Recombinant cellobiose isomerase, engineering bacterium, preparation method and application in lactulose production

Abstract

The invention provides a recombinant cellobiose isomerase, an engineering bacterium, a preparation method and application in lactulose production, belongs to the technical field of biology, and mainly provides the recombinant cellobiose isomerase, the amino acid sequence of which is as shown in SEQ ID NO.3; the escherichia coli engineering bacterium expresses the coding gene sequence of the amino acid sequence SEQ ID NO.3; the invention discloses a preparation method of recombinant cellobiose isomerase and application of the recombinant cellobiose isomerase in production of lactulose, and finds that fusion expression of 20 amino acids at the n-terminal of feruloyl esterase and cellobiose epimerase genes can realize extracellular secretion of the cellobiose epimerase for the first time; through research on fermentation conditions, the prepared cellobiose epimerase converts lactose into lactulose, and the conversion rate can reach 56%.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant cellobiose isomerase, an engineering bacterium, a preparation method and an application in producing lactulose. Background Art

[0002] Lactulose, as an important functional oligosaccharide, is a disaccharide formed by the condensation of galactose and fructose through 1,4-β glycosidic bonds. It is a synthetic substance and does not exist naturally. This compound is widely used in more than 100 countries around the world, mainly for the treatment of chronic constipation and hepatic encephalopathy, and has a huge market demand. The application of lactulose is not limited to the treatment of constipation. It also has many physiological functions such as promoting the proliferation of beneficial bacteria, inhibiting the growth of pathogenic bacteria, restoring the balance of intestinal flora and enhancing intestinal peristalsis. In addition, lactulose is also used in food production as a prebiotic additive, which helps to improve the nutritional value of food. With the increasing attention to environmental protection and sustainability, the biological production of lactulose has become a promising development field due to its environmentally friendly and sustainable characteristics. This method can not only reduce the impact on the environment, but also provide a greener and more sustainable way for the production of lactulose. With the advancement of technology and the growth of market demand, the biological production of lactulose is expected to play a more important role in the future.

[0003] Cellobiose isomerase (CE enzyme) plays a key role in catalyzing the conversion of lactose to lactulose, which is an eco-friendly industrial production route. CE enzyme mainly catalyzes the reducing end of β-1,4-glycosidically linked oligosaccharides to achieve C2-diastereomerization, usually producing epi-lactose. There are reports in the prior art of using cellobiose isomerase to prepare lactulose from lactose. With the deepening of research and the advancement of technology, cellobiose isomerase is expected to play a more critical role in the future industrial production of lactulose.

[0004] There have been reports on heterologous expression of cellobiose epimerase (CE enzyme) in Escherichia coli in the prior art, but the expression was mainly carried out intracellularly, and the E. coli system still faces some uncertainties in the production of CE enzyme. For example, endogenous proteases may degrade CE enzymes, resulting in reduced activity; the stability of CE enzymes in bacteria may also be lower than that in native hosts, affecting their industrial application potential; in addition, high expression levels may be toxic to E. coli, affecting cell growth and metabolism, reducing fermentation efficiency and increasing production costs.

[0005] Therefore, research and development of cellobiose isomerase that can be secreted extracellularly and can efficiently convert lactose into lactulose is of great significance for the production and application of cellobiose isomerase.

[0006] Although there are reports in the prior art on the use of the N-terminal sequence of ferulic acid esterase as a guide peptide, the use of the N-terminal sequence of ferulic acid esterase as a guide peptide to bind to exogenous proteins does not always result in the excretion of exogenous proteins from cells. The N-terminal sequence of ferulic acid esterase is used as a guide peptide to bind to different exogenous proteins, and the effect is unpredictable. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention provides a recombinant cellobiose isomerase, an engineered bacterium, a preparation method and an application in producing lactulose.

[0008] The technical solution of the present invention is as follows:

[0009] A recombinant cellobiose isomerase, the amino acid sequence of which is shown in SEQ ID NO.3.

[0010] The coding gene sequence of the above recombinant cellobiose isomerase is shown in SEQ ID NO.4.

[0011] A recombinant bacterium comprises a gene sequence encoding the amino acid sequence SEQ ID NO.3.

[0012] Preferably according to the present invention, the recombinant bacteria comprises the gene sequence SEQ ID NO.4 encoding the above amino acid sequence SEQ ID NO.3.

[0013] An Escherichia coli engineering bacterium uses Escherichia coli BL21 (DE3) as a host bacterium and pET-22 (b) + as a vector to express the coding gene sequence of the above amino acid sequence SEQ ID NO.3.

[0014] Preferably according to the present invention, the gene sequence is SEQ ID NO.4.

[0015] Preferably, according to the present invention, the method for constructing the Escherichia coli engineered bacteria includes any of the following schemes:

[0016] Solution 1:

[0017] ① Using the genomic DNA of Lactobacillus amylovora CGMCC11056 as a template, the primers N20-F and N20-R were used to amplify the ferulic acid esterase N20 sequence encoding gene, and after the product was purified, the encoding gene of the N20 sequence was obtained; using the genomic DNA of Caldicellulosiruptor bescii DSM 6725 as a template, the primers CE-F and CE-R were used to amplify the target gene cellobiose epimerase gene, and after the product was purified, the encoding gene of cellobiose epimerase CE was obtained;

[0018] The sequence of the primer N20-F is shown in SEQ ID NO.6, and the sequence of the primer N20-R is shown in SEQ ID NO.7;

[0019] The sequence of the primer CE-F is shown in SEQ ID NO.8, and the sequence of the primer CE-R is shown in SEQ ID NO.9;

[0020] ② The coding gene of the N20 sequence obtained in step ① and the coding gene of cellobiose epimerase CE are overlapped and extended by PCR to obtain an N20-CE DNA fragment, the N20 sequence is added to the N-terminus of the cellobiose epimerase CE sequence, and then the N20-CE DNA fragment is connected to the pET22b vector fragment treated with restriction enzymes NdeI and XhoI by homologous recombination to obtain a recombinant vector pET22b-N20CE;

[0021] ③ The recombinant vector pET22b-N20CE was transformed into Escherichia coli BL21 (DE3) to obtain the recombinant strain BL21 (DE3) / pET-22b-N20CE;

[0022] Option 2:

[0023] The gene sequence SEQ ID NO.4 was connected to the pET22b vector fragment treated with restriction enzymes NdeI and XhoI by homologous recombination to obtain the recombinant vector pET22b-N20CE, which was transformed into Escherichia coli BL21 (DE3) and the recombinant strain BL21 (DE3) / pET-22b-N20CE was obtained by screening.

[0024] The use of the above-mentioned recombinant bacteria or Escherichia coli engineered bacteria in the preparation of cellobiose isomerase or recombinant cellobiose isomerase, the amino acid sequence of the cellobiose isomerase is shown in SEQ ID NO.1, and the amino acid sequence of the recombinant cellobiose isomerase is shown in SEQ ID NO.3.

[0025] A method for preparing recombinant cellobiose isomerase comprises the following steps:

[0026] The E. coli engineered bacteria prepared by the above method were used to prepare seed solution, which was then inoculated into 2×YT medium. After culturing for 2.5 to 4.5 hours, IPTG with a final concentration of 0.3 to 0.9 mM and Fe 0.08 to 0.12 mM were added. 2+ and 25 mM or more glycine, continue fermentation and culture for 30 to 54 hours, separate the solid and liquid, and extract the recombinant cellobiose isomerase in the liquid.

[0027] Preferably, according to the present invention, the method for preparing the recombinant cellobiose isomerase comprises the following steps:

[0028] The E. coli engineered bacteria prepared by the above method were used to prepare seed solution, which was then inoculated into 2×YT medium. After 2.5 hours, IPTG with a final concentration of 0.7 mM and 1 mM Fe 2 + and 25 mM glycine, and the fermentation culture was continued for 36 hours, and the solid-liquid separation was performed, and the recombinant cellobiose isomerase was extracted from the liquid.

[0029] Preferably, the engineered E. coli prepared by the above method is inoculated into LB medium, cultured overnight at 37°C and 200 rpm to obtain a seed solution, which is inoculated into 2×YT medium at a volume ratio of 1%, cultured at 37°C and 200 rpm for 2.5 hours, and IPTG and FeCl2 with a final concentration of 0.7 mM are added. 2+ and 25 mM glycine, and continued fermentation at 37°C, 200 rpm for 36 hours, solid-liquid separation, and recombinant cellobiose isomerase was extracted in the liquid.

[0030] According to the preferred embodiment of the present invention, the Fe 2+ Provided by FeSO4.7H2O.

[0031] Preferably according to the present invention, the solid-liquid separation method is above 10000 rpm and centrifuged for more than 5 min.

[0032] According to a preferred embodiment of the present invention, the method for extracting recombinant cellobiose isomerase in a liquid is to add ammonium sulfate to the liquid to 60% saturation, stand at 4°C overnight, centrifuge at 4°C and 10,000 rpm for more than 5 minutes, discard the supernatant, and obtain a precipitate.

[0033] Use of the recombinant cellobiose isomerase or the recombinant cellobiose isomerase prepared by the above method in the preparation of lactulose.

[0034] The beneficial effects of the present invention include at least the following:

[0035] 1. The present invention firstly discovered that the extracellular secretion of cellobiose epimerase (SEQ ID NO.1) can be achieved by using the 20 amino acids at the N-terminus of ferulic acid esterase (MSRITIERDGLTLVGDREEPFSEQID NO.2) as a signal peptide, fusion expression with the cellobiose epimerase gene produced from Caldicellulosiruptor bescii, and transforming Escherichia coli with a vector.

[0036] The amino acid sequence of the recombinant cellobiose epimerase after the n-terminal 20 amino acids of ferulic acid esterase (MSRITIERDGLTLVGDREEPF SEQ ID NO.2) are combined with the cellobiose epimerase (SEQ ID NO.1) is (SEQ ID NO.3).

[0037] 2. By improving the fermentation conditions, the present invention can harvest 1835.53 mg of enzyme protein per liter of fermentation liquid, and its specific activity reaches 19.25 U / mg.

[0038] 3. The cellobiose epimerase recombinant prepared by the present invention can convert lactose into lactulose, and the conversion rate can reach 56%. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The plasmid map and protein analysis electrophoresis diagram of the cellobiose epimerase of the recombinant Escherichia coli strain;

[0040] In the figure: A is the plasmid map of the recombinant Escherichia coli strain cellobiose epimerase; B is the SDS-PAGE analysis of the recombinant Escherichia coli strain. M: marker; 1: whole cell protein sample; 2: intracellular protein sample; 3: extracellular protein sample.

[0041] Figure 2 This is the relative enzyme activity of E. coli BL21 (DE3) / pET-22b-N20CE extracellular protein samples under different fermentation conditions;

[0042] In the figure: A is different culture media; B is different induction start time; C is different IPTG concentrations; D is different lactose concentrations; E is different metal ions; F is different glycine concentrations.

[0043] Figure 3 This is the SDS-PAGE analysis of E. coli BL21 (DE3) / pET-22b-N20CE extracellular protein samples under different fermentation conditions;

[0044] In the figure: A is different culture media; B is different induction start time; C is different IPTG concentrations; D is different lactose concentrations; E is different metal ions; F is different glycine concentrations.

[0045] Figure 4 The relative enzyme activity and SDS-PAGE analysis of the extracellular protein samples of Escherichia coli BL21 (DE3) / pET-22b-N20CE at different culture times under the above-mentioned optimized conditions;

[0046] In the figure: A is the relative enzyme activity at different culture times; B is the SDS-PAGE analysis at different culture times.

[0047] Figure 5 This is the SDS-PAGE analysis and protein volume analysis of the extracellular protein samples of Escherichia coli BL21 (DE3) / pET-22b-N20CE purified at different ammonium sulfate concentrations;

[0048] In the figure: A is the relative enzyme activity of purification at different ammonium sulfate concentrations; B, C are SDS-PAGE analysis diagrams of purification at different ammonium sulfate concentrations.

[0049] Figure 6 This is a graph of lactulose conversion obtained by liquid phase analysis of the reaction of Escherichia coli BL21 (DE3) / pET-22b-N20CE extracellular protein samples with different enzyme additions and 40% lactose substrate concentration for different times at 75°C. DETAILED DESCRIPTION

[0050] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0051] The contents not described in detail in the examples are all based on the conventional techniques in the art; the experimental materials and reagents not described in detail are all common commercially available products.

[0052] 1.1 Materials and methods:

[0053] 1.1.1 Strains, plasmids and culture media

[0054] At 37°C, Lactobacillus amylovora CGMCC11056 and Caldicellulosiruptor bescii DSM 6725 were inoculated into LB medium for shaking culture. Escherichia coli XL1-Blue was used for DNA-related operations, and the target gene fragment was inserted into the plasmid pET-22b(+) and transformed into Escherichia coli BL21(DE3) to achieve protein expression. Ampicillin was added to a final concentration of 100 μg / mL.

[0055] Caldicellulosiruptor bescii DSM 6725: purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0056] The fermentation medium includes LB (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L), TB (tryptone 11.8 g / L, yeast extract 23.6 g / L, dipotassium hydrogen phosphate 9.4 g / L, monopotassium phosphate 2.2 g / L, glycerol 4 mg / L), SB (tryptone 32.0 g / L, yeast extract 20.0 g / L, sodium chloride 5.0 g / L), 2×YT (tryptone 16.0 g / L, yeast extract 10.0 g / L, sodium chloride 5.0 g / L) and SOB (tryptone 20.0 g / L, yeast extract 5 g / L, sodium chloride 0.5 g / L, potassium chloride 0.2 g / L, magnesium chloride 1.0 g / L, magnesium sulfate 1.2 g / L), which is used for the production of target protein.

[0057] The plasmid extraction kit was provided by Tiangen Biochemical Technology Co., Ltd.; 2×PhantaMasterMix, genome extraction, product purification and recovery kit, and ClonExpressⅡ one-step cloning kit were supplied by Nanjing Novogene Biotechnology Co., Ltd. NdeI and XhoI restriction endonucleases were from Takara, and the SDS-PAGE gel rapid preparation kit was provided by Sangon Biotechnology (Shanghai) Co., Ltd.; pET-22b(+), Escherichia coli BL21(DE3), and Escherichia coli BL21(DE3) were provided by Sangon Biotechnology (Shanghai) Co., Ltd.

[0058] 2.2 Construction of recombinant plasmid

[0059] Firstly, the genomic DNA of Lactobacillus amylovora CGMCC11056 containing ferulic acid esterase and the genomic DNA of Caldicellulosiruptor bescii DSM 6725 containing cellobiose epimerase CE were extracted using a genomic extraction kit and stored at -20°C. Subsequently, primers N20-F (5′-AACTTTAAGAAGGAGATATACATATGTCCCGCATTACAATTGAAAG-3′SEQ ID NO.6) and N20-R (5′-AAAAGGCTCTTCACGATCTC-3′SEQ ID NO.7) were designed and used to perform PCR amplification on the template (containing ferulic acid esterase genomic DNA), and after purification, the N20 DNA fragment including the homology arm of pET-22 (b+) was obtained; at the same time, CE-F (5′-GAGATCGTGAAGAGCCTTTTGATATCACTAAGTTCAAGGA SEQ ID NO.8) and CE-R (TTTAGTATTCGCACAACCAGAGCTCGTGGTGGTGGTGGTGGTGGTGACTCTAGGCCGACGATTG-3′SEQ ID NO.9) were used to perform PCR amplification on the template (containing CE genomic DNA), and after purification, the product obtained The CE DNA fragment containing the N20 fragment and the homology arm of the pET-22 (b+) vector was obtained. By overlapping extension PCR, the N20 signal peptide sequence was fused to the N-terminus of the CE sequence. The pET-22b(+) vector was digested with NdeI and XhoI. The N20-CE DNA fragment was homologously recombined with the pET-22b(+) vector to obtain the recombinant plasmid pET-22b-N20CE.

[0060] The recombinant plasmid was transformed into E. coli XL1-BLue competent cells by heat shock transformation, and then statically cultured at 37°C for 12 hours on LB solid plates containing 100 μg / mL ampicillin. Positive transformants were selected and cultured at 37°C in LB liquid medium containing ampicillin resistance. Primers N20-F and CE-R were used to extract and identify the plasmid. Finally, the successfully constructed recombinant plasmid (pET-22b-N20CE) was sequenced and analyzed.

[0061] 2.3 Expression of cellobiose epimerase protein

[0062] The constructed expression vector pET-22b-N20CE was introduced into Escherichia coli BL21 (DE3) by thermal transformation, and then spread cultured overnight on a solid LB plate containing 100 μg / mL ampicillin to obtain recombinant Escherichia coli BL21 (DE3) / pET-22b-N20CE containing the recombinant plasmid. Next, the recombinant Escherichia coli BL21 (DE3) / pET-22b-N20CE cells were inoculated in LB liquid culture medium containing 100 μg / mL ampicillin, and cultured at 37°C at a speed of 200 rpm. When the cells grew to an optical density value OD600 of 0.5, isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.5 mmol / L was added to induce protein expression, and cultured at 37°C for 36 hours. After induction, the cells were centrifuged at a centrifugal force of 13000×g for 5 minutes, and the supernatant and cell pellet were collected respectively. The supernatant was filtered using a 0.22 μm pore size filter to obtain an extracellular protein sample. The cell pellet was resuspended with an equal volume of 100 mmol / L sodium phosphate buffer (PBS, pH 7.0) and the cells were disrupted by an ultrasonic disruptor (Jiangsu Tianling, China), and then the whole cell protein sample was prepared. The cell disruption solution was also centrifuged at 13,000 × g for 5 minutes, and the supernatant was taken as the intracellular protein sample. The whole cell, intracellular and extracellular protein samples were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which included mixing the samples with SDS-PAGE sample buffer [0.25 M Tris-HCl (pH 6.8), 50% glycerol, 10% SDS, 5% 2-mercaptoethanol, 0.05% bromophenol blue] and boiling for 5 minutes. The protein separation was performed using an SDS-PAGE system with 5% stacking gel and 12% separating gel, and the gel was finally stained with Coomassie Brilliant Blue R-250 (Sangon, Shanghai, China).

[0063] 2.4 Determination of cellobiose isomerase activity

[0064] The enzymatic activity of cellobiose epimerase is determined by mixing 1.0 mL of enzyme solution with 1.0 mL of 50 g / L lactose solution, incubating at 75°C for 30 minutes, and then terminating the reaction in a boiling water bath for 10 minutes. The activity unit (U) of cellobiose epimerase is defined as the amount of enzyme required to convert 1 μmol of lactose to lactulose per minute in a 30-minute incubation reaction at 75°C.

[0065] 2.5 Optimization of fermentation conditions for cellobiose epimerase production

[0066] In order to improve the production of cellobiose epimerase in recombinant Escherichia coli BL21 (DE3) / pET-22b-N20CE, this study systematically studied the fermentation conditions. First, the recombinant Escherichia coli BL21 (DE3) / pET-22b-N20CE strain was live (i.e., inoculated in LB medium containing 100 μg / mL ampicillin and cultured overnight), and then transferred to various fermentation media (LB, TB, SB, 2 × YT and SOB) containing 100 μg / mL ampicillin at a ratio of 1% (v / v), and the culture volume was 1L. To explore the effects of different inducers (IPTG and lactose) and their concentrations on protein extracellular secretion, the strain was cultured at 37°C at 200rpm until OD600 reached 0.5, and 0.3 to 0.9 mM IPTG and 0.05, 0.1, 0.25, 0.5, 1, 2, 5, and 10 g / L lactose were added to induce protein expression. At the same time, the induction timing was optimized, and different time points (2h, 2.5h, 3h, 3.5h, 4h, and 4.5h) were set for the addition of the optimal inducer.

[0067] Furthermore, by adding different metal ion solutions (Mn 2+ 、Zn 2+ , Ca 2+ Mg 2+ , Fe 2+ ), with a concentration of 0.1 mM, to promote the secretion of extracellular proteins. In addition, 50 to 150 mmol / L of glycine were added to the culture medium to enhance the secretion efficiency of extracellular proteins. After induction of expression, the supernatant was collected for quantitative analysis of protein concentration and extracellular protein expression by centrifugation at 13000×g for 5 minutes.

[0068] The metal ion solution is respectively MnSO4 7H2O, ZnCL2, CaCl2, FeSO4.7H2O, MgSO 4. 7H2O, and filter the solution using a 0.22μm pore size filter membrane.

[0069] Based on the above optimized conditions, the recombinant strain was cultured in 2×YT medium containing 25 mmol / L glycine. When the induction time reached 2.5 h, IPTG with a final concentration of 0.7 mM and metal Fe with a final concentration of 0.1 mM were added. 2+ , and induce the production of cellobiose epimerase at 37°C. Samples were taken at 6, 12, 18, 24, 30, 36, 42, 48, and 54 h. The extracellular protein content was determined, and the extracellular protein samples were prepared for SDS-PAGE analysis.

[0070] 2.6 Preliminary purification of cellobiose epimerase

[0071] In order to specifically apply cellobiose epimerase, the bacterial culture was collected under optimized fermentation conditions in this study, and the extracellular protein was purified by ammonium sulfate graded precipitation method (4.1 M; concentration gradient from 20% to 90% saturation). During the purification process, after standing at 4°C overnight, the solution was centrifuged at 13000×g for 5 minutes, the supernatant was discarded, and the precipitate was resuspended with an equal volume of phosphate buffered saline (PBS), and the sample was prepared for SDS-PAGE analysis.

[0072] 2.7 Production of lactulose using purified cellobiose epimerase

[0073] On the basis of the above research, the inventors fermented and cultured the recombinant cellobiose isomerase, and purified the protein according to the determined optimal conditions. In the experiment, the inventors prepared a 40% lactose solution as a substrate, and added different concentrations of enzyme solution (0.38g / L, 0.75g / L, 1.15g / L and 1.53g / L, respectively) at 75°C pH7.0 for different time periods (3h, 6h, 9h, 12h and 24h). After the reaction, the inventors took 1mL of the sample and treated it in a boiling water bath for 5 minutes to terminate the reaction activity of the enzyme, then diluted the sample 10 times and filtered it through a 0.22μm filter membrane to ensure the purity of the sample and the accuracy of the analysis.

[0074] In order to accurately analyze the samples, the inventors used a Shimadzu Sμperior LC-20A series of high-performance liquid chromatography systems, which included a RID-10A detector, an LC-20AT pump, and a SIL-20A autosampler. The chromatographic analysis conditions were set to use a glycosyl PakTM column (size 6.5×300mm), inject 20μL at a high temperature of 80°C, use water as the mobile phase, and control the flow rate at 0.2mL / min. By analyzing standard samples of different concentrations of lactose ((10g / L, 20g / L, 30g / L, 40g / L, 50g / L) and lactulose (5g / L, 10g / L, 15g / L, 20g / L, 25g / L), the inventors drew a calibration curve to ensure the accuracy and reliability of the experimental data.

[0075] 2.7 Statistics and Analysis

[0076] Each experiment was repeated at least three times. The data were analyzed using GraphPad Prism 8 software. The results are expressed as mean ± standard deviation.

[0077] 3 Results

[0078] 3.1 Secretory expression of cellobiose epimerase

[0079] This study focused on the cellobiose epimerase from Caldicellulosiruptor bescii DSM 6725. By fusing the first 20 amino acids (N20) of ferulic acid esterase at the front end of the gene sequence as a guide signal peptide, a recombinant expression plasmid pET-22b-N20CE was constructed. This plasmid was transformed into Escherichia coli BL21 (DE3) host cells, and the recombinant strain BL21 (DE3) / pET-22b-N20CE (such as Figure 1 After IPTG induction, the whole cell, intracellular and extracellular protein samples of the recombinant strain were prepared and analyzed by 12% SDS-PAGE. Figure 1 Middle B) shows that the recombinant strain BL21(DE3) / pET-22b-N20CE expressed a target protein band of about 47.2 kDa, confirming the successful expression of cellobiose epimerase in the recombinant strain. In addition, the secretion of extracellular protein showed that the effective fusion of the signal peptide (N20) significantly enhanced the secretion ability of the target protein to the extracellular space, and it was found that N20 could guide the solubilization and secretion of cellobiose epimerase protein.

[0080] Amino acid sequence of N20: MSRITIERDGLTLVGDREEPF SEQ ID NO.2

[0081] Amino acid sequence of cellobiose epimerase (CE) SEQ ID NO.1

[0082] MDITKFKEDLKAHLEEKIVPFWQSLKDDEFGGYYGYMDFNLNIHRKAQKGCILNSRILWFFSACYNVLKNEKCKELAFHAFEFLKNKFWDKEYEGLFWNVSHKGVPVDMTKHVYVQAFGIYGLSEYYEASGDKEALQMAKKLFEILETKCKRENGYTEQFERNWQEKENRFLSENGVIASKTMNTHLHVLESYTN LYKVLRTKDVYEALEWIVRLFVDKIYKKGTGHFKVFCDDNWNELIKAVSYGHDIEASWLLDEAARYLKDEKLKEEVEKLTLEVAQVTLKEAFDGQSLI NEMVEDRVDRSKIWWVEAETVVGFFNAYQKSKEEKFLDAAIKTWKFIEEHLVDKRKNSEWLWKVSEDLKALDMPIVEPWKCPYHNGRMCLEIIKRVG.

[0083] The amino acid sequence of N20 fused with cellobiose epimerase (CE) (recombinant cellobiose epimerase) is SEQ ID NO.3

[0084] MSRITIERDGLTLVGDREEPFDITKFKEDLKAHLEEKIVPFWQSLKDDEFGGYYGYMDFNLNIHRKAQKGCILNSRILWFFSACYNVLKNEKCKELAFHAFE FLKNKFWDKEYEGLFWNVSHKGVPVDMTKHVYVQAFGIYGLSEYYEASGDKEALQMAKKLFEILETKCKRENGYTEQFERNWQEKENRFLSENGVIASKTMNT HLHVLESYTNLYKVLRTKDVYEALEWIVRLFVDKIYKKGTGHFKVFCDDNWNELIKAVSYGHDIEASWLLDEAARYLKDEKLKEEVEKLTLEVAQVTLKEAFD GQSLINEMVEDRVDRSKIWWVEAETVVGFFNAYQKSKEEKFLDAAIKTWKFIEEHLVDKRKNSEWLWKVSEDLKALDMPIVEPWKCPYHNGRMCLEIIKRVG.

[0085] The coding gene sequence corresponding to the fusion of N20 and cellobiose epimerase (CE) SEQ ID NO.4

[0086]

[0087] When the inventors conducted a study on the extracellular secretion of related enzymes, they combined the first 20 amino acids of feruloyl esterase (N20 SEQID NO.2) as a guide signal peptide with the enzyme. The inventors found that the n-terminal 20 amino acids of feruloyl esterase (MSRITIERDGLTLVGDREEPF SEQ ID NO.2) were combined with cellobiose epimerase (SEQ ID NO.1) and transformed into Escherichia coli using a vector, which could achieve the extracellular secretion of cellobiose epimerase and have enzyme activity. However, when the first 20 amino acids of feruloyl esterase (SEQ ID NO.2) were combined with L-arabinose isomerase (amino acid sequence shown in SEQ ID NO.5) from Lactobacillus rhamnosus as a guide signal peptide and transformed into Escherichia coli using a vector, it could not be secreted and expressed extracellularly.

[0088] The amino acid sequence of L-arabinose isomerase is shown in SEQ ID NO.5

[0089] MALSQKYEFWFVAGSQALYGEETLREVKADGQTMADRLNSEGKLPYPVVFKGVMTTAAGITQFMKDVNYHDNVAGVITWMHTFSPAKNWIRGTRLLQKPLLHLATQFHNNIPYDTIDF DYMNLNQSAHGDREYGYINARLGIHNKVVYGYWDDPDVQTEIGQWQDVAVAYDESFQTRVARFGDNMRNVAVTDGDRVEAQIKLGWTVDYYGVGDLVEVVNAVSEADIDNAYEALKQDY DLVQGDNSLEKYTHSVRYQLREYLGIKKFLDDRGYNAFTTNFEDLWGLEQLPGLAAQLLMRDGYGFGAEGDWKTAALSRVIKVMAHNQKTSFMEDYTLDLRKGHEAILGSHMLEVDPSI ASNKPRVEVHPLDIGGKADPARLVFDGSQGDAVDVTLSDFRDGFKLISYPVAAHKPEAATPHLPVAKQLWTPKVGLKQGATTWIQNGGGHHTVFSFAITEQQIEDLAVLYGIKSVAID.

[0090] 3.2 Optimization of fermentation conditions for high-level secretion of cellobiose isomerase

[0091] In order to significantly increase the extracellular production of cellobiose isomerase in E. coli BL21 (DE3) / pET-22b-N20CE, the inventors studied the fermentation conditions. In the experiment, the recombinant strain was cultured in five different fermentation media - LB, TB, SB, 2×YT and SOB, and the extracellular protein content in each culture medium was detected after 24 hours of induction. Figure 2 Middle A and Figure 3 As shown in Figure A, 2×YT medium performed outstandingly in promoting the extracellular protein concentration of E. coli BL21(DE3) / pET-22b-N20CE, which was further confirmed by SDS-PAGE analysis. Therefore, the inventors selected 2×YT as the basic fermentation medium for subsequent optimization.

[0092] When exploring the optimal induction time, the inventors found that the extracellular protein concentration reached a peak at the 2.5h induction time point (i.e., induction started after 2.5h of culture). Figure 2 Medium B and Figure 3 Based on this finding, the inventors also optimized the inducer and concentration of cellobiose isomerase. Although lactose is a good inducer ( Figure 2 Middle C and Figure 3 C), but considering the affinity of cellobiose isomerase for lactose, the inventors used different concentrations of IPTG (0.3 to 0.9 mM) for treatment. After 24 hours, the inventors found that as the concentration of IPTG increased, the amount of extracellular protein secretion gradually increased ( Figure 2 Medium D and Figure 3 D), but the enzyme activity reached the highest level at 0.8 mM, and the extracellular protein secretion was the highest under 0.7 mM IPTG treatment, so the inventors chose 0.7 mM IPTG as the optimal induction concentration.

[0093] Further experiments showed that certain metal ions had a significant effect on the extracellular secretion of proteins. In 2×YT medium, 0.7 mM IPTG and 0.1 mM different metal ions (Mn 2+ 、Zn 2+ , Ca 2+ Mg 2+ , Fe 2+ ) and then induced at 37°C for 24 hours. Figure 2 ZhongE and Figure 3 Fe 2+ The extracellular secretion of cellobiose isomerase was significantly affected. In further optimization, the inventors prepared 2×YT medium containing different concentrations of glycine (25 to 150 mmol / L) and 0.7 mM IPTG and 0.1 mM Fe 2+The culture was terminated at 37℃ for 24 hours. The results showed that the extracellular protein secretion concentration reached the maximum at a glycine concentration of 70mmol / L ( Figure 2 Medium F and Figure 3 F), but considering the cost-effectiveness, the inventors chose a glycine concentration of 25 mmol / L to promote extracellular secretion.

[0094] Finally, the inventors investigated the effect of culture time on protein secretion expression after adding the optimal conditions of the above optimization conditions. Under the above optimization conditions, the inventors set fermentation times of different lengths (6 to 54 hours, which refers to the time for continued fermentation culture after adding the inducer). Figure 4 As shown in A and B, the amount of extracellular secreted protein increased with the extension of culture time and reached the maximum at 36 hours. After more than 36 hours, the enzyme activity and secretion amount decreased, which may be due to cell metabolism and protein degradation.

[0095] By optimizing the fermentation conditions, the inventors not only increased the extracellular production of cellobiose isomerase, but also laid a solid foundation for future industrial applications. The application of these optimization strategies is expected to further promote the efficiency and application scope of biocatalysts in industrial production.

[0096] 3.3 Purification of cellobiose epimerase by ammonium sulfate precipitation

[0097] In order to improve the efficiency of extracellular cellobiose isomerase in industrial applications, this study used ammonium sulfate precipitation to purify the target protein. The experimental results showed that Figure 5 As shown in Figure A, as the concentration of ammonium sulfate increases, the activity of the enzyme increases and reaches a peak at an ammonium sulfate concentration of 80%. However, further increasing the concentration of ammonium sulfate leads to a decrease in the activity of the purified enzyme. This trend is Figure 5 It is clearly visible in B and C. Considering the cost and subsequent processing factors, the comprehensive Figure 5 Based on the data of A, B and C, the inventors selected 60% ammonium sulfate concentration as the optimal purification condition.

[0098] 3.4 Production of lactulose using purified cellobiose epimerase

[0099] Fermentation preparation is carried out under the optimal fermentation conditions obtained above, and the specific method is as follows:

[0100] Escherichia coli BL21 (DE3) / pET-22b-N20CE was inoculated into LB medium and cultured overnight at 37 ° C and 200 rpm to obtain seed solution, which was inoculated into 2 × YT medium at a 1% (v / v) inoculation rate and cultured at 37 ° C and 200 rpm for 2.5 hours. IPTG with a final concentration of 0.7 mM and 0.1 mM Fe2 + and 25 mM glycine, and continue fermentation at 37°C and 200 rpm for 36 hours, with a fermentation volume of 1 L. The fermented bacterial liquid was then centrifuged at 10,000 rpm for 5 minutes, and the precipitate was discarded to obtain the fermentation supernatant of the recombinant cellobiose epimerase.

[0101] Finally, the optimized ammonium sulfate precipitation method was used for purification. Ammonium sulfate was added to the fermentation supernatant to 60% saturation. After standing at 4°C overnight, the supernatant was discarded to obtain a precipitate at 10000rmp at 4°C for 5 minutes. The precipitate was resuspended with 10ml of 3.2M ammonium sulfate, and finally 152.961mg / mL of protease solution was obtained. The total volume after resuspension was 12ml, and its specific activity reached 19.25U / mg. Different enzyme solutions (0.38g / L, 0.75g / L, 1.15g / L and 1.53g / L) were used to react at 75°C for different times (3h, 6h, 9h, 12h and 24h), and liquid phase analysis ( Figure 6 ) showed that at 75°C, the amount of enzyme added was proportional to the efficiency of lactose conversion to lactulose, and as the reaction time increased, the lactulose conversion rate tended to increase. When the enzyme addition amount was 1.53g / L, the reaction was carried out at 75°C for 24 hours, and the highest efficiency of lactose conversion to lactulose was obtained with an enzyme addition amount of 1.53g / L, reaching 56%.

[0102] The present invention successfully achieves the extracellular expression of cellobiose epimerase CE from Caldicellulosiruptor bescii DSM 6725 by using N20 signal peptide, and significantly increases its secretion amount. The molecular weight of extracellular and intracellular N20-CE is consistent, indicating that the protein secreted by the atypical signal peptide is not cleaved by protease. In addition, the extracellular expression of cellobiose epimerase in Escherichia coli has not been reported, which has significant research significance.

[0103] The secretory strain constructed by the present invention allows the performance of the enzyme to be measured directly from the fermentation broth, which greatly speeds up the screening of mutants and improves the efficiency of research and production. This rapid screening capability not only helps to quickly identify mutants with improved performance, but also provides a powerful tool for protein engineering and enzymology research. The construction of a secretory Escherichia coli strain not only simplifies the purification process of the CE enzyme, but also provides an efficient platform for the mutation modification and performance optimization of the enzyme. This achievement is expected to promote the application of CE enzymes in industrial production and bring innovative solutions to the food industry and the pharmaceutical field. With further research and optimization, the industrial production of secretory CE enzymes will bring revolutionary progress to the field of biomanufacturing.

[0104] This study achieved the mass production of extracellular proteins by optimizing the conditions, which not only overcame the degradation of intracellular proteases, but also reduced the contamination of host proteins. In addition, the extracellular proteins did not need to be broken into cells or extracted from internal organelles, but could be directly collected from the culture medium, which simplified the downstream extraction and purification steps. In terms of application, it also achieved efficient conversion of lactulose. Lactulose was obtained by reacting with 40% lactose at 75°C and pH 7.0 for 24 hours, with a conversion rate of up to 56%, and the mass of lactulose reached 224g / L. Further exploration of strategies such as immobilization technology or enzyme modification is expected to improve the lactulose conversion efficiency of the enzyme, which is more in line with the needs of industrial applications.

[0105] The recombinant cellobiose isomerase constructed in this study, combined with the optimization of fermentation conditions and the efficient ammonium sulfate purification method, not only successfully achieved the efficient conversion of lactose, but also reduced production costs, providing key technical support for the industrial application of lactulose, and promoting the efficient utilization of lactose resources and the greening of industrial production processes.

[0106] In this study, the signal peptide of ferulic acid esterase of Lactobacillus amylovora was fused to the cellobiose epimerase gene and expressed in Escherichia coli BL21 (DE3), thereby achieving the extracellular secretion of cellobiose epimerase. By optimizing the fermentation conditions, i.e., adding IPTG with a final concentration of 0.7 mM and 0.1 mM Fe in 2×YT medium after 2.5 hours of induction, the cellobiose epimerase was secreted into the culture medium. 2 + and 25mM glycine, fermented and cultured at 37°C and 200rpm for 36 hours to maximize the secretion of extracellular protein. Cellobiose isomerase was further purified, and in lactose (40%), by adding 1.53g / L enzyme solution for 24 hours, the conversion rate of lactose to lactulose can reach up to 56%. This strategy significantly improves the extracellular efficient production of cellobiose isomerase, providing a solid foundation for the application of this enzyme in the food industry, nutritional health products and pharmaceutical fields. The recombinant Escherichia coli not only has improved enzyme production and concentration, but also shows a high conversion efficiency, and has the potential for industrial production of lactulose.

Claims

1. A recombinant cellobiose isomerase, the amino acid sequence of which is shown in SEQ ID NO.

3.

2. The coding gene sequence of the recombinant cellobiose isomerase according to claim 1 is shown in SEQ ID NO.

4.

3. A recombinant bacterium, characterized in that: A gene sequence encoding the amino acid sequence SEQ ID NO.3 according to claim 1; Preferably, the recombinant bacteria comprises the gene sequence SEQ ID NO.4 encoding the above amino acid sequence SEQ ID NO.

3.

4. An engineered Escherichia coli, characterized in that: Using Escherichia coli BL21 (DE3) as the host bacteria and pET-22 (b) + as the vector, the coding gene sequence of the above amino acid sequence SEQ ID NO.3 was expressed; Preferably, the gene sequence is SEQ ID NO.4; Preferably, the method for constructing the Escherichia coli engineered bacteria includes any of the following schemes: Option 1: ① Using the genomic DNA of Lactobacillus amylovora CGMCC11056 as a template, the primers N20-F and N20-R were used to amplify the ferulic acid esterase N20 sequence encoding gene, and after the product was purified, the encoding gene of the N20 sequence was obtained; using the genomic DNA of Caldicellulosiruptor bescii DSM 6725 as a template, the primers CE-F and CE-R were used to amplify the target gene cellobiose epimerase gene, and after the product was purified, the encoding gene of cellobiose epimerase CE was obtained; The sequence of the primer N20-F is shown in SEQ ID NO.6, and the sequence of the primer N20-R is shown in SEQ ID NO.7; The sequence of the primer CE-F is shown in SEQ ID NO.8, and the sequence of the primer CE-R is shown in SEQ ID NO.9; ② The coding gene of the N20 sequence obtained in step ① and the coding gene of cellobiose epimerase CE are overlapped and extended by PCR to obtain an N20-CE DNA fragment, the N20 sequence is added to the N-terminus of the cellobiose epimerase CE sequence, and then the N20-CE DNA fragment is connected to the pET22b vector fragment treated with restriction enzymes NdeI and XhoI by homologous recombination to obtain a recombinant vector pET22b-N20CE; ③ The recombinant vector pET22b-N20CE was transformed into Escherichia coli BL21 (DE3) to obtain the recombinant strain BL21 (DE3) / pET-22b-N20CE; Option 2: The gene sequence SEQ ID NO.4 was connected to the pET22b vector fragment treated with restriction enzymes NdeI and XhoI by homologous recombination to obtain the recombinant vector pET22b-N20CE, which was transformed into Escherichia coli BL21 (DE3) and the recombinant strain BL21 (DE3) / pET-22b-N20CE was obtained by screening.

5. Use of the recombinant bacterium according to claim 3 or the engineered Escherichia coli bacterium according to claim 4 in the preparation of cellobiose isomerase or recombinant cellobiose isomerase, wherein the amino acid sequence of the cellobiose isomerase is shown in SEQ ID NO.1, and the amino acid sequence of the recombinant cellobiose isomerase is shown in SEQ ID NO.

3.

6. A method for preparing recombinant cellobiose isomerase, characterized in that: The steps include: The Escherichia coli engineered bacteria of claim 4 are used to prepare seed solution, which is then inoculated into 2×YT medium. After culturing for 2.5 to 4.5 hours, IPTG with a final concentration of 0.3 to 0.9 mM, 0.08 to 0.12 mM Fe 2+ and 25 mM or more glycine, continue fermentation and culture for 30 to 54 hours, separate the solid and liquid, and extract the recombinant cellobiose isomerase in the liquid.

7. The preparation method according to claim 6, characterized in that: The steps include: The E. coli engineered bacteria of claim 4 were used to prepare seed solution, which was then inoculated into 2×YT medium. After 2.5 hours, IPTG and 1 mM Fe were added to the medium at a final concentration of 0.7 mM. 2 + and 25 mM glycine, and the fermentation culture was continued for 36 hours, and the solid-liquid separation was performed, and the recombinant cellobiose isomerase was extracted from the liquid.

8. The preparation method according to claim 7, characterized in that: The steps include: The E. coli engineered bacteria of claim 4 were inoculated into LB medium, cultured overnight at 37°C and 200 rpm to obtain a seed solution, which was inoculated into 2×YT medium at a volume ratio of 1% inoculum, cultured at 37°C and 200 rpm for 2.5 hours, and IPTG and 0.1 mM Fe 2+ and 25 mM glycine, and continued fermentation at 37°C, 200 rpm for 36 hours, solid-liquid separation, and recombinant cellobiose isomerase was extracted in the liquid.

9. The preparation method according to any one of claims 6 to 8, characterized in that: In the preparation method, the Fe 2+ Provided by FeSO4.7H2O; Preferably, the solid-liquid separation method is centrifugation at 10,000 rpm or more for more than 5 minutes; Preferably, the method for extracting recombinant cellobiose isomerase in a liquid is to add ammonium sulfate to the liquid to a saturation of 60%, stand at 4°C overnight, centrifuge at 4°C and above 10,000 rpm for more than 5 minutes, discard the supernatant, and obtain a precipitate.

10. Use of the recombinant cellobiose isomerase according to claim 1 or the recombinant cellobiose isomerase prepared by the method according to claim 6 in the preparation of lactulose.

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