A beta-galactosidase mutant and use thereof

Abstract

The application discloses a beta-galactoside enzyme mutant and application thereof. The application takes wild-type beta-galactoside enzyme Bgal1-3 as a parent to provide a mutant with improved thermal stability, enzyme activity and / or oligogalactose production capacity compared with the parent. Meanwhile, the application takes the mutant M9 of Bgal1-3 as a parent to provide a mutant with improved hydrolysis activity compared with the parent. The beta-galactoside enzyme mutant can be used for converting lactose to produce low / zero-lactose or oligogalactose-rich dairy products, and is beneficial to the development of low / zero-lactose or oligogalactose-rich dairy product related industry.

Description

Technical Field

[0001] This invention belongs to the field of enzyme technology. More specifically, it relates to a β-galactosidase mutant and its applications. Background Technology

[0002] Lactose intolerance is a common physiological problem caused by the inability of individuals to break down lactose, leading to adverse reactions such as flatulence, abdominal pain, and diarrhea after consuming lactose-containing dairy products. Utilizing β-galactosidase to degrade lactose in dairy products can effectively solve the problem of lactose intolerance. Furthermore, galactooligosaccharides (GOS), as a typical representative of prebiotics, have functions such as regulating intestinal flora balance, improving immunity, anti-aging, lowering blood pressure, and improving lipid metabolism. Low / lactose-free dairy products rich in GOS, as novel health foods, not only solve the lactose intolerance problem for some people but also benefit their health, thus generating huge market demand.

[0003] β-galactosidase (EC.3.2.1.23), commonly known as lactase, is an important glycoside hydrolase. It is required in the production of low- / lactose-free dairy products and low- / lactose-free dairy products rich in GOS. β-galactosidase possesses both hydrolytic and transglycosylation activities. Its hydrolytic activity breaks down lactose into galactose and glucose, while its transglycosylation activity converts lactose into galacto-oligosaccharides. However, existing β-galactosidases have limitations in GOS yield and thermal stability, hindering the development of β-galactosidase-related food industries.

[0004] For example, the β-galactosidases currently used in the production of low / lactose-free dairy products are mainly room-temperature enzymes, whose optimal temperature (37°C) is ideal for microbial growth and reproduction, potentially posing food safety risks. While low-temperature (4°C) hydrolysis can reduce microbial contamination, the hydrolytic efficiency of β-galactosidase is also low at low temperatures. High-temperature hydrolysis can effectively address these issues, but it also places higher demands on the thermal stability of β-galactosidase. Therefore, there is a need for β-galactosidases with better thermal stability. Building upon good thermal stability, providing a β-galactosidase with a moderate optimal reaction temperature and high lactose hydrolytic activity would be even more beneficial for the production of low / lactose-free dairy products. Furthermore, in the production of low / lactose-free dairy products rich in GOS, the hydrolytic activity of most lactases is far higher than their transglycosylation activity, resulting in low GOS yields.

[0005] In summary, continuous optimization of β-galactosidase is necessary to promote the development of related food industries. While there are reports on improving the thermostability, activity, or GOS yield of β-galactosidase through amino acid mutations, different β-galactosidases have different amino acid sequences, and there is no universal method to improve their thermostability, activity, or GOS yield. Summary of the Invention

[0006] To address the shortcomings of the prior art, this invention provides a β-galactosidase mutant with improved thermal stability, enzyme activity, and / or ability to produce galactooligosaccharides compared to the parent, which can be used in the production of low-lactose or lactose-free dairy products containing galactooligosaccharides.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] This invention uses wild-type β-galactosidase Bgal1-3 and the mutant M9 of wild-type Bgal1-3 disclosed in patent application CN119162154A as parents. By mutating specific amino acid sites in their amino acid sequences, mutants with improved thermostability, enzyme activity, and / or GOS production capacity compared to the parents were obtained. This is beneficial for the preparation of low-lactose or lactose-free dairy products rich in GOS. Therefore, this invention seeks protection for the aforementioned β-galactosidase mutants.

[0009] This invention provides a β-galactosidase mutant, which is obtained by mutating one or more of the following amino acids in the Bgal1-3 amino acid sequence of β-galactosidase: 277, 331, 414, 420, 421, 422, 457, 459, 460, 462, 464, 467, 492, 497, 503, 510, 512, 548, 557, 572, 597, 598, 603, 604, 609, 610, 611, 612, 613, 615, 624, and 676.

[0010] Alternatively, it can be obtained by mutating one or more amino acids at positions 14, 19, 21, 38, 49, 57, 60, 61, 71, 115, 121, 140, 141, 145, 153, 181, 195, 196, 197, 227, 229, 230, 231, 232, 235, 237, 242, 398, 409, 411, 416, 448, 450, 451, 457, 460, 473, 489, 490, 491, 509, 570, 571, 572, 604, 607, 608, 609, 610, 611, 612, and 613 of the β-galactosidase Bgal1-3 mutant M9.

[0011] Specifically, the GenBank accession number for the protein sequence of the wild-type β-galactosidase Bgal1-3 is AGK30046.1; the mutant M9 is obtained by mutating the following amino acid sequences of the wild-type β-galactosidase Bgal1-3: glutamine at position 80 is replaced by glycine, serine at position 277 by valine, valine at position 378 by threonine, tyrosine at position 398 by cysteine, glutamate at position 462 by glutamine, lysine at position 578 by arginine, phenylalanine at position 633 by leucine, serine at position 676 by phenylalanine, and arginine at position 689 by leucine.

[0012] As one alternative implementation, the mutant is a mutant with improved thermal stability; the mutant is one in which the glutamic acid at position 420 of the amino acid sequence of β-galactosidase Bgal1-3 is mutated to lysine or arginine.

[0013] Alternatively, the histidine at position 460 could be mutated to tryptophan;

[0014] Alternatively, the glutamic acid at position 462 could be mutated to asparagine, aspartic acid, cysteine, valine, or proline.

[0015] Alternatively, the threonine at position 574 could be mutated to lysine;

[0016] Alternatively, proline at position 598 could be mutated to lysine;

[0017] Alternatively, the histidine at position 460 may be mutated to tryptophan, and the glutamic acid at position 462 may be mutated to glutamine, glycine, asparagine, aspartic acid, histidine, proline, cysteine, or valine.

[0018] Alternatively, the serine at position 277 could be mutated to valine, and the histidine at position 460 to tryptophan, glutamine, or cysteine.

[0019] Alternatively, the histidine at position 460 could be mutated to tryptophan, the glutamic acid at position 462 to cysteine, and the serine at position 676 to phenylalanine.

[0020] Alternatively, the 277th serine can be mutated to valine, the 457th threonine to isoleucine, the 460th histidine to tryptophan, the 462nd glutamic acid to aspartic acid, the 603rd threonine to serine, the 604th alanine to aspartic acid, and the 612th methionine to valine.

[0021] Alternatively, the following mutations can be made: serine at position 277 can be mutated to valine; threonine at position 457 can be mutated to valine; histidine at position 460 can be mutated to tryptophan; glutamic acid at position 462 can be mutated to aspartic acid or proline; arginine at position 548 can be mutated to proline; leucine at position 557 can be mutated to phenylalanine; threonine at position 603 can be mutated to serine; and alanine at position 604 can be mutated to aspartic acid.

[0022] As one alternative implementation, the mutant is a mutant with increased enzyme activity, wherein the mutant is formed by mutating the 457th threonine of β-galactosidase Bgal1-3 to isoleucine or valine.

[0023] Alternatively, the histidine at position 460 could be mutated to glutamine, serine, glycine, isoleucine, phenylalanine, or tyrosine.

[0024] Alternatively, the alanine at position 467 could be mutated to threonine;

[0025] Alternatively, the arginine at position 548 could be mutated to asparagine, glutamine, tyrosine, or phenylalanine.

[0026] Alternatively, the threonine at position 603 could be mutated to a serine;

[0027] Alternatively, the alanine at position 604 could be mutated to aspartic acid, glycine, glutamic acid, lysine, or phenylalanine.

[0028] Alternatively, arginine at position 548 could be mutated to tyrosine, and methionine at position 597 to isoleucine.

[0029] Alternatively, alanine at position 604 could be mutated to aspartic acid, and arginine at position 609 to leucine.

[0030] Alternatively, threonine at position 603 could be mutated to serine, and alanine at position 604 could be mutated to aspartic acid.

[0031] Alternatively, threonine at position 603 could be mutated to serine, alanine at position 604 to aspartic acid, and glycine at position 624 to aspartic acid.

[0032] Alternatively, the 277th serine can be mutated to valine, the 457th threonine to isoleucine, the 462nd glutamic acid to glutamine, the 603rd threonine to serine, the 604th alanine to aspartic acid, and the 612th methionine to valine.

[0033] As one alternative implementation, the mutant is a mutant with enhanced ability to produce galactooligosaccharides, wherein the mutant is formed by mutating tryptophan at position 331 of β-galactosidase Bgal1-3 to phenylalanine.

[0034] Alternatively, the phenylalanine at position 414 could be mutated to tyrosine or tryptophan;

[0035] Alternatively, the glutamic acid at position 420 could be mutated to alanine, phenylalanine, glycine, asparagine, proline, glutamine, serine, threonine, tryptophan, or tyrosine.

[0036] Alternatively, the leucine at position 421 could be mutated to phenylalanine, proline, arginine, tryptophan, or histidine;

[0037] Alternatively, the glutamic acid at position 462 could be mutated to arginine;

[0038] Alternatively, tryptophan at position 492 could be mutated to phenylalanine or tyrosine;

[0039] Alternatively, the tyrosine residue at position 497 could be mutated to phenylalanine, tryptophan, aspartic acid, glutamine, histidine, isoleucine, asparagine, serine, threonine, glycine, alanine, methionine, cysteine, leucine, or valine.

[0040] Alternatively, the phenylalanine at position 503 could be mutated to tyrosine, tryptophan, asparagine, glutamine, histidine, serine, alanine, or cysteine.

[0041] Alternatively, the cysteine ​​at position 510 could be mutated to tyrosine;

[0042] Alternatively, the histidine at position 512 could be mutated to tyrosine;

[0043] Or, the arginine at position 548 could be mutated to tryptophan;

[0044] Alternatively, the leucine at position 557 could be mutated to phenylalanine, tryptophan, or histidine.

[0045] Alternatively, the tyrosine residue at position 572 could be mutated to glycine, serine, alanine, isoleucine, or leucine.

[0046] Or, the methionine at position 597 could be mutated to isoleucine;

[0047] Alternatively, tryptophan at position 610 could be mutated to tyrosine, phenylalanine, glycine, serine, alanine, proline, isoleucine, or leucine.

[0048] Alternatively, the glycine at position 611 could be mutated to tryptophan, tyrosine, phenylalanine, serine, alanine, isoleucine, leucine, or glutamic acid.

[0049] Alternatively, the serine at position 613 could be mutated to asparagine, aspartic acid, histidine, glutamic acid, methionine, tyrosine, phenylalanine, or glycine.

[0050] Alternatively, the glutamic acid at position 615 could be mutated to isoleucine;

[0051] Alternatively, the phenylalanine at position 414 could be mutated to tyrosine, and the tyrosine at position 497 to phenylalanine, isoleucine, or valine.

[0052] Alternatively, the phenylalanine at position 414 could be mutated to tyrosine, and the phenylalanine at position 503 could be mutated to tyrosine.

[0053] Alternatively, glutamic acid at position 420 could be mutated to glutamine, and leucine at position 421 could be mutated to tryptophan or arginine.

[0054] Alternatively, the glutamic acid at position 420 could be mutated to glutamine, tryptophan, phenylalanine, tyrosine, or histidine, and the leucine at position 557 could be mutated to phenylalanine.

[0055] Alternatively, leucine at position 421 could be mutated to tryptophan, arginine, tyrosine, histidine, or phenylalanine, and leucine at position 557 could be mutated to phenylalanine.

[0056] Alternatively, leucine at position 421 could be mutated to phenylalanine, and tryptophan at position 610 could be mutated to phenylalanine.

[0057] Alternatively, leucine at position 421 could be mutated to phenylalanine, and glycine at position 611 to tryptophan;

[0058] Alternatively, leucine at position 421 could be mutated to phenylalanine, and serine at position 613 could be mutated to leucine.

[0059] Alternatively, leucine at position 421 could be mutated to tryptophan, and glycine at position 611 could be mutated to tryptophan;

[0060] Alternatively, leucine at position 421 could be mutated to tryptophan, and tryptophan at position 610 could be mutated to phenylalanine;

[0061] Alternatively, glycine at position 422 could be mutated to tryptophan, phenylalanine, tyrosine, or histidine, and leucine at position 557 could be mutated to phenylalanine.

[0062] Alternatively, threonine at position 457 could be mutated to valine, and methionine at position 597 to isoleucine.

[0063] Alternatively, threonine at position 457 could be mutated to valine, and leucine at position 557 to phenylalanine.

[0064] Alternatively, threonine at position 457 could be mutated to valine, and tryptophan at position 610 to phenylalanine.

[0065] Alternatively, tyrosine at position 497 could be mutated to isoleucine or valine, and phenylalanine at position 503 could be mutated to tyrosine.

[0066] Alternatively, arginine at position 548 could be mutated to tyrosine, and tryptophan at position 610 could be mutated to phenylalanine.

[0067] Alternatively, arginine at position 548 could be mutated to tyrosine, and glycine at position 611 to tryptophan;

[0068] Alternatively, arginine at position 548 could be mutated to tryptophan, and leucine at position 557 to phenylalanine.

[0069] Alternatively, arginine at position 548 could be mutated to tryptophan, and tryptophan at position 610 could be mutated to phenylalanine.

[0070] Alternatively, arginine at position 548 could be mutated to tryptophan, and glycine at position 611 could be mutated to tryptophan;

[0071] Alternatively, leucine at position 557 could be mutated to phenylalanine, and tryptophan at position 610 could be mutated to phenylalanine.

[0072] Alternatively, leucine at position 557 could be mutated to phenylalanine, and glycine at position 611 could be mutated to phenylalanine or tryptophan.

[0073] Alternatively, leucine at position 557 could be mutated to phenylalanine, and methionine at position 612 could be mutated to leucine.

[0074] Alternatively, leucine at position 557 could be mutated to phenylalanine, and serine at position 613 could be mutated to leucine, tryptophan, or phenylalanine.

[0075] Alternatively, leucine at position 557 could be mutated to phenylalanine, and glutamic acid at position 615 to isoleucine.

[0076] Alternatively, leucine at position 557 could be mutated to tryptophan, and tryptophan at position 610 could be mutated to phenylalanine.

[0077] Alternatively, leucine at position 557 could be mutated to tryptophan, and glycine at position 611 could be mutated to tryptophan;

[0078] Alternatively, tyrosine at position 572 could be mutated to isoleucine or leucine, and tryptophan at position 610 could be mutated to phenylalanine.

[0079] Alternatively, tyrosine at position 572 could be mutated to isoleucine or leucine, and glycine at position 611 could be mutated to tryptophan.

[0080] Alternatively, the methionine at position 597 could be mutated to isoleucine, and the methionine at position 612 to leucine.

[0081] Alternatively, tryptophan at position 610 could be mutated to phenylalanine, and glycine at position 611 could be mutated to tryptophan or phenylalanine.

[0082] Alternatively, tryptophan at position 610 could be mutated to phenylalanine, and serine at position 613 could be mutated to leucine, tryptophan, or phenylalanine.

[0083] Alternatively, tryptophan at position 610 could be mutated to phenylalanine, and glutamic acid at position 615 could be mutated to isoleucine.

[0084] Alternatively, glycine at position 611 could be mutated to tryptophan, and serine at position 613 could be mutated to leucine, tryptophan, or phenylalanine.

[0085] Alternatively, glycine at position 611 could be mutated to tryptophan, and glutamic acid at position 615 could be mutated to isoleucine.

[0086] Alternatively, phenylalanine at position 414 could be mutated to tyrosine, tyrosine at position 497 to isoleucine or valine, and phenylalanine at position 503 to tyrosine.

[0087] Alternatively, glutamic acid at position 420 could be mutated to glutamine, leucine at position 421 to tryptophan or arginine, and leucine at position 557 to phenylalanine.

[0088] Alternatively, leucine at position 421 could be mutated to tryptophan, glutamine at position 464 to lysine, and leucine at position 557 to phenylalanine.

[0089] Alternatively, leucine at position 421 could be mutated to tryptophan, alanine at position 467 to cysteine ​​or isoleucine, and leucine at position 557 to phenylalanine.

[0090] Alternatively, leucine at position 421 could be mutated to arginine, glutamine at position 464 to lysine, and leucine at position 557 to phenylalanine.

[0091] Alternatively, leucine at position 421 could be mutated to arginine, alanine at position 467 to cysteine ​​or isoleucine, and leucine at position 557 to phenylalanine.

[0092] Alternatively, leucine at position 421 could be mutated to phenylalanine or tryptophan, tryptophan at position 610 to phenylalanine, and glycine at position 611 to tryptophan.

[0093] Alternatively, arginine at position 548 could be mutated to tryptophan, tryptophan at position 610 to phenylalanine, and glycine at position 611 to tryptophan.

[0094] Alternatively, leucine at position 557 could be mutated to tryptophan or phenylalanine, tryptophan at position 610 to phenylalanine, and glycine at position 611 to tryptophan.

[0095] Alternatively, leucine at position 557 could be mutated to phenylalanine, threonine at position 603 to serine, and alanine at position 604 to aspartic acid.

[0096] Alternatively, tyrosine at position 572 could be mutated to isoleucine or leucine, tryptophan at position 610 to phenylalanine, and glycine at position 611 to tryptophan.

[0097] Alternatively, methionine at position 597 could be mutated to isoleucine, threonine at position 603 to serine, and alanine at position 604 to aspartic acid.

[0098] Alternatively, threonine at position 603 could be mutated to serine, alanine at position 604 to aspartic acid, and tryptophan at position 610 to phenylalanine.

[0099] Alternatively, tryptophan at position 610 could be mutated to phenylalanine, glycine at position 611 to tryptophan, and serine at position 613 to leucine, phenylalanine, tryptophan, or isoleucine.

[0100] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to proline, and the methionine at position 597 to isoleucine.

[0101] Alternatively, leucine at position 421 could be mutated to tryptophan, glutamine at position 464 to lysine, alanine at position 467 to glutamic acid, cysteine, or isoleucine, and leucine at position 557 to phenylalanine.

[0102] Alternatively, leucine at position 421 may be mutated to arginine, glutamine at position 464 to lysine, alanine at position 467 to glutamic acid, cysteine, or isoleucine, and leucine at position 557 to phenylalanine.

[0103] Alternatively, leucine at position 557 could be mutated to phenylalanine, tryptophan at position 610 to phenylalanine, glycine at position 611 to tryptophan, and serine at position 613 to tryptophan.

[0104] Alternatively, valine at position 459 could be mutated to serine, leucine at position 557 to phenylalanine, threonine at position 603 to serine, alanine at position 604 to aspartic acid, and glycine at position 624 to aspartic acid.

[0105] Alternatively, valine at position 459 could be mutated to serine, methionine at position 597 to isoleucine, threonine at position 603 to serine, alanine at position 604 to aspartic acid, and glycine at position 624 to aspartic acid.

[0106] Alternatively, the 277th serine position could be mutated to valine, the 457th threonine position to isoleucine, the 462nd glutamic acid position to glutamine, the 597th methionine position to isoleucine, the 603rd threonine position to serine, the 604th alanine position to aspartic acid, and the 612th methionine position to valine.

[0107] Alternatively, the following can be obtained by mutating serine at position 277 to valine, threonine at position 457 to isoleucine, glutamic acid at position 462 to glutamine, leucine at position 557 to phenylalanine, threonine at position 603 to serine, alanine at position 604 to aspartic acid, and methionine at position 612 to valine.

[0108] As one alternative implementation, the mutant is a mutant with improved thermal stability and enzyme activity, wherein the mutant is formed by mutating alanine at position 604 of β-galactosidase Bgal1-3 to aspartic acid.

[0109] Or mutate arginine at position 548 to proline;

[0110] Alternatively, arginine at position 548 could be mutated to proline, and methionine at position 612 to leucine.

[0111] Alternatively, threonine at position 457 could be mutated to valine or isoleucine, arginine at position 548 to proline, and methionine at position 612 to leucine.

[0112] Alternatively, the following can be obtained by mutating serine at position 277 to valine, threonine at position 457 to valine, histidine at position 460 to tryptophan, glutamic acid at position 462 to cysteine, leucine at position 557 to phenylalanine, threonine at position 603 to serine, and alanine at position 604 to aspartic acid.

[0113] As one alternative implementation, the mutant is a mutant with improved thermal stability and ability to produce galactooligosaccharides, wherein the mutant is a mutant in which the glutamic acid at position 420 of β-galactosidase Bgal1-3 is mutated to lysine or arginine.

[0114] Alternatively, the glutamic acid at position 462 could be mutated to histidine;

[0115] Or, the methionine at position 597 could be mutated to isoleucine;

[0116] Or, the serine at position 613 could be mutated to leucine;

[0117] Alternatively, arginine at position 548 could be mutated to proline, and methionine at position 597 to isoleucine.

[0118] Alternatively, arginine at position 548 could be mutated to proline, and tryptophan at position 610 could be mutated to phenylalanine.

[0119] Alternatively, the serine at position 277 could be mutated to valine, the glutamic acid at position 462 to glutamine, and the leucine at position 557 to phenylalanine.

[0120] Alternatively, the serine at position 277 could be mutated to valine, the glutamic acid at position 462 to glutamine, and the methionine at position 597 to isoleucine.

[0121] Alternatively, the serine at position 277 could be mutated to valine, the glutamate at position 462 to glutamine, and the tryptophan at position 610 to phenylalanine.

[0122] Alternatively, threonine at position 457 could be mutated to valine, arginine at position 548 to proline, and methionine at position 597 to isoleucine.

[0123] Alternatively, threonine at position 457 could be mutated to valine, arginine at position 548 to proline, and tryptophan at position 610 to phenylalanine.

[0124] Alternatively, arginine at position 548 could be mutated to proline, leucine at position 557 to phenylalanine, and methionine at position 612 to leucine.

[0125] Alternatively, arginine at position 548 could be mutated to proline, methionine at position 597 to isoleucine, and methionine at position 612 to leucine.

[0126] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to aspartic acid, and the leucine at position 557 to phenylalanine or isoleucine.

[0127] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to aspartic acid, and the tryptophan at position 610 to phenylalanine.

[0128] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to cysteine, and the leucine at position 557 to phenylalanine.

[0129] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to cysteine, and the methionine at position 597 to isoleucine.

[0130] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to cysteine, and the tryptophan at position 610 to phenylalanine.

[0131] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to cysteine, and the glycine at position 611 to tryptophan.

[0132] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to proline, and the leucine at position 557 to phenylalanine.

[0133] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to proline, and the tryptophan at position 610 to phenylalanine.

[0134] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamate at position 462 to glutamine, and the leucine at position 557 to phenylalanine.

[0135] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamate at position 462 to glutamine, and the methionine at position 597 to isoleucine.

[0136] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamate at position 462 to glutamine, and the tryptophan at position 610 to phenylalanine.

[0137] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamate at position 462 to glutamine, and the glycine at position 611 to tryptophan.

[0138] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to cysteine, the arginine at position 548 to tyrosine, glutamine, asparagine, phenylalanine, or proline, and the tryptophan at position 610 to phenylalanine.

[0139] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to cysteine, the alanine at position 604 to phenylalanine, and the tryptophan at position 610 to phenylalanine.

[0140] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to cysteine, the tryptophan at position 610 to phenylalanine, and the glycine at position 611 to tryptophan.

[0141] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to glutamine, the leucine at position 557 to phenylalanine, and the alanine at position 604 to phenylalanine.

[0142] Alternatively, the 277th serine position could be mutated to valine, the 460th histidine position to tryptophan, the 462nd glutamate position to glutamine, the 597th methionine position to isoleucine, and the 604th alanine position to phenylalanine.

[0143] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamate at position 462 to glutamine, the alanine at position 604 to phenylalanine, and the tryptophan at position 610 to phenylalanine.

[0144] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to glutamine, the alanine at position 604 to phenylalanine, and the glycine at position 611 to tryptophan.

[0145] Alternatively, the 277th serine position could be mutated to valine, the 462nd glutamic acid position to glutamine, the 557th leucine position to phenylalanine, the 603rd threonine position to serine, and the 604th alanine position to phenylalanine.

[0146] Alternatively, the 277th serine position could be mutated to valine, the 462nd glutamic acid position to glutamine, the 597th methionine position to isoleucine, the 603rd threonine position to serine, and the 604th alanine position to phenylalanine.

[0147] Alternatively, the 277th serine position could be mutated to valine, the 462nd glutamic acid position to glutamine, the 603rd threonine position to serine, the 604th alanine position to phenylalanine, and the 610th tryptophan position to phenylalanine.

[0148] Alternatively, the 277th serine position could be mutated to valine, the 457th threonine position to isoleucine or valine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to cysteine, the 548th arginine position to proline, and the 610th tryptophan position to phenylalanine.

[0149] Alternatively, the serine at position 277 could be mutated to valine, the threonine at position 457 to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to cysteine, the arginine at position 548 to proline, and the glycine at position 611 to tryptophan.

[0150] Alternatively, the 277th serine position could be mutated to valine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to cysteine, the 610th tryptophan position to phenylalanine, the 611th glycine position to tryptophan, and the 613th serine position to either phenylalanine or tryptophan.

[0151] Alternatively, the 277th serine position could be mutated to valine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to cysteine, the 548th arginine position to proline, phenylalanine, tyrosine, asparagine, or glutamine, the 610th tryptophan position to phenylalanine, and the 611th glycine position to tryptophan.

[0152] Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to cysteine, the alanine at position 604 to phenylalanine, aspartic acid, glutamic acid, lysine, or glycine, the tryptophan at position 610 to phenylalanine, and the glycine at position 611 to tryptophan.

[0153] Alternatively, the 277th serine position could be mutated to valine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to cysteine, the 610th tryptophan position to phenylalanine, the 611th glycine position to tryptophan, and the 676th serine position to tryptophan.

[0154] Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 460th histidine to tryptophan, the 462nd glutamic acid to cysteine, the 548th arginine to proline, the 610th tryptophan to phenylalanine, and the 611th glycine to tryptophan.

[0155] Alternatively, the 277th serine position could be mutated to valine, the 457th threonine position to valine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to cysteine, the 548th arginine position to proline, the 610th tryptophan position to phenylalanine, and the 612th methionine position to leucine.

[0156] Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 460th histidine to tryptophan, the 462nd glutamic acid to cysteine, the 557th leucine to phenylalanine, the 610th tryptophan to phenylalanine, and the 611th glycine to tryptophan.

[0157] Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 460th histidine to tryptophan, the 462nd glutamic acid to cysteine, the 610th tryptophan to phenylalanine, the 611th glycine to tryptophan, and the 613th serine to either phenylalanine or tryptophan.

[0158] Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 460th histidine to tryptophan, the 462nd glutamic acid to cysteine, the 610th tryptophan to phenylalanine, the 611th glycine to tryptophan, and the 676th serine to phenylalanine, glutamic acid, asparagine, or threonine.

[0159] Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 462nd glutamic acid to glutamine, the 548th arginine to proline, the 603rd threonine to serine, the 604th alanine to glutamic acid, and the 610th tryptophan to phenylalanine.

[0160] Alternatively, the 277th serine position could be mutated to valine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to cysteine, the 548th arginine position to proline, the 610th tryptophan position to phenylalanine, the 611th glycine position to tryptophan, and the 613th serine position to either phenylalanine or tryptophan.

[0161] Alternatively, the 277th serine position could be mutated to valine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to cysteine, the 557th leucine position to phenylalanine, the 610th tryptophan position to phenylalanine, the 611th glycine position to tryptophan, and the 613th serine position to either phenylalanine or tryptophan.

[0162] Alternatively, the following mutations could be made: serine at position 277 could be mutated to valine; threonine at position 457 could be mutated to valine; histidine at position 460 could be mutated to tryptophan; glutamic acid at position 462 could be mutated to aspartic acid; arginine at position 548 could be mutated to proline; methionine at position 597 could be mutated to isoleucine; threonine at position 603 could be mutated to serine; and alanine at position 604 could be mutated to glutamic acid.

[0163] Alternatively, the following mutations could be made: serine at position 277 could be mutated to valine; threonine at position 457 to valine; histidine at position 460 to tryptophan; glutamic acid at position 462 to aspartic acid, proline, or cysteine; arginine at position 548 to proline; threonine at position 603 to serine; alanine at position 604 to glutamic acid; and tryptophan at position 610 to phenylalanine.

[0164] Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 460th histidine to tryptophan, the 462nd glutamic acid to cysteine, the 548th arginine to proline, the 610th tryptophan to phenylalanine, the 611th glycine to tryptophan, and the 613th serine to either phenylalanine or tryptophan.

[0165] Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 460th histidine to tryptophan, the 462nd glutamic acid to cysteine, the 557th leucine to phenylalanine, the 610th tryptophan to phenylalanine, the 611th glycine to tryptophan, and the 613th serine to either phenylalanine or tryptophan.

[0166] Alternatively, the 277th serine position could be mutated to valine, the 457th threonine position to isoleucine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to proline, the 557th leucine position to phenylalanine, the 603rd threonine position to serine, the 604th alanine position to glutamic acid, and the 612th methionine position to valine.

[0167] Alternatively, the following mutations could be made: serine at position 277 could be mutated to valine; threonine at position 457 to isoleucine; histidine at position 460 to tryptophan; glutamic acid at position 462 to proline; methionine at position 597 to isoleucine; threonine at position 603 to serine; alanine at position 604 to glutamic acid; and methionine at position 612 to valine.

[0168] Alternatively, the 277th serine position could be mutated to valine, the 457th threonine position to isoleucine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to aspartic acid or cysteine, the 557th leucine position to phenylalanine, the 603rd threonine position to serine, the 604th alanine position to glutamic acid, and the 612th methionine position to valine.

[0169] Alternatively, the following mutations can be made: serine at position 277 is mutated to valine, threonine at position 457 is mutated to isoleucine, histidine at position 460 is mutated to tryptophan, glutamic acid at position 462 is mutated to cysteine, methionine at position 597 is mutated to isoleucine, threonine at position 603 is mutated to serine, alanine at position 604 is mutated to glutamic acid, and methionine at position 612 is mutated to valine.

[0170] As one optional implementation, the mutant is a mutant with improved thermal stability, enzyme activity, and ability to produce galactooligosaccharides. The mutant is formed by mutating serine at position 277 of β-galactosidase Bgal1-3 to valine, threonine at position 457 to valine or isoleucine, histidine at position 460 to tryptophan, glutamate at position 462 to cysteine, tryptophan at position 610 to phenylalanine, and glycine at position 611 to tryptophan.

[0171] Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 460th histidine to tryptophan, the 462nd glutamic acid to cysteine, the 610th tryptophan to phenylalanine, the 611th glycine to tryptophan, and the 676th serine to cysteine.

[0172] Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 462nd glutamic acid to glutamine, the 548th arginine to proline, the 557th leucine to phenylalanine, the 603rd threonine to serine, and the 604th alanine to glutamic acid.

[0173] Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 462nd glutamic acid to glutamine, the 548th arginine to proline, the 597th methionine to isoleucine, the 603rd threonine to serine, and the 604th alanine to glutamic acid.

[0174] Alternatively, the 277th serine position could be mutated to valine, the 457th threonine position to isoleucine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to proline or cysteine, the 603rd threonine position to serine, the 604th alanine position to glutamic acid, and the 612th methionine position to valine.

[0175] Alternatively, the following mutations could be made: serine at position 277 could be mutated to valine; threonine at position 457 to valine; histidine at position 460 to tryptophan; glutamic acid at position 462 to proline or cysteine; arginine at position 548 to proline; methionine at position 597 to isoleucine; threonine at position 603 to serine; and alanine at position 604 to glutamic acid.

[0176] As one alternative implementation, the mutant is a mutant with increased β-galactosidase hydrolysis activity, wherein the mutant is obtained by mutating the 14th glutamic acid of mutant M9 to aspartic acid, arginine, glutamine, alanine or lysine.

[0177] Alternatively, the valine at position 19 could be mutated to tryptophan;

[0178] Alternatively, the 21st leucine residue could be mutated to alanine, glycine, glutamic acid, or valine.

[0179] Alternatively, the tyrosine residue at position 398 could be mutated to aspartic acid.

[0180] Or, the isoleucine at position 399 may be mutated to glutamine;

[0181] Alternatively, the tyrosine residue at position 450 could be mutated to phenylalanine;

[0182] Alternatively, the glycine at position 451 could be mutated into methionine, leucine, asparagine, or isoleucine;

[0183] Alternatively, the histidine at position 460 could be mutated to tyrosine or isoleucine;

[0184] Alternatively, the alanine at position 604 could be mutated to glutamic acid, lysine, aspartic acid, or proline.

[0185] Alternatively, the methionine at position 612 could be mutated into leucine or valine;

[0186] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, and glycine at position 451 could be mutated to asparagine.

[0187] Alternatively, glycine at position 451 could be mutated to methionine, and tyrosine at position 450 could be mutated to phenylalanine.

[0188] Alternatively, glycine at position 451 could be mutated to methionine, and isoleucine at position 509 could be mutated to valine.

[0189] Alternatively, glycine at position 451 could be mutated to methionine, and threonine at position 457 could be mutated to isoleucine or valine.

[0190] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and isoleucine at position 399 to glutamine.

[0191] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and tyrosine at position 450 to phenylalanine.

[0192] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and methionine at position 441 to isoleucine.

[0193] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and histidine at position 473 to tyrosine.

[0194] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and arginine at position 571 to histidine.

[0195] Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, and glutamic acid at position 115 to glycine, serine, or threonine.

[0196] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and serine at position 81 to asparagine.

[0197] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and histidine at position 460 to tyrosine.

[0198] Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, and alanine at position 604 to aspartic acid, glutamic acid, proline, glycine, or lysine.

[0199] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and valine at position 19 to tryptophan.

[0200] Alternatively, tyrosine at position 398 can be mutated to aspartic acid, glycine at position 451 to asparagine, and threonine at position 140 to arginine, lysine, leucine, or valine.

[0201] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and threonine at position 448 to cysteine ​​or glycine.

[0202] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and cysteine ​​at position 489 to glycine.

[0203] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and tyrosine at position 409 to serine, leucine, or threonine.

[0204] Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, and threonine at position 233 to aspartic acid, glutamine, or asparagine.

[0205] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and glutamine at position 237 to serine or lysine.

[0206] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and lysine at position 71 to threonine or tyrosine.

[0207] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and aspartic acid at position 6 to glutamine.

[0208] Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, and histidine at position 235 to alanine or aspartic acid.

[0209] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and isoleucine at position 490 to phenylalanine.

[0210] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and serine at position 141 to proline.

[0211] Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, and leucine at position 181 to lysine, aspartic acid, or arginine.

[0212] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and threonine at position 230 to aspartic acid or tyrosine.

[0213] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, or glycine at position 491 to isoleucine.

[0214] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and isoleucine at position 231 to leucine or valine.

[0215] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and phenylalanine at position 242 to glutamine.

[0216] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and isoleucine at position 229 to valine.

[0217] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and serine at position 613 to alanine.

[0218] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and valine at position 145 to isoleucine.

[0219] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and leucine at position 570 to tyrosine.

[0220] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and glutamine at position 371 to glycine.

[0221] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and glycine at position 608 to serine or glutamic acid.

[0222] Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, and proline at position 153 to leucine, aspartic acid, serine, or isoleucine.

[0223] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and glycine at position 121 to glutamine.

[0224] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and alanine at position 197 to phenylalanine.

[0225] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and lysine at position 57 to glycine.

[0226] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and aspartic acid at position 372 to tyrosine.

[0227] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and threonine at position 195 to arginine.

[0228] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and leucine at position 227 to isoleucine.

[0229] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and tryptophan at position 610 to phenylalanine.

[0230] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and tyrosine at position 38 to glycine.

[0231] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and arginine at position 609 to glycine.

[0232] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and aspartic acid at position 232 to isoleucine.

[0233] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and aspartic acid at position 196 to serine or threonine.

[0234] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and leucine at position 607 to glutamic acid.

[0235] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and proline at position 392 to valine.

[0236] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and tyrosine at position 572 to leucine.

[0237] Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, and methionine at position 612 may be mutated to leucine, valine, asparagine, tryptophan, isoleucine, glutamine, serine, tyrosine, phenylalanine, cysteine, histidine, or arginine.

[0238] Alternatively, tyrosine at position 398 can be mutated to aspartic acid, glycine at position 451 to asparagine, leucine at position 181 to lysine, and histidine at position 235 to alanine or aspartic acid.

[0239] Alternatively, tyrosine at position 398 can be mutated to aspartic acid, glycine at position 451 to asparagine, serine at position 141 to proline, and histidine at position 235 to alanine or aspartic acid.

[0240] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, leucine at position 181 to lysine, and serine at position 141 to proline.

[0241] Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and histidine at position 235 to aspartic acid, alanine, glycine, glutamic acid, isoleucine, leucine, phenylalanine, cysteine, methionine, valine, asparagine, proline, lysine, arginine, threonine, serine, threonine, tryptophan, or tyrosine.

[0242] Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, or leucine at position 181 to asparagine, lysine, methionine, histidine, aspartic acid, phenylalanine, isoleucine, cysteine, glycine, or alanine.

[0243] Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and serine at position 141 to proline, threonine, tyrosine, glutamine, or histidine.

[0244] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and arginine at position 571 to histidine.

[0245] Alternatively, tyrosine at position 398 can be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and glutamic acid at position 115 to glycine or aspartic acid.

[0246] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and serine at position 81 to asparagine.

[0247] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and histidine at position 460 to tyrosine.

[0248] Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and alanine at position 604 to aspartic acid, glutamic acid, proline, glycine, or lysine.

[0249] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and valine at position 19 to tryptophan.

[0250] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and threonine at position 233 to asparagine.

[0251] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and glutamine at position 237 to alanine.

[0252] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and isoleucine at position 231 to valine.

[0253] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and arginine at position 165 to lysine.

[0254] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and glutamic acid at position 239 to threonine or valine.

[0255] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and aspartic acid at position 200 to arginine.

[0256] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and aspartic acid at position 238 to glutamine or glutamic acid.

[0257] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and aspartic acid at position 232 to isoleucine.

[0258] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and glutamine at position 188 to glutamic acid or aspartic acid.

[0259] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and alanine at position 207 to glutamic acid.

[0260] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and aspartic acid at position 200 to glutamic acid.

[0261] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and isoleucine at position 229 to methionine.

[0262] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and threonine at position 234 to glutamine.

[0263] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and asparagine at position 173 to glycine.

[0264] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and threonine at position 304 to cysteine.

[0265] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and glutamine at position 237 to aspartic acid.

[0266] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and aspartic acid at position 232 to leucine.

[0267] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and glutamine at position 172 to alanine.

[0268] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and aspartic acid at position 440 to glutamic acid.

[0269] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and proline at position 275 to alanine.

[0270] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and threonine at position 230 to glutamic acid.

[0271] Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and isoleucine at position 229 to valine.

[0272] Alternatively, the tyrosine at position 398 can be mutated to aspartic acid, the glycine at position 451 to asparagine, the methionine at position 612 to leucine, the serine at position 141 to proline, the leucine at position 181 to lysine, and the histidine at position 235 to alanine or aspartic acid.

[0273] The present invention also provides a gene encoding the β-galactosidase mutant. Using the gene, the corresponding mutant can be obtained through recombinant expression.

[0274] The present invention also provides a biomaterial capable of expressing the β-galactosidase mutant.

[0275] Specifically, the biological material is a recombinant vector or recombinant bacteria containing a gene encoding the β-galactosidase mutant.

[0276] Optionally, the recombinant vector is an expression vector.

[0277] Optionally, when constructing the recombinant bacteria, Escherichia coli, yeast, Bacillus, or Lactobacillus can be selected as the host bacteria.

[0278] This invention seeks protection for the use of the mutant in the production of low-lactose or lactose-free dairy products, or in the preparation of products for the production of low-lactose or lactose-free dairy products.

[0279] Accordingly, the use of biological materials capable of expressing the mutant in the production of low-lactose or lactose-free dairy products, or in the preparation of products for the production of low-lactose or lactose-free dairy products, should also be within the scope of protection of this invention.

[0280] The present invention also claims protection for the use of mutants with enhanced ability to produce galactooligosaccharides in the production of low-lactose or lactose-free dairy products containing galactooligosaccharides, or in the preparation of products for the production of low-lactose or lactose-free dairy products containing galactooligosaccharides.

[0281] Accordingly, the use of mutants capable of expressing the enhanced ability to produce galactooligosaccharides in the production of low-lactose or lactose-free dairy products containing galactooligosaccharides, or in the preparation of products for the production of low-lactose or lactose-free dairy products containing galactooligosaccharides, should also be within the scope of protection of this invention.

[0282] The present invention has the following beneficial effects:

[0283] This invention provides a mutant of wild-type β-galactosidase Bgal1-3 with improved thermostability, enzyme activity, and / or galactooligosaccharide production compared to the parent. Simultaneously, this invention provides a mutant of β-galactosidase Bgal1-3, M9, with improved hydrolytic activity compared to the parent. The β-galactosidase mutants described in this invention can be used to convert lactose and produce low / lactose-free or galactooligosaccharide-rich dairy products, which is beneficial to the development of related industries for low / lactose-free or galactooligosaccharide-rich products. Attached Figure Description

[0284] Figure 1 The results show the thermal stability analysis of wild-type Bgal1-3 and its mutant M7; A in the figure shows the melting temperature (Tm) of wild-type Bgal1-3 and its mutant M7; B in the figure shows the half-life of wild-type Bgal1-3 and its mutant M7 at pasteurization temperature (63℃).

[0285] Figure 2 Thin-layer chromatography results of galactooligosaccharides (GOS), glucose, lactose, and galactose in the reaction system after different reaction times with milk as a substrate for wild-type Bgal1-3, Bgal1-3 single mutant Y497F, and mutant M7.

[0286] Figure 3Figure 1 shows the synthesis of galacto-oligosaccharides (GOS) in skim milk by wild-type Bgal1-3 and its mutant M7 at 63℃. Figure A shows the GOS yield and residual lactose in the reaction system obtained by anion exchange chromatography after different reaction times with milk as substrate by wild-type Bgal1-3 and its mutant M7. Figure B shows the TLC detection results of milk reaction samples of wild-type Bgal1-3 and its mutant M7.

[0287] Figure 4 The results show the lactose hydrolysis capacity analysis of wild-type Bgal1-3, Bgal1-3 mutant M9, and mutant M15. Detailed Implementation

[0288] 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.

[0289] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0290] Example 1: Mutation of β-galactosidase Bgal1-3

[0291] This invention uses wild-type β-galactosidase Bgal1-3 (GenBank ID AGK30046.1) as the parent. By performing saturation mutations on one or more amino acids at positions 277, 331, 414, 420, 421, 422, 457, 459, 460, 462, 464, 467, 492, 497, 503, 510, 512, 548, 557, 572, 597, 598, 603, 604, 609, 610, 611, 612, 613, 615, 624, and 676, mutants with improved thermostability, enzyme activity, and / or galactooligosaccharide production compared to the parent were obtained. Combinatorial mutations were obtained by sequentially mutating corresponding amino acid sites.

[0292] 1. Site-directed saturation mutation

[0293] The recombinant plasmid pET-28a-Tac-Bgal1-3 containing the gene sequence encoding β-galactosidase Bgal1-3 was used as a template to perform single-site and combined saturation mutagenesis of the above amino acid sites. The PCR primers used for site-directed saturation mutagenesis are shown in Table 1, and the PCR reaction system used is shown in Table 2.

[0294] Table 1. PCR primers used for site-directed saturation mutagenesis of Bgal1-3.

[0295]

[0296]

[0297]

[0298] Note: SDM is the codon 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).

[0299] Table 2. PCR reaction system used for saturation mutagenesis.

[0300]

[0301] 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 was 10 s / Kb), for a total of 30 cycles, and finally 72℃ for another 3 min extension.

[0302] PCR amplification products were detected by electrophoresis on a 1% agarose gel. After confirming the size of the amplified fragments by electrophoresis, the products were purified and recovered using the FastPure Gel DNA Extraction mini kit from Nanjing Novizan Biotechnology Co., Ltd. The recovered products were then used for homologous recombination using the OK Clon DNA Ligation Kit from Guangzhou Aiji Biotechnology Co., Ltd., reacting at 50℃ for 10 min. The reaction system was prepared according to the kit instructions. After ligation, 5 μL of the ligation product was mixed with 100 μL of L. coli DH5α competent cells, heat-shocked at 42℃ for 45 s, placed on ice for 2 min, and then 900 μL of LB medium was added. The transformants were cultured at 37℃ and 200 rpm for 1 h. The culture was then plated onto LB solid medium supplemented with 20 μM IPTG and 50 μg / mL kanamycin and incubated at 30℃ for 36 h to obtain a site-directed saturated mutant library, from which single colonies were screened.

[0303] 2. Preparation of crude enzyme solution of mutant

[0304] 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, and the supernatant was the crude enzyme solution of the mutant.

[0305] 3. Determination of half-inactivation temperature, GOS yield, and enzyme activity.

[0306] Half-deactivation temperature (T) 50 10 The temperature at which β-galactosidase loses 50% of its initial activity within 10 minutes is defined as the temperature at which it loses 50% of its initial activity. The method for determination is to incubate the enzyme (mutant) at different temperatures for 10 minutes and then measure the remaining enzyme activity.

[0307] 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℃.

[0308] 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, particle size 3.5 μm), 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 Table 3 below.

[0309] Table 3

[0310]

[0311] The detector potential waveform program is shown in Table 4 below:

[0312] Table 4

[0313] 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 -

[0314] 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 Table 5 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.

[0315] Table 5

[0316] 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

[0317] 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, carry the peak area of ​​each component into the standard curve of each component to quantify galactose, glucose and lactose respectively.

[0318] 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.

[0319] The enzyme activity was determined according to the method described in patent application CN119162154A.

[0320] Based on the results of half-inactivation temperature, GOS yield, and enzyme activity assays, this invention classifies the mutants according to their performance changes. Among them, mutants with improved thermal stability relative to the parent (wild-type Bgal1-3) and their half-inactivation temperatures (T...) are classified. 50 10 The results of the determination of enzyme activity are shown in Table 6.

[0321] As shown in Table 6, the single mutants H460W and E462D, as well as the combined mutants S277V-H460Q and S277V-T457V-H460W-E462P-R548P-L557F-T603S-A604D, showed improved thermal stability compared to their parents, and their enzyme activity did not decrease; in fact, it even increased.

[0322] Table 6. Bgal1-3 mutants with improved thermal stability, their half-inactivation temperatures, and enzyme activities.

[0323]

[0324]

[0325] Note: Taking E420R as an example, E420R is a single-point mutation mutant of Bgal1-3, and its corresponding mutation is that the E (glutamic acid) at position 420 of the Bgal1-3 amino acid sequence is changed to R (arginine); S277V-H460W is a combined mutation mutant of Bgal1-3, and its corresponding mutation is that the S (serine) at position 277 of the Bgal1-3 amino acid sequence is changed to V (valine), and its H (histidine) at position 460 is changed to W (tryptophan).

[0326] The results of enzyme activity determination for mutants with increased enzyme activity relative to the parent (wild-type Bgal1-3) are shown in Table 7.

[0327] Table 7. Mutants with increased enzyme activity relative to their parents and their enzyme activities.

[0328]

[0329] The results of the determination of GOS production and enzyme activity of mutants with enhanced ability to produce galactooligosaccharides relative to their parents (wild-type Bgal1-3) are shown in Table 8.

[0330] Table 8. Mutants with increased galactooligosaccharide production compared to their parents and their GOS production.

[0331]

[0332]

[0333]

[0334]

[0335]

[0336] The results of the determination of the mutants with improved thermal stability and enzyme activity relative to the parent (wild-type Bgal1-3) and their half-inactivation temperature and enzyme activity are shown in Table 9.

[0337] Table 9. Mutants with improved thermal stability and enzyme activity compared to their parents, along with their half-inactivation temperatures and enzyme activities.

[0338]

[0339] The results of measuring the heat stability and galactooligosaccharide production capacity of mutants that are improved relative to the parents (wild-type Bgal1-3), as well as their half-inactivation temperature and GOS production, are shown in Table 10.

[0340] Table 10 Mutants with improved thermal stability and GOS production capacity compared to their parents, along with their half-inactivation temperatures and GOS yields.

[0341]

[0342]

[0343]

[0344] The results of measuring the heat stability, enzyme activity, and galactooligosaccharide production capacity of mutants relative to the parent (wild-type Bgal1-3), as well as their half-inactivation temperature, enzyme activity, and GOS production, are shown in Table 11.

[0345] Table 11. Mutants with improved thermal stability, enzyme activity, and GOS production capacity compared to their parents, and the results of performance determination.

[0346]

[0347]

[0348] As shown in Table 11, among the combined mutants, S277V-T457V-H460W-E462C-W610F-G611W-S676C exhibits relatively outstanding thermostability, enzyme activity, and GOS yield. 50 10 The temperature was 67℃, which is 18.8℃ higher than that of the wild type. The total activity was 26.7% higher than that of the wild type, and the GOS yield was 64.4%, which is 42.2% higher than that of the wild type. Therefore, it was named mutant M7 and further analysis was conducted.

[0349] Furthermore, based on the wild-type Bgal1-3 mutant M9 disclosed in patent application CN119162154A, this invention obtained a mutant with increased enzyme activity relative to M9 by performing saturation mutations on specific amino acid sites.

[0350] A site-directed mutagenesis library was constructed using the recombinant plasmid pET-28a-Tac-Bgal1-3-M9 containing the gene sequence encoding mutant M9 as a template, and enzyme activity was measured (enzyme activity measurement temperature was 63℃). The construction method is as described in Part 1 of this embodiment, and the PCR primers used are shown in Table 12.

[0351] Table 12 PCR primers used for site-directed mutagenesis of M9.

[0352]

[0353]

[0354]

[0355]

[0356] The results of enzyme activity assays for mutants with increased enzyme activity compared to the parent (mutant M9) are shown in Table 13.

[0357] Table 13 Mutants with increased enzyme activity compared to mutant M9 and their enzyme activities.

[0358]

[0359]

[0360]

[0361]

[0362]

[0363] Among the mutants shown in Table 13, the M9-Y398D-G451N-M612L-S141P-L181K-H235D mutant showed a significant increase in enzyme activity. This mutant was named M15 and further analyzed. The half-inactivation temperature and crude enzyme activity at 63℃ for mutant M15 are shown in Table 14.

[0364] Table 14. Half-inactivation temperature and crude enzyme activity of mutant M15 at 63℃.

[0365]

[0366] Example 2: Expression, purification, and thermostability analysis of wild-type Bgal1-3 and its mutant M7

[0367] The expression, purification, and thermostability analysis methods for wild-type Bgal1-3 and mutant M7 were based on the patent application with publication number CN119162154A. The purified enzymes of wild-type Bgal1-3 and mutant M7 were obtained using wild-type Bgal1-3 and mutant M7 expression strains, and their thermostability was analyzed.

[0368] Thermostability analysis results of wild-type Bgal1-3 and mutant M7 are as follows: Figure 1 As shown; Figure 1 In this figure, A represents the measured melting temperature (Tm). Figure 1 The value of B in the figure represents the half-life determined at pasteurization temperature (63℃). The Tm of mutant M7 was measured to be 73.6℃, ​​which is 20.5℃ higher than that of wild-type Bgal1-3. Figure 1 (A) The half-life of wild-type Bgal1-3 is only 0.78 min, while the half-life of mutant M7 is 269.22 min. Figure 1The B in Bgal1-3 is 345.2 times that of Bgal1-3. This indicates that the mutant M7 has significantly improved thermal stability and can be used at higher temperatures.

[0369] Example 3: Determination of GOS content in milk

[0370] Following the method described in patent application CN119162154A, wild-type Bgal1-3, Bgal1-3 single mutant Y497F, and mutant M7 were expressed and purified using expression strains to obtain the corresponding purified enzymes. Using the purified enzymes from each parent and mutant as test enzymes, and commercially available skim milk as a substrate, their ability to synthesize GOS in dairy products was tested.

[0371] Equal volumes of skim milk were taken, and purified enzymes with a final concentration of 3 U / mL were added to each. After mixing, the mixture was reacted at 40°C. Samples were taken at intervals during the reaction. The changes in the sugar content (GOS, glucose, lactose, and galactose) in the system were detected by thin-layer chromatography (TLC). Subsequently, the GOS yield in milk was quantitatively detected by high-performance anion chromatography to analyze the ability of the above enzymes to synthesize GOS in dairy products.

[0372] The formula for calculating the GOS content in milk is: GOS content (g / L) = GOS yield (%) × 50 (g / L).

[0373] 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.

[0374] Thin-layer chromatography (TLC) results of GOS, glucose, lactose, and galactose in wild-type Bgal1-3, Bgal1-3 single mutant Y497F, and mutant M7 after reacting with milk as a substrate for different time periods are shown below. Figure 2 As shown. By Figure 2 It can be seen that, compared with wild-type Bgal1-3, single mutant Y497F and mutant M7 can synthesize more GOS while consuming about 90% of lactose.

[0375] Using commercially available skim milk as a substrate, the efficacy of wild-type Bgal1-3 and its mutant M7 in preparing GOS-rich low-lactose milk under pasteurization conditions (63℃ for 30 min) was evaluated. The enzyme dosage was 10 U / mL, and GOS synthesis and lactose consumption within 1 hour of reaction were analyzed. The synthesis of galactooligosaccharides (GOS) in skim milk by wild-type Bgal1-3 and its mutant M7 at 63℃ is shown below. Figure 3 As shown; Figure 3In this context, A represents the yield of GOS and the amount of lactose remaining in the reaction system obtained by anion exchange chromatography after different reaction times with wild-type Bgal1-3 and its mutant M7 using milk as a substrate. Figure 3 In the figure, B represents the TLC detection result of milk reaction samples from wild-type Bgal1-3 and its mutant M7. Figure 3 It was found that mutant M7 could still function normally in milk at 63℃, while wild-type Bgal1-3 stopped synthesizing GOS or consuming lactose after 5 minutes of reaction. Wild-type Bgal1-3 reached a GOS production of 7 g / L in milk at 63℃ and then stopped consuming lactose when the lactose conversion rate was approximately 18%. Mutant M7, however, reached its highest GOS concentration of 21.8 g / L after 10 minutes of reaction, with a lactose conversion rate of 80%; after 30 minutes, the lactose conversion rate reached 90%, and the synthesized GOS concentration reached 20.1 g / L. These results indicate that wild-type Bgal1-3, due to its poor thermal stability, cannot continuously convert lactose in milk to GOS under pasteurization conditions (63℃, 30 minutes), while mutant M7 can be used to prepare low-lactose milk rich in GOS, and can combine pasteurization and GOS milk preparation steps into one, shortening the production cycle and showing good potential for industrial application.

[0376] Table 15 shows the quantitative GOS yield in the reaction system obtained by anion exchange chromatography after different β-galactosidases and their mutants reacted with milk as a substrate for different time periods, as well as the calculated GOS content and lactose conversion rate in the milk.

[0377] Table 15. GOS yield and lactose conversion rate of wild-type and mutant β-galactosidase.

[0378]

[0379] Example 4: Determination of lactose hydrolysis capacity

[0380] Following the method described in patent application CN119162154A, wild-type Bgal1-3, Bgal1-3 mutant M9, and mutant M15 were expressed and purified using expression strains to obtain the corresponding purified enzymes. Using the purified enzymes from each parent and mutant as test enzymes, and commercially available skim milk as a substrate, the sugar content in the system was detected by thin-layer chromatography (TLC). The hydrolytic capacity of Bgal1-3 mutant M9 and mutant M15, as well as the commercial enzyme Maxilact LGi 5000, for lactose in dairy products was determined.

[0381] Equal volumes of skim milk were taken, and purified enzymes with a final concentration of 20 U / mL were added to each. After mixing, the mixture was reacted at 63℃, and the sugar components in the system were detected by thin-layer chromatography. The hydrolytic ability of the enzymes on lactose was analyzed, and the results are as follows. Figure 4 As shown; the markers used in the figure are mixed solutions of glucose, galactose, and lactose with a concentration of 0.5 g / 100 mL. (Combined) Figure 4 It can be seen that under the above conditions, at 63℃ for 20 minutes, the lactose residue in the hydrolyzed milk sample of mutant M15 is less than 0.5g / 100mL, which meets the lactose-free standard.

[0382] 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 β-galactosidase mutant, characterized in that, The mutant is obtained by mutating one or more of the following amino acids in the amino acid sequence of β-galactosidase Bgal1-3: 277, 331, 414, 420, 421, 422, 457, 459, 460, 462, 464, 467, 492, 497, 503, 510, 512, 548, 557, 572, 597, 598, 603, 604, 609, 610, 611, 612, 613, 615, 624, and 676. Alternatively, it can be obtained by mutating one or more amino acids at positions 14, 19, 21, 38, 49, 57, 60, 61, 71, 115, 121, 140, 141, 145, 153, 181, 195, 196, 197, 227, 229, 230, 231, 232, 235, 237, 242, 398, 409, 411, 416, 448, 450, 451, 457, 460, 473, 489, 490, 491, 509, 570, 571, 572, 604, 607, 608, 609, 610, 611, 612, and 613 of the β-galactosidase Bgal1-3 mutant M9. The wild-type β-galactosidase Bgal1-3 has the GenBank accession number AGK30046.

1. The mutant M9 was obtained by mutating the following amino acid sequences of the wild-type β-galactosidase Bgal1-3: glutamine at position 80 to glycine, serine at position 277 to valine, valine at position 378 to threonine, tyrosine at position 398 to cysteine, glutamate at position 462 to glutamine, lysine at position 578 to arginine, phenylalanine at position 633 to leucine, serine at position 676 to phenylalanine, and arginine at position 689 to leucine.

2. The mutant according to claim 1, characterized in that, The mutant is a β-galactosidase mutant with improved thermal stability; the mutant is formed by mutating glutamic acid at position 420 of the amino acid sequence of β-galactosidase Bgal1-3 to lysine or arginine. Alternatively, the histidine at position 460 could be mutated to tryptophan; Alternatively, the glutamic acid at position 462 could be mutated to asparagine, aspartic acid, cysteine, valine, or proline. Alternatively, the threonine at position 574 could be mutated to lysine; Alternatively, proline at position 598 could be mutated to lysine; Alternatively, the histidine at position 460 may be mutated to tryptophan, and the glutamic acid at position 462 may be mutated to glutamine, glycine, asparagine, aspartic acid, histidine, proline, cysteine, or valine. Alternatively, the serine at position 277 could be mutated to valine, and the histidine at position 460 to tryptophan, glutamine, or cysteine. Alternatively, the histidine at position 460 could be mutated to tryptophan, the glutamic acid at position 462 to cysteine, and the serine at position 676 to phenylalanine. Alternatively, the 277th serine can be mutated to valine, the 457th threonine to isoleucine, the 460th histidine to tryptophan, the 462nd glutamic acid to aspartic acid, the 603rd threonine to serine, the 604th alanine to aspartic acid, and the 612th methionine to valine. Alternatively, the following mutations can be made: serine at position 277 can be mutated to valine; threonine at position 457 can be mutated to valine; histidine at position 460 can be mutated to tryptophan; glutamic acid at position 462 can be mutated to aspartic acid or proline; arginine at position 548 can be mutated to proline; leucine at position 557 can be mutated to phenylalanine; threonine at position 603 can be mutated to serine; and alanine at position 604 can be mutated to aspartic acid.

3. The mutant according to claim 1, characterized in that, The mutant is a β-galactosidase mutant with enhanced enzyme activity, wherein the mutant is formed by mutating the 457th threonine of β-galactosidase Bgal1-3 to isoleucine or valine. Alternatively, the histidine at position 460 could be mutated to glutamine, serine, glycine, isoleucine, phenylalanine, or tyrosine. Alternatively, the alanine at position 467 could be mutated to threonine; Alternatively, the arginine at position 548 could be mutated to asparagine, glutamine, tyrosine, or phenylalanine. Alternatively, the threonine at position 603 could be mutated to serine; Alternatively, the alanine at position 604 could be mutated to aspartic acid, glycine, glutamic acid, lysine, or phenylalanine. Alternatively, arginine at position 548 could be mutated to tyrosine, and methionine at position 597 to isoleucine. Alternatively, alanine at position 604 could be mutated to aspartic acid, and arginine at position 609 to leucine. Alternatively, threonine at position 603 could be mutated to serine, and alanine at position 604 could be mutated to aspartic acid. Alternatively, threonine at position 603 could be mutated to serine, alanine at position 604 to aspartic acid, and glycine at position 624 to aspartic acid. Alternatively, the 277th serine can be mutated to valine, the 457th threonine to isoleucine, the 462nd glutamic acid to glutamine, the 603rd threonine to serine, the 604th alanine to aspartic acid, and the 612th methionine to valine.

4. The mutant according to claim 1, characterized in that, The mutant is a β-galactosidase mutant with enhanced ability to produce galactooligosaccharides, wherein the mutant is formed by mutating tryptophan at position 331 of β-galactosidase Bgal1-3 to phenylalanine. Alternatively, the phenylalanine at position 414 could be mutated to tyrosine or tryptophan; Alternatively, the glutamic acid at position 420 could be mutated to alanine, phenylalanine, glycine, asparagine, proline, glutamine, serine, threonine, tryptophan, or tyrosine. Alternatively, the leucine at position 421 could be mutated to phenylalanine, proline, arginine, tryptophan, or histidine; Alternatively, the glutamic acid at position 462 could be mutated to arginine; Alternatively, tryptophan at position 492 could be mutated to phenylalanine or tyrosine; Alternatively, the tyrosine residue at position 497 could be mutated to phenylalanine, tryptophan, aspartic acid, glutamine, histidine, isoleucine, asparagine, serine, threonine, glycine, alanine, methionine, cysteine, leucine, or valine. Alternatively, the phenylalanine at position 503 could be mutated to tyrosine, tryptophan, asparagine, glutamine, histidine, serine, alanine, or cysteine. Alternatively, the cysteine ​​at position 510 could be mutated to tyrosine; Alternatively, the histidine at position 512 could be mutated to tyrosine; Or, the arginine at position 548 could be mutated to tryptophan; Alternatively, the leucine at position 557 could be mutated to phenylalanine, tryptophan, or histidine. Alternatively, the tyrosine residue at position 572 could be mutated to glycine, serine, alanine, isoleucine, or leucine. Or, the methionine at position 597 could be mutated to isoleucine; Alternatively, tryptophan at position 610 could be mutated to tyrosine, phenylalanine, glycine, serine, alanine, proline, isoleucine, or leucine. Alternatively, the glycine at position 611 could be mutated to tryptophan, tyrosine, phenylalanine, serine, alanine, isoleucine, leucine, or glutamic acid. Alternatively, the serine at position 613 could be mutated to asparagine, aspartic acid, histidine, glutamic acid, methionine, tyrosine, phenylalanine, or glycine. Alternatively, the glutamic acid at position 615 could be mutated to isoleucine; Alternatively, the phenylalanine at position 414 could be mutated to tyrosine, and the tyrosine at position 497 to phenylalanine, isoleucine, or valine. Alternatively, the phenylalanine at position 414 could be mutated to tyrosine, and the phenylalanine at position 503 could be mutated to tyrosine. Alternatively, glutamic acid at position 420 could be mutated to glutamine, and leucine at position 421 could be mutated to tryptophan or arginine. Alternatively, the glutamic acid at position 420 could be mutated to glutamine, tryptophan, phenylalanine, tyrosine, or histidine, and the leucine at position 557 could be mutated to phenylalanine. Alternatively, leucine at position 421 could be mutated to tryptophan, arginine, tyrosine, histidine, or phenylalanine, and leucine at position 557 could be mutated to phenylalanine. Alternatively, leucine at position 421 could be mutated to phenylalanine, and tryptophan at position 610 could be mutated to phenylalanine. Alternatively, leucine at position 421 could be mutated to phenylalanine, and glycine at position 611 to tryptophan; Alternatively, leucine at position 421 could be mutated to phenylalanine, and serine at position 613 could be mutated to leucine. Alternatively, leucine at position 421 could be mutated to tryptophan, and glycine at position 611 could be mutated to tryptophan; Alternatively, leucine at position 421 could be mutated to tryptophan, and tryptophan at position 610 could be mutated to phenylalanine; Alternatively, glycine at position 422 could be mutated to tryptophan, phenylalanine, tyrosine, or histidine, and leucine at position 557 could be mutated to phenylalanine. Alternatively, threonine at position 457 could be mutated to valine, and methionine at position 597 to isoleucine. Alternatively, threonine at position 457 could be mutated to valine, and leucine at position 557 to phenylalanine. Alternatively, threonine at position 457 could be mutated to valine, and tryptophan at position 610 to phenylalanine. Alternatively, tyrosine at position 497 could be mutated to isoleucine or valine, and phenylalanine at position 503 could be mutated to tyrosine. Alternatively, arginine at position 548 could be mutated to tyrosine, and tryptophan at position 610 could be mutated to phenylalanine. Alternatively, arginine at position 548 could be mutated to tyrosine, and glycine at position 611 to tryptophan; Alternatively, arginine at position 548 could be mutated to tryptophan, and leucine at position 557 to phenylalanine. Alternatively, arginine at position 548 could be mutated to tryptophan, and tryptophan at position 610 could be mutated to phenylalanine. Alternatively, arginine at position 548 could be mutated to tryptophan, and glycine at position 611 could be mutated to tryptophan; Alternatively, leucine at position 557 could be mutated to phenylalanine, and tryptophan at position 610 could be mutated to phenylalanine. Alternatively, leucine at position 557 could be mutated to phenylalanine, and glycine at position 611 could be mutated to phenylalanine or tryptophan. Alternatively, leucine at position 557 could be mutated to phenylalanine, and methionine at position 612 could be mutated to leucine. Alternatively, leucine at position 557 could be mutated to phenylalanine, and serine at position 613 could be mutated to leucine, tryptophan, or phenylalanine. Alternatively, leucine at position 557 could be mutated to phenylalanine, and glutamic acid at position 615 to isoleucine. Alternatively, leucine at position 557 could be mutated to tryptophan, and tryptophan at position 610 could be mutated to phenylalanine. Alternatively, leucine at position 557 could be mutated to tryptophan, and glycine at position 611 could be mutated to tryptophan; Alternatively, tyrosine at position 572 could be mutated to isoleucine or leucine, and tryptophan at position 610 could be mutated to phenylalanine. Alternatively, tyrosine at position 572 could be mutated to isoleucine or leucine, and glycine at position 611 could be mutated to tryptophan. Alternatively, the methionine at position 597 could be mutated to isoleucine, and the methionine at position 612 to leucine. Alternatively, tryptophan at position 610 could be mutated to phenylalanine, and glycine at position 611 could be mutated to tryptophan or phenylalanine. Alternatively, tryptophan at position 610 could be mutated to phenylalanine, and serine at position 613 could be mutated to leucine, tryptophan, or phenylalanine. Alternatively, tryptophan at position 610 could be mutated to phenylalanine, and glutamic acid at position 615 could be mutated to isoleucine. Alternatively, glycine at position 611 could be mutated to tryptophan, and serine at position 613 could be mutated to leucine, tryptophan, or phenylalanine. Alternatively, glycine at position 611 could be mutated to tryptophan, and glutamic acid at position 615 could be mutated to isoleucine. Alternatively, phenylalanine at position 414 could be mutated to tyrosine, tyrosine at position 497 to isoleucine or valine, and phenylalanine at position 503 to tyrosine. Alternatively, glutamic acid at position 420 could be mutated to glutamine, leucine at position 421 to tryptophan or arginine, and leucine at position 557 to phenylalanine. Alternatively, leucine at position 421 could be mutated to tryptophan, glutamine at position 464 to lysine, and leucine at position 557 to phenylalanine. Alternatively, leucine at position 421 could be mutated to tryptophan, alanine at position 467 to cysteine ​​or isoleucine, and leucine at position 557 to phenylalanine. Alternatively, leucine at position 421 could be mutated to arginine, glutamine at position 464 to lysine, and leucine at position 557 to phenylalanine. Alternatively, leucine at position 421 could be mutated to arginine, alanine at position 467 to cysteine ​​or isoleucine, and leucine at position 557 to phenylalanine. Alternatively, leucine at position 421 could be mutated to phenylalanine or tryptophan, tryptophan at position 610 to phenylalanine, and glycine at position 611 to tryptophan. Alternatively, arginine at position 548 could be mutated to tryptophan, tryptophan at position 610 to phenylalanine, and glycine at position 611 to tryptophan. Alternatively, leucine at position 557 could be mutated to tryptophan or phenylalanine, tryptophan at position 610 to phenylalanine, and glycine at position 611 to tryptophan. Alternatively, leucine at position 557 could be mutated to phenylalanine, threonine at position 603 to serine, and alanine at position 604 to aspartic acid. Alternatively, tyrosine at position 572 could be mutated to isoleucine or leucine, tryptophan at position 610 to phenylalanine, and glycine at position 611 to tryptophan. Alternatively, methionine at position 597 could be mutated to isoleucine, threonine at position 603 to serine, and alanine at position 604 to aspartic acid. Alternatively, threonine at position 603 could be mutated to serine, alanine at position 604 to aspartic acid, and tryptophan at position 610 to phenylalanine. Alternatively, tryptophan at position 610 could be mutated to phenylalanine, glycine at position 611 to tryptophan, and serine at position 613 to leucine, phenylalanine, tryptophan, or isoleucine. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to proline, and the methionine at position 597 to isoleucine. Alternatively, leucine at position 421 could be mutated to tryptophan, glutamine at position 464 to lysine, alanine at position 467 to glutamic acid, cysteine, or isoleucine, and leucine at position 557 to phenylalanine. Alternatively, leucine at position 421 may be mutated to arginine, glutamine at position 464 to lysine, alanine at position 467 to glutamic acid, cysteine, or isoleucine, and leucine at position 557 to phenylalanine. Alternatively, leucine at position 557 could be mutated to phenylalanine, tryptophan at position 610 to phenylalanine, glycine at position 611 to tryptophan, and serine at position 613 to tryptophan. Alternatively, valine at position 459 could be mutated to serine, leucine at position 557 to phenylalanine, threonine at position 603 to serine, alanine at position 604 to aspartic acid, and glycine at position 624 to aspartic acid. Alternatively, valine at position 459 could be mutated to serine, methionine at position 597 to isoleucine, threonine at position 603 to serine, alanine at position 604 to aspartic acid, and glycine at position 624 to aspartic acid. Alternatively, the 277th serine position could be mutated to valine, the 457th threonine position to isoleucine, the 462nd glutamic acid position to glutamine, the 597th methionine position to isoleucine, the 603rd threonine position to serine, the 604th alanine position to aspartic acid, and the 612th methionine position to valine. Alternatively, the following can be obtained by mutating serine at position 277 to valine, threonine at position 457 to isoleucine, glutamic acid at position 462 to glutamine, leucine at position 557 to phenylalanine, threonine at position 603 to serine, alanine at position 604 to aspartic acid, and methionine at position 612 to valine.

5. The mutant according to claim 1, characterized in that, The mutant is a β-galactosidase mutant with improved thermostability and enzyme activity. The mutant is formed by mutating alanine at position 604 of β-galactosidase Bgal1-3 to aspartic acid. Or mutate arginine at position 548 to proline; Alternatively, arginine at position 548 could be mutated to proline, and methionine at position 612 to leucine. Alternatively, threonine at position 457 could be mutated to valine or isoleucine, arginine at position 548 to proline, and methionine at position 612 to leucine. Alternatively, the following can be obtained by mutating serine at position 277 to valine, threonine at position 457 to valine, histidine at position 460 to tryptophan, glutamic acid at position 462 to cysteine, leucine at position 557 to phenylalanine, threonine at position 603 to serine, and alanine at position 604 to aspartic acid.

6. The mutant according to claim 1, characterized in that, The mutant is a β-galactosidase mutant with improved thermal stability and ability to produce galactooligosaccharides. The mutant is formed by mutating the glutamic acid at position 420 of β-galactosidase Bgal1-3 to lysine or arginine. Alternatively, the glutamic acid at position 462 could be mutated to histidine; Or, the methionine at position 597 could be mutated to isoleucine; Or, the serine at position 613 could be mutated to leucine; Alternatively, arginine at position 548 could be mutated to proline, and methionine at position 597 to isoleucine. Alternatively, arginine at position 548 could be mutated to proline, and tryptophan at position 610 could be mutated to phenylalanine. Alternatively, the serine at position 277 could be mutated to valine, the glutamic acid at position 462 to glutamine, and the leucine at position 557 to phenylalanine. Alternatively, the serine at position 277 could be mutated to valine, the glutamic acid at position 462 to glutamine, and the methionine at position 597 to isoleucine. Alternatively, the serine at position 277 could be mutated to valine, the glutamate at position 462 to glutamine, and the tryptophan at position 610 to phenylalanine. Alternatively, threonine at position 457 could be mutated to valine, arginine at position 548 to proline, and methionine at position 597 to isoleucine. Alternatively, threonine at position 457 could be mutated to valine, arginine at position 548 to proline, and tryptophan at position 610 to phenylalanine. Alternatively, arginine at position 548 could be mutated to proline, leucine at position 557 to phenylalanine, and methionine at position 612 to leucine. Alternatively, arginine at position 548 could be mutated to proline, methionine at position 597 to isoleucine, and methionine at position 612 to leucine. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to aspartic acid, and the leucine at position 557 to phenylalanine or isoleucine. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to aspartic acid, and the tryptophan at position 610 to phenylalanine. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to cysteine, and the leucine at position 557 to phenylalanine. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to cysteine, and the methionine at position 597 to isoleucine. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to cysteine, and the tryptophan at position 610 to phenylalanine. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to cysteine, and the glycine at position 611 to tryptophan. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to proline, and the leucine at position 557 to phenylalanine. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to proline, and the tryptophan at position 610 to phenylalanine. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamate at position 462 to glutamine, and the leucine at position 557 to phenylalanine. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamate at position 462 to glutamine, and the methionine at position 597 to isoleucine. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamate at position 462 to glutamine, and the tryptophan at position 610 to phenylalanine. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamate at position 462 to glutamine, and the glycine at position 611 to tryptophan. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to cysteine, the arginine at position 548 to tyrosine, glutamine, asparagine, phenylalanine, or proline, and the tryptophan at position 610 to phenylalanine. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to cysteine, the alanine at position 604 to phenylalanine, and the tryptophan at position 610 to phenylalanine. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to cysteine, the tryptophan at position 610 to phenylalanine, and the glycine at position 611 to tryptophan. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to glutamine, the leucine at position 557 to phenylalanine, and the alanine at position 604 to phenylalanine. Alternatively, the 277th serine position could be mutated to valine, the 460th histidine position to tryptophan, the 462nd glutamate position to glutamine, the 597th methionine position to isoleucine, and the 604th alanine position to phenylalanine. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamate at position 462 to glutamine, the alanine at position 604 to phenylalanine, and the tryptophan at position 610 to phenylalanine. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to glutamine, the alanine at position 604 to phenylalanine, and the glycine at position 611 to tryptophan. Alternatively, the 277th serine position could be mutated to valine, the 462nd glutamic acid position to glutamine, the 557th leucine position to phenylalanine, the 603rd threonine position to serine, and the 604th alanine position to phenylalanine. Alternatively, the 277th serine position could be mutated to valine, the 462nd glutamic acid position to glutamine, the 597th methionine position to isoleucine, the 603rd threonine position to serine, and the 604th alanine position to phenylalanine. Alternatively, the 277th serine position could be mutated to valine, the 462nd glutamic acid position to glutamine, the 603rd threonine position to serine, the 604th alanine position to phenylalanine, and the 610th tryptophan position to phenylalanine. Alternatively, the 277th serine position could be mutated to valine, the 457th threonine position to isoleucine or valine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to cysteine, the 548th arginine position to proline, and the 610th tryptophan position to phenylalanine. Alternatively, the serine at position 277 could be mutated to valine, the threonine at position 457 to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to cysteine, the arginine at position 548 to proline, and the glycine at position 611 to tryptophan. Alternatively, the 277th serine position could be mutated to valine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to cysteine, the 610th tryptophan position to phenylalanine, the 611th glycine position to tryptophan, and the 613th serine position to either phenylalanine or tryptophan. Alternatively, the 277th serine position could be mutated to valine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to cysteine, the 548th arginine position to proline, phenylalanine, tyrosine, asparagine, or glutamine, the 610th tryptophan position to phenylalanine, and the 611th glycine position to tryptophan. Alternatively, the serine at position 277 could be mutated to valine, the histidine at position 460 to tryptophan, the glutamic acid at position 462 to cysteine, the alanine at position 604 to phenylalanine, aspartic acid, glutamic acid, lysine, or glycine, the tryptophan at position 610 to phenylalanine, and the glycine at position 611 to tryptophan. Alternatively, the 277th serine position could be mutated to valine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to cysteine, the 610th tryptophan position to phenylalanine, the 611th glycine position to tryptophan, and the 676th serine position to tryptophan. Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 460th histidine to tryptophan, the 462nd glutamic acid to cysteine, the 548th arginine to proline, the 610th tryptophan to phenylalanine, and the 611th glycine to tryptophan. Alternatively, the 277th serine position could be mutated to valine, the 457th threonine position to valine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to cysteine, the 548th arginine position to proline, the 610th tryptophan position to phenylalanine, and the 612th methionine position to leucine. Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 460th histidine to tryptophan, the 462nd glutamic acid to cysteine, the 557th leucine to phenylalanine, the 610th tryptophan to phenylalanine, and the 611th glycine to tryptophan. Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 460th histidine to tryptophan, the 462nd glutamic acid to cysteine, the 610th tryptophan to phenylalanine, the 611th glycine to tryptophan, and the 613th serine to either phenylalanine or tryptophan. Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 460th histidine to tryptophan, the 462nd glutamic acid to cysteine, the 610th tryptophan to phenylalanine, the 611th glycine to tryptophan, and the 676th serine to phenylalanine, glutamic acid, asparagine, or threonine. Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 462nd glutamic acid to glutamine, the 548th arginine to proline, the 603rd threonine to serine, the 604th alanine to glutamic acid, and the 610th tryptophan to phenylalanine. Alternatively, the 277th serine position could be mutated to valine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to cysteine, the 548th arginine position to proline, the 610th tryptophan position to phenylalanine, the 611th glycine position to tryptophan, and the 613th serine position to either phenylalanine or tryptophan. Alternatively, the 277th serine position could be mutated to valine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to cysteine, the 557th leucine position to phenylalanine, the 610th tryptophan position to phenylalanine, the 611th glycine position to tryptophan, and the 613th serine position to either phenylalanine or tryptophan. Alternatively, the following mutations could be made: serine at position 277 could be mutated to valine; threonine at position 457 could be mutated to valine; histidine at position 460 could be mutated to tryptophan; glutamic acid at position 462 could be mutated to aspartic acid; arginine at position 548 could be mutated to proline; methionine at position 597 could be mutated to isoleucine; threonine at position 603 could be mutated to serine; and alanine at position 604 could be mutated to glutamic acid. Alternatively, the following mutations could be made: serine at position 277 could be mutated to valine; threonine at position 457 to valine; histidine at position 460 to tryptophan; glutamic acid at position 462 to aspartic acid, proline, or cysteine; arginine at position 548 to proline; threonine at position 603 to serine; alanine at position 604 to glutamic acid; and tryptophan at position 610 to phenylalanine. Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 460th histidine to tryptophan, the 462nd glutamic acid to cysteine, the 548th arginine to proline, the 610th tryptophan to phenylalanine, the 611th glycine to tryptophan, and the 613th serine to either phenylalanine or tryptophan. Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 460th histidine to tryptophan, the 462nd glutamic acid to cysteine, the 557th leucine to phenylalanine, the 610th tryptophan to phenylalanine, the 611th glycine to tryptophan, and the 613th serine to either phenylalanine or tryptophan. Alternatively, the 277th serine position could be mutated to valine, the 457th threonine position to isoleucine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to proline, the 557th leucine position to phenylalanine, the 603rd threonine position to serine, the 604th alanine position to glutamic acid, and the 612th methionine position to valine. Alternatively, the following mutations could be made: serine at position 277 could be mutated to valine; threonine at position 457 to isoleucine; histidine at position 460 to tryptophan; glutamic acid at position 462 to proline; methionine at position 597 to isoleucine; threonine at position 603 to serine; alanine at position 604 to glutamic acid; and methionine at position 612 to valine. Alternatively, the 277th serine position could be mutated to valine, the 457th threonine position to isoleucine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to aspartic acid or cysteine, the 557th leucine position to phenylalanine, the 603rd threonine position to serine, the 604th alanine position to glutamic acid, and the 612th methionine position to valine. Alternatively, the following mutations can be made: serine at position 277 is mutated to valine, threonine at position 457 is mutated to isoleucine, histidine at position 460 is mutated to tryptophan, glutamic acid at position 462 is mutated to cysteine, methionine at position 597 is mutated to isoleucine, threonine at position 603 is mutated to serine, alanine at position 604 is mutated to glutamic acid, and methionine at position 612 is mutated to valine.

7. The mutant according to claim 1, characterized in that, The mutant is a β-galactosidase mutant with improved thermal stability, enzyme activity, and ability to produce galactooligosaccharides. The mutant is formed by mutating serine at position 277 of β-galactosidase Bgal1-3 to valine, threonine at position 457 to valine or isoleucine, histidine at position 460 to tryptophan, glutamate at position 462 to cysteine, tryptophan at position 610 to phenylalanine, and glycine at position 611 to tryptophan. Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 460th histidine to tryptophan, the 462nd glutamic acid to cysteine, the 610th tryptophan to phenylalanine, the 611th glycine to tryptophan, and the 676th serine to cysteine. Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 462nd glutamic acid to glutamine, the 548th arginine to proline, the 557th leucine to phenylalanine, the 603rd threonine to serine, and the 604th alanine to glutamic acid. Alternatively, the 277th serine can be mutated to valine, the 457th threonine to valine, the 462nd glutamic acid to glutamine, the 548th arginine to proline, the 597th methionine to isoleucine, the 603rd threonine to serine, and the 604th alanine to glutamic acid. Alternatively, the 277th serine position could be mutated to valine, the 457th threonine position to isoleucine, the 460th histidine position to tryptophan, the 462nd glutamic acid position to proline or cysteine, the 603rd threonine position to serine, the 604th alanine position to glutamic acid, and the 612th methionine position to valine. Alternatively, the following mutations can be made: serine at position 277 can be mutated to valine; threonine at position 457 can be mutated to valine; histidine at position 460 can be mutated to tryptophan; glutamic acid at position 462 can be mutated to proline or cysteine; arginine at position 548 can be mutated to proline; methionine at position 597 can be mutated to isoleucine; threonine at position 603 can be mutated to serine; and alanine at position 604 can be mutated to glutamic acid.

8. The mutant according to claim 1, characterized in that, The mutant is a β-galactosidase mutant with enhanced hydrolytic activity, wherein the mutant is formed by mutating the 14th glutamic acid of mutant M9 to aspartic acid, arginine, glutamine, alanine, or lysine. Alternatively, the valine at position 19 could be mutated to tryptophan; Alternatively, the 21st leucine residue could be mutated to alanine, glycine, glutamic acid, or valine. Alternatively, the tyrosine residue at position 398 could be mutated to aspartic acid. Or, the isoleucine at position 399 may be mutated to glutamine; Alternatively, the tyrosine residue at position 450 could be mutated to phenylalanine; Alternatively, the glycine at position 451 could be mutated into methionine, leucine, asparagine, or isoleucine; Alternatively, the histidine at position 460 could be mutated to tyrosine or isoleucine; Alternatively, the alanine at position 604 could be mutated to glutamic acid, lysine, aspartic acid, or proline. Alternatively, the methionine at position 612 could be mutated into leucine or valine; Alternatively, tyrosine at position 398 could be mutated to aspartic acid, and glycine at position 451 could be mutated to asparagine. Alternatively, glycine at position 451 could be mutated to methionine, and tyrosine at position 450 could be mutated to phenylalanine. Alternatively, glycine at position 451 could be mutated to methionine, and isoleucine at position 509 could be mutated to valine. Alternatively, glycine at position 451 could be mutated to methionine, and threonine at position 457 could be mutated to isoleucine or valine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and isoleucine at position 399 to glutamine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and tyrosine at position 450 to phenylalanine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and methionine at position 441 to isoleucine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and histidine at position 473 to tyrosine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and arginine at position 571 to histidine. Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, and glutamic acid at position 115 to glycine, serine, or threonine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and serine at position 81 to asparagine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and histidine at position 460 to tyrosine. Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, and alanine at position 604 to aspartic acid, glutamic acid, proline, glycine, or lysine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and valine at position 19 to tryptophan. Alternatively, tyrosine at position 398 can be mutated to aspartic acid, glycine at position 451 to asparagine, and threonine at position 140 to arginine, lysine, leucine, or valine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and threonine at position 448 to cysteine ​​or glycine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and cysteine ​​at position 489 to glycine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and tyrosine at position 409 to serine, leucine, or threonine. Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, and threonine at position 233 to aspartic acid, glutamine, or asparagine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and glutamine at position 237 to serine or lysine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and lysine at position 71 to threonine or tyrosine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and aspartic acid at position 6 to glutamine. Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, and histidine at position 235 to alanine or aspartic acid. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and isoleucine at position 490 to phenylalanine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and serine at position 141 to proline. Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, and leucine at position 181 to lysine, aspartic acid, or arginine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and threonine at position 230 to aspartic acid or tyrosine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, or glycine at position 491 to isoleucine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and isoleucine at position 231 to leucine or valine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and phenylalanine at position 242 to glutamine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and isoleucine at position 229 to valine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and serine at position 613 to alanine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and valine at position 145 to isoleucine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and leucine at position 570 to tyrosine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and glutamine at position 371 to glycine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and glycine at position 608 to serine or glutamic acid. Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, and proline at position 153 to leucine, aspartic acid, serine, or isoleucine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and glycine at position 121 to glutamine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and alanine at position 197 to phenylalanine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and lysine at position 57 to glycine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and aspartic acid at position 372 to tyrosine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and threonine at position 195 to arginine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and leucine at position 227 to isoleucine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and tryptophan at position 610 to phenylalanine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and tyrosine at position 38 to glycine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and arginine at position 609 to glycine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and aspartic acid at position 232 to isoleucine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and aspartic acid at position 196 to serine or threonine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and leucine at position 607 to glutamic acid. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and proline at position 392 to valine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, and tyrosine at position 572 to leucine. Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, and methionine at position 612 may be mutated to leucine, valine, asparagine, tryptophan, isoleucine, glutamine, serine, tyrosine, phenylalanine, cysteine, histidine, or arginine. Alternatively, tyrosine at position 398 can be mutated to aspartic acid, glycine at position 451 to asparagine, leucine at position 181 to lysine, and histidine at position 235 to alanine or aspartic acid. Alternatively, tyrosine at position 398 can be mutated to aspartic acid, glycine at position 451 to asparagine, serine at position 141 to proline, and histidine at position 235 to alanine or aspartic acid. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, leucine at position 181 to lysine, and serine at position 141 to proline. Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and histidine at position 235 to aspartic acid, alanine, glycine, glutamic acid, isoleucine, leucine, phenylalanine, cysteine, methionine, valine, asparagine, proline, lysine, arginine, threonine, serine, threonine, tryptophan, or tyrosine. Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, or leucine at position 181 to asparagine, lysine, methionine, histidine, aspartic acid, phenylalanine, isoleucine, cysteine, glycine, or alanine. Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and serine at position 141 to proline, threonine, tyrosine, glutamine, or histidine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and arginine at position 571 to histidine. Alternatively, tyrosine at position 398 can be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and glutamic acid at position 115 to glycine or aspartic acid. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and serine at position 81 to asparagine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and histidine at position 460 to tyrosine. Alternatively, tyrosine at position 398 may be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and alanine at position 604 to aspartic acid, glutamic acid, proline, glycine, or lysine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and valine at position 19 to tryptophan. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and threonine at position 233 to asparagine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and glutamine at position 237 to alanine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and isoleucine at position 231 to valine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and arginine at position 165 to lysine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and glutamic acid at position 239 to threonine or valine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and aspartic acid at position 200 to arginine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and aspartic acid at position 238 to glutamine or glutamic acid. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and aspartic acid at position 232 to isoleucine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and glutamine at position 188 to glutamic acid or aspartic acid. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and alanine at position 207 to glutamic acid. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and aspartic acid at position 200 to glutamic acid. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and isoleucine at position 229 to methionine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and threonine at position 234 to glutamine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and asparagine at position 173 to glycine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and threonine at position 304 to cysteine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and glutamine at position 237 to aspartic acid. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and aspartic acid at position 232 to leucine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and glutamine at position 172 to alanine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and aspartic acid at position 440 to glutamic acid. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and proline at position 275 to alanine. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and threonine at position 230 to glutamic acid. Alternatively, tyrosine at position 398 could be mutated to aspartic acid, glycine at position 451 to asparagine, methionine at position 612 to leucine, and isoleucine at position 229 to valine. Alternatively, the tyrosine at position 398 can be mutated to aspartic acid, the glycine at position 451 to asparagine, the methionine at position 612 to leucine, the serine at position 141 to proline, the leucine at position 181 to lysine, and the histidine at position 235 to alanine or aspartic acid.

9. The use of any of the mutants described in claims 1 to 8 in the production of low-lactose or lactose-free dairy products, or in the preparation of products for the production of low-lactose or lactose-free dairy products.

10. The use of the mutant of claim 4, 6 or 7 in the production of low-lactose or lactose-free dairy products containing galactooligosaccharides, or in the preparation of products for the production of low-lactose or lactose-free dairy products containing galactooligosaccharides.

Images