CN114149987B - Artificially modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis - Google Patents
Artificially modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis Download PDFInfo
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- 108010005774 beta-Galactosidase Proteins 0.000 title claims abstract description 74
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 title claims abstract description 45
- 239000008101 lactose Substances 0.000 title claims abstract description 44
- 101100067708 Caenorhabditis elegans galt-1 gene Proteins 0.000 title claims abstract description 41
- 238000006460 hydrolysis reaction Methods 0.000 title claims abstract description 22
- 230000007062 hydrolysis Effects 0.000 title claims abstract description 21
- 102000005936 beta-Galactosidase Human genes 0.000 title claims abstract description 20
- 235000013336 milk Nutrition 0.000 claims abstract description 24
- 239000008267 milk Substances 0.000 claims abstract description 24
- 210000004080 milk Anatomy 0.000 claims abstract description 24
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 4
- 102000004190 Enzymes Human genes 0.000 claims description 33
- 108090000790 Enzymes Proteins 0.000 claims description 33
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 230000003301 hydrolyzing effect Effects 0.000 claims description 5
- 239000013612 plasmid Substances 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 3
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 17
- 235000020190 lactose-free milk Nutrition 0.000 abstract description 9
- 238000006731 degradation reaction Methods 0.000 abstract description 7
- 230000015556 catabolic process Effects 0.000 abstract description 6
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 abstract description 2
- 102100026189 Beta-galactosidase Human genes 0.000 description 53
- 229940088598 enzyme Drugs 0.000 description 32
- 102000004196 processed proteins & peptides Human genes 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 241001522143 Thermus scotoductus Species 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 108010093096 Immobilized Enzymes Proteins 0.000 description 2
- 108010059881 Lactase Proteins 0.000 description 2
- 102000004882 Lipase Human genes 0.000 description 2
- 108090001060 Lipase Proteins 0.000 description 2
- 239000004367 Lipase Substances 0.000 description 2
- 102000004139 alpha-Amylases Human genes 0.000 description 2
- 108090000637 alpha-Amylases Proteins 0.000 description 2
- 229940024171 alpha-amylase Drugs 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 229940116108 lactase Drugs 0.000 description 2
- 235000019421 lipase Nutrition 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
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- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000228245 Aspergillus niger Species 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 241000235649 Kluyveromyces Species 0.000 description 1
- 201000010538 Lactose Intolerance Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- XPYGGHVSFMUHLH-UUSULHAXSA-N falecalcitriol Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](CCCC(O)(C(F)(F)F)C(F)(F)F)C)=C\C=C1\C[C@@H](O)C[C@H](O)C1=C XPYGGHVSFMUHLH-UUSULHAXSA-N 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000009928 pasteurization Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000012070 whole genome sequencing analysis Methods 0.000 description 1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
- C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
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Abstract
The invention discloses an artificially modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis, wherein the beta-galactosidase GaLT1 is an artificially modified amphiphilic short peptide sequence which is connected with an N-terminal amino acid sequence of a wild beta-galactosidase GaLT0 from T.scotoduct, and the amino acid sequence is shown as SEQ ID NO. 1. Compared with wild beta-galactosidase GaLT0, the temperature stability of the beta-galactosidase GaLT1 is obviously improved, and the half-life at 55 ℃ is improved by 10 times; in addition, the degradation rate of milk lactose of beta-galactosidase GaLT1 is also obviously improved, and 95% of lactose in milk can be degraded in 2 hours at 55 ℃ to reach the national standard of lactose-free milk products.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an artificially modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis.
Background
Lactose is a disaccharide, consisting of glucose and galactose. Milk has rich lactose content. The lack of or even the lack of secretion of lactase by the mucous membrane of the small intestine in some people results in an inability to hydrolyze lactose effectively, a condition known as lactose intolerance. Lactose-free milk products (lactose content <0.5%, w/w, health supervision No. 2007] 300) for this consumer group have appeared on the market. A common method of preparing these lactose-free milk products is to degrade lactose in the milk with lactase (i.e. beta-gaLactosidase, EC: 3.2.1.23).
Beta-galactosidases are widely distributed in animals, plants and microorganisms. At present, two commercial beta-galactosidases exist, but the price is relatively high and the hydrolysis time is relatively long. For example, commercial Aspergillus niger derived beta-galactosidase achieves complete degradation of lactose in milk at 55℃and requires hydrolysis for 12 hours (Rosolen et al 2015); commercial kluyveromyces-derived beta-galactosidase requires 24 hours at 2 ℃ to achieve complete degradation of lactose in milk (Horner et al 2011).
Milk lactose hydrolysis by beta-galactosidase falls into three strategies. First, the hydrolysis is carried out for a long time at a low temperature (below 10 ℃). Enzymes are often added to milk as food additives and the shelf life of the milk is used for hydrolysis. This places severe demands on the food source of the enzyme; secondly, medium-temperature (about 37 ℃) hydrolysis is carried out, and the temperature is the optimal propagation temperature of a plurality of microorganisms, so that the whole enzymolysis process must be carried out under strict sterile conditions, and the cost and the process requirements are high; thirdly, hydrolyzing at high temperature (above 55 ℃) for a short time. The technology can couple the lactose hydrolysis of immobilized enzyme with pasteurization, the two processes are combined, and the efficiency is higher; in addition, the enzyme is often prepared into immobilized enzyme, and exogenous protein is not introduced into milk, so that the safety is high. Thus, the search for high temperature β -galactosidase has been one of the hot spots in the study of the production of lactose-free milk products.
At present, a plurality of high-temperature beta-galactosidases are reported, but the heat stability of some high-temperature beta-galactosidases is not high. For example, the half-life of beta-galactosidase reported by Sandra W et al at an optimum temperature of 50 ℃ is only 10min; the half-life of the beta-galactosidase reported by ArreoLa et al at an optimum temperature of 55℃is only 8min. Some high temperature beta-galactosidases with better thermal stability have lower hydrolytic activity. For example, the beta-galactosidase of chinese patent 201210127007.0 only hydrolyzes 80% of lactose in milk at 65-69 ℃; the beta-galactosidase in Chinese patent 201510358266.8 only hydrolyzes about 70% of lactose in milk at 60 ℃. Therefore, the excavation meets the requirements of high-temperature beta-galactosidase with good heat stability and high lactose hydrolysis rate, and has important significance for the development of lactose-free milk product industry.
There are many methods for improving the thermal stability of enzymes, and fusion of amphiphilic short peptides is a good method, and is not easy to cause the reduction of the catalytic activity of enzymes. At present, the design schemes of amphiphilic short peptides are various, and reports on some success are provided, but reports on some ineffectiveness and even worse are provided at the same time. For example, in chinese patent No. 201810069643.X, the effect of 7 different amphiphilic short peptides on the temperature stability of one glucose oxidase was tried, and it was found that 1 amphiphilic short peptide deteriorated the stability of the enzyme, and the rest had different improvements on the stability of the enzyme, but the highest was only incubated for 30min at 60 ℃. In Chinese patent 2109776686A, 2 different amphiphilic short peptides are fused to the N end of a lipase, so that the heat stability of the enzyme is slightly improved. The lipase has poor heat stability, the half-life period is only 25min, and the half-life period of the enzyme after the fusion of the amphiphilic peptide is improved to 35 min and 45min. The nature and length of the amino acids of the amphiphilic short peptide affect the enzyme properties and do not have obvious rules. This requires that in experiments, multiple amphiphilic short peptides are designed, and that by experimental tests, the relatively best matching amphiphilic short peptide for a particular enzyme is found.
Disclosure of Invention
The invention provides an artificial modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis aiming at the problems existing in the prior art. Compared with the wild type beta-galactosidase GaLT0, the temperature stability of the beta-galactosidase GaLT1 provided by the invention is obviously improved, and the half-life at 55 ℃ is improved by 10 times; in addition, the degradation rate of milk lactose of beta-galactosidase GaLT1 is also obviously improved, and 95% of lactose in milk can be degraded in 2 hours at 55 ℃ to reach the national standard of lactose-free milk products. Therefore, the artificially modified beta-galactosidase GaLT1 has good industrial application value in long-term storage and rapid preparation of lactose-free milk products.
The artificially modified beta-galactosidase GaLT1 is prepared by connecting an artificially modified amphiphilic short peptide sequence with an N-terminal amino acid sequence of wild beta-galactosidase GaLT0 from T.scotoductus, wherein the amphiphilic short peptide sequence is shown as SEQ ID NO. 2.
Further, the amino acid sequence of the beta-galactosidase GaLT1 is shown as SEQ ID NO. 1.
The invention also provides a coding gene of the beta-galactosidase GaLT1, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 3. The sequence is optimized, and can effectively express beta-galactosidase with enzyme activity in escherichia coli.
The expression vector of the beta-galactosidase GaLT1 gene comprises pET22b.
Host strains containing the above expression vectors include E.coli BL21 (DE 3).
The invention also provides a recombinant plasmid for expressing the beta-galactosidase GaLT1, which at least comprises a nucleotide sequence shown as SEQ ID NO. 3.
A recombinant strain for expressing said β -galactosidase GaLT1, which recombinant strain comprises said recombinant plasmid.
The specific enzyme activity of the beta-galactosidase GaLT1 is 145U/mg (lactose is used as a substrate). The enzyme was stored in 50mM phosphate buffer (pH 7.0) at 55℃for 10 hours, and the residual enzyme activity was 74U/mg, i.e., the half-life of the enzyme at 55℃was as high as 10 hours.
The beta-galactosidase GaLT1 is used for hydrolyzing lactose, and has high heat stability and lactose hydrolysis rate.
The optimized lactose hydrolysis condition parameters are as follows: natural milk (pH about 6.7), enzyme amount 20U/mL, reaction temperature 55 ℃, reaction time 2h.
At 55 ℃, natural milk (pH is about 6.7, lactose content is 4.36%, w/w) is subjected to enzymolysis for 2 hours, and the lactose degradation rate of wild-type beta-galactosidase GaLT0 is about 80%; the degradation rate of lactose of the beta-galactosidase GaLT1 is 95% (lactose residue is 0.22%, w/w), and is improved by 19% compared with that of wild beta-galactosidase GaLT0, thus reaching the national standard of lactose-free milk products.
Drawings
FIG. 1 shows SDS-PAGE patterns after expression and purification of the β -galactosidase protein of the invention.
FIG. 2 shows the optimum temperature for the hydrolysis reaction of beta-galactosidase according to the invention.
FIG. 3 (a) shows the temperature stability of the beta-galactosidase of the invention at 50 ℃; (b) The temperature stability of the beta-galactosidase of the invention at 55℃is shown.
FIG. 4 shows the hydrolysis rate of lactose in milk by the beta-galactosidase of the invention.
Detailed Description
Example 1: sequence design and synthesis of artificially modified beta-galactosidase GaLT1
(1) Amphiphilic short peptide design
A total of 8 amphiphilic short peptides were designed in the laboratory:
①AEAEAKAKAEAEAKAKAEAEAKAK；
②AEAEAKAKAEAEAKAK；
③LELELKLKLELELKLK；
④ADADAKAKADADAKAKA；
⑤ADADARARADADARAR；
⑥AEAEAHAHAEAEAHAH；
⑦HNANARARHNANARARHNANARARHNANARAR；
⑧ANANARARANANARAR。
(2) Fusion of the amphiphilic short peptide with a wild-type beta-galactosidase (named GaLT 0) derived from T.scotoductus. The amino acid sequence of this wild-type β -galactosidase GaLT0 was derived from whole genome sequencing of the t.scotoductus strain, numbered wp_015716994.1 in the NCBI database, annotated as α -amylase (alpha-amylase). The related research of the protein is not reported in the literature. The designed 8 amphiphilic short peptides are respectively fused and connected to the N end of a wild beta-galactosidase GaLT0 sequence by entrusted Shanghai biological limited company, and are respectively named GaLT1, gaLT2, gaLT3, gaLT4, gaLT5, gaLT6, gaLT7 and GaLT8. The newly synthesized gene sequences were all optimized for E.coli codon bias.
Example 2: recombinant expression and protein purification of artificially modified beta-galactosidase GaLT1
1. The beta-galactosidase genes fused with different amphiphilic short peptides are cloned onto pET22b respectively, and then transferred into escherichia coli BL21 (DE 3) strain for induced expression according to the standard flow of molecular cloning. The obtained bacterial cells are homogenized and crushed under high pressure, and the supernatant after centrifugation is crude enzyme solution.
2. Purifying the crude enzyme solution by anion exchange chromatography to obtain the artificially modified beta-galactosidase recombinant protein. Protein purity was checked by SDS-PAGE. Through conventional activity test of enzyme, gaLT1 with optimal comprehensive catalytic ability is screened from 8 artificially modified beta-galactosidases. Recombinant expression of the enzyme and protein purification are shown in figure 1. M is high molecular weight standard protein, 1 is supernatant of E.coli disruption solution containing pET22b vector; 2 is the supernatant of the E.coli disruption solution containing pET22b-GaLT0 vector; 3 is the supernatant of the E.coli disruption solution containing pET22b-GaLT1 vector; 4 is purified wild type beta-galactosidase GaLT0; and 5 is purified and artificially modified beta-galactosidase GaLT1.
Example 3: optimum reaction temperature for artificially engineered beta-galactosidase GaLT1 and wild-type enzyme GaLT0
The enzyme reaction system was 500. Mu.L, including 100. Mu.L of 10mM oNPG, 5. Mu.L of the enzymeDiluted pure enzyme solution, 395. Mu.L of 50mM phosphate buffer (pH 7.0). Respectively reacting at 30-70deg.C for 10min, and adding 1M Na with the same volume at the end of the reaction 2 CO 3 The reaction was terminated and absorbance was measured at 405 nm. As shown in FIG. 2, the optimal reaction temperature of the wild-type beta-galactosidase GaLT0 was 50℃and the optimal reaction temperature of the artificially modified beta-galactosidase GaLT1 was increased to 55 ℃.
Example 4: temperature stability of artificially engineered beta-galactosidase GaLT1 and wild-type enzyme GaLT0
The purified artificially modified beta-galactosidase GaLT1 and wild-type enzyme GaLT0 were incubated in water baths (50 mM phosphate buffer, pH 7.0) at 50 and 55℃respectively, and a certain amount of the enzyme was taken out at intervals, and substrates oNPG and the like were added thereto, and their residual enzyme activities were detected at their optimal reaction temperatures, respectively. The enzyme activity of the non-incubated enzyme was defined as 100%. The obtained temperature stability is shown in FIG. 3, and the half-life of the wild-type GaLT0 is 1h at 55 ℃; the half-life period of the artificially modified GaLT1 is 10h, and the thermal stability of the artificially modified GaLT1 is improved by 10 times.
Example 5: artificially modified beta-galactosidase GaLT1 and wild-type enzyme GaLT0 for degrading lactose in natural milk
As measured by High Performance Liquid Chromatography (HPLC), we used natural milk (pH 6.7) with a lactose content of 43.6g/L. The lactose degradation reaction was tested under the following conditions: 1mL of milk, 20U/mL of enzyme, reaction temperature of 55 ℃ and reaction time of 3h. A certain amount of milk was taken out at intervals, and the residual lactose amount was measured by HPLC to calculate the lactose hydrolysis rate. As shown in fig. 4, the artificially modified beta-galactosidase GaLT1 only needs 2 hours, and can hydrolyze 95% of lactose in milk to reach lactose-free milk standard; the lactose hydrolysis rate of wild-type enzyme GaLT0 at 2h was only 80%.
Lactose hydrolysis rate (%) = (initial lactose content in milk-residual lactose content)/initial lactose content in milk x 100%.
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<110> OrganizationName an artificially modified beta-galactosidase GaLT1 and its use in hydrolyzing lactose
Application Project
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<120> Title :
<130> AppFileReference :
<140> CurrentAppNumber :
<141> CurrentFilingDate :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
MAEAEAKAKA EAEAKAKAEA EAKAKGGGGS GGGGSGGGGS MGRILAVWVW LSLALAVPVT 60
FRYTPPSGLE VRSVSLRGSF NSWGETPMQK EDGSWAVTVD LDPGEHQYKF FINGQWPRDM 120
CNDPTFGTPM VDPKAAGCVD DGFGGQNAVI VVQAPVAPTP PAGPVALDFT HDPLDAQYVS 180
HADGKLSVRF RAGEGAVAAA WVEVQGKRLP MHLQLSFPGS EVWRGTLPGG VGAYRILVRT 240
QDGKEEVFGP FNPPERPFAE VAWVGEGVGY QIFPERFYNG DSSNDALALE TDEYRFNQVW 300
QRSSGPKPHL SRWGDPPSPL HCCHQYFGGD LAGVLAKLPY LKALGVSVLY LNPIFDSGSA 360
HGYDTHDYLK VSPKFGDKPL LRKLLDEAHR LGMRVIFDFV PNHTGLGFWA FQDVVKRGPR 420
SPYWNWYFIK RWPFVPGDGS AYEGWWGLGS LPKLNTANPG VKRYLIEVTK YWVRFGFDGV 480
RVDMPGDVLN PHAFFKEMRA ELKAIKPDAY LVAEIWQRDP SWLRGDEFDS LMNYAIGRDI 540
LLRFAKGGSL ALYNARRALA DLARVYALYP EAVAGMGFNL ITSHDTARLL TELGGGGLKD 600
VPSPEARARQ RLAAAMLYAL PGLPVTFQGD ECGFTGERPA DPPHELNRYP FQWEKCHGET 660
LAFYQELAGL RRELAALRSA VFRTYFGEGH LLAFFRGEPG EGEVLAAFNN GVEAVTLPLP 720
PGGWRDPLEG RTYRKEVSLP PLGFRYLVHL GR 752
<212> Type : PRT
<211> Length : 752
SequenceName : SEQ ID NO:1
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
aeaeakakae aeakakaeae akak 24
<212> Type : DNA
<211> Length : 24
SequenceName : SEQ ID NO:2
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
ATGGCAGAAG CCGAGGCGAA AGCGAAAGCT GAAGCTGAAG CAAAAGCAAA AGCGGAAGCG 60
GAAGCCAAAG CCAAAGGTGG TGGTGGTTCC GGTGGTGGTG GTTCCGGTGG TGGTGGTAGC 120
GGAAGAATCC TGGCGGTGTG GGTATGGCTT AGCCTGGCCC TGGCGGTCCC GGTGACCTTC 180
CGCTACACAC CCCCTTCGGG CCTCGAGGTG CGCTCGGTAA GCCTCCGGGG CTCCTTCAAC 240
AGCTGGGGGG AAACCCCCAT GCAGAAGGAG GACGGGTCCT GGGCGGTAAC CGTGGACCTG 300
GATCCAGGGG AGCACCAGTA CAAGTTCTTC ATCAACGGCC AGTGGCCCAG GGACATGTGC 360
AACGATCCCA CCTTCGGCAC GCCCATGGTG GACCCGAAGG CGGCAGGGTG TGTGGACGAT 420
GGCTTTGGGG GTCAGAACGC CGTGATCGTG GTCCAGGCCC CGGTAGCCCC CACCCCTCCT 480
GCGGGGCCCG TGGCCCTGGA CTTCACCCAT GATCCGTTGG ACGCCCAGTA TGTGTCCCAT 540
GCCGACGGCA AGCTTTCCGT GCGCTTCCGG GCAGGGGAGG GGGCGGTGGC GGCCGCCTGG 600
GTCGAGGTGC AGGGAAAGAG GCTCCCCATG CACCTGCAGC TGAGTTTTCC GGGAAGCGAG 660
GTTTGGCGTG GGACCTTACC TGGAGGCGTG GGAGCCTACC GCATCCTGGT GCGGACCCAG 720
GATGGCAAGG AGGAGGTGTT CGGCCCCTTT AACCCTCCCG AAAGGCCCTT CGCCGAGGTG 780
GCATGGGTGG GCGAGGGGGT GGGTTATCAG ATCTTCCCCG AGCGCTTCTA CAACGGGGAT 840
TCCAGCAACG ACGCCTTGGC CCTGGAAACC GACGAGTACC GCTTTAACCA GGTGTGGCAG 900
CGCTCCTCTG GGCCCAAGCC CCATCTTTCC CGCTGGGGCG ATCCCCCCTC GCCCCTGCAC 960
TGCTGCCACC AGTACTTCGG GGGGGATCTT GCCGGGGTGC TGGCCAAGCT TCCTTACCTG 1020
AAGGCCCTGG GGGTTAGCGT CCTCTACCTG AATCCCATCT TTGATTCCGG GTCGGCCCAC 1080
GGCTACGACA CCCACGACTA CCTCAAGGTT TCCCCCAAGT TCGGCGACAA ACCCCTCTTG 1140
CGCAAGCTGC TGGACGAGGC CCACCGCCTC GGCATGCGGG TGATCTTTGA CTTCGTCCCC 1200
AACCACACTG GCCTGGGCTT TTGGGCTTTT CAGGATGTGG TAAAGAGGGG TCCCCGTTCC 1260
CCTTACTGGA ACTGGTACTT CATCAAGCGG TGGCCCTTTG TGCCGGGTGA CGGATCGGCC 1320
TACGAGGGAT GGTGGGGGTT AGGGAGCCTG CCCAAGCTGA ACACCGCAAA CCCCGGGGTG 1380
AAGCGCTACC TGATCGAGGT GACCAAGTAC TGGGTACGCT TCGGCTTTGA CGGGGTGCGG 1440
GTGGATATGC CCGGGGATGT GCTAAATCCT CACGCTTTCT TTAAGGAAAT GCGGGCCGAA 1500
CTGAAGGCCA TCAAGCCCGA CGCCTACCTG GTGGCGGAGA TCTGGCAGAG GGATCCTAGC 1560
TGGCTTCGGG GGGATGAGTT TGACTCCCTG ATGAACTACG CCATCGGCCG GGATATCCTC 1620
CTCCGCTTTG CTAAGGGGGG AAGCCTGGCC CTGTACAACG CCCGCCGAGC CTTGGCGGAC 1680
CTAGCCCGGG TTTACGCCCT TTACCCGGAG GCGGTGGCCG GGATGGGCTT CAACTTGATC 1740
ACCTCCCACG ATACGGCCCG CCTCCTTACC GAGCTTGGGG GCGGGGGCCT GAAGGACGTT 1800
CCCAGCCCGG AAGCCAGGGC CCGGCAGCGG CTTGCGGCGG CCATGCTCTA CGCCCTTCCC 1860
GGCCTCCCCG TAACCTTCCA GGGGGATGAG TGCGGTTTCA CCGGGGAAAG GCCGGCCGAC 1920
CCCCCTCACG AGCTCAACCG GTATCCCTTC CAGTGGGAGA AATGCCATGG GGAAACCCTA 1980
GCCTTTTACC AGGAGCTGGC GGGGCTGCGC CGGGAGCTTG CGGCCCTCAG GAGCGCCGTG 2040
TTCCGGACCT ACTTCGGAGA GGGCCATCTT CTGGCCTTCT TCCGGGGTGA GCCGGGAGAA 2100
GGGGAGGTGC TTGCCGCCTT CAATAACGGG GTGGAGGCCG TCACCTTGCC CTTGCCTCCT 2160
GGGGGCTGGC GGGATCCCCT CGAGGGGCGC ACCTACCGGA AGGAAGTGAG CCTGCCCCCC 2220
CTGGGCTTCC GGTACCTGGT CCACCTGGGG CGGCATCATC ATCATCATCA TTAG 2274
<212> Type : PRT
<211> Length : 2274
SequenceName : SEQ ID NO:3
SequenceDescription :
Claims (6)
1. An artificially modified beta-galactosidase GaLT1, characterized in that:
the amino acid sequence of the beta-galactosidase GaLT1 is shown as SEQ ID NO. 1.
2. A coding gene of beta-galactosidase GaLT1 as described in claim 1, wherein the nucleotide sequence is shown in SEQ ID NO. 3.
3. A recombinant plasmid for expressing the beta-galactosidase GaLT1 according to claim 1, characterized in that the recombinant plasmid comprises at least the nucleotide sequence shown in SEQ ID No. 3.
4. A recombinant strain for expressing the β -galactosidase GaLT1 of claim 1, characterized in that the recombinant strain comprises the recombinant plasmid of claim 3.
5. Use of the β -galactosidase GaLT1 of claim 1, wherein:
the beta-galactosidase GaLT1 is used for hydrolyzing lactose, and has higher heat stability and lactose hydrolysis rate.
6. The use according to claim 5, characterized in that:
the conditions parameters of lactose hydrolysis are: natural milk, enzyme amount 20U/mL, reaction temperature 55 ℃ and reaction time 2h.
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