High-temperature-resistant lactase, application thereof and method for synthesizing galactooligosaccharide through one-pot method

Abstract

The invention belongs to the technical field of genetic engineering, and relates to high-temperature-resistant lactase, application thereof and a method for synthesizing galactooligosaccharide by a one-pot method. The core amino acid sequence of the lactase GAL-Sup is as shown in SEQ ID NO: 1, and the lactase gene with significantly improved temperature stability and expression level is obtained through artificial directed evolution. In the GOS synthesis process of a one-pot method, the GAL-Sup lactase shows extremely high thermal stability in a lactose solution with the substrate concentration of 70%, the tolerable temperature reaches up to 80 DEG C, and the reaction rate is greatly increased. After 500U of enzyme liquid is added into a lactase solution, reaction is carried out at 80 DEG C for 15-24 hours, lactose can be efficiently converted into galactooligosaccharide, and the requirement of high-concentration syrup production is met. The lactase provided by the invention not only improves the thermal stability and activity of the enzyme, but also provides an efficient way for industrial production of galactooligosaccharide.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and relates to a thermostable lactase, in particular to a gene sequence, a recombinant expression vector and a recombinant expression strain, the application of the lactase and a method for synthesizing oligosaccharides by a "one-pot method". Background Art

[0002] Lactase (EC.3.2.1.23) is a β-galactosidase, β-D-galactoside galactose hydrolase, mainly divided into four subfamilies: GH1, GH2, GH35 and GH42, named for its common use in hydrolyzing lactose. The enzyme is widely distributed in a variety of animals, plants and microorganisms, among which microorganisms are its most abundant source, providing sufficient resources for research.

[0003] Lactase has two activities: lactose hydrolysis and transglycosylation. In the presence of high concentrations of substrates such as lactose, lactase mainly exhibits transglycosidic function. The galactose molecule formed by lactase hydrolyzing the β-D-galactose residue at the non-reducing end of lactose is transferred to the substrate, usually the non-reducing end of lactose, and 2-8 galactoses (i.e., degree of polymerization DP 2-8) are connected to form oligosaccharides (Galacto-oligosacchride, GOS).

[0004] GOS is a natural functional water-soluble dietary oligosaccharide widely found in animal milk, formed by transglycosidation by β-galactosidase. GOS can pass through the upper digestive tract intact to the distal colon, and is fermented and utilized by specific intestinal probiotics such as bifidobacteria and lactobacilli to produce short-chain fatty acids (SCFAs) and lactic acid in the colon, maintain intestinal pH, further enhance lipid metabolism and human immunity, and especially promote the healthy development of infants and young children.

[0005] The industrial production of GOS mainly relies on enzymatic synthesis, using lactase from different microorganisms to catalyze lactose to produce GOS. Usually, the solubility of lactose at room temperature (20°C) is only 19g lactose / 100g water. The concentration of lactose solution used for GOS synthesis is between 30%-60% (w / v). Under appropriate reaction conditions, the GOS yield is usually between 30%-50%, and it is difficult to obtain GOS syrup with a higher concentration. Higher concentrations of lactose will increase the amount of GOS synthesized at higher temperatures, but currently obtaining GOS syrups with a concentration of 70% or more is still a bottleneck that is difficult to break through.

[0006] Although lactase can catalyze lactose to produce GOS, the transglycosylation efficiency of different lactases and the structural distribution of GOS products vary greatly. The development of efficient and stable enzyme systems and the process optimization of GOS synthesis are still major challenges.

[0007] Therefore, the present invention focuses on developing new lactases, or optimizing the expression and catalytic efficiency of existing enzymes by genetic engineering means, and finding lactases with ideal properties (such as high activity, high selectivity and high heat resistance) to increase the yield of GOS. The present invention selects lactase from Aspergillus oryzae of the GH35 subfamily with good heat resistance and food safety as a material, improves its high temperature resistance and production level through molecular modification, and develops a "one-pot method" for producing 70% high-concentration GOS syrup. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a high-temperature-resistant and high-transglycosidic activity lactase GAL-Sup and gene sequence, as well as a recombinant expression vector, a recombinant expression strain, a lactase preparation method, and a "one-pot" method for producing high-concentration GOS syrup, which reduces production costs and improves production efficiency.

[0009] In order to achieve the above object, the present invention provides a thermostable lactase GAL-Sup, the amino acid sequence of which is shown in SEQ ID NO: 1. The lactase GAL-Sup of the present invention has an optimum temperature of 70°C and an optimum pH of 5.0.

[0010] The second aspect of the present invention provides a lactase gene gal-sup, which is used to encode the lactase GAL-Sup, and the base sequence of the lactase gene gal-sup has more than 95% identity with the base sequence shown in SEQ ID NO: 2. Preferably, the effect of the present invention can be achieved when the lactase gene gal-sup contains the base sequence shown in SEQ ID NO: 2. Most preferably, the base sequence of the lactase gene gal-sup is as shown in SEQ ID NO: 2.

[0011] The third aspect of the present invention provides a method for obtaining the lactase GAL-Sup according to the present invention. The lactase gene gal-sup and lactase GAL-Sup are artificially designed and optimized based on the amino acid sequence of lactase AoGAL (GenBank accession number: KF857462) of Aspergillus oryzae. The core nucleotide sequence of the optimized lactase gene gal-sup contains the nucleotide sequence shown in SEQ ID NO: 2. Compared with the original lactase gene aogal, the bases corresponding to the key amino acids in the lactase gene gal-sup of the present invention are changed, and the bases are optimized according to the preference of yeast for codons. The codons with higher or highest frequency of use in yeast are used to replace the codons with low frequency of use, thereby significantly improving the expression level of the artificially designed lactase gene gal-sup in yeast cells. The nucleotide sequence of the original lactase gene aogal is shown in SEQ ID NO: 4, and the amino acid sequence of the lactase AoGAL is shown in SEQ ID NO: 3. The invention artificially designs a new lactase gene sequence based on the amino acid sequence of the original lactase, and obtains the lactase gene fragment by an artificial synthesis method. The artificial design of lactase comprises the following steps: by a site-directed mutagenesis method, the serine at position 52 of the original lactase gene is mutated to threonine (S52T), the alanine at position 141 is mutated to tyrosine (A141Y), the glycine at position 256 is mutated to proline (G256P), and the glutamic acid at position 274 is mutated to lysine (E274K).

[0012] The present invention has no particular limitation on the site-directed mutagenesis technique, which can be achieved by conventional technical means in the art. The specific operation steps are well known to those skilled in the art and will not be described in detail herein.

[0013] The fourth aspect of the present invention provides a recombinant expression vector, which includes the above-mentioned lactase gene gal-sup, and may also include other functional units. When the amino acid sequence of lactase GAL-Sup and the base sequence of lactase gene gal-sup are determined, those skilled in the art can select a suitable recombinant expression vector and other functional units, for example, a yeast expression vector. When the sequence of lactase gene gal-sup is determined, those skilled in the art can obtain a suitable recombinant expression vector.

[0014] The present invention has no particular limitation on the method for preparing the recombinant expression vector, which can be determined according to conventional techniques in the art.

[0015] The fifth aspect of the present invention provides a method for constructing the recombinant expression vector, comprising the following steps:

[0016] The restriction endonuclease EcoR I and Xba I sites are introduced into both ends of the lactase gene gal-sup to obtain a lactase gene gal-sup fragment with sticky ends;

[0017] The yeast expression vector pPICZαA was double-digested with restriction endonucleases EcoR I and Xba I to obtain a vector pPICZαA fragment with sticky ends;

[0018] The lactase gene gal-sup fragment with sticky ends and the vector pPICZαA fragment with sticky ends are connected by T4 DNA ligase to obtain the lactase recombinant expression vector pPIC-gal-sup.

[0019] In the present embodiment, other yeast expression vectors may also be selected.

[0020] The sixth aspect of the present invention provides a recombinant expression strain, which contains the above recombinant expression vector pPIC-gal-sup, and whose expression product is the lactase GAL-Sup. When the gene sequence of lactase GAL-Sup is determined, those skilled in the art can obtain a suitable recombinant expression strain.

[0021] Generally speaking, the host cell can be Escherichia coli, Bacillus, Aspergillus, or yeast cells, or other types of cells, such as animal and plant cells. Preferably, the host cell of the recombinant expression strain is yeast. At the same time, the lactase GAL-Sup provided by the present invention is optimized and adopts a strategy that is more suitable for transcription and translation expression in yeast, thereby increasing the expression amount of lactase GAL-Sup in yeast cells.

[0022] The present invention does not particularly limit the method for preparing the recombinant expression strain, which can be determined according to conventional technical means in the art.

[0023] The seventh aspect of the present invention provides a method for preparing the recombinant expression strain, comprising the following steps:

[0024] Linearize the pPIC-gal-sup recombinant expression vector by enzyme digestion (such as restriction endonuclease Sac I);

[0025] The linearized pPIC-gal-sup was introduced into yeast host cells to obtain a recombinant expression strain.

[0026] Optionally, the recombinant expression vector is introduced into a yeast host cell by electroporation transformation to obtain a recombinant expression strain. In some embodiments of the present invention, in other embodiments of the present invention, various yeast strains can be selected.

[0027] The eighth aspect of the present invention provides a method for producing the lactase, which comprises culturing the recombinant expression strain and obtaining lactase from the culture.

[0028] According to a more specific embodiment of the present invention, the method for preparing lactase from the above-mentioned recombinant expression strain comprises the following steps: picking the recombinant expression strain to expand the culture in YPD liquid medium for 16 hours, and harvesting the bacteria; inoculating into 25mL BMMY liquid medium at 28°C and 200rpm at a 2 / 25 inoculation amount, and adding 1% methanol every 24 hours to induce the expression of lactase GAL-Sup. After induced expression for 72 hours, the supernatant is collected by centrifugation to obtain a solution containing lactase GAL-Sup. In some embodiments, the culture can also be carried out by other means, and the fermentation supernatant is regarded as a lactase product.

[0029] The ninth aspect of the present invention provides the use of the lactase GAL-Sup.

[0030] The tenth aspect of the present invention provides a method for synthesizing galacto-oligosaccharides (GOS) using lactose as a substrate using lactase GAL-Sup, which uses a "one-pot method" to prepare GOS, and has the significant advantages of simplified process and improved production efficiency. Specifically, lactase GAL-Sup is contacted with a high-concentration lactose solution, wherein the lactose concentration ranges from 30% to 75%. In order to achieve an efficient conversion reaction, a lactase GAL-Sup enzyme solution containing 400 to 600U is added. The reaction is carried out in a temperature range of 30°C to 90°C, and the pH value is adjusted to the optimal pH condition of the enzyme to maximize the activity of the enzyme. In the "one-pot method", lactase GAL-Sup and lactose are mixed and reacted, and the reaction time ranges from 0 to 24h. This method does not require intermediate separation, simplifies the process flow, and the resulting syrup does not need to be further concentrated to directly reach the concentration of commercially available GOS syrup, greatly improving production efficiency and reducing energy consumption and costs.

[0031] According to a preferred embodiment of the present invention, the lactase GAL-Sup is contacted with a 50-75% lactose solution by a "one-pot method", the activity of the added lactase GAL-Sup is 400-600U, the reaction is carried out at pH 4.0-5.0 and 60-80°C, and the reaction time is 15-24h.

[0032] The method of the present invention concentrates all reaction steps in a single system, reduces the intermediate processing and purification steps of the traditional process, significantly improves the production efficiency and product quality, thereby reducing the production cost, and maintains the high stability and activity of the enzyme under the protection of high-concentration lactose and in a wide temperature range, thereby realizing efficient and economical galacto-oligosaccharide synthesis and improving the economy and operability of industrial applications.

[0033] The present invention does not particularly limit the lactose used to prepare GOS, and various types of lactose such as α-lactose and β-lactose can be selected.

[0034] The lactase GAL-Sup provided by the present invention is derived from the mutation of the original lactase. After exploring the amino acids associated with structural stability, the optimized lactase has greatly improved its high temperature resistance and has a higher expression level. At the same time, the lactase gene gal-sup is artificially designed according to the amino acid sequence of GAL-Sup. According to the preference of yeast codons, codons used at high frequencies in yeast are selected to reduce the free energy of the secondary structure of the gal-sup gene mRNA, thereby further improving the expression level of the artificially synthesized lactase gene gal-sup, and also significantly improving the activity and expression level of lactase, especially temperature stability, and obtaining high-level expression, high enzyme activity, and high temperature resistant lactase and genes. The present invention completes the method for preparing high-concentration galacto-oligosaccharide GOS, laying a foundation for the application of the enzyme in the production of galacto-oligosaccharides.

[0035] The beneficial technical effects of the present invention are as follows:

[0036] 1) The expression intensity of genes is optimized at the molecular level to increase the expression of enzymes, with significant results.

[0037] 2) The lactase expressed by the new gene sequence has stronger high temperature resistance, which is effective in increasing the GOS yield.

[0038] 3) The “one-pot method” directly synthesizes high-concentration GOS that meets commercial concentration requirements. The operation process is simple and does not require further concentration, making it more suitable for industrial applications.

[0039] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0041] Figure 1 The diagram shows the mutation sites introduced into lactase GAL-Sup in Example 1 of the present invention. Figure A shows the mutation sites in the three-dimensional structure of lactase GAL-Sup; Figure B shows the mutated amino acids around the active site.

[0042] Figure 2The results of enzyme digestion tests during the process of cloning the lactase gene into the recombinant expression vector pPICZαA in Example 2 of the present invention are shown in Figure A for verifying the recombinant expression vectors pPIC-aogal and pPIC-gal-sup; Figure B for verifying the recombinant expression vectors pPIC-aogal and pPIC-gal-sup by single digestion with EcoR I; and Figure C for verifying the recombinant expression vectors pPIC-aogal and pPIC-gal-sup by double digestion with EcoR I and Xba I.

[0043] Figure 3 These are the SDS-PAGE test results of the fermentation supernatants of the original lactase gene aogal and the redesigned lactase gene gal-sup recombinant expression strains in Example 3 of the present invention.

[0044] Figure 4 This is the test result of lactase production by the redesigned lactase gene gal-sup recombinant expression strain in Example 3 of the present invention in a 30L fermenter.

[0045] Figure 5 The results of the test of the optimum temperature and the optimum pH of lactase in Example 4 of the present invention are shown in Figure A. The optimum temperature of lactase AoGAL and GAL-Sup is shown in Figure B. The optimum pH of lactase AoGAL and GAL-Sup is shown in Figure B.

[0046] Figure 6 This is a curve showing the change in residual activity of lactase over time after incubation at 70° C. for 0 to 90 min in Example 5 of the present invention.

[0047] Figure 7 The results of the "one-pot method" for synthesizing GOS by lactase GAL-Sup in Example 6 of the present invention are shown in Figure A. Figure A shows the color representation of the reaction catalyzed by lactase AoGAL and GAL-Sup to generate GOS; Figure B shows the thin layer chromatography (TLC) results of lactase AoGAL and GAL-Sup to prepare GOS respectively. DETAILED DESCRIPTION

[0048] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0049] If the specific conditions are not specified in the examples, all the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, all of them are conventional products that can be purchased from the market.

[0050] Example 1

[0051] This example is used to illustrate the design process of introducing mutations into lactase AoGAL. The specific implementation process is as follows:

[0052] According to the three-dimensional structure of lactase AoGAL, the lactase GAL-Sup is introduced with 4 site mutations by protein design from the original lactase AoGAL, and the amino acid sequence of the lactase GAL-Sup is shown in SEQ ID NO: 1. The specific design includes: mutating the serine at position 52 of the original lactase AoGAL to threonine (S52T), mutating the alanine at position 141 to tyrosine (A141Y), mutating the glycine at position 256 to proline (G256P), and mutating the glutamic acid at position 274 to lysine (E274K). The amino acid sequence of the mutated lactase GAL-Sup is shown in SEQ ID NO: 1, and the amino acid sequence of the original lactase AoGAL is shown in SEQ ID NO: 3. The nucleotide sequence is optimized according to the preference of yeast for codons, and the codons with higher or highest frequency of use in the yeast strain are used to replace the codons with low frequency of use, and the lactase gene gal-sup is designed, and its base sequence is shown in SEQ ID NO: 2. The sequence was synthesized by a commercial company.

[0053] Figure 1 A shows the position of mutation in the three-dimensional structure of lactase GAL-Sup; Figure 1 B shows the mutated amino acids around the active site.

[0054] Example 2

[0055] This example is used to illustrate the construction of the original lactase gene aogal and the optimized lactase gene gal-sup recombinant expression strain.

[0056] Add restriction sites EcoR I and Xba I at both ends of the genes gal-sup and aogal, and double-digest with EcoR I and Xba I; similarly, the vector pPICZαA is double-digested with EcoR I and Xba I; the double-digested lactase genes gal-sup and aogal are connected with the double-digested pPICZαA respectively through T4 DNA ligase to obtain pPIC-gal-sup and pPIC-aogal recombinant expression vectors. The connection system is: a total volume of 10 μL, which contains 100 ng of lactase gene, 50 ng of pPICZαA and 5U of T4 DNA ligase, and is connected overnight at 4°C. The connection system is transformed into Escherichia coli (DH5α or TOP10) and the recombinant expression vectors pPIC-gal-sup and pPIC-aogal are obtained. The recombinant expression vectors are extracted and enzyme cutting verification is performed. The results are as follows Figure 2As shown in A, B and C. Figure 2 In A: M is DL5000 DNA Marker, lane 1 is pPIC-aogal recombinant expression vector, lane 2 is pPIC-gal-sup recombinant expression vector; Figure 2 In B: M is DL5000 DNA Marker, lane 1 is the recombinant expression vector pPIC-aogal after single digestion with EcoR I, lane 2 is the recombinant expression vector pPIC-gal-sup after single digestion with EcoR I; Figure 2 In C: M is DL5000 DNA Marker, lane 1 is the recombinant expression vector pPIC-aogal after double digestion with EcoR I and Xba I, and lane 2 is the recombinant expression vector pPIC-gal-sup after double digestion with EcoR I and Xba I. Figure 2 It can be seen that the lactase genes gal-sup and aogal were successfully connected to the pPICZαA expression vector. The recombinant expression vectors pPIC-gal-sup and pPIC-aogal were transformed into yeast X-33 to obtain recombinant expression strains containing the lactase gene gal-sup or aogal.

[0057] Example 3

[0058] This example is used to illustrate the fermentation production of lactase GAL-Sup and AoGAL in shake flasks.

[0059] (1) The recombinant strains containing the gal-sup and aogal genes in Example 2 were inoculated into 5 mL of YPD medium and cultured for 16 h to obtain seed solution.

[0060] (2) Inoculate the above seed liquid into a shake flask containing 25 mL of BMMY fermentation medium at a 2 / 25 inoculation rate and culture at 28°C and 200 rpm. The formula of BMMY fermentation medium is: 1% yeast powder, 2% peptone, 10% 1M phosphate buffer (pH 6.0). Supplement 1% methanol every 24 hours, and stop the shake flask fermentation at 96 hours. The expression level of the product lactase was detected by SDS-PAGE. Figure 3 Shown are the test results of the fermentation broth. Based on all the tests, two samples that were successfully expressed and could represent the average expression level of lactase were selected for retesting (in the figure: M is the protein standard, lane 1 is the expression product of pPIC-aogal; lane 2 is the expression product of pPIC-gal-sup). Figure 3This indicates that the lactase gene aogal and the redesigned lactase gene gal-sup were successfully expressed, and the yield of the lactase gene gal-sup was significantly higher than that of the original lactase gene aogal. After the lactase GAL-Sup was induced to express in a 30L fermenter for 190h (cycle of 215h), the lactase activity in the supernatant reached 2324U / mL, and the protein content reached more than 10mg / mL. Figure 4 shown.

[0061] Example 4

[0062] This example is used to test the optimum temperature and optimum pH of lactase GAL-Sup.

[0063] (1) Under standard assay conditions, lactase activity was determined by measuring the hydrolysis rate of oNPG (o-nitropheny-D-galactopyranoside). The specific steps are as follows: 0.1 mL of fermentation supernatant diluted appropriately was added to a mixture of 0.1 mL of sodium dihydrogen phosphate-citric acid buffer (pH 5.0) and 0.1 mL of 10 mM oNPG, reacted at a specific temperature for 10 min, and 0.2 mL of 10% Na2CO3 was added to terminate the reaction and stabilize the color of the reaction. The control group was first added with the stop solution. Three experimental groups were set up in parallel. At OD 420 The absorbance of the mixture was measured at 4 °C. The lactase activity was calculated based on the measured absorbance and the enzyme activity definition. One unit (U) of enzyme activity is defined as the amount of enzyme required to produce 1 μmol oNP (o-nitrophenol) per minute under standard assay conditions.

[0064] (2) The determination system of the optimum temperature of the original lactase AoGAL and the redesigned lactase GAL-Sup is as follows: add 0.1 mL of lactase solution diluted appropriately to a mixture of 0.1 mL of disodium hydrogen phosphate-citrate buffer (pH 5.0) and 0.1 mL of 10 mM oNPG. The above reaction system was placed in a temperature gradient water bath at 30°C, 40°C, 50°C, 60°C, 70°C and 80°C for 10 minutes. After the reaction, the activity of lactase was determined by the above oNPG method, and the linear relationship between the effect of temperature and lactase activity was plotted. The results are shown in Figure 2. Figure 5 As shown in A, the optimum temperature of the original lactase AoGAL is 65° C. The optimum temperature of the lactase GAL-Sup introduced with amino acid redesign mutations is 70° C. The lactase GAL-Sup of the present invention has a higher optimum temperature and is more tolerant to temperature.

[0065] (3) The optimal pH determination system for the original lactase AoGAL and the redesigned lactase GAL-Sup is as follows: add 0.1 mL of disodium hydrogen phosphate-citric acid buffer of different pH to a mixture of 0.1 mL of 10 mM ONPG and 0.1 mL of lactase solution diluted to an appropriate multiple. Use disodium hydrogen phosphate-citric acid buffer with a pH gradient of pH 3.0, pH 4.0, pH 5.0, pH 6.0, pH 7.0 and pH 8.0. After 10 minutes of reaction, the above-mentioned oNPG method was used to determine the activity of lactase, and the linear relationship of the effect of pH on lactase activity was plotted. The results are shown in Figure 5 As shown in Figure B, the optimal pH of the original lactase AoGAL is 4.0. The optimal pH of the lactase GAL-Sup introduced with amino acid redesign is 5.0. Both optimal pHs are acidic, so the lactase GAL-Sup of the present invention is an acidic lactase, which is suitable for the natural pH of sugar.

[0066] Example 5

[0067] This example is used to test the temperature stability of lactase GAL-Sup.

[0068] The lactase enzyme solution was incubated in a 70°C water bath, and then the residual activity at different times was measured under the standard measurement conditions of Example 4. For the determination of thermal stability, the enzyme activity at the optimum temperature was defined as 100%, the residual enzyme activity was calculated, and a linear relationship between the incubation time and the residual activity of lactase was plotted. The results are shown in Figure 6 As shown, the original lactase AoGAL has a maximum enzyme activity of 70-99% in the range of 45-60°C, and is rapidly inactivated after 30 minutes at a temperature as high as 70°C, while GAL-Sup has a maximum enzyme activity of 73-97% in the range of 45-65°C, and still maintains 67% of the maximum enzyme activity after 30 minutes, and the half-life is increased to 2 times of the original. Therefore, the lactase GAL-Sup of the present invention has good thermal stability and high temperature resistance.

[0069] Example 6

[0070] This example is used to detect the transglycosylation efficiency of GOS synthesized by the "one-pot method" of lactase.

[0071] Thin layer chromatography (TLC) was used to detect transglycosidic products to evaluate the transglycosidic efficiency of lactase and the yield of GOS. Lactase AoGAL was added to a solution with a lactose concentration of 70% (70g anhydrous lactose + 30g water) and reacted at an optimal temperature of 65°C. Lactase GAL-Sup was added to a solution with a lactose concentration of 70% (mass fraction ratio, 70g anhydrous lactose + 30g water) and the synthesis reaction was carried out at a high temperature of 80°C. The enzyme solution added to both contained 500U, and the synthesis reaction lasted for 18h. After being taken out, it was immediately denatured at 100°C for 10min to terminate the reaction. The final reaction solution was diluted 10 times, and 1μL was spotted on a dry silica gel GF254 plate and placed in a layer development tank for layer development to separate and characterize the products and analyze the residual lactose. The products of the lactose transglycosylation reaction were separated by a migration phase of n-butanol-acetic acid-water (2:1:1, vol / vol). Spray with sulfuric acid-methanol (5:95, vol / vol), completely dry, and then carbonize and color at 95-100°C for 5-10 minutes to visually identify mono-oligosaccharides and oligosaccharides.

[0072] The results are as follows Figure 7 As shown in A, the figure shows the color performance of the reaction catalyzed by lactase AoGAL and GAL-Sup to produce oligosaccharides. 1 is the final reaction solution of lactase AoGAL to prepare GOS; 2 is the final reaction solution of lactase GAL-Sup to prepare GOS. In the process of lactase AoGAL to prepare GOS, some sugar crystals precipitated, and the final reaction solution was lighter in color and had a lower viscosity, indicating that the amount of GOS produced was small; while in the whole process of lactase GAL-Sup to prepare GOS, no sugar crystals precipitated, and the final reaction solution was dark in color and had a high viscosity, showing a higher GOS yield and reaction efficiency. Overall, lactase GAL-Sup showed a stronger transglycosidation ability than AoGAL in the process of synthesizing GOS.

[0073] Figure 7 B shows the results of thin layer chromatography (TLC) of GOS prepared by lactase AoGAL and GAL-Sup, respectively. The present invention uses glucose, galactose and lactose as standard products. The lane positions are as follows Figure 7 As shown in the first three lanes of B. Lane 1 is the synthesis of GOS by lactase AoGAL, and lane 2 is the synthesis of GOS by lactase GAL-Sup. Compared with lactase AoGAL, the lactase GAL-Sup provided by the present invention can synthesize more GOS at a higher temperature, and the proportion of polysaccharides with a degree of polymerization greater than 2 in the GOS mixture is larger. Therefore, the transglycosidic activity of the lactase GAL-Sup of the present invention is higher than that of lactase AoGAL, and the GOS yield is also higher, and it is more suitable for industrial production of GOS.

[0074] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A thermostable lactase GAL-Sup, characterized in that: The amino acid sequence of the thermostable lactase GAL-Sup is shown in SEQ ID NO:

1.

2. A lactase gene gal-sup, used to encode the lactase according to claim 1, characterized in that: The base sequence of the lactase gene gal-sup has more than 95% identity with the base sequence shown in SEQ ID NO:

2. Preferably, the base sequence of the lactase gene gal-sup is as shown in SEQ ID NO:

2.

3. The method for obtaining lactase GAL-Sup according to claim 1, characterized in that: The method comprises the following steps: using a site-directed mutagenesis method, the serine at position 52 of the original lactase gene is mutated into threonine (S52T), the alanine at position 141 is mutated into tyrosine (A141Y), the glycine at position 256 is mutated into proline (G256P), and the glutamic acid at position 274 is mutated into lysine (E274K); the original lactase is the lactase AoGAL of Aspergillus oryzae.

4. A recombinant expression vector, characterized in that: Comprising the lactase gene gal-sup as claimed in claim 2.

5. The method for constructing the recombinant expression vector according to claim 4, characterized in that: The steps include: Introducing restriction endonuclease EcoR I and Xba I restriction sites at both ends of the lactase gene gal-sup as claimed in claim 2 to obtain a lactase gene gal-sup fragment with sticky ends; The yeast expression vector pPICZαA was double-digested with restriction endonucleases EcoR I and Xba I to obtain a vector pPICZαA fragment with sticky ends; The lactase gene gal-sup fragment with sticky ends and the vector pPICZαA fragment with sticky ends are connected by T4 DNA ligase to obtain the lactase recombinant expression vector pPIC-gal-sup.

6. A recombinant expression strain, characterized in that: Comprising the recombinant expression vector as claimed in claim 4; the host cell of the recombinant expression strain is yeast.

7. The method for preparing the recombinant expression strain according to claim 6, characterized in that: The steps include: Linearizing the recombinant expression vector according to claim 4 by enzyme digestion; The linearized recombinant expression vector is introduced into the host cell to obtain a recombinant expression strain.

8. The method for producing lactase according to claim 1, characterized in that: Cultivate the recombinant expression strain according to claim 6 and obtain lactase from the culture.

9. Use of the lactase according to claim 1 in the production of galacto-oligosaccharides.

10. A method for synthesizing high-concentration galacto-oligosaccharides by a "one-pot method", comprising the following steps: The lactase GAL-Sup is contacted with a 50-75% lactose solution by a "one-pot method", the activity of the added lactase GAL-Sup is 400-600U, the reaction is carried out at pH 4.0-5.0 and 60-80°C, and the reaction time is 15-24h.

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