 50 μg / mL before use.

[0041] 3. Enzyme activity detection of the original enzyme

[0042] The final concentration of the 1.8 mL reaction system consisted of 50 mM HEPES buffer (pH 7.5), 50 g / L lactose, and 25 g / L wet bacterial cells, totaling 1.8 mL. Reaction conditions: The reaction was terminated at 80℃ for 10 min, followed by an ice bath for 10 min. The peak area of ​​lactulose in the reaction solution was determined by HPLC. A standard curve of peak area versus concentration was prepared under the same conditions using mixed aqueous solutions of lactulose, ipilactose, and lactose standards at a mass ratio of 1:0.5:1 with different total mass concentrations. The lactulose content in the reaction solution was calculated, and the enzyme activity was thus obtained. The results are shown in Table 1.

[0043] HPLC detection conditions: Agilent 1260 HPLC system, Agilent autosampler, Shodex VG-50-4E column, Agilent differential detector, mobile phase 75% ( v / v Acetonitrile, 20% v / v A mixed solution of methanol and 5% ultrapure water was used. The column temperature was set at 40℃, and the flow rate was 1 mL / min. The external standard method was employed to determine the yield of lactulose based on the peak retention time and peak area. The liquid chromatography chromatogram of a mixed aqueous solution prepared by dissolving 1 mg of lactulose, lactose, and epilactose standards in 1 mL of deionized water at a mass ratio of 1:0.5:1 is shown below. Figure 2 As shown, the peak elution times for lactulose were 8.4 min, lactose was 10.3 min, and epilactose was 9.3 min.

[0044] Enzyme activity is defined as the amount of enzyme required per second to isomerize lactose into 1 μmol of lactulose at 80°C and pH 7.5.

[0045] Table 1. Isomerization activity determination of DiCE, DtCE, DbCE and PsCE at 80℃

[0046]

[0047] As shown in Table 1, at 80℃, E. coli The enzyme activity of BL21(DE3) / pET28b / DiCE is 42.4 U / g. E.coli The enzyme activity of BL21(DE3) / pET28b / DtCE was 19.1 U / g, while the other two showed no enzyme activity. The one that performed best at 80℃ was selected. E.coli BL21(DE3) / pET28b / DiCE underwent targeted evolution modification.

[0048] Example 2: Directed Evolution of the Primitive Enzyme DiCE

[0049] 1. Construction of mutant libraries

[0050] Using plasmid pET28b / DiCE as a template, error-prone PCR forward and reverse primers for directed evolution were designed and synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0051] Forward primer: ATGGACCTGAAAACGCTGAAAAACG;

[0052] Reverse primer GTCTTCGTTAATACGTTTAATTGCTTCAATG.

[0053] Error-prone PCR system: 10× Taq Buffer (Mg 2+ 5 μL of free MgCl2, 3.6 μL of 25 mM MgCl2, 1.1 μL of 4.5 mM MnCl2, 1 μL of dNTP Mix (10 mM each), 1 μL each of error-prone forward and reverse PCR primers, and 10 ng of plasmid template. Taq Add 0.5 μL of DNA Polymerase (5 U / μL) and bring the total volume to 50 μL with ultrapure water.

[0054] Error-prone PCR reaction conditions: pre-denaturation 95℃ 3 min; (denaturation: 95℃ 30 s, annealing: 65℃ 30 s, extension: 72℃ 1 min) 30 cycles; final 72℃ 5 min.

[0055] Take 5 μL of PCR product and add it to 100 μL of ice bath. E.coli In a suspension of BL21(DE3) competent cells, the cells were placed on ice for 30 min. The transformation product was then heat-shocked at 42℃ for 90 s and rapidly cooled on ice for 2 min. 600 μL of LB liquid medium was added to the tube, and the cells were incubated at 37℃ and 200 r / min for 30 min. 100 μL of the above bacterial suspension was spread onto an LB solid medium plate containing 50 μg / mL kanamycin. After the bacterial suspension was completely absorbed by the medium, the cells were incubated upside down at 37℃ for 12 h. The colonies that grew were the positive transformants.

[0056] 2. High-throughput screening of positive transformants

[0057] (1) After sterilization, 900 mL of LB liquid culture medium was added to each well of the 96-well plate. The positive transformants obtained in step 1 were picked up with a sterile toothpick and placed in the plate. The plate was then incubated at 37°C and 200 r / min for 8 h.

[0058] (2) Take another new 96-well plate and add LB liquid medium. Use a pipette to transfer the bacterial solution from step (1) at 4% ( v / v The cells were transferred into new 96-well plates. The original 96-well plates were stored at 4°C. The new 96-well plates were incubated at 37°C and 200 r / min for 2 h. Then, IPTG was added to each well to a final concentration of 0.1 M, and expression was induced at 28°C and 200 r / min for 12 h.

[0059] (3) Centrifuge the 96-well plate after induction of expression at 8000 r / min for 10 min, retain the bacterial cells, add 300 μL of 50mM HEPES (pH7.5) buffer to resuspend the bacterial cells, incubate at 80℃ for 20 min, then add 200 μL of 25 g / L lactose aqueous solution, react at 80℃ for 10 min, and stop the reaction by incubating on ice for 10 min.

[0060] (4) Screening mutants in ELISA plates using the tryptophan-cysteine ​​hydrochloride colorimetric method. Preparation of the tryptophan-cysteine ​​hydrochloride colorimetric reagent: Weigh 1.25 g of cysteine ​​hydrochloride and 40 mg of tryptophan, add 30 mL of ultrapure water to dissolve, then add 0.5 mL of 1M HCl, stir until completely dissolved, and bring the volume to 50 mL. Store at room temperature. The reaction system is: 140 μL of 75% concentrated sulfuric acid, 50 μL of the reaction solution from step (3), and 10 μL of tryptophan-cysteine ​​hydrochloride colorimetric reagent. React at 46℃ for 30 min. Using the ELISA plate... E. coli Using BL21(DE3) / pET28b / DiCE as a control, mutant strains with a darker color than the wild type were screened, and the concentrations in the screening reaction solution were determined using a UV spectrophotometer. A 518 Value. Through screening 837 recombinant bacteria strains, three strains of A518 with absorbance values ​​higher than [value missing]. E. coli The recombinant strain BL21(DE3) / pET28b / DiCE was sequenced. Sequencing results showed that the recombinant strain contained mutant enzymes DiCE / S184P, DiCE / K7G, and DiCE / N238F, respectively.

[0061] 3. Enzyme activity assay

[0062] Enzyme activity was determined using the HPLC method described in Example 1. E. coli BL21(DE3) / pET28b / DiCE-S184P, E. coli BL21(DE3) / pET28b / DiCE-K7G, E. coliThe residual enzyme activities of BL21(DE3) / pET28b / DiCE-N238F were 75.5, 27.5, and 58.5 U / g (wet cell), respectively, all higher than the original enzyme DiCE's 26.8 U / g (wet cell). These results indicate that sites 184, 7, and 238 are potential sites that can improve the enzyme's thermostability.

[0063] Table 2. Enzyme activity and residual enzyme activity determination of each mutant

[0064]

[0065] Example 3: Saturation mutation at site 184 of DiCE

[0066] To determine the optimal amino acid at position 184 of DiCE, degenerate primers were designed based on pET28b / DiCE and the DiCE / S184P obtained in Example 2, using rapid PCR technology to perform a saturation mutation at position 184. The primers were:

[0067] Forward primer TATGGCATTATCACC NNK AAAAGCATGAATAGC (The underscores represent mutant bases; N represents A / T / C / G, and K represents G / T)

[0068] Reverse primer GCTATTTCATGCTTTT MNN GGTGATAATGCCATA (underlined bases represent mutant bases, N represents A / T / C / G, and M represents C / A)

[0069] PCR reaction system: 2×PhantaMax Buffer (containing Mg) 2+ 25 μL, 1 μL of dNTP Mix (10 mM each), 1 μL each of the forward and reverse primers for the above saturation mutation, 50 ng of template, 1 μL of Phanta Max Super-Fidelity DNA Polymerase, and add ultrapure water to 50 μL.

[0070] PCR amplification conditions were: 98℃ for 5 min; (98℃ for 30 s, 70℃ for 30 s, 72℃ for 39 s) for 30 cycles; 72℃ for 10 min.

[0071] PCR product transformation E. coli BL21(DE3) competent cells were used to perform high-throughput screening of 692 mutant strains using the method described in Example 2. The enzyme activities of the four initially screened positive mutants were then detected using the same method. The results are shown in Table 3. E. coliCompared to BL21(DE3) / pET28b / DiCE-S184P, E. coli BL21(DE3) / pET28b / DiCE-S184K, E. coli BL21(DE3) / pET28b / DiCE-S184T, E. coli The enzyme activity of BL21(DE3) / pET28b / DiCE-S184Y was significantly increased, with the highest enzyme activity of the mutant DiCE / S184K, which was 80.5 U / g (wet cell).

[0072] Table 3 Enzyme activity of recombinant bacteria with DiCE 184 site mutation

[0073]

[0074] Example 4: 7-site iterative saturation mutation of DiCE / S184K

[0075] To determine the optimal amino acid at position 7 of DiCE / S184K, degenerate primers were designed using pET28b / DiCE / S184K as a template. A single-point mutation was introduced at position 7 using rapid PCR. The primers used were:

[0076] Forward primer AAAACGCTG NNK AACGAAGTT (underlined bases are mutant bases)

[0077] Reverse primer AACTTCGTT MNN CAGCGTTTT (underlined bases are mutant bases)

[0078] PCR reaction system: 2×PhantaMax Buffer (containing Mg) 2+ 25 μL, 1 μL of dNTP Mix (10 mM each), 1 μL each of the forward and reverse primers for the above saturation mutation, 50 ng of template, 1 μL of Phanta Max Super-Fidelity DNA Polymerase, and add ultrapure water to 50 μL.

[0079] PCR amplification conditions were: 98℃ for 5 min; (98℃ for 30 s, 56℃ for 30 s, 72℃ for 39 s) for 30 cycles; 72℃ for 10 min.

[0080] PCR product transformation E. coli BL21(DE3) competent cells were used to perform high-throughput screening on 727 mutant strains using the method described in Example 2. Three positive mutants were selected, and their residual enzyme activity was then measured. The results are shown in Table 4. It can be seen that... E.coli Compared to BL21(DE3) / pET28b / DiCE-S184K, E. coli BL21(DE3) / pET28b / DiCE-S184K-K7G, E. coli BL21(DE3) / pET28b / DiCE-S184K-K7Y、 E. coli The enzyme activities of BL21(DE3) / pET28b / DiCE-S184K-K7A were significantly increased, with the mutant showing the highest enzyme activity being [missing information]. E. coli BL21(DE3) / pET28b / DiCE-S184K-K7G has a residual enzyme activity of 99.6 U / g (wet cell).

[0081] Table 4 Enzyme activity of the DiCE / S180K 7-site mutant recombinant bacteria

[0082]

[0083] Example 5: 238-bit iterative saturation mutation of DiCE-S184K-K7G

[0084] Using pET28b / DiCE-S184K-K7G obtained in Example 4 as a template, degenerate primers were designed, and a single-point mutation was introduced at position 238 using rapid PCR technology. The primers were:

[0085] Forward primer AAACTGAAC NNK ATTATAGACGC (underlined bases are mutant bases)

[0086] Reverse primer GCGTCTATAAT MNN GTTCAGTTT (underlined bases are mutant bases)

[0087] PCR reaction system: 2×PhantaMax Buffer (containing Mg) 2+ 25 μL, 1 μL of dNTP Mix (10 mM each), 1 μL each of the forward and reverse primers for the above saturation mutation, 50 ng of template, 1 μL of Phanta Max Super-Fidelity DNA Polymerase, and add ultrapure water to 50 μL.

[0088] PCR amplification conditions were 98℃ for 5 min; (98℃ for 30 s, 59℃ for 30 s, 72℃ for 39 s) for 30 cycles; 72℃ for 10 min.

[0089] PCR product transformation E. coliBL21(DE3) competent cells were used to screen 535 mutant strains using the method described in Example 2. Four positive mutants were selected, and their residual enzyme activity was then measured. The results are shown in Table 5. E. coli BL21(DE3) / pET28b / DiCE-S184K-K7G was used as a control. E. coli BL21(DE3) / pET28b / DiCE-S184K-K7G-N238P, E. coli BL21(DE3) / pET28b / DiCE-S184K-K7G-N238F、 E. coli BL21(DE3) / pET28b / DiCE-S184K-K7G-N238I、 E. coli The enzyme activities of BL21(DE3) / pET28b / DiCE-S184K-K7G-N238R were all significantly increased, with the mutant showing the highest enzyme activity. E. coli The residual enzyme activity of BL21(DE3) / pET28b / DiCE-S184K-K7G-N238P is 124.7 U / g (wet cell). The amino acid sequence and nucleotide sequence of DiCE / S184K / N238P are shown in SEQ ID No. 6 and SEQ ID No. 7, respectively.

[0090] Table 5 Enzyme activity assay of the three-point mutant recombinant bacteria

[0091]

[0092] Example 6: Induction and purification of the original enzyme DiCE and the mutant enzyme DiCE-S184K-K7G-N238P

[0093] 1. Recombinant bacterial induction expression

[0094] E.coli BL21(DE3) / pET28b / DiCE, E.coli BL21(DE3) / pET28b / DiCE-S184K-K7G-N238P were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 150 r / min. OD 600 Seed culture was obtained by adjusting the growth rate to 0.6-0.8. The seed culture was then inoculated at a volume concentration of 2% into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and cultured at 37℃ and 150 r / min. OD 600When the concentration reaches 0.6-0.8, add IPTG to the culture medium to a final concentration of 1 mM. Induce expression at 28℃ and 150 r / min for 10 h. Centrifuge at 4℃ and 8000 r / min for 10 min, discard the supernatant, wash the wet cells twice with 0.85% physiological saline, and collect the wet cells.

[0095] 2. Obtain crude enzyme solution

[0096] Resuspend 1 g of wet bacterial cells in 20 mL of 50 mM HEPES (pH 7.5) buffer, sonicate at 200 W for 20 min with 2 s working intervals and 3 s intervals, centrifuge the mixture at 8000 r / min for 10 min, and collect the supernatant as the crude enzyme solution, which is used as the loading solution.

[0097] 2. Separation and purification of DiCE and DiCE-S184K-K7G-N238P

[0098] Purification was performed using a Nickel-NTA affinity chromatography column (Bio-Scale Mini Profinity IMAC pre-packed column, 40 mm long × 12.6 mm inner diameter). The column was first equilibrated with equilibration buffer (20 mM phosphate buffer, 300 mM NaCl, 20 mM imidazole, pH 8.0). 20 mL (4 column volumes) of sample buffer was loaded at a rate of 1 mL / min. Elution was then performed with elution buffer (50 mM phosphate buffer, 300 mM NaCl, 500 mM imidazole, pH 8.0) at a rate of 1 mL / min. Based on the signal responses of the UV and conductivity detectors, the corresponding eluent was collected when both signals increased simultaneously, and collection was stopped when the conductivity detector signal remained unchanged and the UV detector signal decreased. These were the purified enzyme solutions. The protein concentrations of the purified enzyme solutions were tested using a BCA kit, yielding results of 1.58 mg / mL and 2.04 mg / mL, respectively.

[0099] Example 7: Determination of the optimal temperature for DiCE and DiCE-S184K-K7G-N238P

[0100] The pure enzyme solutions of DiCE and DiCE-S184K-K7G-N238P prepared by the method in Example 6 were used as the conversion enzymes, and the optimal reaction temperature of the enzymes was determined. The final concentration composition of 1 mL of reaction system was: 100 g / L lactose and 0.5 mg / mL pure enzyme solution based on protein content, followed by the addition of 50 mM HEPES (pH 7.5) buffer to a total volume of 1 mL. The reaction was carried out at different temperatures (60, 65, 70, 75, 80, 85, 90 °C) for 10 min, and the relative enzyme activity was detected by HPLC as described in Example 1. The results are shown in [Figure 1]. Figure 3 The optimal reaction temperature for the mutant enzyme DiCE-S184K-K7G-N238P is 85℃, which is 5℃ higher than that of the original enzyme DiCE.

[0101] Example 8: Determination of thermal stability of DiCE and DiCE-S184K-K7G-N238P

[0102] The pure enzyme solutions of DiCE and DiCE-S184K-K7G-N238P prepared by the method in Example 6 were placed in a water bath at 80°C for 120 min, and samples were taken every 15 min. The residual enzyme activity was determined using the reaction system and method in Example 7, and the enzyme half-life was calculated. The results are shown in […]. Figure 4 The half-life of DiCE at 80℃ is 42.3 min, while the half-life of the mutant enzyme DiCE-S184K-K7G-N238P at 80℃ is 184.2 min, which is 4.3 times that of DiCE.

[0103] Example 9: Calculation of kinetic parameters of DiCE and DiCE-S184K-K7G-N238P

[0104] Different concentrations (50, 100, 150, 200, 300, 400, 600, and 800 mM) of lactose, 0.4 mg / mL pure enzyme solution (based on protein content), and an appropriate amount of 50 mM HEPES (pH 7.5) buffer were added to a total reaction volume of 1 mL. The reaction was terminated by incubating at 70°C for 20 min and then on ice for 10 min. Enzyme activity was detected using the HPLC method described in Example 1. The kinetic parameters of DiCE and DiCE-S184K-K7G-N238P were obtained by fitting with Origin software. The results are shown in Table 6. DiCE's... K m 103.1 mM k cat 3117.1 s -1 , k cat / K m It is 30.3 s-1 ·mM -1 DiCE-S184K-K7G-N238P K m 82.1mM, k cat It is 7023.3 s -1 , k cat / K m It is 85.5 s -1 ·mM -1 DiCE-S184K-K7G-N238P exhibits better affinity for the substrate lactose and higher catalytic efficiency.

[0105] Table 6. Kinetic parameters of DiCE and DiCE-S184K-K7G-N238P

[0106]

[0107] Example 10: Lactulose synthesis by recombinant bacteria containing DiCE and DiCE-S184K-K7G-N238P.

[0108] Prepared according to the method in step 1 of Example 6 E.coli BL21 (DE3) / pET28b / DiCE and E.coli Wet bacterial cells of BL21(DE3) / pET28b / DiCE / S184K / K7G / N238P were used as a biocatalyst to bioconvert lactose into lactulose.

[0109] The final concentration of the 100 mL reaction system consisted of 350 g / L lactose and 135 g / L wet bacterial cells, with an appropriate amount of 50 mM HEPES buffer (pH 7.5) added to a total volume of 100 mL. The reaction system was incubated at 85 °C and 200 r / min for 3 h. After filtering the reaction solution through a 0.22 μm membrane, the concentrations of lactose, lactulose, and epilactose in the filtrate were determined by HPLC as described in Example 1, and a transformation progress graph was plotted.

[0110] Depend on Figure 6 The transformation process showed that the substrate conversion rate of the recombinant bacteria containing DiCE-S184K-K7G-N238P was 75.5%, the lactulose yield was 66.6%, and the ipilactose yield was 8.9%. In comparison, the substrate conversion rate of the recombinant bacteria containing DiCE was 65.1%, the lactulose yield was 54.8%, and the ipilactose yield was 10.3%. Figure 5The recombinant strain DiCE-S184K-K7G-N238P exhibited a higher transformation level than DiCE, while generating fewer byproducts.

[0111] Example 11: Isolation and purification of lactulose

[0112] Preparation of borate resin: Take 50 g of macroporous resin type 203 (provided by Xi'an Lanxiao Technology), swell it with 100 mL of dimethylformamide at room temperature for 24 h, separate by suction filtration, dry at 50℃, and then mix it with 400 mL of organic solvent (anhydrous methanol: glutaraldehyde: water = 50:1:3) in an Erlenmeyer flask. v / v / v The mixture was stirred in the dark for 12 h, filtered, and washed three times with anhydrous methanol to obtain the pretreated resin. 8 g of the pretreated resin was placed in a reaction flask, and 25 mL of anhydrous methanol and 8 g of 3-aminophenylboronic acid powder were added. The flask was tightened and the mixture was shaken in a shaker at 45°C for 24 h. 10 mL of sodium cyanoborohydride was added to the reaction flask, and the mixture was reduced and amination at low temperature (4°C) for 24 h. The filtered resin was washed successively with acetone, deionized water, and methanol, and then dried under vacuum at 60°C for 12 h to obtain 7.5 g of borate resin.

[0113] In Example 10 E.coli The BL21(DE3) / pET28b / DiCE-S184K-K7G-N238P reaction solution was centrifuged at 8000 r / min for 20 min to remove bacterial cells. 15 g of boric acid resin was added to 100 mL of the supernatant, and the mixture was allowed to stand at 28℃ for 2 h before filtering. The resulting resin was washed twice with ultrapure water, and then 50 mL of ultrapure water was added. The pH was adjusted to 2 with HCl, and the resin was removed again using a filter (the resin can be packed into a column, washed, and reused). The resulting acidic solution was adjusted to pH 7.5 with NaOH to obtain a lactulose solution. The lactulose purity of the lactulose solution described in Example 1 was determined by HPLC, and almost no lactose or ipilactose peaks were observed. The lactulose product was obtained by vacuum distillation until no liquid flowed out, with a recovery rate of 90%.

Claims

1. A thermoresistant cellobiose epimerase mutant, characterized in that, The mutant was obtained by performing a single point mutation or a combination mutation on the 184th, 7th, or 238th amino acid sequence shown in SEQ ID NO.

1.

2. The mutant as described in claim 1, characterized in that, The mutant is an amino acid sequence shown in SEQ ID NO: 1 that is mutated to one of the following: (1) the 184th serine is mutated to lysine, proline, glycine, threonine, or tyrosine; (2) the 184th serine is mutated to lysine, and the 7th lysine is mutated to glycine, tyrosine, or alanine; (3) the 184th serine is mutated to lysine, the 7th lysine is mutated to glycine, and the 238th asparagine is mutated to proline, phenylalanine, isoleucine, or arginine.

3. The mutant as described in claim 1, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.

6.

4. A recombinant genetically engineered bacterium containing the encoding gene of the thermostable cellobiose epimerase mutant as described in claim 1.

5. The application of the heat-resistant cellobiose epimerase mutant of claim 1 in the catalytic preparation of lactose from lactose.

6. The application as described in claim 5, characterized in that, The application is as follows: using wet bacterial cells obtained by fermentation culture of recombinant genetically engineered bacteria containing cellobiose epimerase mutant encoding gene, or pure enzyme solution extracted by ultrasonic disruption of wet bacterial cells, as a biocatalyst, lactose as substrate, and a buffer solution with pH 6-8 as the reaction medium to form a reaction system, the reaction is carried out at 65-85℃ and 100-300 r / min. After the reaction is complete, the reaction solution is separated and purified to obtain lactulose.

7. The application as described in claim 6, characterized in that, In the reaction system, the final concentration of lactose added is 300-400 g / L; the amount of catalyst added in the form of wet bacterial cells is 100-150 g / L; and the amount of catalyst added in the form of pure enzyme is 1.0-1.8 mg / mL based on protein content.

8. The application as described in claim 6, characterized in that, The reaction medium was a 50 mM HEPES buffer solution at pH 7.5; the reaction conditions were 85℃ and 200 r / min.

9. The application as described in claim 6, characterized in that, The wet bacterial cells can be prepared as follows: Recombinant genetically engineered bacteria containing the cellobiose epimerase mutant encoding gene, stored at -80℃, are streaked onto LB solid medium containing a final concentration of 50 μg / mL kanamycin. The culture is incubated at 37℃ for 12 h inverted position. Single colonies are picked and inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance. The culture is then carried out at 37℃ and 150-220 r / min until OD500. 600 =0.6-0.8; the culture medium was transferred at a volume concentration of 2% to LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and cultured at 37℃ and 150-220 r / min until OD. 600 =0.6-0.8, add isopropyl thiogalactoside to a final concentration of 0.1 mM to induce expression, and induce fermentation at 28℃ and 150-200 r / min for 8-10 h. Centrifuge, discard the supernatant, and collect the wet cells.

10. The application as described in claim 6, characterized in that, The pure enzyme solution was prepared as follows: Resuspension of 1 g of wet bacterial cells from recombinant genetically engineered bacteria containing the cellobiose epimerase mutant coding gene in 20 mL of pH 7.5, 50 mM HEPES buffer was performed. The mixture was sonicated at 200 W for 20 min, with 2 s intervals and 3 s intervals between cycles. The mixture was then centrifuged at 8000 r / min for 10 min, and the supernatant was collected as the crude enzyme solution, used as the loading solution. Purification was performed using a Nickel-NTA affinity chromatography column. The column was first equilibrated with equilibration buffer. Four column volumes of the loading solution were loaded at 1 mL / min. Elution was then performed with elution buffer at 1 mL / min. The elution buffer was collected when both the UV detector signal and the conductivity detector signal increased simultaneously. Collection was stopped when the conductivity detector signal remained unchanged and the UV detector signal decreased. This was the pure enzyme solution. The equilibration buffer consisted of: 20 mM phosphate buffer, 300 mM NaCl, 20 mM imidazole, pH 7.

5. 8.0; The eluent composition is: 50 mM phosphate buffer, 300 mM NaCl, 500 mM imidazole, pH 8.0.

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