High-stability lactase and application thereof in production of galactooligosaccharide

Abstract

The invention belongs to the field of gene engineering, and relates to high-stability lactase and application thereof in production of galactooligosaccharide. The core amino acid sequence of the lactase NK-Lac is as shown in SEQ ID NO: 1, and the temperature and pH stability and the expression efficiency of the lactase NK-Lac are remarkably enhanced through artificial directed evolution. Through shake flask fermentation, the recombinant strain shows higher protein yield and enzymatic activity, and especially shows excellent catalytic efficiency at a lactose substrate concentration of 60%. 15U of enzyme liquid is added into a lactose solution to react at 50 DEG C for 12-21 hours, so that lactose can be efficiently converted to generate high-concentration GOS syrup. After the reaction, thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) are adopted to analyze the generation condition of the GOS, and the yield and the purity of the GOS are verified. The method not only meets the production requirements of the high-concentration GOS syrup, but also simplifies the industrial preparation process, and provides a stable and efficient solution.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to a lactase with high pH stability, with particular attention paid to its gene sequence, recombinant expression vector and recombinant expression strain. At the same time, the present invention also covers the application of the lactase and the process of synthesizing oligosaccharides. Background Art

[0002] Lactase (EC.3.2.1.23), also known as β-D-galactoside galactosyl hydrolase, is an enzyme widely found in microorganisms, plants and animals. It mainly catalyzes the hydrolysis of lactose into glucose and galactose. As people pay more attention to healthy diet, galacto-oligosaccharides (GOS) have received widespread attention due to their benefits in promoting intestinal health, enhancing immunity and improving nutrient absorption. GOS is a non-digestible sugar converted from lactose by enzyme catalysis. It has excellent biological activity and good taste and has been widely used in infant food, dairy products and functional foods.

[0003] However, existing lactases still face a series of challenges in the process of synthesizing high-concentration GOS. First, the catalytic efficiency of many lactases at high substrate concentrations is significantly reduced, resulting in unsatisfactory yields. In addition, the temperature and pH stability of existing enzymes is poor, and they are easily inactivated, which limits their application in industrial production. For example, the transglycosidic activity of traditional lactase is usually limited to narrow reaction conditions, and it is difficult to effectively catalyze under wider temperature and pH conditions, which affects the synthesis efficiency of GOS. Secondly, current research focuses on the optimization of the single performance of lactase, and lacks systematic research on the comprehensive regulation of enzyme activity, stability and reaction conditions. This makes it difficult for many research results to be transformed into practical industrial applications and cannot meet the market demand for high-concentration GOS. In addition, 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 a higher concentration of GOS syrup.

[0004] Therefore, developing a lactase with superior transglycosidic activity and stability to improve the synthesis efficiency of GOS has become an important issue that needs to be solved urgently.

[0005] In order to solve the above problems, the present invention provides a lactase with high pH stability, which has been artificially directed evolution, and its activity, stability and transglycosidic activity are significantly improved, and is particularly suitable for the synthesis of high-concentration GOS. The lactase exhibits excellent catalytic efficiency at a lactose substrate concentration of 60%, can maintain stability within the optimal pH range, and effectively prolongs the half-life of the enzyme. In addition, the lactase of the present invention can directly achieve the production of high-concentration GOS syrup without going through a concentration step, thereby simplifying the process and improving production efficiency. These advantages make the lactase of the present invention have a wide range of application prospects in the industrial production of GOS, and can meet the market's urgent demand for high-quality oligosaccharides. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a lactase NK-Lac with high pH stability and high transglycosidic activity, as well as a gene sequence, a recombinant expression vector and a recombinant expression strain. At the same time, the present invention also covers a preparation method of the lactase and a process for synthesizing high-concentration galacto-oligosaccharides, thereby reducing production costs and improving production efficiency.

[0007] In order to achieve the above object, the first aspect of the present invention provides a high-stability lactase NK-Lac, the amino acid sequence of the lactase NK-Lac is shown in SEQ ID NO: 1. The lactase NK-Lac of the present invention has an optimum temperature of 60°C and an optimum pH of 6.0, and maintains more than 95% of the optimum enzyme activity in the pH range of 5-7, which is suitable for the catalytic conversion environment of lactose.

[0008] The second aspect of the present invention provides a lactase gene nk-lac, which is used to encode the lactase NK-Lac, and the base sequence of the lactase gene nk-lac 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 nk-lac contains the base sequence shown in SEQ ID NO: 2. Most preferably, the base sequence of the lactase gene nk-lac is as shown in SEQ ID NO: 2.

[0009] The third aspect of the present invention provides a method for obtaining the above-mentioned lactase NK-lac. According to the present invention, the lactase gene nk-lac and lactase NK-Lac are artificially designed and optimized based on the amino acid sequence of lactase BcGAL (GenBank accession number: AB605256) of Bacillus circulans. The core nucleotide sequence of the optimized lactase gene nk-lac contains the nucleotide sequence shown in SEQ ID NO: 2. Compared with the original lactase gene bcgal, the bases corresponding to the key amino acids in the lactase gene nk-lac 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 the yeast strain are used to replace the codons with low frequency of use, thereby significantly improving the expression amount of the artificially designed lactase gene nk-lac in yeast cells. The nucleotide sequence of the original lactase gene bcgal is shown in SEQ ID NO: 4, and the amino acid sequence of the lactase BcGAL 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: mutating the alanine at position 401 of the original lactase gene to tyrosine (A401Y), mutating the glutamic acid at position 480 to histidine (E480H), and mutating the threonine at position 594 to serine (T594S) by a site-directed mutagenesis method.

[0010] 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.

[0011] The fourth aspect of the present invention provides a recombinant expression vector, which includes the above-mentioned lactase gene nk-lac, and may also include other functional units. When the amino acid sequence of lactase NK-lac and the base sequence of lactase gene nk-lac 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 nk-lac is determined, those skilled in the art can obtain a suitable recombinant expression vector.

[0012] 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.

[0013] The fifth aspect of the present invention provides a method for preparing the recombinant expression vector, comprising the following steps:

[0014] The restriction endonuclease EcoR I and Xba I sites were introduced into both ends of the lactase gene nk-lac to obtain a lactase gene nk-lac fragment with sticky ends;

[0015] 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;

[0016] The lactase gene nk-lac 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-nk-lac.

[0017] In the embodiment of the present invention, other yeasts such as Kluyveromyces lactis and Saccharomyces cerevisiae expression vectors can also be selected.

[0018] The sixth aspect of the present invention provides a recombinant expression strain, which contains the above-mentioned recombinant expression vector pPIC-nk-lac, and its expression product is the lactase NK-Lac. When the gene sequence of lactase NK-Lac is determined, those skilled in the art can obtain a suitable recombinant expression strain.

[0019] Generally speaking, the host cell can be Escherichia coli, Bacillus, Aspergillus, or Kluyveromyces, Saccharomyces cerevisiae, 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 NK-Lac provided by the present invention is optimized and adopts a strategy that is more suitable for transcription and translation expression in yeast cells, thereby increasing the expression amount of lactase NK-Lac in yeast cells.

[0020] 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.

[0021] The seventh aspect of the present invention provides a method for preparing the recombinant expression strain, comprising the following steps:

[0022] Linearize the recombinant expression vector pPIC-nk-lac by enzyme digestion (such as restriction endonuclease Sac I);

[0023] The linearized recombinant expression vector pPIC-nk-lac was introduced into the host cell to obtain a recombinant expression strain.

[0024] Alternatively, the recombinant expression vector is introduced into a yeast host cell by electroporation transformation to obtain a recombinant expression strain. In other embodiments of the present invention, various types of yeast strains can be selected.

[0025] The eighth aspect of the present invention provides the above-mentioned method for producing lactase, culturing the above-mentioned recombinant expression strain, and obtaining lactase from the culture. 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 culture medium for 16 hours, and harvesting the bacteria; inoculating into 25mL BMMY liquid culture medium at 28°C and 200rpm at a 2 / 25 inoculation rate, and adding 1% methanol every 24 hours to induce the expression of lactase NK-Lac. After induced expression for 72 hours, the supernatant was collected by centrifugation to obtain a solution containing lactase NK-Lac. In some embodiments, the culture can also be carried out in other ways, and the fermentation supernatant is regarded as a lactase product.

[0026] The ninth aspect of the present invention provides the use of the lactase NK-Lac in the production of galacto-oligosaccharides (GOS).

[0027] The tenth aspect of the present invention provides a method for synthesizing oligogalactose using lactose as a substrate using lactase NK-Lac, which significantly improves production efficiency through the advantages of high temperature stability and high pH stability, comprising the following steps: contacting the lactase NK-Lac described in claim 1 with an activity of 10 to 20 U with a 30% to 60% lactose solution, reacting for 12 to 21 hours under the conditions of pH 5 to 7 and 50 to 70°C to obtain oligogalactose. Preferably, the reaction is carried out under the conditions of an optimum pH of 6.0 and an optimum temperature of 60°C.

[0028] The method for synthesizing galacto-oligosaccharides of the present invention significantly improves the conversion efficiency of GOS, and finally a GOS syrup concentration of up to 60% can be directly obtained without concentration, reaching and exceeding the standard of commercial concentrated syrup. This is due to the excellent transglycosidic activity and strong pH tolerance of NK-Lac lactase, making the preparation process of high-concentration GOS more stable and efficient.

[0029] The present invention not only simplifies the production process, but also ensures the efficiency and consistency of the GOS synthesis process through the high stability of lactase NK-Lac under the optimal pH 6.0 condition, thereby improving the operability and economic benefits of industrial production.

[0030] 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.

[0031] The lactase NK-Lac provided by the present invention is mutated from the original lactase. After exploring the amino acids associated with structural stability, the optimized lactase has greatly improved the temperature and pH stability characteristics, and has a higher expression level. At the same time, the lactase gene nk-lac is artificially designed according to the amino acid sequence of NK-Lac, and the codons used at high frequencies are selected according to the preference of the yeast strain codons, thereby reducing the free energy of the secondary structure of the nk-lac gene mRNA, thereby further improving the expression of the artificially synthesized lactase gene nk-lac, and also significantly improving the activity, expression level, etc. of lactase, especially temperature and pH stability, and obtaining a lactase and gene with high transglycosidic activity and high stability to temperature and pH. The present invention completes the method for preparing high-concentration galacto-oligosaccharide GOS, which lays a foundation for the application of the enzyme in the production of galacto-oligosaccharides.

[0032] The beneficial technical effects of the present invention are as follows:

[0033] 1) Increase the expression of enzymes through molecular optimization and significantly improve the efficiency of GOS synthesis.

[0034] 2) The lactase with the new gene sequence exhibits excellent pH stability and transglycosidic activity, significantly improving the GOS yield.

[0035] 3) This method directly synthesizes high-concentration GOS, simplifies the process, and is more suitable for industrial production.

[0036] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] 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.

[0038] Figure 1 The diagram shows the mutation sites introduced into lactase NK-Lac in Example 1 of the present invention. Figure A shows the mutation sites in the three-dimensional structure of lactase NK-Lac; Figure B shows the mutated amino acids around the active site.

[0039] Figure 2 The SDS-PAGE test results of the fermentation supernatant of the original lactase gene bcgal and the redesigned lactase gene nk-lac recombinant expression strain in Example 3 of the present invention.

[0040] Figure 3 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 BcGAL and NK-Lac is shown in Figure B. The optimum pH of lactase BcGAL and NK-Lac is shown in Figure B.

[0041] Figure 4 The results of the temperature stability and pH stability tests of lactase in Example 5 of the present invention are shown in Figure A. The curve of the residual activity after incubation at 60°C for 0 to 90 minutes versus time; Figure B. The curve of the residual activity after incubation at 37°C and pH 6.0 for 0 to 780 minutes versus time.

[0042] Figure 5 The results of thin layer chromatography (TLC) of lactase synthesizing GOS in Example 6 of the present invention are shown in Figure A. Figure A shows the TLC results of lactase BcGAL and NK-Lac catalyzing the reaction to generate GOS; Figure B shows the TLC analysis results of lactase NK-Lac synthesizing GOS at different reaction times. As the reaction time increases, the yield of GOS shows a corresponding change.

[0043] Figure 6 The results of high performance liquid chromatography (HPLC) analysis of GOS synthesized by lactase NK-Lac at different reaction times are shown. DETAILED DESCRIPTION

[0044] 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.

[0045] Example 1

[0046] This example is used to illustrate the design process of introducing mutations into lactase BcGAL. The specific implementation steps are as follows:

[0047] Based on the three-dimensional structure of lactase BcGAL, lactase NK-Lac was introduced with three key mutation sites through protein design, and the amino acid sequence of the resulting lactase NK-Lac is shown in SEQ ID NO: 1. The design includes: mutating the alanine at position 401 of lactase BcGAL to tyrosine (A401Y), mutating the glutamic acid at position 480 to histidine (E480H), and mutating the threonine at position 594 to serine (T594S). The amino acid sequence of the original lactase BcGAL is shown in SEQ ID NO: 3, and the amino acid sequence of the mutated lactase NK-Lac is shown in SEQ ID NO: 1. The nucleotide sequence was optimized according to the codon preference of yeast, and the codons with higher or highest frequency of use in the yeast strain were used to replace the codons with low frequency of use, and the lactase gene nk-lac was designed, and its base sequence is shown in SEQ ID NO: 2. The sequence was commissioned to be synthesized by a commercial company.

[0048] The mutation sites introduced into lactase NK-Lac are as follows Figure 1 shown. Figure 1 A shows the distribution of mutation sites in the three-dimensional structure of lactase NK-Lac; Figure 1 B presents the mutated amino acids around the active site.

[0049] Example 2

[0050] This example describes the construction process of the original lactase gene bcgal and the optimized lactase gene nk-lac recombinant expression strain.

[0051] First, EcoR I and Xba I restriction sites were added to both ends of the genes nk-lac and bcgal, respectively, followed by double digestion. The vector pPICZαA was also subjected to the same double digestion treatment. The cut lactase genes nk-lac and bcgal were connected to the double-digested pPICZαA by T4 DNA ligase to obtain recombinant expression vectors pPIC-nk-lac and pPIC-bcgal. The total volume of the ligation reaction system was 10 μL, containing 100 ng of lactase gene, 50 ng of pPICZαA and 5 U of T4 DNA ligase, and the reaction was carried out overnight at 4 ° C. The ligation system was transformed into Escherichia coli (DH5α or TOP10) to obtain recombinant expression vectors pPIC-nk-lac and pPIC-bcgal. The recombinant expression vector was transformed into yeast X-33 to obtain a recombinant expression strain containing the lactase gene nk-lac or bcgal.

[0052] Example 3

[0053] This example is used to illustrate the fermentation production of lactase NK-Lac and BcGAL in shake flasks.

[0054] (1) The recombinant expression strains containing the nk-lac and bcgal genes in Example 2 were inoculated into 5 mL of YPD medium respectively and cultured for 16 h to obtain seed solution;

[0055] (2) Inoculate the above seed liquid into a shake flask containing 25 mL of BMMY fermentation medium at a rate of 2 / 25, 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. Based on all the tests, two samples that were successfully expressed and could represent the average expression level of lactase were selected for re-testing. The results are as follows Figure 2 As shown ( Figure 2Middle: M is a protein standard, lane 1 is the expression product of pPIC-bcgal; lane 2 is the expression product of pPIC-nk-lac). Figure 2 This indicates that the lactase gene bcgal and the redesigned lactase gene nk-lac were successfully expressed, and the yield of the lactase gene nk-lac was significantly higher than that of the original lactase gene bcgal. After the lactase NK-Lac was induced to express in a 250 mL conical flask for 72 hours (cycle of 96 hours), the enzyme activity increased by 4-6 times.

[0056] Example 4

[0057] This example is used to test the optimum temperature and optimum pH of lactase NK-Lac.

[0058] (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 6.0) 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.

[0059] (2) The optimal temperature of the original lactase BcGAL and the redesigned lactase NK-Lac was determined as follows: 0.1 mL of a properly diluted lactase solution was added to a mixture of 0.1 mL of pH 6.0 sodium hydrogen phosphate-citrate buffer and 0.1 mL of 10 mM oNPG. The reaction system was placed in a temperature gradient water bath from 30°C to 90°C for 10 min. After the reaction, the lactase activity was determined using the oNPG method, and a curve of the relationship between temperature and lactase activity was plotted. The results are shown in Figure 2. Figure 3 As shown in A, the optimum temperature of the original lactase BcGAL and the mutant lactase NK-Lac is 60°C. In the temperature range of 50°C to 70°C, the enzyme activity retains 50% of the maximum activity, and the temperature range is wide. When the temperature is lower than 30°C or higher than 80°C, the lactase activity can still retain 40% of the maximum activity. The temperature environment is suitable for the catalytic conversion of lactose.

[0060] (3) The optimal pH of the original lactase BcGAL and the redesigned lactase NK-Lac was determined as follows: 0.1 mL of 10 mM ONPG and 0.1 mL of appropriately diluted lactase solution were added with 0.1 mL of sodium hydrogen phosphate-citrate buffer of different pH values ​​(pH 3.0 to pH 8.0). After 10 min of reaction, the lactase activity was determined using the oNPG method, and a curve showing the effect of pH on lactase activity was plotted. The results are shown in Table 1. Figure 3 As shown in Figure B, the optimum pH of the original lactase BcGAL and the mutant lactase NK-Lac is 6.0, that is, NK-Lac is an acidic enzyme. As can be seen from the figure, within the pH range of 5.0-8.0, the enzyme activity retains 50% of the maximum activity. The pH environment is suitable for the catalytic conversion of lactose. When the pH is less than or equal to 3, it is rapidly inactivated, and when the pH is greater than 8, it still retains more than 70% of the maximum enzyme activity.

[0061] Example 5

[0062] This example is used to test the temperature stability and pH stability of lactase NK-Lac.

[0063] (1) The lactase enzyme solution was incubated in a 60°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 FIG. Figure 4 As shown in A, at the optimum temperature of 60°C, BcGAL has only 25% of the maximum activity after 30 minutes of incubation, and is completely inactivated after 60 minutes. However, NK-Lac still has more than 50% of the maximum enzyme activity after 30 minutes of incubation, and the half-life is increased to twice the original. It can be seen that the lactase NK-Lac of the present invention has good thermal stability.

[0064] (2) The lactase solution was added to a pH 6.0 buffer solution and incubated in a 37°C water bath, and then the residual activity at different times was measured under the standard assay conditions of Example 4. For the determination of pH stability, the enzyme activity at the optimum pH 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 2. Figure 4As shown in Figure B, at the optimum pH of 6.0, the enzyme activity of BcGAL showed a significant downward trend after incubation for 30 minutes. When the incubation time reached 270 minutes, the enzyme activity decreased by 45%, and only 55% of the maximum activity was retained. However, after incubation for 480 minutes, the enzyme activity of NK-Lac still retained 90% of the maximum activity. Even when the incubation time was extended to 630 minutes, the enzyme activity only decreased by 15% and retained 85% of the maximum activity, showing extremely tolerant characteristics. This characteristic is particularly important in the preparation of GOS, which can explain that the transglycosidation activity will not be affected by pH from beginning to end, which is conducive to the industrial production of syrup.

[0065] Example 6

[0066] This example is used for preliminary screening and qualitative analysis of GOS produced by lactase transglycosidation.

[0067] Thin layer chromatography (TLC) was used to visualize the transglycosylation products to evaluate the transglycosylation efficiency of lactase and preliminarily determine the optimal process conditions for preparing GOS. 15U of lactase enzyme solution BcGAL and NK-Lac were added to a 60% lactose solution, the pH was adjusted to the optimum 6.0, and the reaction was carried out at the optimum temperature of 60℃ for 10h to compare the differences between BcGAL and NK-Lac in the synthesis of GOS. After being taken out, it was immediately heat denatured at 100℃ for 10min to terminate the reaction. The final reaction solution was diluted appropriately, 1μL was spotted on a dry silica gel GF254 plate, and placed in a layer development tank for layer development, aiming 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℃ for 5-10 minutes to visually identify mono-oligosaccharides and oligosaccharides. Figure 5 As shown in A, from left to right are glucose, galactose and lactose standards, followed by a control group (without adding enzyme solution), two parallel groups of BcGAL synthesizing GOS, and two parallel groups of NK-Lac synthesizing GOS. It is known from the TLC results that the transglycosidic activity of the lactase NK-Lac of the present invention is higher than that of the lactase BcGAL, and the GOS yield is also higher, but the specific GOS composition needs to be determined by high performance liquid chromatography (HPLC). Compared with lactase BcGAL, the activity of the lactase NK-Lac provided by the present invention can be maintained longer in a dynamically changing pH environment, which may be one of the main reasons for its synthesis of more GOS, and therefore it is more suitable for industrial production of syrup.

[0068] In order to explore the optimal reaction time for preparing GOS with lactase NK-Lac, lactase NK-Lac was used to prepare GOS under the above-mentioned conditions, and samples were taken at 2h, 4h, 6h, 9h, 12h, 15h, 18h, 21h and 24h. The results were visualized according to the above method. Figure 5 As shown in Figure B, the development positions of glucose, galactose and lactose standards are all different. When the synthesis time is as long as 2-9h, a small amount of GOS is generated. In the range of 12-21h, the GOS content continues to increase. As the synthesis time increases, the GOS content does not continue to increase. This may be because as the concentration of lactose is degraded and continues to decrease, lactase can no longer be protected by high concentrations of lactose and gradually inactivates. In industrial production, it helps to shorten the preparation time and improve efficiency, thereby saving costs.

[0069] Example 7

[0070] This example is used to accurately and quantitatively analyze the composition and concentration of GOS.

[0071] The concentration of oligosaccharides with different degrees of polymerization in the reaction solution was determined by high performance liquid chromatography (HPLC) and the transglycosidation efficiency of NK-Lac was analyzed. All reactions were carried out in a conical flask at 55°C in a magnetic stirring water bath. The reaction system consisted of 40% lactose (80g lactose dissolved in 120g water, pH adjusted to 6.0) and 75U lactase enzyme solution NK-Lac. Samples were taken every 4h and heated at 100°C for 10min to inactivate the enzyme to terminate the reaction. In order to collect the supernatant in the reaction mixture, the sample was centrifuged at 10,000rpm for 10min and filtered through a 0.2μm sterile syringe filter. 2mL of the reaction mixture diluted with deionized water was taken for HPLC analysis. A control group (no enzyme solution was added) was set up for all experiments, and the experimental group was repeated to ensure the reliability of the results. The content of disaccharides and polysaccharides other than lactose was determined by HPLC, with a ShiNex HE NH25 μm column (4.6×250 mm) from SHIMADZU and an acetonitrile-water (70:30, vol / vol) buffer as the mobile phase. Individual sugars were identified by comparing their retention times with those of the standards, and quantified based on the peak areas calibrated with the sugar standards.

[0072] The results of the analysis showed that no GOS products were detected in any of the samples from the control group. Figure 6As shown, Sam1-6 are reaction solutions with synthesis times of 4h, 8h, 12h, 16h, 20h, and 24h, respectively. As the synthesis time increases, the content of glucose (peaking at around 6min) continues to increase, and the content of galactose (peaking at around 5.8min, next to the glucose peak) and lactose (peaking at around 8.5min) continues to decrease. Oligosaccharide mixtures may contain hundreds of individual compounds, and the differences are reflected in monomer type, arrangement order, bonding method, and chain length. The prebiotic activity and physiological effects of GOS are closely related to its glycosidic bond type, chain length, purity, and concentration. By comparing with standard substances, the specific composition of GOS can be clearly identified. For example, the peak at 9.5min corresponds to allolactose, 10.5min is galactotriose, and 15.5min is galactotetraose. Figure 6 It shows that the yield of GOS reaches the highest level and the types of polysaccharides are the most abundant between 12-20 hours of reaction. At first, it is easier to generate sugar chains with a degree of polymerization of 2 or 4, but after 8 hours, the proportion of short-chain sugars gradually increases, the content of tetrasaccharides decreases, and the content of allolactose continues to increase. This shows that with the extension of synthesis time, the length of sugar chains in the GOS mixture has changed significantly, and long-chain polysaccharides may be gradually broken down into short chains, and the number of isomers also increases. This shows that the entire GOS synthesis process has dynamic characteristics, and the syrup yield gradually increases. It is calculated that the highest yield of syrup is 70%.

[0073] 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 high stability lactase NK-Lac, characterized in that: The amino acid sequence of the lactase NK-Lac is shown in SEQ ID NO:

1.

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

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

2.

3. The method for obtaining lactase NK-lac according to claim 1, characterized in that: The method comprises the following steps: mutating the alanine at position 401 of the original lactase gene into tyrosine (A401Y), mutating the glutamic acid at position 480 into histidine (E480H), and mutating the threonine at position 594 into serine (T594S) by a site-directed mutagenesis method; the original lactase is the lactase BcGAL of Bacillus circulans.

4. A recombinant expression vector, characterized in that: Comprising the lactase gene nk-lac 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 nk-lac as claimed in claim 2 to obtain a lactase gene nk-lac 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 nk-lac 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-nk-lac.

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 galacto-oligosaccharides, comprising the following steps: The lactase NK-Lac according to claim 1 with an activity of 10-20 U is contacted with a 30%-60% lactose solution, and reacted for 12-21 hours under the conditions of pH 5-7 and 50-70° C. to obtain galacto-oligosaccharide.

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