High efficient expression of aspergillus oryzae beta-galactosidase mutants and their application in dairy products

Abstract

The application discloses efficient expression of Aspergillus oryzae beta-galactoside enzyme mutant and application thereof in dairy products, and belongs to the technical fields of enzyme engineering and dairy product processing, and particularly relates to efficient expression of Aspergillus oryzae beta-galactoside enzyme mutant and application thereof in dairy products.The mutant protein can be as follows: A1) a protein with an amino acid sequence of SEQ ID No.2; A2) a fusion protein with the same function obtained by connecting a label to the N terminal and / or C terminal of A1).The mutant protein is subjected to high-density fermentation in a 5L fermentation tank, and the enzyme activity of the fermentation liquor can reach 4628U / mL, and the recovery rate of the mutant enzyme is increased by 1.7 times.Compared with the wild type, the optimal pH of the mutant is increased from 4.5 to 5.5, the optimal temperature is decreased from 60 DEG C to 50 DEG C, the lactose hydrolysis efficiency is increased by 11%, and the mutant is more suitable for the production process of low / zero lactose dairy products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of enzyme engineering and the technical field of dairy processing, and specifically relates to efficient expression of Aspergillus oryzae beta-galactosidase mutants and application thereof in dairy products. BACKGROUND

[0002] Lactose intolerance is a condition in which the body does not produce enough of the enzyme beta-galactosidase (EC 3.2.1.23) to digest lactose, which can cause symptoms such as bloating, nausea, abdominal cramps, and diarrhea. A low / zero lactose diet is an important way to solve lactose intolerance, and beta-galactosidase is an enzyme used in the industrial production of zero-lactose food, which is widely used in the dairy industry. The types of zero-lactose dairy products on the market are single, so the development of new zero-lactose dairy products is of great significance to enrich the market and meet the health needs of the public.

[0003] Beta-galactosidase hydrolyzes lactose to produce monosaccharides with higher sweetness, which can partially replace the added sucrose in dairy products, and has the potential to produce low / no sucrose dairy products. Studies have shown that the sweetness of 70% of lactose in milk (about 45 g / L lactose) is equivalent to the addition of 2% sucrose. Existing sucrose-free dairy products have a sour taste and additional sweeteners. Su Benxian et al. (Chinese Patent No. ZL201910854000.0) prepared sucrose-free probiotic yogurt by adding erythritol, xylitol, maltitol, stevioside or mogroside.

[0004] Commercial beta-galactosidase is mainly derived from fungi such as Kluyveromyces and Aspergillus. Aspergillus oryzae is the most important fungal source of beta-galactosidase, but the substrate inhibition and acidophilic properties of beta-galactosidase from this source hinder its application in dairy processing. Improving the hydrolysis efficiency and enzymatic properties of beta-galactosidase through molecular modification is an effective method to enhance the application adaptability of beta-galactosidase. Segmentuoguo et al. (Chinese Patent No. ZL202210560601.2) reported that the soluble expression level and thermal stability of the beta-galactosidase mutant were significantly improved compared to wild-type beta-galactosidase. Yu Xiaowei et al. (Chinese Patent No. ZL202210151251.4) obtained a beta-galactosidase mutant with a lactose hydrolysis efficiency of 60%, which was improved from 42% of the wild type.

[0005] Purification of β-galactosidase is usually performed by affinity chromatography, but this method has the problems of high cost and reduced activity of target protein due to multiple washes, which limits its application in industrial production. Inserting a protein tag with highly predictable functional properties into the recombinant plasmid to modify the target protein can improve the relative enzyme activity and recovery rate of the mutant, and simplify the purification process. Addai et al. linked the elastin-like peptide gene sequence to the β-galactosidase gene sequence from Lactobacillus sp. B164, constructed a recombinant plasmid and expressed it in E. coli, and the recovery rate of the fusion protein was 2.1 times that of the traditional method.

[0006] β-galactosidase is widely used in milk, and low / zero lactose milk is usually produced by adding β-galactosidase after ultra-high temperature treatment. Hu Zhi and (Chinese Patent No: ZL202011048475.X) used lactase to hydrolyze lactose under ultra-high pressure to prepare low-lactose milk. In fermented dairy products such as yogurt, the ideal β-galactosidase is to complete the hydrolysis of lactose during fermentation. However, the fermentation process is accompanied by a decrease in the pH of the system, so it is required that the β-galactosidase remains stable in the pH range of 4.5-6.5. In addition, lactose is an important carbon source in the fermentation process of lactic acid bacteria, and the addition of β-galactosidase may affect the growth and metabolism of lactic acid bacteria. Studies have shown that the addition of β-galactosidase can affect the fermentation characteristics and fermentation flavor of lactic acid bacteria, prolong the fermentation time of yogurt, or change the texture properties of yogurt. Too fast hydrolysis rate of β-galactosidase can lead to rapid changes in the carbohydrate composition in yogurt, which in turn affects the growth and metabolism of the fermentation agent. However, too slow hydrolysis rate can result in lactose content not reaching the zero-lactose standard before the end of fermentation. Therefore, in order to meet the production needs of "zero-lactose" dairy products, it is of great significance to optimize the hydrolysis efficiency and other enzymatic properties of β-galactosidase through molecular modification and to achieve high expression of β-galactosidase for its application in the dairy industry. SUMMARY

[0007] The technical problem to be solved by the present application is how to optimize the hydrolysis efficiency of β-galactosidase and improve the high expression of microbial β-galactosidase to produce new zero-lactose and sucrose-free dairy products.

[0008] To solve the above technical problems, the present application provides a mutant of Aspergillus oryzae β-galactosidase.

[0009] The mutant of Aspergillus oryzae β-galactosidase provided by the present application is a mutant protein obtained by mutating the threonine Thr at position 955 of the β-galactosidase AoBgal35A to arginine Arg, wherein the amino acid sequence of the β-galactosidase is shown in SEQ ID No. 1.

[0010] The mutant protein is T955R-AoBgal35A.

[0011] In the above, the mutant protein can be any one of the following:

[0012] A1) a protein with an amino acid sequence of SEQ ID No. 2;

[0013] A2) a fusion protein with the same function obtained by connecting a tag at the N-terminus and / or C-terminus of A1).

[0014] The above protein can be artificially synthesized, or a gene encoding the same can be synthesized first and then expressed biologically.

[0015] In the above protein, the protein tag refers to a polypeptide or protein fused and expressed with the target protein by DNA in vitro recombination technology, so as to facilitate the expression, detection, tracking and / or purification of the target protein. The protein tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0016] In the above, the fusion protein with the same function obtained by connecting a tag can be a protein with an amino acid sequence of SEQ ID No. 3.

[0017] A person skilled in the art can easily mutate the nucleotide sequence encoding the protein of the present application by using known methods, such as directed evolution or point mutation.

[0018] The above protein is derived from Aspergillus oryzae.

[0019] The present application also provides a biological material related to the above protein, which can be any one of the following:

[0020] B1) a nucleic acid molecule encoding the above protein;

[0021] B2) an expression cassette containing the nucleic acid molecule of B1);

[0022] B3) a recombinant vector containing the nucleic acid molecule of B1);

[0023] B4) a recombinant vector containing the expression cassette of B2);

[0024] B5) a recombinant microorganism containing the nucleic acid molecule of B1);

[0025] B6) a recombinant microorganism containing the expression cassette of B2);

[0026] B7) a recombinant microorganism comprising the recombinant vector of B3);

[0027] B8) a recombinant microorganism comprising the recombinant vector of B4).

[0028] In the above biomaterials, the nucleic acid molecule of B1) can be any one of the following DNA molecules:

[0029] d1) a DNA molecule with the nucleotide sequence shown in SEQ ID No. 4;

[0030] d1) a DNA molecule with the nucleotide sequence shown in SEQ ID No. 5;

[0031] d3) a DNA molecule hybridizing to the nucleotide sequence defined in d1) or d2) under stringent conditions and encoding the above protein.

[0032] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0033] The vector described herein is well known to those skilled in the art, including but not limited to: plasmid, bacteriophage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e. cos plasmid), Ti plasmid or viral vector. Specifically, the vector can be pPIC9K-T955R-AoBgal35A and pPIC9K-ELP-T955R-AoBgal35A.

[0034] In the above, the recombinant microorganism is a recombinant yeast.

[0035] In the above, the recombinant yeast is a recombinant Pichia pastoris.

[0036] In the above, the recombinant Pichia pastoris is constructed according to the following method.

[0037] The present application also provides a method for constructing a recombinant Pichia pastoris, which comprises introducing the gene encoding any one of the above proteins into the recipient Pichia pastoris.

[0038] The above method further comprises connecting a VPGVG50 tag to the N-terminus and / or C-terminus of any one of the above proteins to obtain a fusion protein having the same function as any one of the above proteins.

[0039] In the present application, the introduction can be the transformation of a vector carrying the DNA molecule of the present application into a host cell by any known transformation method such as chemical transformation or electroporation. The introduced DNA molecule can be single copy or multiple copies. The introduction can be the integration of the foreign gene into the host chromosome or the expression of the plasmid outside the chromosome.

[0040] The present application also provides a method for preparing the Aspergillus oryzae β-galactosidase mutant, which comprises introducing the mutant-encoding gene of any of the above into a recipient microorganism to obtain a recombinant microorganism expressing any of the above mutants, culturing the recombinant microorganism, and obtaining the Aspergillus oryzae β-galactosidase mutant from the fermentation product.

[0041] In a specific embodiment, the method for preparing the Aspergillus oryzae β-galactosidase mutant comprises culturing the above recombinant Pichia pastoris to obtain a fermentation product, and obtaining the Aspergillus oryzae β-galactosidase mutant from the fermentation product.

[0042] The recombinant microorganism of the present application can be cultured in a 5L fermenter at high density, and the enzyme activity of the fermentation broth can reach 4628U / mL, which is higher than the expression level of β-galactosidase from most other sources in Pichia pastoris, such as β-galactosidase from Sulfolobus solfataricus (204.9U / mL), Aspergillus candidus (3600U / mL), Arthrobacter sp. (1926U / mL), and Aspergillus awamori (347.8U / mL).

[0043] The present application also provides the above method or the above recombinant Pichia pastoris for any of the following applications:

[0044] A) for preparing and producing low / no lactose dairy products;

[0045] B) for preparing and producing low / no sucrose dairy products;

[0046] C) for use in whey processing.

[0047] In the above, the dairy products include liquid milk (pasteurized milk, sterilized milk, fermented milk, and lactic acid bacteria beverage); milk powder (full-fat milk powder, skim milk powder, and modified milk powder).

[0048] In the above, the low / no sucrose dairy products refer to the use of the mutant protein to hydrolyze the natural or added lactose in the above dairy products to obtain more free monosaccharides to increase sweetness and replace sucrose.

[0049] The application also provides a preparation method of zero-lactose and sucrose-free yogurt, which comprises adding whey powder into raw milk, inoculating yogurt starter, and adding the mutant protein in any of the above to obtain zero-lactose and sucrose-free yogurt.

[0050] The yogurt starter is Lactobacillus bulgaricus and Streptococcus thermophilus (Yo-mix, purchased from Danisco, item number 3625226).

[0051] In the above method, the basic raw material for preparing the yogurt is 869-969 parts of raw cow milk, 1-120 parts of whey powder, 1-10 parts of lactase, and 0.01-1 parts of starter.

[0052] In the above method, the enzyme amount of the mutant protein is 1-20 U / mL.

[0053] In the above method, the whey powder is derived from dairy product processing by-products, and the lactose content is not less than 65%.

[0054] In the application, the threonine Thr at position 955 of the Aspergillus oryzae-derived beta-galactosidase AoBgal35A is mutated into arginine Arg to construct the mutant T955R. The mutant is subjected to high-density fermentation in a 5L fermenter, and the enzyme activity of the fermentation liquor reaches 4628 U / mL. The recombinant plasmid is inserted with 50 elastin-like polypeptide (VPGVG) tags, and the mutant enzyme recovery rate is increased by 1.7 times by using the reversible phase change characteristics thereof.

[0055] Compared with the wild type, the optimal pH of the mutant is increased from 4.5 to 5.5, the optimal temperature is decreased from 60℃ to 50℃, the lactose hydrolysis efficiency is increased by 11%, and the mutant is more suitable for the production process of low / zero-lactose dairy products. The addition of whey powder increases the lactose content in the system, and more free monosaccharides are hydrolyzed by the mutant enzyme to increase the sweetness, replace sucrose, and prepare low / sucrose-free dairy products with moderate sweetness and without the addition of sweeteners.

[0056] The addition of the mutant of the application can shorten the fermentation time of zero-lactose and sucrose-free yogurt, increase the viable cell count of yogurt, and obtain high-quality yogurt. The dairy products prepared by the mutant enzyme can meet the health needs of consumers for low-lactose and low-sucrose dairy products, and have the advantages of moderate sweetness, mellow taste, and stable quality during the shelf life. Therefore, the Aspergillus oryzae beta-galactosidase mutant of the application has a good application prospect in the dairy industry. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 The enzyme activity (·) and the wet weight (▲) of the mutant ELP-T955R-AoBgal35A produced by high-density fermentation of Pichia pastoris are shown.

[0058] Figure 2Optimum pH (a), pH stability (b), optimum temperature (c) and temperature stability (d) of wild type AoBgal35A (□) and mutant ELP-T955R-AoBgal35A (·).

[0059] Figure 3 High performance liquid chromatography sugar content analysis of whey hydrolysis by mutant ELP-T955R-AoBgal35A.

[0060] Figure 4 Treatment results of thin layer chromatography (left panel) and high performance liquid chromatography sugar content analysis (right panel) of whey hydrolysis by mutant ELP-T955R-AoBgal35A.

[0061] Figure 5 Treatment results of enzyme loading optimization high performance liquid chromatography lactose content analysis of lactose hydrolysis in milk by mutant ELP-T955R-AoBgal35A.

[0062] Figure 6 pH (a), lactose content (b), Streptococcus thermophilus viable count (c) and Lactobacillus bulgaricus viable count (d) during the fermentation process of zero lactose sucrose-free yogurt by mutant ELP-T955R-AoBgal35A. DETAILED DESCRIPTION

[0063] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the application.

[0064] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0065] In the quantitative experiments in the following examples, three repeated experiments were set, unless otherwise specified.

[0066] The pET-ELP vector in the following examples has been described in: Enzymatic synthesis of sugar nucleotides and β-mannosides, Liu Jing, 2021. The biological material can be obtained from the applicant, and is only used for repeating the experiments of the application, and cannot be used for other purposes.

[0067] The eukaryotic expression vector pPIC9K-AoBgal35A used in the following examples has been described in: Cloning, expression and recombinant enzyme properties of the β-galactosidase gene from T. aurantiacus and T. terrestris, Sun Zhenxing, 2017. The biological material is available from the applicant for the purpose of repeating the experiments of the present invention only and cannot be used for other purposes.

[0068] The competent cells of Pichia pastoris GS115 used in the examples have been described in: Lin-Cereghino J, Naranjo CA, Lin-Cereghino GP. Competent Cell Preparation and Transformation of Pichia pastoris. Methods Mol Biol. 2022; 2513: 113-120. The biological material is available from the applicant for the purpose of repeating the experiments of the present invention only and cannot be used for other purposes.

[0069] The data in the following examples were processed using SPSS 11.5 statistical software, and the experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used, and P < 0.05 was considered to be a significant difference.

[0070] Example 1, Construction of Recombinant Plasmid and Preparation of Aspergillus oryzae β-galactosidase Mutant

[0071] Preparation of pPIC9K-T955R-AoBgal35A Recombinant Plasmid: PCR amplification was performed using pPIC9K-AoBgal35A recombinant plasmid as the template. The system was: 10x Fast pfu buffer 10 μL, dNTPs 4 μL, T955R-UP: 5'- ACTACCGCGGACGAAACTATGTGGCACTGAGTCTTT-3' / DN (5'-TCGTCCGCGGTAGTTCAGGATCCCTTCG-3') each 1 μL, template 1 μL, Fast pfu DNA polymerase 1 μL, and pure water to 50 μL. Reaction conditions: 95°C for 3 min, 95°C for 20 s, 55°C for 20 s, 72°C for 4 min, reaction cycle 25 times; 72°C for 10 min. DpnI was digested at 37°C for 4 h, and E. coli DH5α was transformed and plated on LB plates containing ampicillin (100 μg / mL). The transformants were incubated at 37°C overnight, and the plasmid pPIC9K-T955R-AoBgal35A was linearized by restriction enzyme PmeI, ethanol precipitated and recovered, and electroporated into Pichia pastoris GS115 competent cells. The transformation products were uniformly plated on MD plates (YNB 13.4 g / L, biotin 4x10-4 g / L, glucose 10 g / L, agar 15 g / L) in an incubator at 30°C for 72 h. Recombinant Pichia pastoris GS115 / pPIC9K-T955R-AoBgal35A containing plasmid pPIC9K-T955R-AoBgal35A was obtained.

[0072] Preparation of recombinant plasmid pPIC9K-ELP-T955R-AoBgal35A: The elastin-like polypeptide (ELP) fragment was recovered after electrophoretic separation of the pET-ELP vector digested with Nco I and BamH I restriction enzymes and inserted into the recombinant vector pPIC9K-T955R-AoBgal35A.

[0073] The structure of the recombinant plasmid pPIC9K-T955R-AoBgal35A is described as follows: the fragment between the SnaB I and Avr II enzyme sites of the starting vector pPIC9K-AoBgal35A was replaced with the DNA molecule shown in SEQ ID No. 4, and the other nucleotide sequences on the pPIC9K vector were kept unchanged to obtain the recombinant vector. This recombinant vector was named as the recombinant vector pPIC9K-T955R-AoBgal35A, which can express the T955R-AoBgal35A protein with an expected molecular weight of about 108 KD.

[0074] The structure of the recombinant plasmid pPIC9K-ELP-T955R-AoBgal35A is described as follows: the fragment between the SnaB I and Avr II enzyme sites of the starting vector pPIC9K-ELP was replaced with the DNA molecule shown in SEQ ID No. 5, and the other nucleotide sequences on the pPIC9K vector were kept unchanged to obtain the recombinant vector. This recombinant vector was named as the recombinant vector pPIC9K-ELP-T955R-AoBgal35A, which can express the ELP-T955R-AoBgal35A protein with an expected molecular weight of about 133 KD.

[0075] The amino acid sequence of the β-galactosidase mutant T955R-AoBgal35A is SEQ ID No. 2. The amino acid sequence of the β-galactosidase mutant ELP-T955R-AoBgal35A containing an elastin-like polypeptide tag is SEQ ID No. 3.

[0076] Example 2, High-efficiency expression and purification of mutants T955R-AoBgal35A and ELP-T955R-AoBgal35A

[0077] The recombinant Pichia pastoris containing high copy screened mutant T955R-AoBgal35A and ELP-T955R-AoBgal35A obtained in Example 1 was streaked on YPD solid medium (containing yeast extract powder 10 g / L, peptone 20 g / L, glucose 20 g / L, agar 15 g / L, the rest is made up with water) plate, and single colonies were picked to YPD liquid medium (containing yeast extract powder 10 g / L, peptone 20 g / L, glucose 20 g / L, the rest is made up with water) and cultured at 30°C, 200 rpm overnight as seed liquid. The seed liquid was inoculated into 1.35 L BSM medium (calcium sulfate 0.93 g / L, potassium sulfate 18.2 g / L, magnesium sulfate heptahydrate 14.9 g / L, potassium hydroxide 4.13 g / L, glycerol 40 g / L, 85% phosphoric acid 26.7 mL / L, the rest is made up with water) (inoculation amount is 10%), and when the dissolved oxygen decreased to 0%, 50% (w / w) glycerol was added. After the dissolved oxygen remained stable, methanol induction containing 1.2% (v / v) PTM1 trace salt solution was started. The fermentation liquid was centrifuged at 4°C, 12000 rpm for 15 min to collect the supernatant, which was the crude enzyme liquid (containing mutant protein T955R-AoBgal35A or ELP-T955R-AoBgal35A).

[0078] Purification of T955R-AoBgal35A protein: The recombinant protein was purified using Q-Sepharose strong anion exchange column, and the loading flow rate was 0.4 mL / min. The exchange column was previously equilibrated with 10 column volumes of buffer (20 mmol / L sodium phosphate, pH 7.0), and the linear elution was performed using 20 mmol / L (pH 7.0) sodium phosphate buffer containing 0-500 mmol / L NaCl, and the purified T955R-AoBgal35A protein was collected.

[0079] Purification of ELP-T955R-AoBgal35A protein: At 20°C, an equal volume of 4M sodium chloride was added to the crude enzyme liquid, so that the final concentration of sodium chloride was 2M, and then the stirring was continued for 5-10 min. Centrifugation was performed at room temperature, 10000 rpm for 10 min, the centrifugation precipitate was resuspended with buffer, and incubated at 4°C overnight. Centrifugation was performed at 4°C, 14000 rpm for 10 min, and the supernatant (containing fusion protein ELP-T955R-AoBgal35A) was reserved.

[0080] The results are as follows Figure 1As shown, after high-density fermentation in a 5L fermentor and methanol induction for 168 hours, the wet weight of the fermentation broth was 388 g / L, and the enzyme activity of β-galactosidase was 4628 U / mL, which is higher than the expression level of most other β-galactosidases in Pichia pastoris, such as β-galactosidases from the extremely thermophilic archaeon Sulfolobus solfataricus (204.9 U / mL), Aspergillus candidus (3600 U / mL), Arthrobacter sp. (1926 U / mL), and Aspergillus awamori (347.8 U / mL). Therefore, the efficient expression of the mutant of the present invention makes it have good application potential in industrial production.

[0081] In Table 1, the mutant protein T955R-AoBgal35A, which lacks an elastin-like tag, was purified using affinity chromatography, yielding a pure enzyme recovery rate of 54.2% and a purification fold of 1.2-fold. In Table 2, the mutant protein ELP-T955R-AoBgal35A, which carries an elastin-like tag, was purified using a reversible phase change cycle method, achieving significantly improved pure enzyme recovery and purification folds of 96.4% and 2.3-fold, respectively, demonstrating significantly superior purification efficiency compared to affinity chromatography. Therefore, the recombinant plasmid carrying the ELP tag constructed by the present invention can improve the mutant's recovery rate and purification fold, while maintaining mutant stability and increasing specific enzyme activity.

[0082] Table 1. Purification analysis results of mutant protein T955R-AoBgal35A

[0083]

[0084] Table 2. Purification analysis results of mutant protein ELP-T955R-AoBgal35A

[0085]

[0086] Example 3. Enzymatic properties of mutant ELP-T955R-AoBgal35A

[0087] β-galactosidase activity was determined by adding 75 μL of 15 mmol / L oNPG and 150 μL of 50 mmol / L citric acid-trisodium citrate buffer (pH 5.5) to a 1.5 mL centrifuge tube. After preheating at the optimal temperature for 3 minutes, 25 μL of the appropriately diluted enzyme solution was added. After 10 minutes, the reaction was terminated by adding 750 μL of 2 M Na₂CO₃. The amount of o-nitrophenol released was measured at 410 nm. One unit (U) of enzyme activity is defined as the amount of enzyme required to hydrolyze oNPG (source leaf, Catalog No. 2816-24-2) to release 1 μmol o-nitrophenol per minute under the above reaction conditions.

[0088] Optimal pH determination: The ELP-T955R-AoBgal35A protein was diluted with buffer systems of different pH values ​​(citrate, 3.0-6.0; PB, 6.0-8.0; Tris-HCl, 6.0-9.0; CHES, 8.0-10.0), and then the β-galactosidase activity was measured at 60°C. The graph was plotted with the highest enzyme activity point as 100%.

[0089] pH stability assay: Dilute the enzyme solution to approximately 1 mg / mL using the above-mentioned buffer solutions of different pH values ​​and incubate at 50°C for 30 min. Then, quickly cool the sample in an ice-water bath for 30 min. Determine the residual enzyme activity according to the standard method, and calculate the relative enzyme activity based on the untreated pure enzyme solution as 100%.

[0090] Optimum temperature determination: Using a buffer solution with the optimal pH value, determine the optimal temperature of the enzyme reaction at different temperatures (30-75°C). Taking the highest enzyme activity point as 100%, calculate the relative enzyme activity under different temperature conditions.

[0091] Temperature stability assay: The enzyme solution was diluted to approximately 1 mg / mL with a buffer solution of optimal pH, and then incubated at different temperatures (30-75°C) for 30 min. The sample was then quickly cooled in an ice-water bath for 30 min, and the residual enzyme activity was determined. The relative enzyme activity was calculated with the untreated pure enzyme solution as 100%.

[0092] Compared with the wild type, the optimal pH of ELP-T955R-AoBgal35A shifted to the right by 1 pH to pH 5.5 ( Figure 2 a) is more suitable for application in yogurt processing (the pH of yogurt decreases from 6.5 to 4.5 during fermentation).

[0093] The enzyme activity can be maintained at more than 80% in the pH range of 4.0-7.0, and it is stable in a wider range than the wild type (pH 4.5-5.5). Figure 2 (b)

[0094] The optimum temperature of T955R-AoBgal35A is 50℃, which is 5℃ lower than that of the wild type (60℃) Figure 2 The enzyme activity of T955R-AoBgal35A is maintained at more than 80% at 60℃, which is slightly higher than that of the wild type (≤55℃) Figure 2 The decrease in optimum temperature makes it closer to the yogurt fermentation temperature (35-45℃), which is more suitable for application in the yogurt process.

[0095] Example 4, Hydrolysis of lactose solution by mutant protein ELP-T955R-AoBgal35A

[0096] A 5% (w / v) lactose solution (solute: citric acid-sodium citrate (pH 5.0) buffer) was used as the substrate, and the reaction was carried out at 55℃ with an enzyme amount of 5 U / mL. Samples were taken at 5, 15 and 30 min, 1, 2, 4, 6 and 8 h, and then the samples were boiled for 5 min to inactivate the enzyme. The remaining lactose concentration in the hydrolysate was analyzed qualitatively by TLC and quantitatively by HPLC.

[0097] HPLC content analysis of lactose hydrolyzed by mutant ELP-T955R-AoBgal35A Figure 3 The results show that the hydrolysis rate of 5% (w / v) lactose is 95.8% under the conditions of pH 5.0 and 55℃ for 4 h, which is 11.6% higher than that of the wild type (84.2%). Therefore, the mutant protein ELP-T955R-AoBgal35A of the present application has better hydrolysis properties.

[0098] Example 5, Hydrolysis of whey by mutant protein ELP-T955R-AoBgal35A

[0099] A 5% (w / v) whey solution was prepared at pH 5.0, and mutant protein ELP-T955R-AoBgal35A was added to the solution at an enzyme amount of 10 U / mL. After mixing, the solution was placed at room temperature for reaction. Samples were taken at 0, 0.5, 1, 2, 4, 6, 8, 12 and 24 h, and then the samples were boiled for 5 min to inactivate the enzyme. The remaining lactose concentration in the hydrolysate was analyzed qualitatively by TLC and quantitatively by HPLC, with lactose concentration as the index.

[0100] The results are shown in Table 2. Figure 4 Under the conditions of pH 5.0 and room temperature (20-25℃) with an enzyme amount of 10 U / mL, ELP-T955R-AoBgal35A can hydrolyze 98.3% of the lactose in whey after 24 h of reaction.

[0101] Example 6, Preparation of zero-lactose milk by mutant protein ELP-T955R-AoBgal35A

[0102] In the commercially available milk, the mutant protein ELP-T955R-AoBgal35A was added, and the enzyme amount (1, 2, 3, 4, 5 U / mL) was optimized at 25°C. After sampling at a fixed time, the sample was boiled for 5 min to inactivate the enzyme. The lactose content and hydrolysis rate were used as indicators, and quantitative analysis was performed by HPLC. The TLC and HPLC detection methods were the same as in Example 4.

[0103] The results are shown in Figure 5 When the enzyme amount was 2 U / mL, the lactose in the milk was basically completely hydrolyzed after 72 h of hydrolysis, the lactose hydrolysis rate was 94.1%, and the lactose content was 0.18%. The enzyme amount of the mutant protein ELP-T955R-AoBgal35A only needed 10% of the wild type, and better hydrolysis effect could be achieved. In addition, the β-galactosidase from Erwinia E602 could hydrolyze 60% of the lactose in the milk at an enzyme amount of 3 U / mL for 12 h (Xia, et al. Journal of Dairy Science, 2018, 101: 6946-6954), and the β-galactosidase Bgal322 from Psychrobacter LW097 could only hydrolyze 8.4% of the lactose in the milk at an enzyme amount of 5 U / mL for 24 h (Wang, et al. International Journal of Biological Macromolecules, 2013, 60: 109-115). Therefore, the excellent hydrolysis characteristics of the mutant of the present application make it have better industrial application value in the preparation of lactose-free milk.

[0104] Example 7, Preparation of zero-lactose and zero-sucrose yogurt by mutant protein ELP-T955R-AoBgal35A

[0105] Prepare raw materials: pure milk (Mengniu), desalted whey powder (Milkavita), β-galactosidase (enzyme preparation obtained by the present application), and fermentation agent (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus, Yomix, Danisco). Processing technology: material dissolution, homogenization, sterilization, fermentation and hydrolysis, post-ripening and filling. Traditional yogurt with 7% sucrose was used as control group 1, and experimental groups 1-3 were zero-sucrose and zero-lactose yogurt with whey powder (added amounts were 5%, 10% and 15%, respectively) and mutant protein ELP-T955R-AoBgal35A.

[0106] The sensory evaluation of yogurt was scored according to the "Fermented Milk Sensory Evaluation Rules" (RHB 104-2020) to explore the improvement effect of whey powder and lactase addition on the sourness of zero sucrose yogurt. Twenty trained food science students were selected to evaluate the sensory characteristics of zero lactose sucrose-free yogurt with different amounts of whey powder from color, texture, odor, taste, and flavor. The sensory evaluation was based on a nine-point scale, with 1 being the worst and 9 being the best. The sum of the scores of the four indicators was the total score.

[0107] The results are shown in Table 3. With the increase of whey powder addition, the sweetness of yogurt was significantly improved, and zero lactose sucrose-free yogurt with no significant difference in sourness and sweetness from traditional yogurt was prepared. In summary, the zero lactose sucrose-free yogurt prepared from mutant protein ELP-T955R-AoBgal35A and whey powder has moderate sourness and sweetness, smooth mouthfeel, and rich aroma.

[0108] Table 3. Sensory evaluation of zero lactose sucrose-free yogurt

[0109] Group Color Tissue state Odor Taste Acidity Sweetness Total score Control group 1 8.0±1.1 7.3±0.8 4.5±0.9 3.0±1.1 7.3±1.0 8.5±0.7 38.6 Experimental group 1 8.0±1.5 7.5±1.1 5.5±0.6 4.8±0.7 2.0±1.8 2.3±2.1 30.1 Experimental group 2 8.5±2.1 8.0±1.6 6.0±1.1 6.0±0.5 4.8±1.0 5.3±1.1 38.7 Experimental group 3 8.3±1.4 7.8±0.7 6.8±1.3 6.8±0.9 6.9±0.9 7.7±0.7 44.3

[0110] The changes in pH, lactose content, and viable cell count during fermentation and hydrolysis of zero lactose sucrose-free yogurt were monitored. The detection of viable cell count was based on GB 4789.35-2016 "Food Microbiology Testing Lactic Acid Bacteria Testing". The samples were diluted to an appropriate concentration, and the plate count of Streptococcus thermophilus and Lactobacillus bulgaricus in yogurt was performed using MC medium (Beijing Aoboxing Biotechnology Co., Ltd., Catalog No. 02-291) and cysteine hydrochloride modified MRS agar medium (Qingdao Haibo Biological, Catalog No. HB0384-6a), respectively, and incubated at 37℃±1℃ for 72±2h under anaerobic conditions.

[0111] The results showed that the pH reduction rate of the experimental group was significantly higher than that of the control group in the early fermentation period ( Figure 6 a), and the mutant ELP-T955R-AoBgal35A accelerated the acidification of yogurt in the early fermentation period, and the fermentation time of yogurt was 4.5-5h. The lactose concentration of experimental group 3 decreased from 10.1% to 1.9% after 1h of fermentation, and slowly decreased to 0.36% within the next 5h, which was beneficial to the growth of Streptococcus thermophilus with lactose as the main carbon source ( Figure 6 b). At 2h of fermentation, the viable cell count of Streptococcus thermophilus in the mutant group was 9.6log CFU / mL, which was significantly higher than that of the control group 1 (8.9log CFU / mL) Figure 6In the middle c). At the end of fermentation, the viable counts of L. bulgaricus (7.2 log CFU / mL) and S. thermophilus (10.2 log CFU / mL) of the mutant group were significantly higher than 6.5 log CFU / mL and 9.8 log CFU / mL of the control group 1, which promoted the proliferation of yogurt probiotics Figure 6 In the middle d). The mutant ELP-T955R-AoBgal35A of the application slowly hydrolyzed lactose during fermentation, did not compete with probiotics, and could also accelerate the fermentation of yogurt and promote the proliferation of probiotics.

[0112] The physicochemical indicators of yogurt in the 28-day shelf life of the experimental group 3 with the highest sensory score were detected, including lactose concentration, glucose concentration, galactose concentration, total viable count, and titratable acidity. The determination of acidity referred to "Determination of Food Acidity" GB5009.239-2016.

[0113] The results are shown in Table 4. The residual amount of lactose in yogurt during storage was in the range of 0.17%-0.36%, which belonged to the range of lactose-free food (lactose concentration <0.5%); the total viable count was in the range of 6.3×10 8 CFU / mL-2.0×10 9 CFU / mL, the viable count was stable during the shelf life, and met the national standard (>6.0 log CFU / mL); the titratable acidity was in the range of 78-87°T, and no obvious post-acidification occurred. In summary, the zero-lactose, sucrose-free yogurt prepared using the mutant group had stable quality during the shelf life.

[0114] Table 4, Changes in lactose concentration, total viable count, and titratable acidity of zero-lactose, sucrose-free yogurt during the shelf life

[0115]

[0116] The application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the application, and without unnecessary experiments, the application can be implemented in a wider range under equivalent parameters, concentrations, and conditions. Although the application gives a special example, it should be understood that further improvements can be made to the application. In summary, according to the principle of the application, this application intends to include any changes, uses or improvements of the application, including changes made by conventional techniques known in the art, which deviate from the range disclosed in this application.

Claims

1. Aspergillus oryzae β-galactosidase mutant protein, characterized in that The mutant protein is any one of the following: A1) a protein having an amino acid sequence of SEQ ID No. 2; A2) A fusion protein having the same function as A1) is obtained by connecting a tag to the N-terminus and / or C-terminus.

2. The protein-related biomaterial according to claim 1, characterized in that The biological material is any one of the following: B1) a nucleic acid molecule encoding the protein according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1); B4) a recombinant vector containing the expression cassette described in B2); B5) a recombinant microorganism containing the nucleic acid molecule described in B1); B6) a recombinant microorganism containing the expression cassette described in B2); B7) a recombinant microorganism containing the recombinant vector described in B3); B8) A recombinant microorganism containing the recombinant vector described in B4).

3. The biomaterial according to claim 2, characterized in that The recombinant microorganism is a recombinant yeast.

4. The biomaterial according to claim 3, characterized in that The recombinant yeast is recombinant Pichia pastoris.

5. The biomaterial according to any one of claims 2 to 4, characterized in that B1) The nucleic acid molecule is a DNA molecule as shown below: d1) the nucleotide sequence is the DNA molecule shown in SEQ ID No. 4; d2) the nucleotide sequence is the DNA molecule shown in SEQ ID No. 5; d3) A DNA molecule that hybridizes under stringent conditions with the nucleotide sequence defined in d1) or d2) and encodes the protein of claim 1.

6. A method for constructing a recombinant Pichia pastoris, characterized in that: The method comprises introducing a gene encoding the protein according to any one of claims 1 to 5 into the recipient Pichia pastoris.

7. The method according to claim 6, characterized in that: The method further comprises connecting a VPGVG50 tag to the N-terminus and / or C-terminus of the protein according to any one of claims 1 to 5 to obtain a fusion protein having the same function as the protein according to any one of claims 1 to 5.

8. A method for preparing an Aspergillus oryzae β-galactosidase mutant, comprising introducing a mutant encoding gene according to any one of claims 1 to 5 into a recipient microorganism to obtain a recombinant microorganism expressing the mutant according to any one of claims 1 to 5, culturing the recombinant microorganism, and obtaining the Aspergillus oryzae β-galactosidase mutant from the fermentation product.

9. Use of the mutant protein according to claim 1, or the biomaterial according to any one of claims 2 to 5, or the method according to any one of claims 6 to 8 in any of the following: A) Application in the preparation and production of low / lactose-free dairy products; B) Application in the preparation and production of low / no sucrose dairy products; C) Application in whey processing.

10. A method for preparing lactose-free and sucrose-free yogurt, comprising adding whey powder to raw milk and adding the protein according to claim 1 when inoculating a yogurt starter to obtain lactose-free and sucrose-free yogurt.

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