A cellobiose epimerase, its nucleotide sequence, preparation method, and applications.

Abstract

This invention relates to the field of enzyme engineering, and provides a cellobiose epimerase, its nucleotide sequence, preparation method, and application. The cellobiose epimerase includes one of H1, H2, and H3. The amino acid sequence of H1 is shown in SEQ ID NO:1, the amino acid sequence of H2 is shown in SEQ ID NO:2, and the amino acid sequence of H3 is shown in SEQ ID NO:3. This cellobiose epimerase has high quality enzyme activity, which helps to improve the conversion rate and yield of lactulose.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering, specifically to a cellobiose epimerase, its nucleotide sequence, preparation method, and applications. Background Technology

[0002] Cellobiose epimerases (CE, EC 5.1.3.11) can catalyze the reversible epimerization of the D-glucose subunit at the reducing end of β-1,4-linked oligosaccharides (such as β-1,4-mannose, cellulobiose, and lactose) to the D-mannose subunit. Some CE enzymes have also been found to possess structural isomerization activity, converting D-glucose / D-mannose subunits to D-fructose subunits, such as *Caldicellulosiruptor saccharolyticus* (CsCE), *Dictyoglomus turgidum* (DtCE), *Caldicellulosiruptor obsidiansis* (CoCE), and *Dictyoglomus thermophilum* (DhCE). When lactose is used as a substrate, these enzymes can catalyze the production of epilactose and lactulose. Lactulose is a non-digestible disaccharide that helps maintain the intestinal microenvironment and can also be used as an adjunct therapy for constipation and hepatic encephalopathy.

[0003] To increase lactulose production, the activity of cellobiose epimerase can be improved. Therefore, how to improve the activity of cellobiose epimerase has become a crucial issue that urgently needs to be addressed. Summary of the Invention

[0004] This invention provides a cellobiose epimerase, its nucleotide sequence, preparation method, and application. The cellobiose epimerase has high quality enzyme activity, which helps to improve the conversion rate and yield of lactulose.

[0005] The present invention provides a cellobiose epimerase, wherein the cellobiose epimerase comprises one of H1, H2 and H3, wherein the amino acid sequence of H1 is shown in SEQ ID NO:1, the amino acid sequence of H2 is shown in SEQ ID NO:2 and the amino acid sequence of H3 is shown in SEQ ID NO:3.

[0006] The present invention provides a recombinant DNA molecule that encodes a cellobiose epimerase as described above.

[0007] Optionally, the recombinant DNA molecule is a DNA sequence selected from: a) SEQ ID No:5, SEQ ID No:6, or SEQ ID No:7; b) a DNA sequence that encodes the same polypeptide as the sequence listed in a) due to the degeneracy of the genetic codon.

[0008] The present invention provides a recombinant expression vector comprising the recombinant DNA molecule as described above.

[0009] Optionally, the expression vector in the recombinant expression vector is pET-28b.

[0010] The present invention provides a recombinant bacterium, wherein the recombinant bacterium includes the recombinant expression vector described above.

[0011] Optionally, the recombinant bacteria are obtained by transforming the host bacterial species into the recombinant expression vector as described above.

[0012] The present invention provides a method for producing cellobiose epimerase, comprising: culturing the recombinant bacteria as described above under conditions favorable for the production of cellobiose epimerase to produce the cellobiose epimerase.

[0013] The present invention provides a composition comprising the cellobiose epimerase as described above or the cellobiose epimerase prepared according to the production method described above.

[0014] The present invention provides a method for preparing lactulose, comprising: obtaining the lactulose from the fermentation product of the recombinant bacteria as described above or obtaining it by reacting lactose with the composition as described above.

[0015] This invention provides a cellobiose epimerase, its nucleotide sequence, preparation method, and application. Compared with wild-type CsCE enzyme, the cellobiose epimerase with the above-mentioned amino acid sequence has higher quality enzyme activity, which helps to improve the conversion rate and yield of lactulose. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Technical roadmap for the modification of cellobiose epimerase (CsCE enzyme);

[0018] Figure 2Bar chart showing the mass enzyme activity of wild-type CsCE enzyme (WT), CsCE enzyme (H1), CsCE enzyme (H2), and CsCE enzyme (H3);

[0019] Figure 3 This is a schematic diagram illustrating the mechanism of the colorimetric method for measuring enzyme activity. Detailed Implementation

[0020] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present invention, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Normally, CsCE enzyme is a cellobiose epimerase produced by pyrolytic cellulolytic bacteria (Caldicellulosiruptors accharolyticus). It can catalyze the production of lactulose from lactose as a substrate. Lactulose can stabilize the intestinal microenvironment and can also be used as an adjunct therapy for constipation and hepatic encephalopathy. CsCE enzyme has good structural isomerism. To increase lactulose production, the quality and enzyme activity of CsCE enzyme can be improved.

[0022] Based on this, the first aspect of the present invention provides a cellobiose epimerase, which includes one of H1, H2, and H3; the amino acid sequence of H1 is shown in SEQ ID NO:1; the amino acid sequence of H2 is shown in SEQ ID NO:2; and the amino acid sequence of H3 is shown in SEQ ID NO:3.

[0023] The study found that, compared with wild-type CsCE enzyme, CsCE enzyme with the above-mentioned amino acid sequence SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 had higher quality enzyme activity, which helped to improve the conversion rate and yield of lactulose. This may be because, compared with wild-type CsCE enzyme, the CsCE enzyme with the above-mentioned amino acid sequence SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 had higher quality enzyme activity. The number of hydrogen bonds between the CsCE enzyme (protein) of NO:3 and the substrate (lactose) is significantly increased, which is significantly higher than that between the wild-type CsCE enzyme and the substrate (lactose). The increase in the number of hydrogen bonds facilitates the conversion of the substrate (lactose) to lactulose. Furthermore, the microenvironment of the active site of the CsCE enzyme in this embodiment of the invention is changed. Not only does the interaction between the substrate and the CsCE enzyme change, but the number of hydrogen bonds between the substrate and solvent molecules is also significantly higher than that of the wild-type CsCE enzyme. In addition, the substitution of amino acids in the CsCE enzyme of this embodiment of the invention leads to a significant reduction in the distance between the catalytic residue His377 and the O1 of the substrate (lactose), which is more conducive to the catalytic conversion of the substrate (lactose) to lactulose.

[0024] The amino acid sequence of the above wild-type CsCE enzyme is shown in SEQ ID NO:4.

[0025] A second aspect of the present invention provides a recombinant DNA molecule that encodes the aforementioned cellobiose epimerase.

[0026] Specifically, the recombinant DNA molecule described above is a DNA sequence selected from the following: a) SEQ ID No: 5 or SEQ ID No: 6 or SEQ ID NO: 7; b) a DNA sequence that encodes the same polypeptide as the sequence listed in a) due to the degeneracy of the genetic codon.

[0027] It is understandable that, since the amino acid composition of the codon-optimized CsCE enzyme remains unchanged, the recombinant DNA molecule described above also includes the codon-optimized DNA sequence shown in a) or b). That is, cellobiose epimerases can also include CsCE enzymes obtained after codon optimization.

[0028] Codon optimization is a technique that improves protein expression levels in organisms by increasing the translation efficiency of target genes. It typically involves redesigning genes by avoiding rare codons, utilizing preferred codons, simplifying mRNA secondary structure, optimizing repetitive sequences, eliminating restriction enzyme sites, and adjusting GC content to improve translation efficiency and thus increase protein expression levels.

[0029] Codon bias refers to the phenomenon where different organisms, and even different protein-coding genes within the same organism, use degenerate codons at different frequencies. For example, in *E. coli*, tyrosine is encoded by two codons, UAU and UAC, but *E. coli* prefers to use UAU to guide amino acid synthesis. Some rare codon clusters within degenerate codons can inhibit the expression of the target protein or cause frameshift mutations because the translation complex pauses when encountering rare codons during mRNA translation. Therefore, codon optimization of the CsCE enzyme according to the rare codon bias observed in *E. coli* translation can further enhance the expression level of the CsCE enzyme.

[0030] Figure 1 The technical roadmap for the modification of cellobiose epimerase (CsCE enzyme) is shown, including the mechanism diagram of the colorimetric assay for enzyme activity. Figure 3 As shown.

[0031] A third aspect of the present invention provides a recombinant expression vector comprising the above-described recombinant DNA molecule.

[0032] The recombinant expression vector in this invention can be a recombinant plasmid. Plasmids have the advantages of stability, reliability, and ease of operation; therefore, recombinant plasmids can be constructed by recombining the above-mentioned recombinant DNA molecules into plasmids. Like plasmids, the resulting recombinant plasmids also have the ability to self-replicate and maintain a constant copy number in daughter cells.

[0033] The embodiments of this invention do not limit the method of recombining recombinant DNA molecules into plasmids; those skilled in the art can choose the method as needed. Specifically, those skilled in the art can digest the recombinant DNA molecules and plasmids with restriction endonucleases to obtain recombinant DNA molecules and plasmids with corresponding sticky ends, and then use DNA ligase to ligate the recombinant DNA molecules and plasmids with corresponding sticky ends. Further, the digestion can be single or double digestion, that is, the restriction endonuclease can be one or two.

[0034] For example, a recombinant expression vector containing a gene expression cassette can be obtained by double digestion with restriction endonucleases NdeI and XhoI, followed by T4 ligase.

[0035] Alternatively, Gibson assembly technology can be used to introduce terminal homologous sequences into recombinant DNA molecules and plasmids, and then the gene expression cassette and plasmid can be ligated to obtain a recombinant expression vector.

[0036] Furthermore, in the scheme of the present invention, the expression vector in the recombinant expression vector is pET-28b.

[0037] The coding sequence of pET-28b used in this embodiment of the invention is shown in SEQ ID NO:8. The expression vector pET-28b has a low basal expression level in the E. coli expression system and will not affect the expression of the recombinant DNA molecule of this invention. It can regulate the expression level of CsCE enzyme according to the concentration of inducers, such as isopropyl thiogalactoside (IPTG). It can also provide configurations for various fusion tags, marker genes, or other expression systems. Therefore, choosing pET-28b as the expression vector in this embodiment of the invention helps to improve the expression level of CsCE enzyme.

[0038] A fourth aspect of the present invention provides a recombinant bacterium, comprising the above-described recombinant expression vector.

[0039] In this embodiment of the invention, a recombinant expression vector carrying the aforementioned recombinant DNA molecule (i.e., the encoding gene for the highly active cellobiose epimerase) is introduced into bacteria, causing the bacteria to express the encoding gene for the highly active cellobiose epimerase in the recombinant DNA molecule, thus obtaining recombinant bacteria. The construction and culture methods of the recombinant bacteria are simple and easy to implement, and the entire process is easy to control precisely, stably producing large amounts of CsCE enzyme.

[0040] Furthermore, recombinant bacteria can be obtained by transforming host bacterial strains into recombinant expression vectors.

[0041] The embodiments of the present invention do not limit the method of transforming the recombinant expression vector into the host bacterial species. For example, the recombinant expression vector (such as a recombinant plasmid) can be introduced into the host bacterial species by heat shock transformation to obtain recombinant bacteria.

[0042] Specifically, the host bacterial strain can be E. coli BL21(DE3), a strain of Escherichia coli B that lacks Lon protease and the outer membrane protease OmpT, thus reducing the degradation of heterologous proteins expressed in cells and exhibiting high transformation efficiency. Therefore, selecting E. coli BL21(DE3) as the host bacterial strain in this embodiment of the invention helps to increase the expression level of CsCE enzyme.

[0043] CsCE enzyme can catalyze the conversion of lactose into lactulose. The aforementioned recombinant DNA molecules, recombinant expression vectors, or recombinant bacteria can improve the quality and enzyme activity of CsCE enzyme. Therefore, the aforementioned recombinant DNA molecules, recombinant expression vectors, or recombinant bacteria can be used in lactulose preparation. By improving the quality and enzyme activity of CsCE enzyme, more lactose is converted into lactulose, thereby increasing the yield of lactulose preparation.

[0044] A fifth aspect of this invention provides a method for producing cellobiose epimerase, comprising: culturing the recombinant bacteria described above under conditions favorable for cellobiose epimerase production to generate cellobiose epimerase. This production method can produce cellobiose epimerase with high quality and enzyme activity.

[0045] A sixth aspect of this invention provides a composition comprising the above-described cellobiose epimerase or a cellobiose epimerase prepared according to the above-described production method. This composition can be used to prepare lactulose and can improve the yield of lactulose preparation.

[0046] Specifically, the above composition may include one or more of food compositions, feed compositions, and dairy product compositions.

[0047] A seventh aspect of the present invention provides a method for preparing lactulose, comprising obtaining lactulose from the fermentation product of recombinant bacteria or obtaining lactulose by catalyzing the reaction of lactose using the above-described composition. Since the recombinant bacteria provided in the embodiments of the present invention include the above-described recombinant expression vector, i.e., including the above-described recombinant DNA molecule, the recombinant bacteria can produce the cellobiose epimerase of the embodiments of the present invention during fermentation. This cellobiose epimerase has higher quality enzyme activity and can better catalyze the conversion of lactose to lactulose, thereby improving the yield of lactulose preparation.

[0048] The technical solution of the present invention will be further explained and described below with reference to specific embodiments.

[0049] For other experimental methods that do not specify particular conditions, they are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, all reagents used are commercially available or readily available from public sources.

[0050] The primer sequences involved in the following examples are shown in Table 1.

[0051] Table 1

[0052]

[0053] Example 1

[0054] 1. Constructing recombinant DNA molecules

[0055] 1) The whole genome of Caldicellulosiruptor saccharolyticus DSM 8903 was extracted as a template. Primers CsCE-F and CsCE-R were designed based on the CsCE gene sequence (Gene Accession: YP_0011791132.1). Their DNA sequences are shown in Table 1. The restriction enzyme sites NdeI and XhoI were introduced. The gene fragment expressing CsCE was amplified by PCR. Its amino acid sequence is shown in SEQ ID NO:4.

[0056] 2) Extract pET-28b(+) plasmid, digest the pET-28b(+) plasmid and the CsCE gene fragment with NdeI and XhoI respectively, and then ligate the CsCE gene fragment and the pET-28b(+) plasmid with T4 ligase to obtain the recombinant plasmid pET-28b-CsCE (DNA of recombinant wild-type CsCE).

[0057] 3) Construct the DNA sequences shown in SEQ ID No: 5, SEQ ID No: 6, and SEQ ID No: 7.

[0058] 3.1 Using the recombinant plasmid pET-28b-CsCE as a template, primers L172V / S173L-F, L172V / S173L-R, K356L-F, K356L-R, Q371E-F, and Q371E-R were designed. The sequences of the above primers are shown in Table 1. Using PCR technology, leucine (L) at position 172 of the CsCE gene fragment was mutated to valine (V), serine (S) at position 173 was mutated to leucine (L), and leucine (L) at position 356 was mutated to valine (V). The lysine (K) at position 371 is mutated to leucine (L), and the tryptophan (W) at position 371 is mutated to glutamic acid (E), thereby amplifying pET-28b-CsCE (4 amino acid substitutions). The pET-28b-CsCE (4 amino acid substitutions) introduces mutation sites L172V, S173L, K356L, and Q371E into the CsCE gene fragment. pET-28b-CsCE (4 amino acid substitutions) encodes the amino acid sequence H1 (as shown in SEQ ID NO:1).

[0059] 3.2 Using the recombinant plasmid pET-28b-CsCE as a template, primers K46Y / Q48N-F, K46Y / Q48N-R, E174P-F, E174P-R, N358D / D360S / E362N-F, N358D / D360S / E362N-R, Q371E-F, and Q371E-R were designed. Using PCR, lysine (K) at position 46 of the CsCE gene fragment was mutated to tyrosine (Y), glutamine (Q) at position 48 was mutated to asparagine (N), glutamate (E) at position 174 was mutated to proline (P), and asparagine (N) at position 358 was mutated. The aspartic acid (D) at position 360 was mutated to serine (S), the glutamic acid (E) at position 362 was mutated to asparagine (N), and the glutamine (Q) at position 371 was mutated to glutamic acid (E), thereby amplifying pET-28b-CsCE (7 amino acid substitutions). This pET-28b-CsCE (7 amino acid substitutions) introduces mutation sites K46Y, Q48N, E174P, N358D, D360S, E362N, and Q371E into the CsCE gene fragment. pET-28b-CsCE (7 amino acid substitutions) encodes the amino acid sequence of H2 (as shown in SEQ ID NO:2).

[0060] 3.3 Using the recombinant plasmid pET-28b-CsCE as a template, primers were designed as follows: L172V / S173L-F, L172V / S173L-R, K356L-F, K356L-R, Q371E-F, Q371E-R, K46Y / Q48N-F, K46Y / Q48N-R, E174P-F, E174P-R, and N358D / D360S / E362N-F. N358D / D360S / E362N-R, Q371E-F, and Q371E-R were modified using PCR to mutate the following CsCE gene fragments: lysine (K) at position 46 to tyrosine (Y), glutamine (Q) at position 48 to asparagine (N), leucine (L) at position 172 to valine (V), serine (S) at position 173 to leucine (L), and leucine (L) at position 174. The following mutations were performed: glutamic acid (E) was mutated to proline (P), lysine (K) at position 356 was mutated to leucine (L), asparagine (N) at position 358 was mutated to aspartic acid (D), aspartic acid (D) at position 360 was mutated to serine (S), glutamic acid (E) at position 362 was mutated to asparagine (N), and tryptophan (W) at position 371 was mutated to glutamic acid (E), thus amplifying pET-28b-CsCE (10 amino acid substitutions). This pET-28b-CsCE (10 amino acid substitutions) introduced mutation sites K46Y, Q48N, L172V, S173L, E174P, K356L, N358D, D360S, E362N, and Q371E into the CsCE gene fragment. pET-28b-CsCE (10 amino acid substitutions) encodes the amino acid sequence H3 (as shown in SEQ). (As shown in ID NO:3).

[0061] 4) The pET-28b-CsCE (4 amino acid replacements), pET-28b-CsCE (7 amino acid replacements), and pET-28b-CsCE (10 amino acid replacements) fragments were amplified by PCR. The PCR amplification reaction system is shown in Table 2, and the total volume of the reaction system is 50 μL; the PCR amplification program is shown in Table 3.

[0062] Table 2

[0063]

[0064] Table 3

[0065]

[0066] 2. Constructing recombinant bacteria

[0067] Recombinant plasmids pET-28b-CsCE (4 amino acid replacements), pET-28b-CsCE (7 amino acid replacements), and pET-28b-CsCE (10 amino acid replacements) were introduced into competent E. coli Trans5α strains using the heat shock method. The transformed E. coli Trans5α strains were then plated on LB agar plates containing kanamycin (Kan) and incubated upside down at 37°C for 12-16 h.

[0068] Single colonies were picked from LB agar plates for colony PCR, and the bands were detected by agarose gel electrophoresis. Single colonies with the correct band size were selected for sequencing.

[0069] Plasmids were extracted from the successfully sequenced bacterial cultures and transformed into E. coli BL21(DE3). Colony PCR and sequencing were performed again to verify the results. Finally, the following recombinant plasmids were obtained: pET-28b-CsCE (4 amino acid replacements) with E. coli BL21(DE3), pET-28b-CsCE (7 amino acid replacements) with E. coli BL21(DE3), and pET-28b-CsCE (10 amino acid replacements) with E. coli BL21(DE3) (evolved recombinant bacteria).

[0070] 3. Codon optimization

[0071] Using the recombinant plasmid pET-28b-CsCE (with 4 amino acid substitutions) as a template, the base sequence of CsCE (with 4 amino acid substitutions) was optimized according to the rare codon preference of E. coli translation, resulting in the codon-optimized CsCE (with 4 amino acid substitutions) gene fragment, the nucleotide sequence of which is shown in SEQ ID NO:5. The CsCE (with 4 amino acid substitutions) gene fragment was ligated into plasmid pET-28b(+) to obtain the recombinant plasmid pET-28b-CsCE-opt (with 4 amino acid substitutions). The recombinant plasmid pET-28b-CsCE-opt (with 4 amino acid substitutions) was transformed into E. coli Trans5α for expansion culture, followed by extraction and then transformation into E. coli BL21(DE3) to obtain the recombinant plasmid pET-28b-CsCE-opt (with 4 amino acid substitutions) with E. coli BL21(DE3) as the host bacterium.

[0072] Using the recombinant plasmid pET-28b-CsCE (7 amino acid substitutions) as a template, the base sequence of CsCE (7 amino acid substitutions) was optimized according to the rare codon preference of E. coli translation, resulting in the codon-optimized CsCE (7 amino acid substitutions) gene fragment, whose nucleotide sequence is shown in SEQ ID NO:6. The CsCE (7 amino acid substitutions) gene fragment was ligated into plasmid pET-28b(+) to obtain the recombinant plasmid pET-28b-CsCE-opt (7 amino acid substitutions). The recombinant plasmid pET-28b-CsCE-opt (7 amino acid substitutions) was transformed into E. coli Trans5α for expansion culture, followed by extraction and then transformation into E. coli BL21(DE3) to obtain the recombinant plasmid pET-28b-CsCE-opt (7 amino acid substitutions) with E. coli BL21(DE3) as the host bacterium.

[0073] Using the recombinant plasmid pET-28b-CsCE (10 amino acid replacements) as a template, the base sequence of CsCE (10 amino acid replacements) was optimized according to the rare codon preference of E. coli translation, resulting in the codon-optimized CsCE (7 amino acid replacements) gene fragment, namely the CsCE (10 amino acid replacements) gene fragment, the nucleotide sequence of which is shown in SEQ ID NO:7. The CsCE (10 amino acid replacements) gene fragment was ligated into plasmid pET-28b(+) to obtain the recombinant plasmid pET-28b-CsCE-opt (10 amino acid replacements). The recombinant plasmid pET-28b-CsCE-opt (10 amino acid replacements) was transformed into E. coli Trans5α for expansion culture, followed by extraction, and then transformed into E. coli BL21(DE3) to obtain the recombinant plasmid pET-28b-CsCE-opt (10 amino acid replacements) with E. coli BL21(DE3) as the host bacterium.

[0074] Example 2

[0075] 1) Extract pure enzyme solution of CsCE enzyme (H1) with the amino acid sequence shown in SEQ ID No: 1.

[0076] The recombinant plasmid pET-28b-CsCE (with 4 amino acid substitutions) of E. coli BL21(DE3) obtained in step 3) of Example 1 was inoculated into LB liquid medium containing kanamycin (Kan) and cultured overnight at 37°C and 200 rpm to obtain activated bacterial solution. Then, the activated bacterial solution was inoculated into LB medium containing kanamycin (Kan) at an inoculation rate of 1% (v / v) and cultured at 37°C and 200 rpm until OD. 600The pH was 0.6-0.8. Isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.2 mM, and the mixture was induced at 25°C and 200 rpm for 18 h to obtain the fermentation broth. The fermentation broth was then centrifuged, and the cells were collected for protein purification. The collected cells were then resuspended and mixed with Lysis Buffer (50 mM Na₂HPO₄, 200 mM NaCl, 10 mM imidazole, pH 7.5), and sonicated in an ice bath to obtain the crude enzyme solution. The crude enzyme solution was then centrifuged at 12000 rpm and 4°C for 15 min to remove cell debris, yielding the crude enzyme supernatant. The crude enzyme supernatant was then passed through a nickel (Ni) ion affinity chromatography column. The column was first washed with Washing Buffer (50 mM Na₂HPO₄, 200 mM NaCl, 100 mM imidazole, pH 7.5), and then with Elution Buffer (50 mM Na₂HPO₄, 200 mM NaCl, 100 mM imidazole, pH 7.5). Elute with Na2HPO4, 200 mM NaCl, 250 mM imidazole (pH 7.5), and collect the eluent containing the target protein. Dialyze the eluent containing the target protein using a 50 mM 1,4-piperazine diethanesulfonic acid (PIPES) buffer at pH 7.5. Dialyze three times, six hours each time. After dialysis, collect the purified enzyme solution from the dialysis bag into an EP tube and store at 4°C for later use.

[0077] 2) Extract pure enzyme solution of CsCE enzyme (H2) with the amino acid sequence shown in SEQ ID No: 2.

[0078] Following the method described above for collecting pure enzyme solution of CsCE enzyme (H1) with amino acid sequence as shown in SEQ ID No: 1, pure enzyme solution of CsCE enzyme (H2) with amino acid sequence as shown in SEQ ID No: 2 was collected.

[0079] 3) Extract pure enzyme solution of CsCE enzyme (H3) with the amino acid sequence shown in SEQ ID No: 3.

[0080] Following the method described above for collecting pure enzyme solution of CsCE enzyme (H1) with amino acid sequence as shown in SEQ ID No: 1, pure enzyme solution of CsCE enzyme (H3) with amino acid sequence as shown in SEQ ID No: 3 was collected.

[0081] Comparative Example 1

[0082] The pure enzyme solution of wild-type CsCE enzyme (WT) was collected according to the method described above for collecting pure enzyme solution of CsCE enzyme (H1) with an amino acid sequence as shown in SEQ ID No: 1.

[0083] Test case

[0084] The following parameters of the above-mentioned wild-type CsCE enzymes (WT), (H1), (H2), and (H3) were tested.

[0085] 1) Expression level

[0086] Bacterial cells were obtained at 37°C and 200 rpm according to the method in step 1) of Example 2. Enzymes were extracted from all cultured bacteria to obtain equal volumes of pure enzyme solutions; the protein concentration in the pure enzyme solutions was detected using a BCA protein quantification kit. The expression level of CsCE enzyme refers to the mass of CsCE enzyme produced per liter of fermentation broth under induction conditions, calculated using the following formula: Where c is the protein concentration in the pure enzyme solution, V1 is the volume of the pure enzyme solution, and V2 is the volume of the fermentation broth.

[0087] 2) Quality enzyme activity

[0088] In a 50 mM PIPES (pH 7.5) buffer environment, the pure enzyme solution was mixed with 400 mM lactose at a volume ratio of 1:1, and then reacted at 80°C for 10 min. The reaction was then immediately terminated by boiling for 5 min to obtain the enzyme reaction solution.

[0089] The enzyme reaction solution was diluted 10-fold with Wahaha purified water, and 15% trichloroacetic acid was added to precipitate the protein. After centrifugation at 8000 rpm for 2 min at room temperature, the solution was filtered through a 0.22 μm aqueous membrane and transferred to a HPLC vial for later use. A high-performance liquid chromatography (HPLC) system (Waters Co.) equipped with an RI detector (Waters Alliance 2414, Waters Co.) and an NH4P-50-4E column (4.6 mm × 250 mm; Shodex, Tokyo, Japan) was used. The column temperature was set to 40℃, and elution was performed with acetonitrile / methanol / water (75:20 / 5, v / v / v) at a flow rate of 1 mL / min. The concentration of the product in the reaction solution was quantified using lactulose, ipilactose, and lactose standards.

[0090] Mass enzyme activity refers to the amount of enzyme required to produce 1 μmol of product per minute under optimal conditions, which is 1 U. It is calculated using the following formula: Where c1 is the product concentration, c2 is the enzyme concentration, V is the reaction volume, and t is the reaction time.

[0091] In this invention, mass enzyme activity refers to structural isomer activity.

[0092] Structural isomer activity: The amount of enzyme required to produce 1 μmol of lactulose per minute is 1 U. This is calculated using the following formula: Where c3 is the lactulose concentration, c2 is the enzyme concentration, V is the reaction volume, and t is the reaction time.

[0093] Epimeric activity: The amount of enzyme required to produce 1 μmol of ipilactose per minute is 1 U. This is calculated using the following formula: Where c4 is the concentration of ipilactose, c2 is the enzyme concentration, V is the reaction volume, and t is the reaction time.

[0094] Michaelis constant (K) m The catalytic constant (kcat) was evaluated based on the increase in lactulose, the product of lactose with a substrate of 60 mM–900 mM lactose. The data were fitted using a nonlinear Michaelis-Menten equation. The calculation formula is as follows:

[0095]

[0096]

[0097] In equations (1) and (2), Vmax is the maximum reaction rate; K m - Michaelis constant; S - substrate concentration; kcat - catalytic constant; [E] - enzyme concentration.

[0098] Test results

[0099] Table 4. Expression levels of wild-type CsCE enzyme (WT), CsCE enzyme (H1), and CsCE enzyme (H2).

[0100]

[0101] Table 5. Enzymatic properties of wild-type CsCE enzyme (WT), CsCE enzyme (H1), and CsCE enzyme (H2).

[0102]

[0103] As shown in Table 4, the expression levels of CsCE enzyme (H1) (216 mg / L), CsCE enzyme (H2) (202 mg / L) and CsCE enzyme (H3) (222 mg / L) did not change significantly compared with wild-type CsCE enzyme (WT) (208 mg / L).

[0104] like Figure 2 As shown, the structural isomers of H1, H2, and H3 all exhibited significantly enhanced enzymatic activity (structural isomer activity). H1, at 80℃ and pH 7.5, achieved an enzymatic activity (structural isomer activity) of 19.00 U / mg, which is 3.66 times higher than that of wild-type CsCE (WT). Figure 2Under the same conditions, the mass enzyme activity (structural isomer activity) of H2 reached 20.72 U / mg, which is 4.08 times higher than that of wild-type CsCE (WT). Under the same conditions, the mass enzyme activity (structural isomer activity) of H3 reached 23.19 U / mg, which is 4.68 times higher than that of wild-type CsCE (WT).

[0105] As shown in Table 5, the catalytic efficiency of H1 (kcat / K m The value is 0.17 s. -1 mM -1 It is 2.125 times that of WT, but its K m The value remained essentially unchanged, indicating that H1's ability to convert lactulose per minute was significantly enhanced. The catalytic efficiency of H2 was 0.099 s⁻¹. -1 mM -1 It is 1.24 times that of WT. Its kcat value (18.93 s) -1 The significant increase in K indicates that its ability to convert lactulose per minute is also significantly enhanced, but K m The increase in the concentration (192.0 mM) led to a decrease in substrate affinity, resulting in a slight increase in catalytic activity against structural isomers. This is consistent with the ratio of its structural isomer catalytic activity (20.72 U / mg) to its epimeric catalytic activity (58.87 U / mg). The catalytic efficiency of H3 was 0.17 s⁻¹. -1 mM -1 It is 2.125 times that of WT, similar to H1. Its kcat value (36.84s) -1 The significant increase in K indicates that its ability to convert lactulose per minute is also significantly enhanced, but K m The increase in the value (217.8.0 mM) led to a decrease in its substrate affinity, which is similar to the results for H2.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cellobiose epimerase, characterized in that, The cellobiose epimerase is selected from one of H1, H2, and H3, wherein the amino acid sequence of H1 is shown in SEQ ID NO:1, the amino acid sequence of H2 is shown in SEQ ID NO:2, and the amino acid sequence of H3 is shown in SEQ ID NO:

3.

2. A recombinant DNA molecule, characterized in that, The recombinant DNA molecule encodes the cellobiose epimerase described in claim 1.

3. The recombinant DNA molecule according to claim 2, characterized in that, The recombinant DNA molecule is a DNA sequence selected from the following: a) SEQ ID No:5 or SEQ ID No:6 or SEQ ID No:7; b) DNA sequences that encode the same polypeptide as the sequences listed in a) due to the degeneracy of the genetic codon.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the recombinant DNA molecule as described in claim 2 or 3.

5. The recombinant expression vector according to claim 4, characterized in that, The recombinant expression vector is pET-28b.

6. A recombinant bacterium, characterized in that, The recombinant bacteria comprises the recombinant expression vector as described in claim 4 or 5.

7. The recombinant bacteria according to claim 6, characterized in that, The recombinant bacteria are obtained by transforming the host bacterial strain into the recombinant expression vector as described in claim 4 or 5.

8. A method for producing cellobiose epimerase, characterized in that, include: The recombinant bacteria of claim 6 or 7 are cultured under conditions favorable for the production of cellobiose epimerase to produce the cellobiose epimerase.

9. A composition, characterized in that, The composition comprises the cellobiose epimerase of claim 1 or the cellobiose epimerase prepared according to the production method of claim 8.

10. A method for preparing lactulose, characterized in that, include: The lactulose is obtained from the fermentation product of the recombinant bacteria of claim 6 or 7, or by reacting lactose with the composition of claim 9.

Images