A cellobiose epimerase and uses thereof

Abstract

The application discloses a recombinant domain fusion enzyme and application thereof in preparation of D-allulose, wherein the recombinant domain fusion enzyme is formed by splicing and recombining a 5' domain and a partial 3' domain of D-tagatose 3-epimerase and an active center domain of xylose isomerase. The application uses the domain fusion enzyme of the 5' domain and the partial 3' domain of D-tagatose 3-epimerase and the active center domain of xylose isomerase as a biological catalyst, and the yield of D-allulose generated by catalyzing isomerization of 260 g / L glucose is 35.9%. The method disclosed by the application is rapid and efficient, reaction conditions are simple, substrate conversion rate is high, and byproducts are few, so that the pressure of separation and purification is reduced, and the method is suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a recombinant domain fusion enzyme and its application in the preparation of D-allulose. Background Technology

[0002] D-Allulose is a hexose found in very low amounts in nature, and is an epimer of D-fructose at the C-3 site. D-Allulose is poorly digested and absorbed, providing almost no energy for life activities, thus making it a very useful low-calorie sweetener.

[0003] D-allulose 3-epimerase or D-tagatose 3-epimerase can achieve the interconversion between D-fructose and D-allulose. D-fructose can then be generated from glucose through an epimerization reaction catalyzed by glucose isomerase or xylose isomerase. Therefore, a two-enzyme system composed of D-allulose 3-epimerase or D-tagatose 3-epimerase, along with glucose isomerase or xylose isomerase, can generate D-allulose from glucose in a single step. However, the process involves a large amount of the intermediate product D-fructose, which significantly affects the conversion efficiency. This is unavoidable in traditional two-enzyme reaction systems or fusion enzyme reaction systems composed of two enzymes linked by peptide linkages.

[0004] By fusing the functional domains of various enzyme molecules using genes to construct entirely new enzyme molecules, we can create recombinant domain fusion enzymes. Unlike simply combining two enzyme molecules into a single macromolecule by linking peptide chains, recombinant domain fusion enzymes have more similar functional domains in structure, resulting in a stronger "proximity effect." This leads to a more pronounced promoting effect on the forward enzymatic reaction and a reduction in reaction free energy. Furthermore, the enzyme molecular weight is lower than that of simple fusion enzymes linked by peptide chains, avoiding problems such as insoluble expression and difficulty in proper folding of large proteins caused by the linking of different enzyme molecules.

[0005] Chinese Patent 202010805264.X discloses a recombinant fusion enzyme and its application in the synthesis of methyl glyoxylate. The enzyme uses a large recombinant enzyme constructed by linking glycolate oxidase, catalase and hemoglobin through a linker peptide as a biocatalyst to catalyze the oxidation of 200 mM methyl glycolate. After 6 hours of reaction, the yield of methyl glyoxylate reaches 95.4%.

[0006] Chinese Patent 202111188472.0 discloses a fusion enzyme for producing nicotinamide mononucleotide and its application. It constructs a large recombinant enzyme by linking nicotinamide phosphoribosyltransferase and ribophosphate pyrophosphate kinase through a linker peptide, and transforms it into Escherichia coli or Bacillus subtilis as a cell catalyst or purifies the recombinant enzyme as a catalyst. Using nicotinamide as a substrate, it ferments to produce nicotinamide mononucleotide, and the yield of nicotinamide mononucleotide can be increased by more than 50%.

[0007] Chinese Patent 202210352448.4 discloses a fusion protein that catalyzes the synthesis of D-allulose from glucose and its construction method. It utilizes flexible and rigid linker peptides of different lengths to connect a glucose isomerase gene and a D-allulose 3-epimerase, selecting the most effective macromolecular recombinant enzyme as a catalyst. Using 100 g / L glucose as a substrate, the D-allulose yield is no less than 5.69 g / L. Although this represents an improvement compared to a two-enzyme reaction system, the conversion efficiency remains low. Summary of the Invention

[0008] This invention discloses a recombinant domain fusion enzyme and its application in the preparation of D-allulose. The recombinant domain fusion enzyme is constructed by combining the 5' domain and part of the 3' domain of D-tagatose 3-epimerase with the active site domain of xylose isomerase. Using this domain fusion enzyme as a biocatalyst, the isomerization reaction of 260 g / L glucose to produce D-allulose yields 35.9%. The method described in this invention is rapid and efficient, with simple reaction conditions, high substrate conversion, and few byproducts, reducing the pressure of separation and purification, and is suitable for large-scale industrial production.

[0009] The technical solution adopted in this invention is: This invention provides a recombinant fusion enzyme composed of the 5' domain (DTE-5') and part of the 3' domain (DTE-3') of D-tagatose 3-differentiated enzyme, and the active site domain (XYI-VE) of xylose isomerase. The amino acid sequence of the recombinant domain fusion enzyme (DTE-5'-XYI-VE-DTE-3') is shown in SEQ ID No. 1. Furthermore, the D-tagatose 3-epimerase (DTE) is derived from Rhodotorula globulus, and its amino acid sequence is shown in SEQ ID No. 2, while the nucleotide sequence of the encoding gene is shown in SEQ ID No. 3. The xylose isomerase (XYI) is derived from *Gyrodactylus hyrodactylus*, and its amino acid sequence is shown in SEQ ID No. 4. The nucleotide sequence of the encoding gene is shown in SEQ ID No. 5. Furthermore, the 5' domain (DTE-5') of the D-tagatose 3-differentiatorase consists of amino acid residues from position 1 to position 138 of the D-tagatose 3-differentiatorase (DTE), as shown in SEQ ID No. 6; the 3' domain (DTE-3') of the D-tagatose 3-differentiatorase consists of amino acid residues from position 155 to position 295 of the D-tagatose 3-differentiatorase (DTE), as shown in SEQ ID No. 7; and the active site domain (XYI-VE) of the xylose isomerase consists of amino acid residues from position 32 to position 205 of the xylose isomerase (XYI), as shown in SEQ ID No. 8. Furthermore, the nucleotide sequence of the gene encoding the recombinant domain fusion enzyme (DTE-5'-XYI-VE-DTE-3') is shown in SEQ ID No. 9; This invention also relates to a vector containing a recombinant domain fusion enzyme gene and a recombinant genetically engineered bacterium constructed from the vector. The construction of the vector containing the recombinant fusion enzyme gene and the recombinant genetically engineered bacterium includes the following steps: The construction of the nucleotide sequence encoding the recombinant domain fusion enzyme (DTE-5'-XYI-VE-DTE-3') includes the following steps: sequentially amplifying the encoding genes for the 5' domain (DTE-5') of D-taglatose 3-differentiated isomerase, the active site domain (XYI-VE) of xylose isomerase, and the partial 3' domain (DTE-3') of D-taglatose 3-differentiated isomerase, respectively; using overlap PCR, assembling the three encoding genes into a fusion gene fragment; and ligating the fusion gene fragment to the NdeI and NotI restriction enzyme sites of the vector pET22b using a one-step cloning method to obtain a recombinant plasmid. The recombinant plasmid is transformed into *Escherichia coli* BL21(DE3) to obtain the recombinant genetically engineered bacterium containing the recombinant fusion enzyme gene. The preferred recombinant genetically engineered bacteria is recombinant Escherichia coli. E. coliBL21(DE3) / pET22b-DTE-5'-XYI-VE-DTE-3'. Recombinant *E. coli* was inoculated into LB liquid medium containing 50 μg / mL ampicillin resistance and cultured at 37°C and 200 rpm for 12 h. Then, 1% (v / v) inoculum was added to fresh LB liquid medium containing 50 μg / mL ampicillin resistance and cultured at 37°C and 150 rpm until the bacterial OD600 reached 0.6-0.8. IPTG was added to a final concentration of 24 μg / mL, and the culture was induced at 25°C for 16 h. The culture was then centrifuged at 4°C and 8000 rpm for 20 min, the supernatant was discarded, and the precipitate was collected. The precipitate was washed twice with pH 7.5, 20 mM phosphate buffer to obtain wet cells. The wet cells were resuspended in pH 7.5, 100 mM phosphate buffer and sonicated on an ice-water mixture for 5 min. The sonication conditions were: power 200 W, 1 s of disruption, 2 s pause, to obtain crude enzyme solution.

[0010] In the reaction system of this invention, the catalyst can be crude enzyme solution after cell disruption, or it can be resting cells of engineered bacteria expressing recombinant enzymes, or it can be purified enzyme, or an immobilized enzyme.

[0011] This invention also provides an application of a recombinant domain fusion enzyme (DTE-5'-XYI-VE-DTE-3') in the preparation of D-allulose. The application involves using a crude enzyme solution obtained through fermentation culture of engineered bacteria containing the encoding gene of the fusion enzyme (DTE-5'-XYI-VE-DTE-3'), resting cells, purified enzyme, or immobilized enzyme as a biocatalyst. The substrate ethyl glucose is added, and a transformation system is constructed using a 50mM buffer solution at pH 6.0–10.0 as the reaction medium. The reaction is carried out at 50–65°C for 8–24 hours. After the reaction is complete, the supernatant is subjected to ion exchange and decolorization, followed by separation using macroporous resin to obtain D-allulose and unreacted substrate glucose. The glucose can be recovered and reintroduced into the reaction system.

[0012] Furthermore, the buffer solution is preferably 50 mM Tris-HCl (pH 7.0). The reaction temperature is preferably 55°C. The reaction time is preferably 12 hours.

[0013] Furthermore, in the conversion system, the crude enzyme solution is added at a final concentration of 1-5 g / L based on the total protein content, preferably 2 g / L; the concentration of the substrate glucose in the conversion system is 260 g / L.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes a recombinant domain fusion enzyme formed by combining the 5' and part of the 3' domain of D-tagatose 3-epimerase with the active site domain of xylose isomerase. This fusion enzyme exhibits a single enzyme activity and can directly and efficiently prepare D-allulose from glucose. Under controlled reaction conditions and substrate concentrations, almost no D-fructose byproduct is generated, improving reaction efficiency and reducing the burden of separation and purification. Under optimized reaction conditions (reaction buffer pH 7.0, reaction temperature 55℃, reaction time 12 hours), with a substrate glucose concentration of 260 g / L, the isomerization reaction produces 93.3 g / L of D-allulose, with a yield of 35.9%. The absence of fructose byproduct provides a significant cost advantage and makes it suitable for large-scale industrial production. Detailed Implementation

[0015] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0016] Example 1: Construction of expression vector and engineered bacteria Primers were designed based on the nucleotide sequence shown in SEQ ID No. 9, and NdeI and NotI restriction enzyme sites were introduced into the primers, respectively: Upstream primer: 5'-CATATGATGAAGAAAACGCTTAAACTT-3'; Downstream primer: 5'-GCGGCCGCTCACGGTTGCCAGTCTACA-3'; Using pET-22b plasmid as an expression vector, Escherichia coli was constructed E. coli BL21(DE3) / pET22b-cgt1 Construction of expression plasmid: Under the initiation of the above primers, the target gene was used as a template and amplified using high-fidelity Pfu DNA polymerase to obtain the cyclodextrin glycosyltransferase gene sequence. After sequencing, the amplified fragment was processed with NdeI and NotI restriction endonucleases (TaKaRa), and the fragment was ligated with the vector pET-22b treated with the same restriction endonucleases using T4 DNA ligase (TaKaRa) to construct the expression vector pET22b-cgt1.

[0017] Preparation of competent cells: Glycerol tubes were obtained from a -80°C freezer and stored in the cell. E. coliStrawberry strain BL21(DE3) was streaked onto antibiotic-free LB agar plates and cultured at 37°C for 10 h to obtain single colonies. Single colonies from LB agar plates were picked and inoculated into test tubes containing 5 mL of LB medium, and cultured at 37°C and 180 rpm for 9 h. 200 μL of the bacterial culture was taken from the test tube and inoculated into 50 mL of LB medium, and cultured at 37°C and 180 rpm until the OD600 reached 0.4-0.6. The bacterial culture was pre-chilled on ice, transferred to sterile centrifuge tubes, placed on ice for 10 min, and centrifuged at 4°C and 5000 rpm for 10 min. The supernatant was discarded, taking care to prevent contamination. The cell pellet was resuspended in pre-chilled 0.1 mol / L CaCl2 aqueous solution and placed on ice for 30 min. The pellet was centrifuged at 4°C and 5000 rpm for 10 min, the supernatant was discarded, and the cell pellet was resuspended in pre-chilled 0.1 mol / L CaCl2 aqueous solution containing 15% glycerol. 100 μL of the supernatant was taken. μL of the resuspended cells were aliquoted into sterile 1.5 mL centrifuge tubes and stored at -80°C. The cells were removed as needed.

[0018] Construction of recombinant *E. coli*: First, *E. coli* BL21(DE3) (Invitrogen) competent cells stored at -80℃ were incubated at 0℃ for 10 min. Then, 5 µL of plasmid pET22b-cgt1 was added in a clean bench, incubated at 0℃ for 30 min, heat-shocked in a 42℃ water bath for 90 s, and incubated at 0℃ for 2 min. 600 µL of LB medium was added, and the cells were cultured at 37℃ and 200 rpm for 1 h. The cultured cells were then plated on LB agar plates containing 50 μg / ml ampicillin resistance and cultured at 37℃ for 8-12 h. Clones were randomly picked, plasmids were extracted, and sequenced for identification. Recombinant *E. coli* containing the recombinant plasmid expression were screened to obtain the cells. E. coli BL21(DE3) / pET22b-cgt1.

Claims

1. A recombinant domain fusion enzyme, characterized in that, The recombinant domain fusion enzyme is constructed by combining the 5' domain and part of the 3' domain of D-tagatose 3-epimerase with the active site domain of xylose isomerase.

2. The D-tagatose 3-epimerase (DTE) is derived from Rhodotorula globosum, with the amino acid sequence shown in SEQ ID No. 2 and the nucleotide sequence of the encoding gene shown in SEQ ID No. 3; the xylose isomerase (XYI) is derived from Zygomycetes globosum, with the amino acid sequence shown in SEQ ID No. 4 and the nucleotide sequence of the encoding gene shown in SEQ ID No.

5.

3. The nucleotide sequence of the gene encoding the recombinant domain fusion enzyme (DTE-5'-XYI-VE-DTE-3') is shown in SEQ ID No.

9.

4. A vector containing a recombinant fusion enzyme gene and a recombinant genetically engineered bacterium constructed from the vector, wherein the construction of the vector containing the recombinant fusion enzyme gene and the recombinant genetically engineered bacterium includes the following steps: The construction of the nucleotide sequence encoding the recombinant domain fusion enzyme (DTE-5'-XYI-VE-DTE-3') includes the following steps: sequentially amplifying the encoding genes for the 5' domain (DTE-5') of D-taglatose 3-differentiated isomerase, the active site domain (XYI-VE) of xylose isomerase, and the partial 3' domain (DTE-3') of D-taglatose 3-differentiated isomerase, respectively; using overlap PCR, assembling the three encoding genes into a fusion gene fragment; and ligating the fusion gene fragment to the NdeI and NotI restriction enzyme sites of the vector pET22b using a one-step cloning method to obtain a recombinant plasmid. The recombinant plasmid is transformed into *Escherichia coli* BL21(DE3) to obtain the recombinant genetically engineered bacterium containing the recombinant fusion enzyme gene.

5. Application of recombinant domain fusion enzyme (DTE-5'-XYI-VE-DTE-3') in the preparation of D-allulose: The application involves using crude enzyme solution obtained by fermentation culture of engineered bacteria containing the encoding gene of fusion enzyme (DTE-5'-XYI-VE-DTE-3'), resting cells, purified enzyme, or immobilized enzyme as a biocatalyst. The substrate ethyl glucose is added, and a transformation system is constructed using a 50mM buffer solution with pH 6.0–10.0 as the reaction medium. The reaction is carried out at 50–65℃ for 8–24 hours. After the reaction is complete, the supernatant is subjected to ion exchange and decolorization, followed by separation using macroporous resin to obtain D-allulose and unreacted substrate glucose.