Method for producing composite rare sugar syrup through whole-cell catalysis

Abstract

The invention relates to the technical field of rare sugar preparation, and discloses a method for producing composite rare sugar syrup through whole-cell catalysis, which comprises the following steps: by taking a fructose-containing substrate as a raw material, carrying out double-enzyme coupling reaction by utilizing tagatose-4-isomerase and psicose-3-isomerase to obtain the composite rare sugar syrup. And preparing the composite rare sugar syrup containing the D-tagatose and the D-psicose. By constructing the recombinant bacteria containing different enzyme genes and double-enzyme combined reaction paths of different production processes, the rare sugar syrup solution containing D-tagatose and D-psicose can be prepared from fructose or high fructose corn syrup raw materials, and the conversion rate and the utilization rate of fructose are remarkably improved.

Description

Technical Field

[0001] The invention relates to the technical field of rare sugar preparation, in particular to a method for producing composite rare sugar syrup by whole-cell catalysis. Background Art

[0002] Rare sugars are a type of monosaccharides and sugar alcohols that exist in nature but in very small quantities. They taste similar to sucrose, but have the advantages of low calories, high stability, and no cariogenicity. They can make up for the shortcomings of traditional sweeteners and play an important role in improving the diet of special groups of people. D-allulose and D-tagatose are typical representatives of rare sugars and are currently widely studied. They not only have the characteristics of low calories, but also have important physiological functions such as inhibiting blood sugar increase and body fat accumulation, scavenging free radicals, anti-oxidation and prebiotics; at the same time, they can also effectively improve the flavor and taste of food, and have an auxiliary role in the treatment of specific diseases such as diabetes and obesity.

[0003] Rare sugars are found in low concentrations in nature and are usually mass-produced through chemical or biological methods. However, the current industrial production processes for D-psicose and D-tagatose are relatively complicated, and the costs of raw material procurement and separation and purification are relatively high, which also leads to high market prices for the final products, seriously hindering the promotion and application of these rare sugars. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for producing composite rare sugar syrup by whole-cell catalysis, wherein tagatose-4-isomerase and psicose-3-isomerase are subjected to dual enzyme coupling, and fructose, which is in sufficient supply and relatively cheap in the market, is used as a raw material, and finally converted through a one-step reaction to achieve the production of rare sugar syrups with different ratios of D-tagatose and D-psicose. The production process route is simple in steps, the process is green and environmentally friendly, the cost of raw materials is low and the utilization rate of raw materials is high, and two types of rare sugar components can be obtained at the same time. The rare sugar composite syrup is expected to further promote the promotion and application of rare sugar raw materials.

[0005] The purpose of the present invention is achieved through the following technical solutions: The invention provides a method for producing composite rare sugar syrup by whole-cell catalysis, comprising the following steps: using a substrate containing fructose as a raw material, utilizing tagatose-4-isomerase and psicose-3-isomerase to carry out a double enzyme coupling reaction, and preparing a composite rare sugar syrup containing D-tagatose and D-psicose.

[0006] Preferably, the tagatose-4-isomerase is a tagatose-4-isomerase mutant, wherein the serine at position 125 of the tagatose-4-isomerase having an amino acid sequence as shown in SEQ ID NO.1 is mutated to aspartic acid, the glutamic acid at position 167 is mutated to serine, the serine at position 185 is mutated to glutamine, the valine at position 267 is mutated to methionine, the serine at position 268 is mutated to threonine, the threonine at position 272 is mutated to aspartic acid, the tryptophan at position 306 is mutated to methionine, the lysine at position 337 is mutated to tryptophan, the arginine at position 386 is mutated to valine, and the tyrosine at position 403 is mutated to isoleucine.

[0007] The patent publication number is CN 108884454 A, and the invention name is "Hexuronate C4-isomerase variant with improved D-tagatose conversion activity and method for producing D-tagatose using the same" has disclosed the above tagatose-4-isomerase mutant.

[0008] Preferably, the amino acid sequence of the tagatose-4-isomerase mutant is shown in SEQ ID NO.2.

[0009] Preferably, the DPE is derived from Staphylococcus aureus, named SaDPE, with Genbank number SQA09501.1, and its amino acid sequence is shown in SEQ ID NO.3.

[0010] Preferably, the expression vector used in the construction of the recombinant bacteria is a pET28a(+) plasmid, a pET25b(+) plasmid or a pET-Duet-1 plasmid.

[0011] An expression vector carrying a tagatose-4-isomerase gene or a psicose-3-isomerase gene.

[0012] Preferably, the starting strain in the process of constructing the recombinant bacteria is Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum or Saccharomyces.

[0013] A starting strain carrying a tagatose-4-isomerase gene or a psicose-3-isomerase gene or the above expression vector.

[0014] Preferably, the method for constructing the recombinant bacteria is: connecting the target gene fragment to an expression vector, and then introducing the obtained recombinant expression vector into the starting strain, so as to obtain the recombinant bacteria.

[0015] Preferably, the substrate is fructose or fructose-glucose syrup; in 1 mL of the reaction system, the concentration of fructose in the substrate is 50-500 g / L.

[0016] Preferably, in the dual enzyme coupling reaction, the pH is 8.0-9.0, the temperature is 60-75° C., and the reaction time is 3-12 h.

[0017] Preferably, the dual enzyme coupling reaction is a "one-pot" reaction, that is, recombinant bacteria containing a tagatose-4-isomerase mutant and a recombinant bacteria containing a psicose-3-isomerase are added simultaneously for reaction, or recombinant bacteria containing a tagatose-4-isomerase mutant and a psicose-3-isomerase are added simultaneously for reaction.

[0018] Preferably, the one-pot reaction is as follows: in a 1 mL reaction system, fructose (50-500 g / L), tagatose isomerase recombinant bacteria (0.12-0.15 g, including tagatose isomerase mutants), psicose isomerase recombinant bacteria (0.04 g-0.05 g, including psicose isomerase) and 50 mmol·L -1 Tris-HCl buffer (pH 9.0) was added with 3`5mmol·L -1 Ni 2+ and 1.5~3mmol·L -1 Mg 2+ Add the reaction mixture and react at 60-75°C and 500-800 rpm for 3-6 hours. After the reaction is completed, the reaction solution is centrifuged at 12000 rpm for 3-10 minutes, and the supernatant is filtered through a 0.22 μm filter membrane and analyzed by HPLC.

[0019] As a preferred method, the one-pot reaction is as follows: in a 1 mL reaction system, fructose (50-500 g / L), double-plasmid transformed recombinant bacteria (0.12-0.15 g, containing tagatose isomerase mutant and psicose isomerase) and 50 mmol·L -1 Tris-HCl buffer (pH 9.0) and add 3-5 mmol·L -1 Ni 2+ and 1.5~3mmol·L -1 Mg 2+ Add the reaction mixture and react at 60-75°C and 500-800 rpm for 3-6 hours. After the reaction is completed, the reaction solution is centrifuged at 12000 rpm for 3-10 minutes, and the supernatant is filtered through a 0.22 μm filter membrane and analyzed by HPLC.

[0020] Preferably, the dual enzyme coupling reaction is a "step-by-step" reaction, i.e., recombinant bacteria containing a tagatose-4-isomerase mutant are first added for reaction, and then recombinant bacteria containing psicose-3-isomerase are added for reaction, or, recombinant bacteria containing psicose-3-isomerase are first added for reaction, and then recombinant bacteria containing a tagatose-4-isomerase mutant are added for reaction.

[0021] Preferably, the stepwise reaction is as follows: in a 1 mL reaction system, fructose (50-500 g / L), tagatose isomerase recombinant bacteria (0.12-0.15 g, including tagatose isomerase mutants) and 50 mmol·L -1 Tris-HCl buffer (pH 9.0) was added with 3-5 mmol / L -1 Ni 2+ The reaction mixture was reacted at 60-75°C and 500-800 rpm for 3-6 hours. After the reaction, recombinant psicose isomerase bacteria (0.04-0.05 g, containing psicose isomerase) and 1-2 mmol·L -1 Mg 2+ The reaction was continued for 1 to 3 hours at 60 to 75°C and 500 to 800 rpm. After the reaction was completed, the reaction solution was centrifuged at 12,000 rpm for 3 to 10 minutes, and the supernatant was filtered through a 0.22 μm filter membrane and analyzed by HPLC.

[0022] As a preferred method, the “step-by-step method” reaction is as follows: in a 1 mL reaction system, fructose (50-500 g / L), tagatose isomerase recombinant bacteria (0.12-0.15 g, including tagatose isomerase mutants) and 50 mmol·L -1 Tris-HCl buffer (pH 9.0) was added with 3-5 mmol / L -1 Ni 2+ The reaction mixture was added to the reaction mixture, and the reaction mixture was reacted at 60-75°C and 500-800 rpm for 3-6 hours. After the reaction was completed, the reaction solution was centrifuged at 12000 rpm for 3-10 minutes, and the supernatant obtained by centrifugation was added with recombinant bacteria of psicose isomerase (0.04-0.05 g, containing psicose isomerase) and 1-2 mmol·L -1 Mg 2+ The reaction was continued for 1 to 3 hours at 60 to 75°C and 500 to 800 rpm. After the reaction was completed, the reaction solution was centrifuged at 12,000 rpm for 3 to 10 minutes, and the supernatant was filtered through a 0.22 μm filter membrane and analyzed by HPLC.

[0023] Preferably, the “step-by-step method” reaction is as follows: in a 1 mL reaction system, fructose (50-500 g / L), recombinant psicose isomerase bacteria (0.04-0.05 g, containing psicose isomerase) and water are contained, sodium bicarbonate or sodium carbonate is used to adjust the pH to 8.0, and 1.5-3 mmol·L -1 Mg 2+ to the reaction mixture, and the reaction mixture was reacted at 70°C and 600 rpm for 1 to 3 hours. After the reaction was completed, tagatose isomerase recombinant bacteria (0.12 to 0.15 g, containing tagatose isomerase mutants) and 3 to 5 mmol·L -1 Ni 2+ The reaction was continued for 3 to 6 hours at 60 to 75°C and 500 to 800 rpm. After the reaction was completed, the reaction solution was centrifuged at 12,000 rpm for 3 minutes, and the supernatant was filtered through a 0.22 μm filter membrane and analyzed by HPLC.

[0024] Preferably, the “step-by-step method” reaction is as follows: in a 1 mL reaction system, fructose (50-500 g / L), recombinant psicose isomerase bacteria (0.04-0.05 g, containing psicose isomerase) and water are contained, sodium bicarbonate or sodium carbonate is used to adjust the pH to 8.0, and 1.5-3 mmol·L -1 Mg 2+ The reaction mixture was added to the reaction mixture, and the reaction mixture was reacted at 70°C and 600 rpm for 1 to 3 hours. After the reaction was completed, the reaction solution was centrifuged at 12000 rpm for 3 to 10 minutes, and the supernatant was taken and 50 mmol·L of the supernatant was added relative to 1 / 2 volume of the supernatant. -1 50% Tris-HCl buffer (pH 9.0), then add tagatose isomerase recombinant bacteria (0.12-0.15 g, containing tagatose isomerase mutant) and 3-5 mmol·L -1 Ni 2+ The reaction was continued for 3 to 6 hours at 60 to 75°C and 500 to 800 rpm. After the reaction was completed, the reaction solution was centrifuged at 12,000 rpm for 3 to 10 minutes, and the supernatant was filtered through a 0.22 μm filter membrane and analyzed by HPLC.

[0025] After the reaction, centrifugal filtration, decolorization and / or desalination can be performed, or the dual enzyme reaction product can be decolorized and impurities removed using activated carbon or diatomaceous earth, and then concentrated to obtain a syrup rich in rare sugars.

[0026] As a preferred embodiment, the reaction conditions of tagatose isomerase are as follows: 1 mL of the reaction system contains fructose (50-500 g / L), tagatose isomerase recombinant bacteria (0.12-0.15 g, containing tagatose isomerase) and 50 mmol·L-1 Tris-HCl buffer (pH 9.0) was added with 3-5 mmol / L -1 Ni 2+ The reaction mixture was added to the reaction mixture, and the reaction mixture was reacted at 60-75°C and 500-800 rpm for 3-6 hours. After the reaction, the reaction solution was centrifuged at 12000 rpm for 3-10 minutes, and the supernatant was filtered through a 0.22 μm filter membrane and analyzed by HPLC.

[0027] Preferably, the reaction conditions of psicose isomerase are as follows: 1 mL of the reaction system contains fructose (50-500 g / L), psicose isomerase recombinant bacteria (0.04-0.05 g, containing psicose isomerase) and water, the pH is adjusted to 8.0 with sodium bicarbonate or sodium carbonate, and 1.5-3 mmol·L -1 Mg 2+ The reaction mixture was added to the reaction mixture, and the reaction mixture was reacted at 60-75°C and 500-800 rpm for 1-3 hours. After the reaction was completed, the reaction solution was centrifuged at 12000 rpm for 3-10 minutes, and the supernatant was filtered through a 0.22 μm filter membrane and analyzed by HPLC.

[0028] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a method for preparing a composite rare sugar syrup using whole-cell catalysis. By constructing a recombinant bacterium containing different enzyme genes and a dual-enzyme combined reaction pathway with different production processes, a rare sugar syrup solution containing D-tagatose and D-psicose can be prepared from fructose or fructose-glucose syrup raw materials. This is a new method for producing a rare sugar-rich syrup.

[0029] (2) Compared with the mainstream D-galactose conversion route, the tagatose preparation route adopted by the present invention from fructose raw materials has a stable raw material supply and significantly reduced procurement costs. At the same time, compared with the reported single enzyme conversion method of tagatose or allulose, the dual enzyme conversion also significantly improves the utilization rate of raw materials, further reducing the cost of raw material use.

[0030] (3) The present invention prepares composite rare sugar syrup based on a one-step enzymatic pathway, and the reaction system, reaction conditions and post-treatment process are simple, and the production process is flexible, and different production processes can be selected according to the actual needs of the product. It is environmentally friendly and has good prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the result of the one-pot reaction in Example 4 using fructose as a substrate.

[0032] Figure 2This is the result of the stepwise reaction using fructose as substrate in Example 9.

[0033] Figure 3 This is the result of the stepwise reaction using high fructose syrup as substrate in Example 10. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is described below with specific embodiments, but the protection scope of the present invention is not limited thereto: Example 1 Expression of isomerase gene The amino acid sequences of tagatose isomerase (DTE) and psicose isomerase (DTE) were obtained from the NCBI database or literature reports, codon optimization was performed according to the preference of Escherichia coli, and the amino acid sequences were synthesized by a total synthesis method through conventional genetic engineering operations. The amino acid sequence of tagatose isomerase (DTE) is shown in SEQ ID NO.1, and the amino acid sequence of psicose isomerase (DTE) is shown in SEQ ID NO.3.

[0035] (1) Suzhou GeneWeiZi Company was commissioned to optimize the sequence and chemically synthesize the tagatose isomerase (DTE) gene and the psicose isomerase (DTE) gene, and then connected to the vector pET-28a(+) using the NdeI and XhoI restriction sites to obtain the recombinant plasmids pET-28a(+)-DTE and pET-28a(+)-DPE. The obtained recombinant plasmids were transformed into Escherichia coli BL21(DE3) competent cells, and the transformation products were spread on LB solid medium (containing 100 mg mL -1 Kanamycin sulfate) and cultured upside down in a 37°C constant temperature incubator for 12 to 16 hours to obtain transformants.

[0036] (2) A single clone was picked and inoculated into 5 mL of LB liquid culture medium (containing 50 μg / mL kanamycin sulfate), and cultured overnight at 37° C. and 200 rpm to prepare a seed solution.

[0037] (3) The seed solution was transferred to 100 mL of TB liquid culture medium containing 50 μg / mL kanamycin sulfate at an inoculum volume of 1% (v / v), and cultured at 37°C and 200 rpm for 18 to 20 h to obtain a fermentation broth. The prepared fermentation broth was centrifuged at 8000 rpm and 4°C for 5 min, the supernatant was discarded, and the wet cells were washed twice with 50 mM Tris-HCl buffer (pH 9.0) to obtain recombinant bacterial cells containing DTE gene and DPE gene for later use.

[0038] (4) Resuspend the recombinant bacteria in Tris-HCl (pH 9.0) buffer, place in an ice-water mixture, and ultrasonically disrupt. The disruption parameters are set as follows: 90% intensity, 2s ultrasonic pause 3s condition ultrasonic disruption for 10 min. After disruption, centrifuge at 9000 rpm for 30 min at 4°C. After centrifugation, take the supernatant and precipitate for SDS-PAGE analysis of soluble expression.

[0039] SEQ ID NO.1: Tagatose-4-isomerase amino acid sequence MVLKVFKDHFGRGYEVYEKSYREKDSLSFFLTKGEEGKILVVAGEKAPEGLSFFKKQRVEG VSFFFCERNHENLEVLRKYFPDLKPVRAGLRASFGTGDRLGITTPAHVRALKDSGLFPIFAQ QSVRENERTGRTWRDVLDDATWGVFQEGYSEGFGADADHVKRPEDLVSAAREGFTMFTID PSDHVRNLSKLSEREKNEMFEEILKKERIDRIYLGKKYTVLGERLEFDEKNLRDAALVYYD AIAHVDMMYQILKDETPDFDFEVSVDETETPTSPLFHIFVVEELRRRGVEFTNLALRFIGEW EKGIDYKGDLAQFEREIKMHAEIARMFEGYKISLHSGSDKFSVYPAFASATGGLFHVKTAGT SYLEAVKVISMVNPELFREIYRCALDHFEEDRKSYHISADLSKVPEVEKVKDEDLPGLFEDI NVRQLIHVTYGSVLKDASLKERLFKTLEQNEELFYETVAKHIKRHVDLLKGSEQ ID NO.3: Psicose-3-isomerase amino acid sequence MNIGCHGLVWTGNFDAEGIRLAVHKTREAGFDLIEFPLMDPFTFDVSVAKDALEEYDLAVS ASLGLSEATDVTSADPAIAAAAGEALLVKAVDVLADLGGEHFCGVIYSAMKKYMEPVTPEGL ASSKAAIARVADHAAARGVSVSLEVVNRYETNVLNTARQALAYIAEVDRPNLGIHIDTYHM NIEESDMFTPVLDAAPALRYVHIGESHRGYLGTGTVDFDTFFKALGRIGYGGPIVFESFSSAV VAPDLSRMLGIWRNLWNDNDELGAHANAYIRDKLVAVDSIRLH Example 2 Tagatose isomerase mutant Suzhou GeneWeizhi Co., Ltd. was commissioned to optimize and chemically synthesize the sequence shown in SEQ ID NO.2, and the gene sequence was also commissioned to Suzhou GeneWeizhi Co., Ltd. for synthesis.

[0040] Design site-directed mutagenesis primers and perform PCR amplification to obtain a vector containing the gene encoding the mutant. PCR amplification of the gene encoding the site-directed mutant: Rapid PCR technology was used, with the recombinant plasmid pET-28a(+)-DTE carrying the gene encoding tagatose isomerase as the template. The reaction system was: forward primer (10 μmol·L -1 )1μL, reverse primer (10μmol·L -1 )1μL, 2*TranStartFastPfu Fly PCR Supermix 25μL, template DNA 1μL, add ddH2O to 50μL.

[0041] The PCR amplification program was set as follows: 98°C pre-denaturation for 1 min; 98°C denaturation for 10 s, 57°C annealing for 30 s, 72°C extension for 2 min, 32 cycles, and finally 72°C extension for 1 min, followed by 4°C insulation. The PCR product was detected by 1% agarose gel electrophoresis. DMT was used to digest the PCR product to remove the residual template plasmid and reduce the probability of false positives in the transformation results. Reaction system: 50 μL of PCR product, 2 μL of DMT enzyme.

[0042] At the same time, the Omega PCR product direct recovery kit was used to recover the enzyme digestion product, and the Vazyme C112 homologous recombination kit was used to connect the vector. 10 μL of the connection product was transferred into BL21 (DE3) competent cells, and the transformation liquid was spread on the LB plate containing Kan (50 μg / mL) resistance and cultured at 37°C overnight. Single clones were picked, the bacterial liquid was enriched, the plasmid was extracted and sequenced, and the sequencing work was completed by Suzhou Jinweizhi Company.

[0043] The tagatose isomerase mutant II is a tagatose-4-isomerase whose amino acid sequence is shown in SEQ ID NO.1, wherein the serine at position 125 is mutated to aspartic acid, the glutamic acid at position 167 is mutated to serine; the serine at position 185 is mutated to glutamine; the valine at position 267 is mutated to methionine; the serine at position 268 is mutated to threonine; the threonine at position 272 is mutated to aspartic acid; the tryptophan at position 306 is mutated to methionine; the lysine at position 337 is mutated to tryptophan; the arginine at position 386 is mutated to valine, and the tyrosine at position 403 is mutated to isoleucine; and its amino acid sequence is shown in SEQ ID NO.2.

[0044] SEQ ID NO.2: Amino acid sequence of tagatose-4-isomerase mutant MVLKVFKDHFGRGYEVYEKSYREKDSLSFFLTKGEEGKILVVAGEKAPEGLSFFKKQRVEG VSFFFCERNHENLEVLRKYFPDLKPVRAGLRASFGTGDRLGITTPAHVRALKDSGLFPIFAQ QDVRENERTGRTWRDVLDDATWGVFQEGYSEGFGADADHVKRPSDLVSAAREGFTMFTI DPQDHVRNLSKLSEREKNEMFEEILKKERIDRIYLGKKYTVLGERLEFDEKNLRDAALVYY DAIAHVDMMYQILKDETPDFDFEMTVDEDETPTSPLFHIFVVEELRRRGVEFTNLALRFIGE MEKGIDYKGDLAQFEREIKMHAEIARMFEGYWISLHSGSDKFSVYPAFASATGGLFHVKTA GTSYLEAVKVISMVNPELFVEIYRCALDHFEEDRKSIHISADLSKVPEVEKVKDEDLPGLFE DINVRQLIHVTYGSVLKDASLKERLFKTLEQNEELFYETVAKHIKRHVDLLKG Example 3 Construction of recombinant bacteria (1) Recombinant strain of tagatose isomerase mutant: Suzhou Jinweizhi Company was commissioned to optimize and chemically synthesize the amino acid sequence shown in SEQ ID NO.2, and connected to the vector pET-28a(+) using NdeI and XhoI restriction sites to obtain the recombinant plasmid pET-28a(+)-DPE. The obtained recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and the transformation product was spread on LB solid medium (containing 50μg / mL kanamycin sulfate), and inverted in a 37℃ constant temperature incubator for 12-16h to obtain recombinant bacteria containing tagatose isomerase mutants.

[0045] Pick a single clone and inoculate it into 5mL (containing 50μg / mL kanamycin sulfate) LB liquid medium, and culture it overnight at 37°C and 200rpm to prepare a seed solution. Then transfer the seed solution to 100mL TB liquid medium containing 50μg / mL kanamycin sulfate according to the inoculation amount of 1% (v / v), and culture it at 37°C and 200rpm for 18-20h to obtain a fermentation broth. The prepared fermentation broth was centrifuged at 8000rpm and 4°C for 5min, the supernatant was discarded, and the wet bacteria were washed twice with 50mM Tris-HCl buffer (pH 9.0) to obtain recombinant bacterial cells containing tagatose isomerase gene (tagatose isomerase mutant recombinant bacteria) for standby use.

[0046] (2) Recombinant bacteria for psicose isomerase: Suzhou Jinweizhi Company was commissioned to optimize and chemically synthesize the amino acid sequence shown in SEQ ID NO.3, and connected to the vector pET-28a(+) using NdeI and XhoI restriction sites to obtain the recombinant plasmid pET-28a(+)-DPE. The obtained recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and the transformation product was spread on LB solid medium (containing 50μg / mL kanamycin sulfate), and inverted in a 37°C constant temperature incubator for 12 to 16 hours to obtain a recombinant bacterium containing psicose isomerase.

[0047] A single clone was picked and inoculated into 5 mL of LB liquid medium (containing 50 μg / mL kanamycin sulfate), and cultured overnight at 37°C and 200 rpm to prepare a seed solution. The seed solution was then transferred to 100 mL of TB liquid medium containing 50 μg / mL kanamycin sulfate at an inoculation amount of 1% (v / v), and cultured at 37°C and 200 rpm for 18 to 20 hours to obtain a fermentation broth. The prepared fermentation broth was centrifuged at 8000 rpm and 4°C for 5 minutes, the supernatant was discarded, and the wet bacteria were washed twice with 50 mM Tris-HCl buffer (pH 9.0) to obtain recombinant bacterial cells containing the psicose isomerase gene (psicose isomerase recombinant bacteria) for standby use.

[0048] (3) Double plasmid transformation recombinant bacteria: The psicose isomerase was linked to the vector pET-25b(+) by PCR and homologous recombination to obtain the recombinant plasmid pET-25b(+)-DPE containing the sequence shown in SEQ ID NO.3; at the same time, the sequence shown in SEQ ID NO.2 was constructed on the vector pET-28a(+) to obtain the recombinant plasmid pET-28a(+)-DTE. The above two recombinant plasmids (containing the tagatose isomerase mutant gene and the psicone isomerase gene, respectively) were transformed into Escherichia coli BL21(DE3) competent cells, and the transformation product was spread on LB solid medium (containing 50 μg mL -1 Kanamycin sulfate and 100 μg mL -1 Ampicillin), and cultured upside down in a 37°C constant temperature incubator for 12 to 16 hours to obtain recombinant transformed bacteria.

[0049] A single clone was picked and inoculated into 5 mL of LB liquid medium (containing 50 μg / mL kanamycin sulfate and 100 μg / mL ampicillin), and cultured overnight at 37°C and 220 rpm to prepare a seed solution. The seed solution was transferred to 100 mL of TB liquid medium containing 50 μg / mL kanamycin sulfate and 100 μg / mL ampicillin at an inoculum of 1% (v / v), and cultured at 37°C and 220 rpm for 3 to 4 hours, and IPTG was added thereto at a final concentration of 0.1 mM, and cultured at 16°C and 180 rpm for 18 to 20 hours to obtain a fermentation broth. The prepared fermentation broth was centrifuged at 8000 rpm and 4°C for 5 min, the supernatant was discarded, and the wet bacteria were washed twice with 50 mM Tris-HCl buffer (pH 9.0) to obtain recombinant bacterial cells containing tagatose isomerase gene and psicose isomerase gene (double-plasmid transformed recombinant bacteria) for later use.

[0050] Example 4 "One Pot" Reaction I In a 1 mL reaction system, fructose (100 g / L), tagatose isomerase mutant recombinant bacterial cells (0.12 g, containing tagatose isomerase mutant), psicose isomerase recombinant bacterial cells (0.05 g, containing psicose isomerase) and 50 mmol·L -1 Tris-HCl buffer (pH 9.0) was added with 3 mmol·L -1 Ni 2+ and 1.5mmol·L -1 Mg 2 + The reaction mixture was added and reacted at 70°C and 600 rpm for 3 h. After the reaction was completed, the reaction solution was centrifuged at 12000 rpm for 3 min, and the supernatant was filtered through a 0.22 μm filter membrane and analyzed by HPLC.

[0051] Example 5 "One Pot" Reaction II In a 1 mL reaction system, fructose (100 g / L), double-plasmid transformed recombinant bacterial cells (0.17 g, containing tagatose isomerase mutant and psicose isomerase) and 50 mmol·L -1 Tris-HCl buffer (pH 9.0) was added with 3 mmol·L -1 Ni 2+ and 1.5mmol·L -1 Mg 2+ The reaction mixture was added and reacted at 70°C and 600 rpm for 3 h. After the reaction was completed, the reaction solution was centrifuged at 12000 rpm for 3 min, and the supernatant was filtered through a 0.22 μm filter membrane and analyzed by HPLC.

[0052] Example 6 "Stepwise Method" Reaction Ⅰ In a 1 mL reaction system, fructose (100 g / L), recombinant bacterial cells of tagatose isomerase mutant (0.12 g, containing tagatose isomerase mutant) and 50 mmol·L -1 Tris-HCl buffer (pH 9.0) was added with 3 mmol·L -1 Ni 2+ The reaction mixture was added to the reaction mixture, and the reaction mixture was reacted at 70°C and 600 rpm for 3 hours. After the reaction was completed, recombinant bacterial cells of psicose isomerase (0.05 g, containing psicose isomerase) were directly added, and the reaction was continued for 1 hour at 70°C and 600 rpm. After the reaction was completed, the reaction solution was centrifuged at 12000 rpm for 3 minutes, and the supernatant was filtered through a 0.22 μm filter membrane and analyzed by HPLC.

[0053] Example 7 "Stepwise Method" Reaction II In a 1 mL reaction system, fructose (100 g / L), recombinant bacterial cells of tagatose isomerase mutant (0.12 g, containing tagatose isomerase mutant) and 50 mmol·L -1 Tris-HCl buffer (pH 9.0) was added with 3 mmol·L -1 Ni 2+ to the reaction mixture, and the reaction mixture was reacted at 70°C and 600 rpm for 3 hours. After the reaction was completed, the reaction solution was centrifuged at 12000 rpm for 3 minutes, and the recombinant bacterial cells of D-psicose isomerase (0.05 g, containing D-psicose isomerase) were added to the supernatant obtained by centrifugation, and the reaction was continued for 1 hour at 70°C and 600 rpm. After the reaction was completed, the reaction solution was centrifuged at 12000 rpm for 3 minutes, and the supernatant was filtered through a 0.22 μm filter membrane and then detected and analyzed by HPLC.

[0054] Example 8 "Stepwise Method" Reaction III In a 1 mL reaction system, fructose (100 g / L), recombinant psicose isomerase bacterial cells (0.05 g, containing psicose isomerase) and water (pH adjusted to 8.0 with sodium bicarbonate or sodium carbonate) were added. -1 Mg 2+ to the reaction mixture, and the reaction mixture was reacted at 70°C and 600rpm for 1h. After the reaction, tagatose isomerase mutant recombinant bacterial cells (0.12g, containing tagatose isomerase mutant) were directly added, and the reaction was continued for 3h at 70°C and 600rpm. After the reaction, the reaction solution was centrifuged at 12000rpm for 3min, and the supernatant was filtered through a 0.22μm filter membrane and analyzed by HPLC.

[0055] Example 9 "Stepwise Method" Reaction IV In a 1 mL reaction system, fructose (100 g / L), recombinant psicose isomerase bacterial cells (0.05 g, containing psicose isomerase) and water (pH adjusted to 8.0 with sodium bicarbonate or sodium carbonate) were added. -1 Mg 2+to the reaction mixture, and the reaction mixture was reacted at 70°C and 600rpm for 1h. After the reaction, the reaction solution was centrifuged at 12000rpm for 3min, the supernatant was taken and 0.5mL of Tris-HCl buffer (pH 9.0, 50mM) was added, and then tagatose isomerase mutant recombinant bacterial cells (0.12g, containing tagatose isomerase mutant) were added, and the reaction was continued for 3h under the reaction conditions of 70°C and 600rpm. After the reaction, the reaction solution was centrifuged at 12000rpm for 3min, the supernatant was taken and filtered through a 0.22μm filter membrane, and then detected and analyzed by HPLC.

[0056] Example 10 "Stepwise Method" Reaction IV In a 1 mL reaction system, there were fructose syrup (F55, 250 g / L), recombinant bacterial cells of psicose isomerase (0.05 g, containing psicose isomerase) and 50 mmol·L -1 Tris-HCl buffer (pH 9.0) was added with 1.5 mmol·L -1 Mg 2+ to the reaction mixture, and the resulting reaction mixture was reacted at 70°C and 600rpm for 1h. After the reaction was completed, the reaction solution was centrifuged at 12000rpm for 3min, the supernatant was taken, and then tagatose isomerase mutant recombinant bacterial cells (0.12g, containing tagatose isomerase mutant) were added, and the reaction was continued for 3h under the reaction conditions of 70°C and 600rpm. After the reaction was completed, the reaction solution was centrifuged at 12000rpm for 3min, the supernatant was taken and filtered through a 0.22μm filter membrane, and then detected and analyzed by HPLC.

[0057] Comparative Example 1 In a 1 mL reaction system, fructose (100 g / L), recombinant bacterial cells of tagatose isomerase mutant (0.12 g, containing tagatose isomerase mutant) and 50 mmol·L -1 Tris-HCl buffer (pH 9.0) was added with 3 mmol·L -1 Ni 2+ The reaction mixture was added to the reaction mixture, and the reaction mixture was reacted at 70°C and 600 rpm for 3 h. After the reaction was completed, the reaction solution was centrifuged at 12000 rpm for 3 min, and the supernatant was filtered through a 0.22 μm filter membrane and analyzed by HPLC.

[0058] Comparative Example 2 In a 1 mL reaction system containing fructose (100 g / L), recombinant psicose isomerase bacterial cells (0.05 g, containing psicose isomerase) and water, the pH was adjusted to 8.0 using sodium bicarbonate or sodium carbonate, and 1.5 mmol·L-1 Mg 2+ The reaction mixture was added to the reaction mixture, and the reaction mixture was reacted at 70°C and 600 rpm for 1 hour. After the reaction was completed, the reaction solution was centrifuged at 12000 rpm for 3 minutes, and the supernatant was filtered through a 0.22 μm filter membrane and analyzed by HPLC.

[0059] Comparative Example 3 In a 1 mL reaction system, fructose syrup (F55, 250 g / L), tagatose isomerase mutant recombinant bacterial cells (0.12 g, containing tagatose isomerase mutant) and 50 mmol·L -1 Tris-HCl buffer (pH 9.0) was added with 3 mmol·L -1 Ni 2+ The reaction mixture was added to the reaction mixture, and the reaction mixture was reacted at 70°C and 600 rpm for 3 h. After the reaction was completed, the reaction solution was centrifuged at 12000 rpm for 3 min, and the supernatant was filtered through a 0.22 μm filter membrane and analyzed by HPLC.

[0060] Comparative Example 4 In a 1 mL reaction system containing fructose syrup (F55, 250 g / L), recombinant bacterial cells of psicose isomerase (0.05 g, containing psicose isomerase) and water, the pH was adjusted to 8.0 with sodium bicarbonate or sodium carbonate, and 1.5 mmol·L -1 Mg 2+ The reaction mixture was added to the reaction mixture, and the reaction mixture was reacted at 70°C and 600 rpm for 1 hour. After the reaction was completed, the reaction solution was centrifuged at 12000 rpm for 3 minutes, and the supernatant was filtered through a 0.22 μm filter membrane and analyzed by HPLC.

[0061] HPLC detection conditions: Chromatographic column: Waters Sugar-Pak column, 10 μm, 6.5*300 mm; Mobile phase: calcium disodium EDTA (50 mg L -1 ) aqueous solution; flow rate 0.5mL·min -1 , column temperature 90°C, differential detector, detection temperature 50°C, injection volume 10μL.

[0062] Method for detecting the content of rare sugars: The amount of reaction products in the sample is determined by high performance liquid chromatography (HPLC); conversion rate (%) = concentration of rare sugars in the product × 100 / concentration of added substrate fructose. Fructose utilization rate (%) = total product concentration × 100 / concentration of added substrate fructose.

[0063] Table 1 During the complex enzyme conversion, after the reaction of the former isomerase, the fructose substrate concentration decreases, and the reaction product and reaction conditions after the reaction will affect the reaction conversion rate of the latter isomerase, resulting in a low conversion rate of a single rare sugar relative to that of a single enzyme reaction. Moreover, tagatose isomerase is more sensitive to the influence of fructose substrate concentration and reaction conditions than psicose isomerase, resulting in a large difference in tagatose conversion rates between Examples 4-10 and Comparative Examples 1 and 3. In addition, in the "one-pot" or "step-by-step" reaction process, the reaction of tagatose isomerase first will also lead to a decrease in tagatose conversion rate. This is due to the influence of the total reaction time and the changes in the reaction environment, resulting in some tagatose being converted into other sugars, and there is a situation where the fructose utilization rate is slightly higher than the total conversion rate of complex sugars.

[0064] As shown in Table 1, the present invention prepares complex rare sugars through dual enzyme coupling reaction, and can simultaneously obtain a syrup product containing two rare sugars, and the conversion rate of the composite enzyme is generally significantly higher than the conversion rate of a single enzyme. Since the fructose substrate concentration is the same, fructose can only be converted into tagatose or allulose or other sugars. Therefore, compared with the conversion of a single enzyme, the present invention has a significant effect in improving the total conversion rate of fructose. At the same time, the constructed "one-pot method" and "step-by-step method" dual enzyme coupling reaction paths can significantly improve the total conversion rate of complex sugars and the utilization rate of fructose.

[0065] In addition, if Figure 1 , Figure 2 and Figure 3 Shown are HPLC graphs of the reaction products in Examples 4, 9, and 10, respectively, indicating that the composite syrups containing different proportions of tagatose and psicose obtained using different production processes.

[0066] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the specification of the present invention, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for producing composite rare sugar syrup by whole-cell catalysis, characterized in that: The method comprises the following steps: taking a substrate containing fructose as a raw material, utilizing tagatose-4-isomerase and psicose-3-isomerase to carry out a double enzyme coupling reaction, and preparing a composite rare sugar syrup containing D-tagatose and D-psicose.

2. The method for producing composite rare sugar syrup by whole-cell catalysis according to claim 1, characterized in that: The tagatose-4-isomerase is a tagatose-4-isomerase mutant, wherein the amino acid sequence of the tagatose-4-isomerase shown in SEQ ID NO.1 is mutated from serine at position 125 to aspartic acid, from glutamic acid at position 167 to serine, from serine at position 185 to glutamine, from valine at position 267 to methionine, from serine at position 268 to threonine, from threonine at position 272 to aspartic acid, from tryptophan at position 306 to methionine, from lysine at position 337 to tryptophan, from arginine at position 386 to valine, and from tyrosine at position 403 to isoleucine.

3. The method for producing composite rare sugar syrup by whole-cell catalysis according to claim 2, characterized in that: The amino acid sequence of the tagatose-4-isomerase mutant is shown in SEQ ID NO.

2.

4. The method for producing composite rare sugar syrup by whole-cell catalysis according to claim 1, characterized in that: The substrate is fructose or fructose-glucose syrup.

5. The method for producing composite rare sugar syrup by whole-cell catalysis according to claim 1 or 4, characterized in that: In a 1 mL reaction system, the concentration of fructose in the substrate is 50~500 g / L.

6. The method for producing composite rare sugar syrup by whole-cell catalysis according to any one of claims 1 to 4, characterized in that: In the dual enzyme coupling reaction, the pH is 8.0-9.0, the temperature is 60-75°C, and the reaction time is 3-12 h.

7. The method for producing composite rare sugar syrup by whole-cell catalysis according to claim 1, characterized in that: In the dual enzyme coupling reaction, a recombinant bacterium containing tagatose-4-isomerase and a recombinant bacterium containing psicose-3-isomerase are added simultaneously for reaction, or a recombinant bacterium containing tagatose-4-isomerase and psicose-3-isomerase are added simultaneously for reaction.

8. The method for producing composite rare sugar syrup by whole-cell catalysis according to claim 1, characterized in that: In the dual enzyme coupling reaction, the recombinant bacteria containing tagatose-4-isomerase is first added for reaction, and then the recombinant bacteria containing psicose-3-isomerase is added for reaction; alternatively, the recombinant bacteria containing psicose-3-isomerase is first added for reaction, and then the recombinant bacteria containing tagatose-4-isomerase is added for reaction.

9. The method for producing composite rare sugar syrup by whole-cell catalysis according to claim 7 or 8, characterized in that: The expression vector used in the process of constructing the recombinant bacteria is pET28a(+) plasmid, pET25b(+) plasmid or pET-Duet-1 plasmid.

10. The method for producing composite rare sugar syrup by whole-cell catalysis according to claim 7 or 8, characterized in that: The starting strain in the process of constructing the recombinant bacteria is Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum or yeast.

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