Recombinant Escherichia coli for producing D-tagatose by using redox enzyme driven by cofactor regeneration as well as construction method and application of recombinant Escherichia coli

Abstract

The invention discloses recombinant escherichia coli for producing D-tagatose by using redox enzyme driven by cofactor regeneration as well as a construction method and application of the recombinant escherichia coli, and belongs to the technical field of biological engineering. The method comprises the following steps: introducing xylose reductase xyrB, glucose dehydrogenase Gox2015, galactitol dehydrogenase RlGDH mutant T193G / G98C and water-producing NADH oxidase SpNox into escherichia coli, so as to construct recombinant escherichia coli; according to the method disclosed by the invention, a way for synthesizing the D-tagatose by regenerating and driving oxidoreductase through cofactors is designed and constructed, and lactose in whey can be completely converted into the D-tagatose and sodium gluconate by combining the way with beta-galactosidase, so that high-valued utilization of a dairy product processing by-product whey is realized.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a recombinant Escherichia coli strain that produces D-tagatose via cofactor regeneration-driven oxidoreductase, its construction method, and its application. Background Technology

[0002] The production of dairy products such as cheese, yogurt, and casein generates approximately 200 million tons of whey annually, with an annual growth rate of 4%. In small and medium-sized cheese manufacturers in developing countries, whey is still treated as waste, either directly discharged into water resources or sent to landfills. Untreated whey, due to its high chemical oxygen demand (COD) and biological oxygen demand (BOD), can have negative environmental impacts. Previous research has focused more on whey protein recovery, with most lactose being wasted, and its disposal causing serious environmental problems. In recent years, the high-value utilization of lactose, which accounts for approximately 70% of whey dry matter, has received increasing attention. Lactose in whey, or its hydrolysis products (D-glucose and D-galactose), can be converted into valuable bioproducts such as biogas, bioplastics, or rare sugars through microorganisms or enzymes. D-galactose, a hydrolysis product of whey lactose, is a direct substrate for D-tagatose synthesis; its conversion into D-tagatose through biological methods can bring significant socio-economic benefits. D-Tag sugar is a natural, low-calorie functional sweetener with 92% the sweetness of sucrose but only 38% of its calories. Due to its various bioactivities, including lowering blood sugar, anti-oxidation, improving gut microbiota, and others, it has wide applications in the food and pharmaceutical industries. Furthermore, D-Tag sugar is listed as a Generally Recognized As Safe (GRAS) material by the U.S. Food and Drug Administration (FDA).

[0003] In the past, whey or lactose was mainly converted to D-tagatose via a two-step enzymatic process using β-galactosidase and L-arabinose isomerase. However, due to the thermodynamic equilibrium limitations of L-arabinose isomerase, the equilibrium ratio of D-galactose to D-tagatose is typically between 7:3 and 4:6, resulting in a low conversion rate of D-tagatose from lactose and insignificant processing benefits for whey or lactose. Improving the conversion rate of lactose to D-tagatose is crucial for achieving the industrial biosynthesis of D-tagatose and enhancing the economic efficiency of the whey powder processing industry. Summary of the Invention

[0004] The purpose of this invention is to provide a recombinant Escherichia coli strain for producing D-tagatose using cofactor regeneration-driven oxidoreductase, along with its construction method and application, to address the problems existing in the prior art. Based on a cofactor regeneration cycle, a highly efficient three-step enzymatic conversion process for lactose—"hydrolysis-dual-enzyme reduction-dual-enzyme oxidation"—is established to completely convert lactose into D-tagatose and sodium gluconate. This establishes a highly efficient multi-enzyme catalytic system for the preparation of D-tagatose, which is of great significance for improving the economic benefits of the whey powder processing industry.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a recombinant *Escherichia coli* strain for producing D-tagatose, comprising recombinant *E. coli* I and recombinant *E. coli* II. Recombinant *E. coli* I simultaneously expresses xylose reductase xyrB, glucose dehydrogenase Gox2015, a galactitol dehydrogenase mutant T193G / G98C, and an aqueous NADH oxidase SpNox; recombinant *E. coli* II simultaneously expresses a galactitol dehydrogenase mutant T193G / G98C and an aqueous NADH oxidase SpNox. The amino acid sequence of the xylose reductase xyrB is shown in SEQ ID NO.1, the amino acid sequence of the glucose dehydrogenase Gox2015 is shown in SEQ ID NO.2, the amino acid sequence of the galactitol dehydrogenase mutant T193G / G98C is shown in SEQ ID NO.4, and the amino acid sequence of the water-producing NADH oxidase SpNox is shown in SEQ ID NO.3.

[0006] The present invention also provides a method for constructing the recombinant Escherichia coli, comprising the method shown in (1) or (2): (1) The genes encoding xylose reductase xyrB and glucose dehydrogenase Gox2015 were co-expressed in an expression vector to construct a recombinant plasmid for the reduction module; The coding genes for galactitol dehydrogenase mutant T193G / G98C and water-producing NADH oxidase SpNox were co-expressed in an expression vector to construct an oxidative module recombinant plasmid. The recombinant plasmid of the reduction module and the recombinant plasmid of the oxidation module were co-transformed into E. coli T7 competent cells to obtain the recombinant E. coli I. (2) The coding genes of galactitol dehydrogenase mutant T193G / G98C and water-producing NADH oxidase SpNox were co-expressed in the expression vector to construct an oxidative module recombinant plasmid; The recombinant plasmid of the oxidation module was transformed into Escherichia coli T7 competent cells to obtain the recombinant Escherichia coli II.

[0007] This invention also provides the application of the recombinant Escherichia coli in the production of D-tagatose. Furthermore, the recombinant Escherichia coli can be used as a catalyst in the production of D-tagatose in the form of whole cell lysate, cell lysis lysate, or in the form of a bacterial agent.

[0008] The present invention also provides a method for producing D-tagatose, comprising the following steps: (1) Using the hydrolysate of whey powder after hydrolysis by β-galactosidase as a substrate, or using D-galactose as a substrate and D-glucose as a cofactor NADPH regenerator, the supernatant of the recombinant Escherichia coli I is added as a catalyst, and the reaction is carried out under the condition of oxygen introduction, so that D-galactose and D-glucose are completely converted into D-tagatose and sodium gluconate; (2) D-tagatose was separated and purified from the reaction solution.

[0009] Preferably, in step (1), the final concentration of the cell-breaking supernatant added is 10 mg / mL; the reaction conditions are: temperature 30℃, shaking speed 180 rpm, pH value of the reaction system 8.0, and reaction time 24 h. In step (2), the separation and purification method includes: adding CaCl2 to the reaction solution to convert sodium gluconate into calcium gluconate precipitate, centrifuging to separate the precipitate, and then desalting the supernatant by ion exchange to obtain D-tagatose.

[0010] The present invention also provides a method for producing D-tagatose, comprising the method shown in (1) or (2) below: (1) Using galactitol as a substrate, the recombinant Escherichia coli II was added as a catalyst and the whole cell reaction was carried out for 24 h to obtain D-tagatose; (2) Using D-galactose as substrate and D-glucose as cofactor NADPH regenerator, the recombinant Escherichia coli I was added as catalyst, the pH was adjusted and maintained at 8.0, and the whole cell reaction was carried out at 30℃ and 180 rpm for 24 h to obtain D-tagatose.

[0011] The present invention also provides a recombinant Escherichia coli for producing galactitol, wherein the recombinant Escherichia coli simultaneously expresses xylose reductase xyrB and glucose dehydrogenase Gox2015; The amino acid sequence of the xylose reductase xyrB is shown in SEQ ID NO.1, and the amino acid sequence of the glucose dehydrogenase Gox2015 is shown in SEQ ID NO.2.

[0012] The present invention also provides a method for preparing the recombinant Escherichia coli, comprising the following steps: The genes encoding xyrB and glucose dehydrogenase Gox2015 were co-expressed in an expression vector to construct a recombinant plasmid for the reduction module. The recombinant plasmid of the reduction module was transformed into Escherichia coli T7 competent cells to obtain the recombinant Escherichia coli.

[0013] This invention also provides the application of the recombinant Escherichia coli in the production of galactitol. Furthermore, the recombinant Escherichia coli can be used as a catalyst in the production of galactitol in the form of whole cell lysate, cell lysis lysate, or in the form of a bacterial agent.

[0014] The present invention also provides a method for producing galactitol, comprising the following steps: using D-galactose as a substrate, D-glucose as a cofactor NADPH regenerator, the recombinant Escherichia coli as a catalyst, adjusting the pH value and maintaining the pH value at 8.0, reacting the whole cells for 6 h to obtain galactitol.

[0015] The present invention discloses the following beneficial effects: This invention establishes a highly efficient three-step enzyme cascade conversion process for lactose based on cofactor cycling: hydrolysis, dual-enzyme reduction, and dual-enzyme oxidation. The conversion rate of lactose to D-tagatose reaches 100% using whole-cell or cell-disrupted crude enzyme solutions. The establishment of a highly efficient, high-substrate-concentration, and high-conversion-rate multi-enzyme catalytic system for D-tagatose preparation enables the high-value utilization of whey, a byproduct of dairy processing, and is of great significance for improving the economic benefits of the whey powder processing industry. 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 embodiments will be briefly introduced below. Obviously, the drawings described below are only 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 Results of whole-cell reactions of the reduction modules with different substrate concentrations; Figure 2 Results of whole-cell reactions of the oxidation module with different substrate concentrations; Figure 3 Results of whole-cell reactions of the reduction-oxidation combination module with different substrate concentrations; Figure 4 Results of the cell-disrupting supernatant reaction of the reduction-oxidation combination module with different substrate concentrations; Figure 5 A flowchart illustrating the three-step enzymatic process for preparing D-tagatose from whey lactose via hydrolysis, dual-enzyme reduction, and dual-enzyme oxidation. Figure 6 Results of the efficient preparation of D-tagatose from whey powder using a three-step enzymatic method of "hydrolysis-double enzyme reduction-double enzyme oxidation"; Figure 7 The HPLC chromatogram of D-tagatose standard sample is shown. Figure 8HPLC chromatogram of D-tagatose preparation from whey powder via a three-step enzymatic process of hydrolysis-dual-enzyme reduction-dual-enzyme oxidation. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] The materials used in the embodiments of the present invention include: Escherichia coli (E. coli) Escherichia coli The T7 strain, expression vectors pETDuet-1 and pCDFDuet-1 were purchased from Novagen. Restriction endonucleases, DNA polymerases, and other reagents were purchased from TaKaRa. Glucose, D-galactose, galactitol, and D-tagatose were purchased from Sigma-Aldrich. β-galactosidase (400 U / mg) was obtained from *E. coli*. E. coliPurchased from Shanghai Yuanye Biotechnology Co., Ltd. Whey powder purchased from Shandong Fengtai Biotechnology Co., Ltd. Other consumables and reagents purchased from Shanghai Bioengineering Technology Co., Ltd.

[0024] Example 1: Construction of the restoration module Literature search and enzyme library screening revealed: from Gluconobacter oxydans The NADPH-dependent glucose dehydrogenase Gox2015 (GenBank ID:WP_011253528.1) is strictly dependent on the cofactor NADPH, has no effect on NADH, and has an enzyme activity of 178 U / mg, making it an ideal NADPH regeneration enzyme for the reducing module. (Source: [Original Source Name]) Aspergillus niger Xylose reductase xyrB (GenBank ID: NT_166524.1) is strictly dependent on the cofactor NADPH and has no effect on NADH. Furthermore, this enzyme has an activity of 136 U / mg for D-galactose, making it an ideal reductase for the reduction of D-galactose to galactitol in the reductive module. Therefore, these two enzymes were selected to construct the reductive module.

[0025] Will xyrB and Gox2015 The codon-optimized nucleotide sequence of the gene was synthesized by GenScript Biotech Co., Ltd., and then processed using restriction endonucleases. Nde I and Xho I was inserted into the multiple cloning site of plasmid pETDuet-1 to obtain the recombinant plasmid pETDuet- xyrB and pETDuet- Gox2015 To facilitate protein purification, a 6×His-tag was added to the N-terminus of the target gene. Then, pETDuet- Gox2015 As a template, Nco I and Hin Primers designed for the dIII restriction site (Gox2015-F: 5'-TATACCATGGGTccggcgccgtacaaagatcgtttcg-3', SEQ ID NO.9; Gox2015-R: 5'-CCCAGCTTttagctgctccagttgttttcgaa-3', SEQ ID NO.10) were used for amplification. Gox2015 Gene fragments were cloned into pETDuet- through enzyme digestion and ligation. xyrB The MCS 1 site was used to obtain the recombinant plasmid pETDuet- Gox2015-xyrB The recombinant plasmid pETDuet- from the restoration module will be used. Gox2015-xyrB Recombinant Escherichia coli III was obtained by transforming it into E. coli T7 competent cells and converting equimolar amounts of D-galactose and D-glucose into galactitol and gluconic acid through cofactor regeneration.

[0026] Example 2: Construction of the oxidation module Literature searches and enzyme library screening revealed that there are few reports on galactitol dehydrogenases, and their enzyme activities are generally low. However, from... Rhizobium leguminosarumand galactitol dehydrogenase Rl The GDH double mutant T193G / G98C (patent number: ZL202411799966.6) exhibits a high catalytic activity of 50 U / mg for galactitol, making it an ideal enzyme for D-tagatose production. Water-producing NADH oxidase can irreversibly convert NADH to NAD. + It also directly reduces dissolved oxygen back to water, making it an ideal source of NAD+. + Regeneration enzyme. From Streptococcus pyogenes Aquagenic NADH oxidase Sp Nox (GenBank ID: WP_011184515.1) possesses an enzyme activity as high as 344 U / mg and strong temperature stability, maintaining 100% activity even after 6 hours of reaction at temperatures below 50°C. This enzyme does not require the addition of FAD in vitro and retains 70% of its peak activity within a temperature range of 30-70°C and pH 6.5-8.5. It has been used to couple various reductases for cofactor regeneration and synthesis of target compounds. Therefore, these two enzymes were selected to construct the oxidation module.

[0027] Will RlGDH T193G / G98C and SpNox The codon-optimized nucleotide sequence of the gene was synthesized by GenScript Biotech Co., Ltd., and then processed using restriction endonucleases. Nde I and Xho I was inserted into the multiple cloning site of plasmid pETDuet-1 to obtain recombinant plasmid pETDeut- RlGDH T193G / G98C and pETDeut- SpNox To facilitate protein purification, a 6×His-tag was added to the N-terminus of the target gene. Then, using... Nco I and Hind Primers were designed for the restriction enzyme sites (spNox-F: 5'-TATACCATGGGTggctctaaaatcgttgttgttggc-3', SEQ ID NO.11; spNox-R: 5'-CCCAGCTTttaatctttcgcgcccagagccgc-3', SEQ ID NO.12) to amplify the enzymes. SpNox Gene fragments were cloned into pETDeut- through enzyme digestion and ligation. RlGDH T193G / G98CThe recombinant plasmid pETDeut- was obtained by targeting the MCS 1 site of the plasmid. RlGDH T193G / G98C - SpNox. The recombinant plasmid pETDeut- of the oxidation module RlGDH T193G / G98C - SpNox The strain was co-transformed into Escherichia coli T7 competent cells to obtain a recombinant strain that converts galactitol into D-tagatose driven by cofactor regeneration, abbreviated as recombinant Escherichia coli II.

[0028] Example 3: Construction of a reduction-oxidation combined module A dual-plasmid co-expression strategy was employed to coordinate the expression of the reductive and oxidative modules of the tagatose synthesis pathway in *E. coli* T7 using compatibility plasmids of different copies. The oxidative module recombinant plasmid pETDuet- SpNox - RlGDH T193G / G98C genes through Nco I and Xho I enzyme digestion and ligation were performed to clone the recombinant plasmid pCDFDuet-1, resulting in the recombinant plasmid pCDFDuet-1. RlGDH T193G / G98C - SpNox The recombinant plasmid pETDuet- from the restoration module will be used. Gox2015-xyrB Recombinant plasmid pCDFDuet- with oxidation module RlGDH T193G / G98C - SpNox Recombinant strains were obtained by co-transforming Escherichia coli T7 competent cells. These strains, known as recombinant Escherichia coli I, are characterized by cofactor regeneration-driven reductogenic oxidase converting equimolar amounts of D-galactose and D-glucose into D-tagatose and gluconic acid.

[0029] Example 4: Whole-cell catalytic reaction Whole-cell reactions were conducted in 100 mL systems in 500 mL Erlenmeyer flasks with baffles. The reactions used either 20 g / L D-galactose as substrate and 25 g / L D-glucose as NADPH regenerator, or 180 g / L D-galactose as substrate and 220 g / L D-glucose as NADPH regenerator. 20 CDW / L of recombinant *E. coli* III (i.e., *E. coli* T7 competent cells carrying the recombinant plasmid pETDuet-Gox2015-xyrB, with 20 g dry weight of recombinant *E. coli* III per liter of reaction solution) from Example 1 was added as a catalyst. The pH was adjusted to 8.0 with 5 M NaOH solution, and the whole-cell reaction time was 6 h. The results are as follows: Figure 1As shown, through the whole-cell reaction of the reduction module, regardless of whether the D-galactose substrate is at a low or high concentration, D-galactose can be completely converted into galactitol and sodium gluconate simultaneously under the drive of NADPH regeneration by D-glucose dehydrogenase.

[0030] Whole-cell oxidation of the system was carried out in a 100 mL system in a 500 mL Erlenmeyer flask with a baffle: 20 g / L and 182 g / L galactitol were used as substrates, respectively, and 20 CDW / L of recombinant Escherichia coli II (i.e., carrying pETDeut-) from Example 2 were added. RlGDH T193G / G98C -SpNox Using *E. coli* T7 competent cells as a catalyst, the whole-cell reaction time was 24 h, and the results were as follows: Figure 2 As shown. When the galactitol substrate concentration is 20 g / L, at Sp Nox regeneration cofactor NAD + Driven by galactitol, the whole-cell reaction of the oxidation module can completely convert it into D-tagatose. However, when the galactitol concentration is increased to 182 g / L, the amount of D-tagatose generated by the whole-cell reaction of the oxidation module is only 53.98 g / L.

[0031] Whole-cell reactions using a 100 mL reduction-oxidation combined module were conducted in a 500 mL Erlenmeyer flask with a baffle: 20 g / L and 180 g / L D-galactose were used as substrates, and 25 g / L and 220 g / L D-glucose were used as cofactors and NADPH regenerators, respectively. 20 CDW / L of recombinant E. coli I from Example 3 (i.e., simultaneously carrying the recombinant plasmid pETDuet-) was added. Gox2015-xyrB and pETDeut- RlGDH T193G / G98C - SpNox Using *E. coli* T7 competent cells as a catalyst, the reaction was carried out at 30℃ and 180 rpm, with the pH adjusted to 8.0 using 5M NaOH solution. The whole-cell reaction time was 24 h. The results are as follows: Figure 3 As shown. When the D-galactose substrate concentration is 20 g / L, during the regeneration of NADPH by D-glucose dehydrogenase and Sp Nox regeneration cofactor NAD + Driven by [the specific mechanism], the whole-cell reaction of the reduction-oxidation module can completely convert it into D-tagatose. However, when the D-galactose concentration is increased to 180 g / L, the amount of D-tagatose generated by the whole-cell reaction of the reduction-oxidation module is only 36.89 g / L.

[0032] Example 5: Reaction of cell-disrupted supernatant In whole-cell reactions, the cell membrane's barrier effect affects oxygen transport, leading to oxygen deficiency, a common substrate for intracellular NADH oxidase, which in turn causes the cofactor NAD+ to be deficient. + Regeneration is limited, which in turn limits the ability of the oxidation module to synthesize D-tagatose (in the whole-cell reaction of the oxidation module, with 182 g / L galactitol as the substrate, the D-tagatose yield was only 53.98 g / L after 24 hours of reaction, and 102 g / L after 24 hours of reaction with oxygen). Therefore, this invention involves sonicating recombinant *E. coli* I to disrupt the cells, centrifuging at 12000 rpm for 20 min, and then using the supernatant for the reaction, while simultaneously introducing oxygen to ensure the necessary oxygen supply for the reaction.

[0033] A 100 mL reduction-oxidation combined module cell lysis supernatant was used in a 500 mL Erlenmeyer flask with a baffle. The reaction was carried out using 180 g / L D-galactose as the substrate and 180 g / L D-glucose as the NADPH regenerator (or 20 g / L D-galactose as the substrate and 20 g / L D-glucose as the NADPH regenerator), with 10 mg / mL recombinant E. coli I cell lysis supernatant as the catalyst. The reaction was conducted at 30℃ and 180 rpm with oxygen, and the pH was adjusted to 8.0 with 5 M NaOH solution. The reaction time was 24 h. The results are as follows: Figure 4 As shown. The reaction was carried out using the cell lysis supernatant, regardless of whether the D-galactose substrate was 20 g / L or 180 g / L, during the regeneration of NADPH by D-glucose dehydrogenase. Sp Nox regeneration cofactor NAD + Driven by this, the reduction-oxidation combined module reaction can completely convert equimolar amounts of D-galactose and D-glucose into D-tagatose and sodium gluconate.

[0034] Example 6: Efficient preparation of D-tagatose and calcium gluconate from whey powder using a three-step enzymatic method of "hydrolysis-dual-enzyme reduction-dual-enzyme oxidation". The preparation process flowchart is shown below. Figure 5The specific procedure was as follows: 130 g / L whey powder (lactose content 100 g / L) was hydrolyzed by β-galactosidase to produce 52.3 g / L D-galactose and D-glucose, with a hydrolysis rate of 99.4%. Subsequently, the hydrolysate was inactivated by boiling water bath and centrifuged to remove β-galactosidase. After vacuum concentration four times, the content of D-galactose and D-glucose was 209.2 g / L. The hydrolysate was transferred to a baffled Erlenmeyer flask, and the supernatant of recombinant Escherichia coli I from Example 3 was added for reaction (final concentration 10 mg / mL). The reaction was carried out at 30°C and 180 rpm with oxygen. The pH was adjusted to 8.0 with 5 M NaOH solution until the pH no longer changed. After 24 h of reaction, D-galactose and D-glucose were completely converted into galactitol and sodium gluconate. An equimolar amount of CaCl2 equal to sodium gluconate was added to the reaction solution and stirred until dissolved. The solution was then allowed to stand at 4°C for 24 hours (sodium gluconate reacts with CaCl2 to precipitate calcium gluconate). The precipitate was obtained by centrifugation at 12000 rpm for 10 min. The supernatant was desalted by ion exchange to obtain high-purity D-tagatose. The results are as follows... Figure 6 As shown, HPLC analysis revealed a D-tagatose content of 205.2 g / L. Thus, this invention provides a highly efficient conversion pathway from whey lactose to D-tagatose and calcium gluconate with high substrate concentrations and a conversion rate of 100%.

[0035] The HPLC conditions were as follows: instrument: Waters 1525 chromatograph; column: MARS MCa HPX-87C; mobile phase: ultrapure water; flow rate: 0.6 mL / min; detector: Waters 2414 refractive index detector.

[0036] Figure 7 This is the HPLC chromatogram of a D-tagatose standard; Figure 8 This is an HPLC chromatogram of the efficient preparation of D-tagatose from whey powder using a three-step enzymatic method of "hydrolysis-dual-enzyme reduction-dual-enzyme oxidation". HPLC results show that 130 g / L whey powder (approximately 100 g / L lactose) was completely converted into D-tagatose.

[0037] In summary, this invention successfully established a three-step enzyme cascade reaction pathway of "hydrolysis – dual-enzyme reduction – dual-enzyme oxidation" based on cofactor regeneration cycle, completely converting lactose into D-tagatose and sodium gluconate. The application of this highly efficient multi-enzyme catalytic system in the preparation of D-tagatose is of great significance for improving the economic benefits of the whey powder processing industry.

[0038] >XyrB amino acid sequence, 299aa (SEQ ID NO.1): MSNLEHTKKVYTLNTGDKIPAVGLGTWQSKPNEVREAVKNALLKGYRHIDTALAYGNEAEVGQGIKDSGVPREEIWVTTKLDNPWHHRVAEGIDSSLKDLGLDYVDLYLVHWPSSTDPNDLKKHLPDWDFIKTWQEMQKLPATGKVRNIGVSNFGIKNLEKLLNDPSCKIVPAVNQIELHPNNPSPKLVAYNTSKGIHSTGYSCLGSTNSPLYKDETLLKLAEKKGKTPQQVLLLWGVQKGWSVIPKSVSKSRIDANFELDGWELTAEEIEQLDNLKDRFKVCGDDWLPVKVFFGDDE 。

[0039] >Amino acid sequence of Gox2015, 267 aa (SEQ ID NO.2): MPAPYKDRFAGKKVLVTGASQGIGEATALRFAEEGAQVALNGRKEDKLIAVREKLPKVSGGEHPIATGDISKEDDVKRLVAESIKAMGGLDVLVCNAGYQIPSPSEDIKLEDFEGVMAVNVTGVMLPCREVIRYWLENGIKGTIIVNSSVHQIIPKPHYLGYSASKGAVGNIVRTLALEYATRGIRVNAVAPGAIVTPINMSWIDDPEQYKAVSSHIPMKRPGESREIADAITFLAAEDSTYITGQTLYVDGGLTLYGDFENNWSS 。

[0040] >Amino acid sequence of SpNox, 458 aa (SEQ ID NO.3): MGSKIVVVGANHAGTACIKTMLTNYGDANEIVVFDQNSNISFLGCGMALWIGEQIAGPEGLFYSDKEELESLGAKVYMESPVQSIDYDAKTVTALVDGKNHVETYDKLIFATGSQPILPPIKGAEIKEGSLEFEATLENLQFVKLYQNSADVIAKLENKDIKRVAVVGAGYIGVELAEAFQRKGKEVVLIDVVDTCLAGYYDRDLTDLMAKNMEEHGIQLAFGETVKEVAGNGKVEKIITDKNEYDVDMVILAVGFRPNTTLGNGKIDLFRNGAFLVNKRQETSIPGVYAIGDCATIYDNATRDTNYIALASNAVRTGIVAAHNACGTDLEGIGVQGSNGISIYGLHMVSTGLTLEKAKRLGFDAAVTEYTDNQKPEFIEHGNFPVTIKIVYDKDSRRILGAQMAAREDMSMGIHMFSLAIQEGVTIEKLALTDIFFLPHFNKPYNYITMAALGAKD 。

[0041] >RlGDH T193G / G98C Amino acid sequence, 258 aa (SEQ ID NO.4): MSYQQKFRLDGERAVVTGGGRAIGLCCTEALAEAGAAVVVIERSEADAEQALALRNRGYDVEVRVGDVTDAARMDAIATELADGGRPATILVNNAGICQSGIPAQDLTDADWLRMMDVNLNGVFWCSRAFGRSMISMKRGAIVNLGSMSGTICNRPQPQTAYNVSKAAVHHLTRSLAAEWAHHGIRVNAVAPGYIETPMVVAVEANRERIPLWLADTPMARMGTPEEVASAVLFLASGAASLMTGAIVNVDAGFTCW 。

[0042] >XyrB nucleotide sequence (1bp - 897bp, direct) 897bp, SEQ ID NO.5: ATGTCAAATCTAGAACACACAAAAAAGGTATACACCCTGAATACCGGCGACAAAATCCCAGCGGTGGGTCTGGGCACCTGGCAATCCAAACCGAACGAGGTTAGAGAAGCGGTCAAAAACGCCCTGTTGAAGGGCTACCGCCACATTGATACCGCTCTGGCGTATGGAAACGAAGCCGAGGTGGGCCAAGGTATCAAAGACAGCGGTGTGCCGCGTGAAGAGATTTGGGTTACCACCAAACTTGACAACCCGTGGCATCATCGTGTTGCGGAGGGTATCGACTCCAGCCTGAAGGACCTCGGCCTAGACTATGTTGATTTGTACCTGGTTCATTGGCCGTCTTCCACCGATCCGAACGACCTCAAGAAGCACCTGCCTGATTGGGATTTCATCAAGACGTGGCAAGAAATGCAGAAGTTACCGGCGACCGGCAAAGTGCGTAATATTGGTGTCAGTAACTTCGGCATCAAGAACCTGGAAAAGCTGCTGAATGATCCGAGCTGCAAAATCGTGCCGGCAGTAAATCAGATTGAACTGCACCCGAATAACCCGTCGCCGAAACTTGTGGCGTATAACACTTCAAAGGGTATCCACAGCACCGGTTATAGCTGTCTGGGTTCTACGAACAGCCCACTATACAAGGACGAGACACTGCTGAAGTTGGCTGAGAAAAAAGGCAAGACGCCGCAGCAGGTTTTGTTGCTGTGGGGTGTCCAGAAGGGTTGGAGCGTGATCCCGAAAAGCGTTTCCAAAAGCCGTATTGACGCAAATTTCGAGCTGGATGGTTGGGAATTGACCGCTGAGGAAATTGAGCAACTGGACAACCTGAAGGACCGCTTTAAAGTGTGCGGTGATGATTGGCTGCCGGTTAAAGTCTTTTTTGGCGACGATGAGTAA。

[0043] >Gox2015 nucleotide sequence (4bp - 822bp, direct) 819bp, SEQ ID NO.6: ATGCCGGCGCCGTACAAAGATCGTTTCGCTGGTAAAAAAGTTCTGGTTACCGGTGCGAGCCAGGGCATCGGTGAAGCGACCGCGCTGCGTTTCGCGGAAGAAGGTGCGCAGGTTGCGCTGAACGGTCGTAAAGAAGATAAACTGATCGCTGTTCGTGAAAAACTGCCGAAAGTTAGCGGCGGTGAACACCCGATCGCGACCGGTGATATCAGCAAAGAAGATGATGTTAAACGTCTGGTTGCGGAAAGCATCAAAGCGATGGGTGGTCTGGATGTTCTGGTTTGCAACGCGGGTTACCAGATCCCGAGCCCGTCTGAAGATATCAAACTGGAAGATTTCGAAGGTGTTATGGCTGTGAACGTTACCGGCGTTATGCTGCCGTGCCGTGAAGTTATCCGTTACTGGCTGGAAAACGGTATCAAAGGCACCATCATCGTGAACTCTAGCGTTCACCAGATCATCCCGAAACCGCACTACCTGGGCTACAGCGCGAGCAAAGGTGCGGTTGGCAACATCGTTCGTACCCTGGCGCTGGAATACGCGACCCGTGGTATCCGTGTTAACGCAGTTGCACCGGGCGCGATCGTGACCCCGATCAACATGAGCTGGATCGATGATCCGGAACAGTACAAAGCGGTTAGCAGCCACATCCCGATGAAACGTCCGGGTGAATCTCGTGAAATTGCGGATGCGATCACCTTCCTGGCGGCGGAAGATAGCACCTACATCACCGGTCAGACCCTGTACGTTGATGGTGGCCTGACCCTGTACGGTGATTTCGAAAACAACTGGAGCAGCTAA。

[0044] > RlGDH T193G / G98C Nucleotide sequence (1bp - 774bp, direct) 774bp, SEQ ID NO.7: ATGTCTTACCAGCAGAAATTCCGTCTGGATGGTGAACGTGCTGTTGTTACCGGTGGTGGTCGTGCGATCGGTCTGTGTTGCACCGAAGCATTAGCTGAAGCGGGTGCTGCCGTAGTAGTAATTGAACGTTCTGAAGCTGATGCTGAACAGGCTCTGGCTCTGCGTAACCGTGGTTATGATGTTGAAGTTCGTGTTGGCGATGTTACCGATGCGGCGCGTATGGATGCGATCGCGACCGAACTGGCTGATGGCGGTCGTCCGGCGACCATCCTGGTTAACAACGCGGGCATCTGTCAGAGCGGTATCCCGGCACAGGATCTGACCGATGCGGATTGGCTGCGTATGATGGATGTTAACCTGAACGGTGTTTTCTGGTGCTCTCGTGCGTTCGGTCGTAGCATGATCTCTATGAAACGTGGCGCGATCGTTAACCTGGGCAGCATGAGCGGCACCATCTGCAACCGTCCGCAGCCGCAGACCGCGTACAACGTTAGCAAAGCGGCGGTTCATCACCTGACCCGTAGCCTGGCGGCTGAATGGGCGCACCACGGTATCCGTGTTAACGCGGTTGCGCCGGGTTACATCGAAACCCCGATGGTTGTTGCGGTTGAAGCGAACCGTGAACGTATCCCGCTGTGGCTGGCGGATACCCCGATGGCGCGTATGGGCACCCCGGAAGAAGTTGCGAGCGCGGTTCTGTTCCTGGCGTCTGGCGCGGCGAGCCTGATGACCGGCGCGATCGTTAACGTTGATGCGGGCTTCACCTGCTGGTAA。

[0045] >spNox nucleotide sequence (1bp - 1374bp, direct) 1374bp, SEQ ID NO.8:

[0046] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A recombinant Escherichia coli for producing D-tagatose, characterized in that, The recombinant Escherichia coli includes recombinant Escherichia coli I and recombinant Escherichia coli II. Recombinant Escherichia coli I simultaneously expresses xylose reductase xyrB, glucose dehydrogenase Gox2015, galactitol dehydrogenase mutant T193G / G98C, and aqueous NADH oxidase SpNox; recombinant Escherichia coli II simultaneously expresses galactitol dehydrogenase mutant T193G / G98C and aqueous NADH oxidase SpNox. The amino acid sequence of the xylose reductase xyrB is shown in SEQ ID NO.1, the amino acid sequence of the glucose dehydrogenase Gox2015 is shown in SEQ ID NO.2, the amino acid sequence of the galactitol dehydrogenase mutant T193G / G98C is shown in SEQ ID NO.4, and the amino acid sequence of the water-producing NADH oxidase SpNox is shown in SEQ ID NO.

3.

2. A method for constructing recombinant Escherichia coli according to claim 1, characterized in that, Including the method shown in (1) or (2): (1) The genes encoding xylose reductase xyrB and glucose dehydrogenase Gox2015 were co-expressed in an expression vector to construct a recombinant plasmid for the reduction module; The coding genes for galactitol dehydrogenase mutant T193G / G98C and water-producing NADH oxidase SpNox were co-expressed in an expression vector to construct an oxidative module recombinant plasmid. The recombinant plasmid of the reduction module and the recombinant plasmid of the oxidation module were co-transformed into E. coli T7 competent cells to obtain the recombinant E. coli I. (2) The coding genes of galactitol dehydrogenase mutant T193G / G98C and water-producing NADH oxidase SpNox were co-expressed in the expression vector to construct an oxidative module recombinant plasmid; The recombinant plasmid of the oxidation module was transformed into Escherichia coli T7 competent cells to obtain the recombinant Escherichia coli II.

3. The application of the recombinant Escherichia coli as described in claim 1 in the production of D-tagatose.

4. A method for producing D-tagatose, characterized in that, Includes the following steps: (1) Using the hydrolysate of whey powder after hydrolysis by β-galactosidase as a substrate, or using D-galactose as a substrate and D-glucose as a cofactor NADPH regenerator, the supernatant of the recombinant Escherichia coli I described in claim 1 as a catalyst, the reaction is carried out under the condition of oxygen introduction, so that D-galactose and D-glucose are completely converted into D-tagatose and sodium gluconate; (2) D-tagatose was separated and purified from the reaction solution.

5. The method as described in claim 4, characterized in that, In step (1), the final concentration of the cell-breaking supernatant added is 10 mg / mL; the reaction conditions are: temperature 30℃, shaking speed 180 rpm, pH value of the reaction system 8.0, and reaction time 24 h. In step (2), the separation and purification method includes: adding CaCl2 to the reaction solution to convert sodium gluconate into calcium gluconate precipitate, centrifuging to separate the precipitate, and then desalting the supernatant by ion exchange to obtain D-tagatose.

6. A method for producing D-tagatose, characterized in that, Including the methods shown in (1) or (2) below: (1) Using galactitol as a substrate, the recombinant Escherichia coli II described in claim 1 was added as a catalyst and the whole-cell reaction was carried out for 24 h to obtain D-tagatose; (2) Using D-galactose as substrate and D-glucose as cofactor NADPH regenerator, the recombinant Escherichia coli I described in claim 1 was added as catalyst, the pH value was adjusted and maintained at 8.0, and the whole cell reaction was carried out at 30°C and 180 rpm for 24 h to obtain D-tagatose.

7. A recombinant Escherichia coli for producing galactitol, characterized in that, The recombinant Escherichia coli simultaneously expresses xylose reductase xyrB and glucose dehydrogenase Gox2015; The amino acid sequence of the xylose reductase xyrB is shown in SEQ ID NO.1, and the amino acid sequence of the glucose dehydrogenase Gox2015 is shown in SEQ ID NO.

2.

8. A method for preparing recombinant Escherichia coli according to claim 7, characterized in that, Includes the following steps: The genes encoding xyrB and glucose dehydrogenase Gox2015 were co-expressed in an expression vector to construct a recombinant plasmid for the reduction module. The recombinant plasmid of the reduction module was transformed into Escherichia coli T7 competent cells to obtain the recombinant Escherichia coli.

9. The application of the recombinant Escherichia coli as described in claim 7 in the production of galactitol.

10. A method for producing galactitol, characterized in that, Includes the following steps: Using D-galactose as a substrate, D-glucose as a cofactor and NADPH regenerator, and the recombinant Escherichia coli as described in claim 7 as a catalyst, the pH value was adjusted and maintained at 8.0, and the whole-cell reaction was carried out for 6 h to obtain galactitol.

Images