A method for improving the ability of beta-galactosidase to produce oligogalactose

Abstract

The application discloses a method for improving the ability of beta-galactosidase to produce oligogalactose. In order to improve the yield of oligogalactose, the application provides a method for improving the ability of beta-galactosidase to produce oligogalactose, with wild-type beta-galactosidases BbgIII-d580, BIF3-d3 and LAC4 as parents. That is, the ability of the obtained beta-galactosidase mutant to produce oligogalactose is improved by single-point mutation or combined mutation of specific amino acid sites of the beta-galactosidase, and the yield of oligogalactose can be improved when the beta-galactosidase mutant is used to produce oligogalactose. In addition to improving the ability of beta-galactosidase to produce oligogalactose, the method also improves the enzyme activity of part of the mutants, achieving multiple effects at once. The application is beneficial to the industrial production of oligogalactose, and the obtained beta-galactosidase mutant can be used to prepare low-lactose or lactose-free dairy products containing oligogalactose.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology. More specifically, it relates to a method for improving the ability of β-galactosidase to produce galactooligosaccharides. Background Technology

[0002] Prebiotics, as important functional factors for regulating the balance of the gut microbiota, can effectively prevent and improve many chronic metabolic diseases. Galacto-oligosaccharides (GOS), a typical representative of prebiotics, have functions such as regulating gut microbiota balance, improving immunity, promoting mineral absorption, improving lactose intolerance, anti-aging, lowering blood pressure in hypertensive individuals, and improving lipid metabolism. They are now widely used in infant formula, functional foods, beverages, and baked goods.

[0003] β-galactosidase (EC.3.2.1.23), commonly known as lactase, is an important glycoside hydrolase with both hydrolytic and transglycosylation activities. Its hydrolytic activity breaks down lactose into galactose and glucose, allowing for the production of low-lactose or lactose-free dairy products, effectively addressing lactose intolerance. Its transglycosylation activity converts lactose into GOS. Low / lactose dairy products rich in GOS, as novel health foods, are not only beneficial to human health but also suitable for a wider range of people, including those with lactose intolerance. This plays a vital role in maintaining national nutrition and health.

[0004] Converting lactose in dairy products to GOS using β-galactosidase is an effective method for preparing low / lactose-free dairy products rich in GOS. However, most existing β-galactosidases have hydrolytic activity far exceeding their transglycosylation activity, resulting in low GOS yields. Furthermore, while some β-galactosidases can synthesize higher yields of GOS, the lactose conversion rate is low at this stage. Even when the lactose conversion rate reaches the low-lactose or lactose-free requirements, GOS undergoes secondary hydrolysis, leading to a still low final GOS yield. To further improve the GOS yield of β-galactosidase and achieve low-lactose or lactose-free requirements to meet the increasing dietary demands, optimization of β-galactosidase is necessary.

[0005] BbgIII-d580, BIF3-d3, and LAC4 are commonly used β-galactosidases. Optimizing them to enable their use in the preparation of low-lactose or lactose-free dairy products with high GOS content is of great significance to the development of the dairy industry. However, there are currently no clear reports on which amino acid sites or mutations in BbgIII-d580, BIF3-d3, and LAC4 can increase GOS yield. Summary of the Invention

[0006] This invention provides a method to improve the ability of three commonly used β-galactosidases, BbgIII-d580, BIF3-d3, and LAC4, to produce galactooligosaccharides.

[0007] The first objective of this invention is to provide a method for improving the ability of β-galactosidase to produce galactooligosaccharides.

[0008] A second objective of this invention is to provide a β-galactosidase mutant obtained by mutation using the method described above.

[0009] A third objective of this invention is to provide a gene encoding the β-galactosidase mutant.

[0010] A fourth objective of this invention is to provide a biomaterial capable of expressing the β-galactosidase mutant.

[0011] A fifth objective of this invention is to provide the application of the β-galactosidase mutant and the recombinant bacteria in the biological material in the production of galactooligosaccharides.

[0012] A sixth object of the present invention is to provide the use of the β-galactosidase mutant, the gene, and the biomaterial in the preparation of formulations for the production of galacto-oligosaccharides.

[0013] A seventh object of the present invention is to provide the application of the β-galactosidase mutant and the recombinant bacteria in the biological material in the production of dairy products containing galacto-oligosaccharides.

[0014] An eighth object of the present invention is to provide the use of the β-galactosidase mutant, the gene, and the biomaterial in the preparation of formulations for the production of dairy products containing galacto-oligosaccharides.

[0015] The ninth object of the present invention is to provide a method for increasing the yield of galactooligosaccharides.

[0016] The above-mentioned objective of this invention is achieved through the following technical solution:

[0017] This invention investigated wild-type β-galactosidases BbgIII-d580, BIF3-d3, and LAC4 as parents and found that single-point or combined mutations at specific amino acid sites of these β-galactosidases resulted in mutants with enhanced oligogalactose production compared to the parents. Therefore, this invention provides a method for improving the oligogalactose production capacity of β-galactosidases.

[0018] Specifically, the β-galactosidase is BbgIII-d580, BIF3-d3, or LAC4; the amino acid sequence of the β-galactosidase BbgIII-d580 is shown in SEQ ID NO.1; the amino acid sequence of the β-galactosidase BIF3-d3 is shown in SEQ ID NO.2; and the GenBank number of the β-galactosidase LAC4 is AAA35265.1.

[0019] Specifically, the method involves: mutating one or more amino acids at positions 592, 602, and 623 of the amino acid sequence of β-galactosidase BbgIII-d580 or BIF3-d3; or mutating amino acids at positions 537 or 597 of the amino acid sequence of β-galactosidase BIF3-d3; or mutating amino acids at positions 582 or 620 of the amino acid sequence of β-galactosidase LAC4.

[0020] More specifically, the method involves: mutating tryptophan at position 592 of β-galactosidase BbgIII-d580 to phenylalanine or tyrosine; or mutating threonine at position 602 to serine or alanine; or mutating phenylalanine at position 623 to tryptophan; or mutating tryptophan at position 592 to phenylalanine and threonine at position 602 to valine, serine, alanine, or isoleucine.

[0021] Alternatively, the 537th serine of β-galactosidase BIF3-d3 may be mutated to alanine; or the 592nd tryptophan may be mutated to tyrosine or phenylalanine; or the 597th tyrosine may be mutated to phenylalanine; or the 602nd threonine may be mutated to serine; or the 623rd phenylalanine may be mutated to tyrosine or tryptophan; or the 592nd tryptophan may be mutated to tyrosine or phenylalanine, and the 602nd threonine may be mutated to serine; or the 597th tyrosine may be mutated to phenylalanine, and the 602nd threonine may be mutated to serine.

[0022] Alternatively, tryptophan at position 582 of β-galactosidase LAC4 may be mutated to phenylalanine; or phenylalanine at position 620 may be mutated to tyrosine or tryptophan.

[0023] The present invention also provides a β-galactosidase mutant with enhanced ability to produce galactooligosaccharides, wherein the mutant is obtained by mutation using the method described in the present invention.

[0024] The present invention also provides a gene encoding the β-galactosidase mutant. Using the gene, the corresponding mutant can be obtained through recombination expression.

[0025] The present invention also provides a biomaterial capable of expressing the β-galactosidase mutant.

[0026] Specifically, the biological material is a recombinant vector or recombinant bacteria containing a gene encoding the β-galactosidase mutant.

[0027] Optionally, the recombinant vector is an expression vector.

[0028] Optionally, when constructing the recombinant bacteria, Escherichia coli, yeast, Bacillus, or Lactobacillus can be selected as the host bacteria.

[0029] The present invention also seeks protection for the β-galactosidase mutant and the use of the recombinant bacteria in the production of galactooligosaccharides.

[0030] The present invention also claims protection for the use of the β-galactosidase mutant, the gene, and the biological material in the preparation of formulations for the production of galacto-oligosaccharides.

[0031] The present invention also claims protection for the use of the β-galactosidase mutant and the recombinant bacteria in the production of dairy products containing galacto-oligosaccharides.

[0032] The present invention also claims protection for the use of the β-galactosidase mutant, the gene, and the biological material in the preparation of formulations for the production of dairy products containing galacto-oligosaccharides.

[0033] Specifically, the dairy product is a low-lactose or lactose-free dairy product.

[0034] In addition to the applications described above, the application of the β-galactosidase mutant and the recombinant bacteria described in this invention in the degradation of lactose or in the production of low-lactose or lactose-free dairy products should also be within the scope of protection of this invention.

[0035] Similarly, the β-galactosidase mutant, the gene, and the biological material described in this invention should also be within the scope of protection of this invention in the preparation of formulations for the degradation of lactose or for the production of low-lactose or lactose-free dairy products.

[0036] The present invention has the following beneficial effects:

[0037] This invention aims to improve the yield of galactooligosaccharides (GOS) produced using β-galactosidases. Using wild-type β-galactosidases BbgIII-d580, BIF3-d3, and LAC4 as parents, a method is provided to enhance the GOS production capacity of β-galactosidases. Specifically, by performing single-point or combined mutations on specific amino acid sites of the β-galactosidases, the resulting β-galactosidase mutants exhibit enhanced GOS production capacity, thus increasing GOS yield. In addition to improving the GOS production capacity of β-galactosidases, the method also enhances the enzyme activity of some mutants, achieving multiple benefits. This invention is beneficial for the industrial production of GOS, and the obtained β-galactosidase mutants can be used to prepare low-lactose or lactose-free dairy products containing GOS. Attached Figure Description

[0038] Figure 1 Thin-layer chromatography (TLC) results of GOS, glucose, lactose, and galactose in different β-galactosidases and their mutants after reacting with milk as a substrate for different durations.

[0039] Figure 2 The GOS yield and residual lactose in the reaction system were quantified by anion exchange chromatography after different β-galactosidases and their mutants reacted with milk as a substrate for different time periods. In the figure, A represents the GOS yield and residual lactose in milk of BbgIII-d580 and its mutant W592F; B represents the GOS yield and residual lactose in milk of BIF3-d3 and its mutant W592F; and C represents the GOS yield and residual lactose in milk of LAC4 and its mutant F620W. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0041] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0042] The detection methods involved in the embodiments of the present invention are as follows:

[0043] β-galactosidase activity assay: Mix 50 μL of enzyme with 450 μL of lactose solution (5%, w / v), incubate at 40℃ for 15 min, then inactivate at 90℃ for 10 min; after shaking and mixing, add 10 μL of the reaction solution to 750 μL of glucose oxidation kit reaction solution, and react at 37℃ for 15 min; after the reaction, take 300 μL of the reaction solution and measure the absorbance at 492 nm wavelength; each reaction is performed in triplicate.

[0044] Construction of the glucose standard curve: Prepare a glucose stock solution (0.1M, pH 7.0) with a concentration of 2.5 g / L using phosphate buffer. Dilute the glucose stock solution to glucose solutions with concentrations of 0.025%, 0.05%, 0.075%, 0.100%, 0.125%, 0.150%, 0.175%, and 0.200% (w / v). Take 10 μL of each diluted glucose solution and add it to 750 μL of glucose kit test solution. Mix well and incubate at 37°C for 15 min. Then take 300 μL and measure the absorbance at a wavelength of 492 nm. Plot the standard curve A = a × C + b with glucose concentration C as the x-axis and absorbance A as the y-axis.

[0045] Enzyme activity unit (U) is defined as the amount of enzyme required to break down lactose into 1 μmol of glucose per minute under the given reaction conditions;

[0046]

[0047] C: Glucose concentration calculated based on the glucose standard curve;

[0048] 20: Convert the enzyme activity in 50 μL of enzyme solution to the enzyme activity in 1 mL of enzyme solution;

[0049] 50: Convert the glucose content in 10 μL of the mixture to the glucose content in 500 μL of the mixture;

[0050] 15: Reaction time 15 min;

[0051] 1000: Converts 1 nmol to 1 μmol.

[0052] Thin-layer chromatography (TLC) was used to determine the sugar content in the solution. First, the TLC plate was activated in an 80℃ oven for 20 min, and 1 μL of sample was applied every 1 cm. The plate was then dried at 37℃ for 3 min, and then placed in a chromatography bar containing 50 mL of chromatography solution. The plate was dried once every 1 h for a total of 4 chromatography cycles. Finally, the colorimetric solution was sprayed evenly onto the TLC plate using a spray bottle, and the plate was placed in an 80℃ oven for 6 min for color development.

[0053] Determination of GOS yield: The reaction was carried out in a 40% lactose solution with an enzyme concentration of 3 U / mL (if the enzyme activity of the crude enzyme solution is too low, even if it is concentrated 10 times, it still cannot reach 3 U / mL, so the enzyme concentration is directly 10 times). The reaction was carried out at 40℃ and 200 rpm for 24 h. Samples were taken every 2 h and placed in a 98℃ water bath for 10 min to inactivate the enzyme. After centrifugation, the total sugar concentration was diluted to 5% with phosphate buffer (pH 7.0) and stored at -20℃. The reaction was also carried out in milk with an enzyme concentration of 3 U / mL (if the enzyme activity is low, the enzyme concentration is 1 U / mL). The reaction was carried out at 40℃ and 200 rpm for 6 h. Samples were taken at appropriate time intervals, and the samples were placed in a 98℃ water bath for 10 min to inactivate the enzyme. After centrifugation, the samples were stored at -20℃.

[0054] After the reaction, the GOS content in the solution was quantified by anion exchange chromatography. The instrument used for quantification was a high-performance ion chromatograph equipped with a pulsed amperometric detector. The chromatographic column was a PA20 anion exchange column (150 mm × 3 mm, 3.5 μm particle size), the guard column was (30 mm × 3 mm), the column temperature was 30 °C, the mobile phase flow rate was 0.4 mL / min, the injection volume was 20 μL, the detector was a pulsed amperometric detector, the working electrode was gold, and the reference electrode was Ag / AgCl. The elution gradient of the mobile phase is shown in the table below.

[0055]

[0056] The detector potential waveform program is shown in the table below:

[0057] Time / s Potential / V integral 0.00 0.1 - 0.20 0.1 start 0.40 0.1 Finish 0.41 -2.0 - 0.42 -2.0 - 0.43 0.6 - 0.44 -0.1 -

[0058] Plot standard working curves for galactose, lactose, and glucose. Measure the mixed standard solutions of galactose, lactose, and glucose separately to prepare a series of mixed standard solutions, as shown in the table below. After determination under the above chromatographic conditions, plot the standard working curves with the concentration of each component as the abscissa and the peak area as the ordinate.

[0059] Galactose (μg / mL) Lactose (μg / mL) Glucose (μg / mL) 0.50 0.475 0.50 1.00 0.95 1.00 2.00 1.90 2.00 5.00 4.95 5.00 10.00 9.50 10.00

[0060] Add 50 μL of 20% acetonitrile solution to 1 mL of sample solution, centrifuge at 10000 r / min for 10 min, filter the upper aqueous phase through a 0.22 μm filter membrane, and after determination under the above chromatographic conditions, input the peak areas of each component into the standard curve of each component to quantify galactose, glucose and lactose respectively.

[0061] The formula for calculating GOS yield is: GOS yield (%) = (initial amount of lactose before reaction - galactose after reaction - glucose after reaction - lactose after reaction) / initial amount of lactose before reaction.

[0062] The formula for calculating the GOS content in milk is: GOS content (g / L) = GOS yield (%) × 50 (g / L).

[0063] The formula for calculating the lactose conversion rate in milk is: Lactose conversion rate (%) = (Initial amount of lactose before reaction - Amount of lactose after reaction) / Initial amount of lactose before reaction.

[0064] The formula for calculating the lactose content in milk is: Lactose content (g / L) = Lactose content after reaction / Initial lactose content before reaction × 50 (g / L).

[0065] Example 1: Site-directed saturation mutagenesis of β-galactosidase

[0066] To increase the yield of galactooligosaccharides, this invention uses wild-type β-galactosidases BbgIII-d580, BIF3-d3, and LAC4 as parents, and obtains mutants with improved galactooligosaccharide production capacity relative to the parents by saturating mutations at specific amino acid sites.

[0067] The amino acid sequence of the β-galactosidase BbgIII-d580 is shown in SEQ ID NO.1, and saturation mutations are performed on amino acids at positions 592, 602 and / or 623.

[0068] The amino acid sequence of the β-galactosidase BIF3-d3 is shown in SEQ ID NO.2, and saturation mutations are performed on amino acids at positions 537, 592, 597, 602 and / or 623.

[0069] The β-galactosidase LAC4 has the GenBank number AAA35265.1, and its 582nd and / or 620th amino acids were subjected to saturation mutations.

[0070] 1. Construction of site-directed saturation mutant libraries

[0071] Suzhou Genewise Biotechnology Co., Ltd. was commissioned to optimize and synthesize the genes encoding β-galactosidase BbgIII-d580, BIF3-d3, and LAC4, respectively. Recombinant plasmids pET-28a-Tac-BbgIII-d580, pET-28a-Tac-BIF3-d3, and pET-28a-Tac-LAC4 containing gene sequences encoding β-galactosidase BbgIII-d580, BIF3-d3, and LAC4 were constructed. Single-point and combined saturation mutagenesis of the above amino acid sites were performed using the constructed recombinant plasmids as templates. The PCR primers used for saturation mutagenesis are shown in Table 1, and the PCR reaction system used is shown in Table 2.

[0072] Table 1. PCR primers used for saturation mutagenesis.

[0073]

[0074]

[0075] Note: SDM represents the codons corresponding to 20 amino acids: alanine (GCG), cysteine ​​(TGC), aspartic acid (GAC), glutamic acid (GAA), phenylalanine (TTC), glycine (GGT), isoleucine (ATC), lysine (AAA), leucine (CTG), methionine (ATG), asparagine (AAC), proline (CCG), glutamine (CAG), arginine (CGT), serine (TCT), threonine (ACC), valine (GTG), tryptophan (TGG), tyrosine (TAC), and histidine (CAC).

[0076] Table 2. PCR reaction system used for saturation mutagenesis.

[0077]

[0078]

[0079] PCR reaction conditions used for saturation mutagenesis: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 10 s, 58℃ annealing for 10 s, 72℃ extension for 1 min 30 s (the extension rate of the enzyme used in this invention is 10 s / Kb), for a total of 30 cycles, and finally 72℃ for another 3 min extension.

[0080] PCR products were detected by 1% agarose gel electrophoresis. After confirming the amplified fragment size was correct, the gel was purified and recovered using the FastPure Gel DNA Extraction mini kit from Nanjing Novizan Biotechnology Co., Ltd. Homologous recombination of the PCR amplified products was performed using the OK Clon DNA Ligation Kit from Guangzhou Aiji Biotechnology Co., Ltd. The homologous recombination reaction system is shown in Table 3, and the reaction was carried out at 50℃ for 10 min.

[0081] Table 3 Homologous recombination reaction system

[0082]

[0083] After ligation, 5 μL of the ligation product was mixed with 100 μL of E. coli DH5α competent cells, and then heat-shocked at 42℃ for 45 s. After placing on ice for 2 min, 900 μL of LB medium was added, and the transformant was cultured at 37℃ and 200 rpm for 1 h. The culture was then spread on LB solid medium supplemented with 20 μM IPTG and 50 μg / mL kana and cultured at 30℃ for 36 h to obtain the site-directed saturation mutant library.

[0084] 2. Preparation of crude enzyme solution of mutant

[0085] Single colonies were picked and cultured overnight at 37°C and 220 rpm in LB liquid medium containing 50 μg / mL kana. 3% of the bacterial culture was transferred to 20 mL of LB medium and cultured at 37°C and 200 rpm until the bacterial OD600 reached 0.8–1.0. 0.2 mL of IPTG (final concentration 1 mM) was added, and the culture was continued at 25°C and 200 rpm for 14 h. The bacterial cells were collected by centrifugation (5000 g, 10 min, 4°C). An equal volume of 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.0) was added to resuspend the bacterial cells. The cells were then disrupted using an ultrasonic cell disruptor and centrifuged at 14000 rpm for 1 min. The supernatant was collected to obtain the crude enzyme solution of the mutant.

[0086] Example 2: Determination of GOS yield, enzyme activity, and relative activity of β-galactosidase mutant

[0087] This invention uses crude enzyme solutions of β-galactosidase BbgIII-d580, BIF3-d3, and LAC4 parents as controls. By comparing the GOS yield of the mutant crude enzyme solution with that of its corresponding parent crude enzyme solution, mutants with enhanced oligogalactoside production capacity relative to the parents were obtained. The selected mutants were sequenced to determine their sequences, and their crude enzyme solutions were used to determine enzyme activity. Relative enzyme activities were calculated with the activities of the β-galactosidase BbgIII-d580, BIF3-d3, and LAC4 parents as 100%.

[0088] The mutants with enhanced galactooligosaccharide production capacity obtained by using β-galactosidase BbgIII-d580 as the parent in this invention, and their GOS yield, enzyme activity and relative activity are shown in Table 4.

[0089] Table 4. GOS yield, enzyme activity, and relative activity of BbgIII-d580 and its mutants

[0090]

[0091] Note: Taking F623W as an example, F623W is a single-point mutation mutant of BbgIII-d580, with the corresponding mutation being that F (phenylalanine) at position 623 of the BbgIII-d580 amino acid sequence is mutated to W (tryptophan); W592F / T602V is a combined mutation mutant of BbgIII-d580, with the corresponding mutation being that W (tryptophan) at position 592 of the BbgIII-d580 amino acid sequence is mutated to F (phenylalanine), and T (threonine) at position 602 is mutated to V (valine).

[0092] The mutants with enhanced oligogalactosidase production capacity obtained by using β-galactosidase BIF3-d3 as the parent in this invention, and their GOS yield, enzyme activity, and relative activity are shown in Table 5.

[0093] Table 5. GOS yield, enzyme activity, and relative activity of BIF3-d3 and its mutants.

[0094]

[0095]

[0096] Compared with the parent, mutants W592F and W592F / T602S showed not only increased GOS production but also significantly improved enzyme activity.

[0097] The mutants with enhanced galactooligosaccharide production capacity obtained by the present invention using β-galactosidase LAC4 as the parent, and their GOS yield, enzyme activity and relative activity are shown in Table 6.

[0098] Table 6. GOS yield, enzyme activity, and relative activity of LAC4 and its mutants.

[0099]

[0100] Example 3: Determination of GOS yield in milk

[0101] Wild-type β-galactosidase BbgIII-d580 and its mutant W592F, wild-type β-galactosidase BIF3-d3 and its mutant W592F, and wild-type β-galactosidase LAC4 and its mutant F620W were recombinantly expressed to obtain the corresponding purified enzymes. Using the purified enzymes of each parent and each mutant as test enzymes and commercially available skim milk as substrate, their ability to synthesize GOS in dairy products was tested.

[0102] Take equal volumes of skim milk, add purified enzyme to a final concentration of 3 U / mL, mix well, and react at 40℃. Take samples at intervals during the reaction. Analyze the changes in sugar content (GOS, glucose, lactose, and galactose) in the system using thin-layer chromatography (TLC). The results are as follows: Figure 1 As shown in the figure; BbgIII WT refers to wild-type BbgIII-d580, and BIF3WT refers to wild-type BIF3-d3.

[0103] Depend on Figure 1 It was found that the mutants BbgIII-d580W592F, BIF3-d3W592F, and LAC4F620W all showed significantly increased GOS production compared to their corresponding wild-type enzymes. Based on this, this invention further quantified the GOS production and residual lactose in milk using anion exchange chromatography and calculated the lactose conversion rate.

[0104] The GOS yield and residual lactose in the reaction system were quantitatively obtained by anion exchange chromatography after reacting different β-galactosidases and their mutants with milk as a substrate for different time periods. Figure 2 As shown; Figure 2 In the table, A through C represent the GOS yield and residual lactose in milk for BbgIII-d580 and its mutant W592F, BIF3-d3 and its mutant W592F, and LAC4 and its mutant F620W, respectively. Figure 2 The results show that BbgIII-d580WT and W592F reached their highest GOS contents at 30 min and 1 h of reaction, respectively, with lactose conversion rates of 69.8% and 76.5%. BIF3-d3WT and W592F both reached their highest GOS contents at 30 min of reaction, with contents of 21.7 g / L and 22.5 g / L, respectively, with lactose conversion rates of 86.5% and 87.1%. LAC4WT and F620W reached their highest GOS contents at 3 h and 6 h of reaction, respectively, with contents of 13.4 g / L and 16.3 g / L, respectively, with lactose conversion rates of 90.2% and 89.5%. These results indicate that all the above mutants can synthesize more GOS in milk.

[0105] This invention further analyzed the GOS content in milk when each parent and mutant converted approximately 90% of lactose in dairy products, and the results are shown in Table 7. As shown in Table 7, compared with the wild-type parent, each single mutant can synthesize more GOS while consuming 90% of lactose.

[0106] Table 7. GOS yield and lactose conversion rate of wild-type and mutant β-galactosidase.

[0107]

[0108] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for improving the ability of β-galactosidase to produce galactooligosaccharides, characterized in that, The β-galactosidase is BbgIII-d580, BIF3-d3, or LAC4; the amino acid sequence of the β-galactosidase BbgIII-d580 is shown in SEQ ID NO.1; the amino acid sequence of the β-galactosidase BIF3-d3 is shown in SEQ ID NO.2; the GenBank number of the β-galactosidase LAC4 is AAA35265.

1. The method involves: mutating one or more amino acids at positions 592, 602, and 623 of the amino acid sequence of β-galactosidase BbgIII-d580 or BIF3-d3; or mutating amino acids at positions 537 or 597 of the amino acid sequence of β-galactosidase BIF3-d3; or mutating amino acids at positions 582 or 620 of the amino acid sequence of β-galactosidase LAC4.

2. The method according to claim 1, characterized in that, The method is as follows: mutate tryptophan at position 592 of β-galactosidase BbgIII-d580 to phenylalanine or tyrosine, or mutate threonine at position 602 to serine or alanine, or mutate phenylalanine at position 623 to tryptophan, or mutate tryptophan at position 592 to phenylalanine, or mutate threonine at position 602 to any one of valine, serine, alanine or isoleucine; Alternatively, the 592nd tryptophan in β-galactosidase BIF3-d3 may be mutated to tyrosine or phenylalanine; or the 597th tyrosine may be mutated to phenylalanine; or the 602nd threonine may be mutated to serine; or the 623rd phenylalanine may be mutated to tyrosine or tryptophan; or the 592nd tryptophan may be mutated to tyrosine or phenylalanine and the 602nd threonine may be mutated to serine; or the 597th tyrosine may be mutated to phenylalanine and the 602nd threonine may be mutated to serine. Alternatively, tryptophan at position 582 of β-galactosidase LAC4 may be mutated to phenylalanine; or phenylalanine at position 620 may be mutated to tyrosine or tryptophan.

3. The β-galactosidase mutant obtained by the method of claim 1 or 2.

4. The gene encoding the β-galactosidase mutant of claim 3.

5. A biomaterial capable of expressing the β-galactosidase mutant of claim 3, characterized in that, The biological material is a recombinant vector or recombinant bacteria containing the gene described in claim 4.

6. The use of the β-galactosidase mutant of claim 3 and the recombinant bacteria of claim 5 in the production of galactooligosaccharides.

7. The use of the β-galactosidase mutant of claim 3, the gene of claim 4, and the biomaterial of claim 5 in the preparation of formulations for the production of galacto-oligosaccharides.

8. The application of the β-galactosidase mutant of claim 3 and the recombinant bacteria of claim 5 in the production of dairy products containing galacto-oligosaccharides, characterized in that, The dairy product is a low-lactose or lactose-free dairy product containing galactooligosaccharides.

9. The use of the β-galactosidase mutant of claim 3, the gene of claim 4, and the biomaterial of claim 5 in the preparation of formulations for producing dairy products containing galacto-oligosaccharides, characterized in that, The dairy product is a low-lactose or lactose-free dairy product containing galactooligosaccharides.

10. A method for increasing the yield of galactooligosaccharides, characterized in that, Galacto-oligosaccharides can be produced using the β-galactosidase mutant of claim 3 or the recombinant bacteria of claim 5.

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