CN114149987A - 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 76
- 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 50
- 239000008101 lactose Substances 0.000 title claims abstract description 50
- 101100067708 Caenorhabditis elegans galt-1 gene Proteins 0.000 title claims abstract description 42
- 238000006460 hydrolysis reaction Methods 0.000 title claims abstract description 21
- 102000005936 beta-Galactosidase Human genes 0.000 title claims abstract description 20
- 230000007062 hydrolysis Effects 0.000 title claims abstract description 20
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- 239000008267 milk Substances 0.000 claims abstract description 30
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- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 6
- 241001522143 Thermus scotoductus Species 0.000 claims abstract description 5
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- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 238000006731 degradation reaction Methods 0.000 abstract description 9
- 230000015556 catabolic process Effects 0.000 abstract description 8
- 102100026189 Beta-galactosidase Human genes 0.000 description 54
- 229940088598 enzyme Drugs 0.000 description 31
- 238000000034 method Methods 0.000 description 7
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- 102000004139 alpha-Amylases Human genes 0.000 description 2
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- 229940024171 alpha-amylase Drugs 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
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- 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
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- 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
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- 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
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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 connected with an N-terminal amino acid sequence of a wild-type beta-galactosidase GaLT0 from T.scotoductus, and the amino acid sequence is shown as SEQ ID NO. 1. Compared with the wild beta-galactosidase GaLT0, the beta-galactosidase GaLT1 has the advantages that the temperature stability is obviously improved, and the half-life period at 55 ℃ is improved by 10 times; in addition, the milk lactose degradation rate of the beta-galactosidase GaLT1 is also obviously improved, 95% of lactose in milk can be degraded at 55 ℃ within 2 hours, and the milk lactose degradation rate reaches the national standard of milk products without lactose.
Description
Technical Field
The invention relates to the technical field of biology, in particular to artificially modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis.
Background
Lactose is a disaccharide consisting of glucose and galactose. The milk is rich in lactose. Lactase in the small intestinal mucosa of some people is not secreted enough or even is not secreted, so that lactose cannot be hydrolyzed effectively, and the symptom is called lactose intolerance. Lactose-free milk products for this consumer group have appeared on the market today (lactose content < 0.5%, w/w, health supervision 2007 # 300). A common method of preparing these lactose-free milk products is to degrade lactose in milk with lactase (i.e., beta-gaLactosidase, EC: 3.2.1.23).
Beta-galactosidase enzymes are widely distributed in animals, plants and microorganisms. At present, two commercial beta-galactosidases are available, but the price is expensive and the hydrolysis time is long. For example, commercial A.niger derived β -galactosidase, which requires hydrolysis for 12h at 55 ℃ to achieve complete degradation of lactose in milk (Rosolen et al 2015); commercial kluyveromyces-derived β -galactosidase required 24h at 2 ℃ for complete degradation of lactose in milk (Horner et al 2011).
Lactose hydrolysis of beta-galactosidase milk is divided into three strategies. One is low temperature (below 10 ℃) long term hydrolysis. Often, enzymes are added to milk as food additives and the milk is hydrolyzed during its shelf life. This places very stringent requirements on the food source of the enzyme; secondly, hydrolysis is carried out at medium temperature (about 37 ℃), which is the optimal propagation temperature of a plurality of microorganisms, so the whole process of enzymolysis must be carried out under strict aseptic conditions, and the cost and process requirements are high; thirdly, hydrolyzing at high temperature (above 55 ℃) for a short time. The technology can couple the lactose hydrolysis of the immobilized enzyme and the pasteurization, integrates the two processes, and has higher efficiency; and the enzyme is often prepared into immobilized enzyme, so that foreign protein is not introduced into the milk, and the safety is high. Therefore, the search for high temperature beta-galactosidase has been one of the hot spots in the research for producing lactose-free milk products.
There are many reports of high temperature beta-galactosidase, but some of them are not highly thermostable. For example, Sandra W et al report that the half-life of beta-galactosidase at the optimum temperature of 50 ℃ is only 10 min; the half-life of beta-galactosidase reported by ArreoLa et al at an optimum temperature of 55 ℃ is only 8 min. Some of the high temperature beta-galactosidases with better thermostability have lower hydrolytic activity. For example, the beta-galactosidase in the Chinese patent 201210127007.0 can only hydrolyze 80% of lactose in milk at 65-69 ℃; the beta-galactosidase in the Chinese patent 201510358266.8 can only hydrolyze about 70% of lactose in milk at 60 ℃. Therefore, the high-temperature beta-galactosidase with good thermal stability and high lactose hydrolysis rate is obtained, and the method has important significance for the development of the lactose-free milk product industry.
The method for improving the thermal stability of the enzyme is various, and the fusion of the amphiphilic short peptide is a better method and is not easy to cause the reduction of the enzyme catalytic activity. At present, the design schemes of the amphiphilic short peptide are various and have many successful reports, but at the same time, there are many ineffective reports and even worse reports. For example, in the Chinese invention patent 201810069643.X, 7 different amphiphilic short peptides are tried to influence the temperature stability of one glucose oxidase, and it is found that 1 amphiphilic short peptide can deteriorate the stability of the enzyme, and the rest amphiphilic short peptides improve the stability of the enzyme to different degrees, but the highest amphiphilic short peptide is only incubated at 60 ℃ for 30 min. Chinese patent 2109776686A reports that 2 different amphiphilic short peptides are respectively fused at the N-terminal of a lipase, and the thermal stability of the lipase is slightly improved. The lipase has poor thermal 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 45 min. The kind and length of amino acid of the amphiphilic short peptide can affect the property of enzyme, and there is no obvious rule. This requires that a variety of amphiphilic short peptides be designed in an experiment, and the amphiphilic short peptide which is relatively best matched with a specific enzyme can be found through the experiment.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides an artificially modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis. Compared with the wild beta-galactosidase GaLT0, the temperature stability of the beta-galactosidase GaLT1 provided by the invention is obviously improved, and the half-life period at 55 ℃ is improved by 10 times; in addition, the milk lactose degradation rate of the beta-galactosidase GaLT1 is also obviously improved, 95% of lactose in milk can be degraded at 55 ℃ within 2 hours, and the milk lactose degradation rate reaches the national standard of milk products without lactose. Therefore, the artificially modified beta-galactosidase GaLT1 has good industrial application value in the aspects of long-term storage and rapid preparation of the lactose-free milk product.
The artificially modified beta-galactosidase GaLT1 is formed by connecting an artificially modified amphiphilic short peptide sequence to an N-terminal amino acid sequence of a 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 in SEQ ID NO. 3. The sequence is optimized, and beta-galactosidase with enzyme activity can be effectively expressed in escherichia coli.
The expression vector of the beta-galactosidase GaLT1 gene comprises pET22 b.
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, wherein the recombinant plasmid at least comprises an amino acid sequence shown as SEQ ID NO. 1.
A recombinant strain for expressing said β -galactosidase GaLT1, the recombinant strain comprising said recombinant plasmid.
The specific enzyme activity of the beta-galactosidase GaLT1 is 145U/mg (lactose is used as a substrate). The enzyme is stored for 10h at 55 ℃ in 50mM phosphate buffer (pH7.0), and the residual enzyme activity is measured to be 74U/mg, namely the half-life period of the enzyme at 55 ℃ is as high as 10 h.
The beta-galactosidase GaLT1 is used in hydrolyzing lactose and has high heat stability and lactose hydrolyzing 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 2 h.
At the temperature of 55 ℃, the lactose degradation rate of wild beta-galactosidase GaLT0 is about 80 percent after 2 hours of enzymolysis of natural milk (pH is about 6.7, lactose content is 4.36 percent, and w/w); the lactose degradation rate of the beta-galactosidase GaLT1 is 95% (lactose residual quantity is 0.22%, w/w), which is 19% higher than that of wild beta-galactosidase GaLT0, and reaches the national standard of milk products without lactose.
Drawings
FIG. 1 shows SDS-PAGE of the beta-galactosidase protein of the present invention after expression and purification.
FIG. 2 shows the optimum temperature for the hydrolysis reaction of beta-galactosidase according to the present 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 present 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 are designed in a laboratory:
①AEAEAKAKAEAEAKAKAEAEAKAK；
②AEAEAKAKAEAEAKAK；
③LELELKLKLELELKLK；
④ADADAKAKADADAKAKA；
⑤ADADARARADADARAR；
⑥AEAEAHAHAEAEAHAH；
⑦HNANARARHNANARARHNANARARHNANARAR；
⑧ANANARARANANARAR。
(2) fusion of the amphiphilic short peptide with a wild-type β -galactosidase derived from t.scotoductus (named GaLT 0). The amino acid sequence of this wild-type β -galactosidase, GaLT0, was derived from whole genome sequencing of a t.scotoductus strain, numbered WP _015716994.1 in the NCBI database and annotated as α -amylase (alpha-amylase). The related research of the protein is not reported in the literature. 8 designed amphiphilic short peptides are respectively fused and connected to the N end of a wild beta-galactosidase GaLT0 sequence by Shanghai Biotechnology Limited company, and are named as GaLT1, GaLT2, GaLT3, GaLT4, GaLT5, GaLT6, GaLT7 and GaLT 8. The newly synthesized gene sequences were all optimized according to the codon preference of E.coli.
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 respectively cloned on pET22b, and then transferred into Escherichia coli BL21(DE3) strain to be induced and expressed according to the standard flow of molecular cloning. The obtained bacterial cells are homogenized and broken under high pressure, and the supernatant after centrifugation is crude enzyme liquid.
2. And 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 enzyme activity tests, the GaLT1 with the best comprehensive catalytic capability is screened from 8 artificially-modified beta-galactosidases. The recombinant expression and protein purification of the enzyme are shown in FIG. 1. M is high molecular weight standard protein, 1 is the supernatant of the Escherichia coli disruption solution containing pET22b vector; 2 is the supernatant of the Escherichia coli crushed liquid containing pET22b-GaLT0 vector; 3 is the supernatant of the Escherichia coli broken liquid containing pET22b-GaLT1 vector; 4 is purified wild-type beta-galactosidase GaLT 0; 5 is purified and artificially modified beta-galactosidase GaLT 1.
Example 3: optimum reaction temperature of artificially modified beta-galactosidase GaLT1 and wild-type enzyme GaLT0
Of enzymesThe reaction system was 500. mu.L, comprising 100. mu.L of 10mM oNPG, 5. mu.L of an appropriately diluted pure enzyme solution, and 395. mu.L of 50mM phosphate buffer (pH 7.0). Respectively reacting at 30-70 deg.C for 10min, and adding 1M Na with the same volume2CO3The reaction was stopped and the absorbance was measured at 405 nm. As shown in FIG. 2, the optimum reaction temperature of wild-type beta-galactosidase GaLT0 was 50 ℃ and the optimum reaction temperature of 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 are respectively incubated in 50 and 55 ℃ water baths (50mM phosphate buffer solution, pH7.0), a certain amount of enzyme is taken out at intervals, substrate oNPG and the like are added, and the residual enzyme activity is respectively detected at the optimal reaction temperature. The enzyme activity of the non-incubated enzyme was defined as 100%. The temperature stability is shown in figure 3, and the half-life of wild-type GaLT0 is 1h at 55 ℃; the half-life of the artificially modified GaLT1 is 10h, and the thermal stability is improved by 10 times.
Example 5: artificially modified beta-galactosidase GaLT1 and wild enzyme GaLT0 for degrading lactose in natural milk
The lactose content of the natural milk (pH6.7) was 43.6g/L as determined by High Performance Liquid Chromatography (HPLC). The test conditions for lactose degradation reactions were: 1mL of milk, 20U/mL of enzyme, the reaction temperature of 55 ℃ and the reaction time of 3 h. A certain amount of milk was taken out at intervals, and the amount of residual lactose 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 the lactose-free milk standard; the lactose hydrolysis rate of the wild-type enzyme GaLT0 at 2h was only 80%.
Lactose hydrolysis ratio (%) - (initial lactose content-residual lactose content in milk)/initial lactose content in milk × 100% SEQ ID NO:1
MAEAEAKAKAEAEAKAKGGGGSGGGGSGGGGSMGRILAVWVWLSLALAVPVTFRYTPP SGLEVRSVSLRGSFNSWGETPMQKEDGSWAVTVDLDPGEHQYKFFINGQWPRDMCNDPT FGTPMVDPKAAGCVDDGFGGQNAVIVVQAPVAPTPPAGPVALDFTHDPLDAQYVSHADG KLSVRFRAGEGAVAAAWVEVQGKRLPMHLQLSFPGSEVWRGTLPGGVGAYRILVRTQDG KEEVFGPFNPPERPFAEVAWVGEGVGYQIFPERFYNGDSSNDALALETDEYRFNQVWQRS SGPKPHLSRWGDPPSPLHCCHQYFGGDLAGVLAKLPYLKALGVSVLYLNPIFDSGSAHGY DTHDYLKVSPKFGDKPLLRKLLDEAHRLGMRVIFDFVPNHTGLGFWAFQDVVKRGPRSPY WNWYFIKRWPFVPGDGSAYEGWWGLGSLPKLNTANPGVKRYLIEVTKYWVRFGFDGVR VDMPGDVLNPHAFFKEMRAELKAIKPDAYLVAEIWQRDPSWLRGDEFDSLMNYAIGRDIL LRFAKGGSLALYNARRALADLARVYALYPEAVAGMGFNLITSHDTARLLTELGGGGLKD VPSPEARARQRLAAAMLYALPGLPVTFQGDECGFTGERPADPPHELNRYPFQWEKCHGET LAFYQELAGLRRELAALRSAVFRTYFGEGHLLAFFRGEPGEGEVLAAFNNGVEAVTLPLPP GGWRDPLEGRTYRKEVSLPPLGFRYLVHLGR
SEQ ID NO:2
AEAEAKAKAEAEAKAKAEAEAKAK
SEQ ID NO:3
ATGGCAGAAGCCGAGGCGAAAGCGAAAGCTGAAGCTGAAGCAAAAGCAAAAGGTGGT GGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGTGGTAGCGGAAGAATCCTGGCGGTGT GGGTATGGCTTAGCCTGGCCCTGGCGGTCCCGGTGACCTTCCGCTACACACCCCCTTCG GGCCTCGAGGTGCGCTCGGTAAGCCTCCGGGGCTCCTTCAACAGCTGGGGGGAAACCC CCATGCAGAAGGAGGACGGGTCCTGGGCGGTAACCGTGGACCTGGATCCAGGGGAGCA CCAGTACAAGTTCTTCATCAACGGCCAGTGGCCCAGGGACATGTGCAACGATCCCACCT TCGGCACGCCCATGGTGGACCCGAAGGCGGCAGGGTGTGTGGACGATGGCTTTGGGGG TCAGAACGCCGTGATCGTGGTCCAGGCCCCGGTAGCCCCCACCCCTCCTGCGGGGCCCG TGGCCCTGGACTTCACCCATGATCCGTTGGACGCCCAGTATGTGTCCCATGCCGACGGC AAGCTTTCCGTGCGCTTCCGGGCAGGGGAGGGGGCGGTGGCGGCCGCCTGGGTCGAGG TGCAGGGAAAGAGGCTCCCCATGCACCTGCAGCTGAGTTTTCCGGGAAGCGAGGTTTG GCGTGGGACCTTACCTGGAGGCGTGGGAGCCTACCGCATCCTGGTGCGGACCCAGGAT GGCAAGGAGGAGGTGTTCGGCCCCTTTAACCCTCCCGAAAGGCCCTTCGCCGAGGTGG CATGGGTGGGCGAGGGGGTGGGTTATCAGATCTTCCCCGAGCGCTTCTACAACGGGGAT TCCAGCAACGACGCCTTGGCCCTGGAAACCGACGAGTACCGCTTTAACCAGGTGTGGC AGCGCTCCTCTGGGCCCAAGCCCCATCTTTCCCGCTGGGGCGATCCCCCCTCGCCCCTG CACTGCTGCCACCAGTACTTCGGGGGGGATCTTGCCGGGGTGCTGGCCAAGCTTCCTTA CCTGAAGGCCCTGGGGGTTAGCGTCCTCTACCTGAATCCCATCTTTGATTCCGGGTCGGC CCACGGCTACGACACCCACGACTACCTCAAGGTTTCCCCCAAGTTCGGCGACAAACCCC TCTTGCGCAAGCTGCTGGACGAGGCCCACCGCCTCGGCATGCGGGTGATCTTTGACTTC GTCCCCAACCACACTGGCCTGGGCTTTTGGGCTTTTCAGGATGTGGTAAAGAGGGGTCC CCGTTCCCCTTACTGGAACTGGTACTTCATCAAGCGGTGGCCCTTTGTGCCGGGTGACG GATCGGCCTACGAGGGATGGTGGGGGTTAGGGAGCCTGCCCAAGCTGAACACCGCAAA CCCCGGGGTGAAGCGCTACCTGATCGAGGTGACCAAGTACTGGGTACGCTTCGGCTTTG ACGGGGTGCGGGTGGATATGCCCGGGGATGTGCTAAATCCTCACGCTTTCTTTAAGGAA ATGCGGGCCGAACTGAAGGCCATCAAGCCCGACGCCTACCTGGTGGCGGAGATCTGGC AGAGGGATCCTAGCTGGCTTCGGGGGGATGAGTTTGACTCCCTGATGAACTACGCCATC GGCCGGGATATCCTCCTCCGCTTTGCTAAGGGGGGAAGCCTGGCCCTGTACAACGCCCG CCGAGCCTTGGCGGACCTAGCCCGGGTTTACGCCCTTTACCCGGAGGCGGTGGCCGGGA TGGGCTTCAACTTGATCACCTCCCACGATACGGCCCGCCTCCTTACCGAGCTTGGGGGC GGGGGCCTGAAGGACGTTCCCAGCCCGGAAGCCAGGGCCCGGCAGCGGCTTGCGGCG GCCATGCTCTACGCCCTTCCCGGCCTCCCCGTAACCTTCCAGGGGGATGAGTGCGGTTTC ACCGGGGAAAGGCCGGCCGACCCCCCTCACGAGCTCAACCGGTATCCCTTCCAGTGGG AGAAATGCCATGGGGAAACCCTAGCCTTTTACCAGGAGCTGGCGGGGCTGCGCCGGGA GCTTGCGGCCCTCAGGAGCGCCGTGTTCCGGACCTACTTCGGAGAGGGCCATCTTCTGG CCTTCTTCCGGGGTGAGCCGGGAGAAGGGGAGGTGCTTGCCGCCTTCAATAACGGGGT GGAGGCCGTCACCTTGCCCTTGCCTCCTGGGGGCTGGCGGGATCCCCTCGAGGGGCGC ACCTACCGGAAGGAAGTGAGCCTGCCCCCCCTGGGCTTCCGGTACCTGGTCCACCTGGG GCGGCATCATCATCATCATCATTAG 。
Organization Applicant
----------------------
Street :
City :
State :
Country :
PostalCode :
PhoneNumber :
FaxNumber :
EmailAddress :
<110> organization name:universityof Anhui
Application Project
-------------------
<120> Title, an artificially modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis
<130> AppFileReference :
<140> CurrentAppNumber :
<141> CurrentFilingDate : ____-__-__
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
MAEAEAKAKA EAEAKAKGGG GSGGGGSGGG GSMGRILAVW VWLSLALAVP VTFRYTPPSG 60
LEVRSVSLRG SFNSWGETPM QKEDGSWAVT VDLDPGEHQY KFFINGQWPR DMCNDPTFGT 120
PMVDPKAAGC VDDGFGGQNA VIVVQAPVAP TPPAGPVALD FTHDPLDAQY VSHADGKLSV 180
RFRAGEGAVA AAWVEVQGKR LPMHLQLSFP GSEVWRGTLP GGVGAYRILV RTQDGKEEVF 240
GPFNPPERPF AEVAWVGEGV GYQIFPERFY NGDSSNDALA LETDEYRFNQ VWQRSSGPKP 300
HLSRWGDPPS PLHCCHQYFG GDLAGVLAKL PYLKALGVSV LYLNPIFDSG SAHGYDTHDY 360
LKVSPKFGDK PLLRKLLDEA HRLGMRVIFD FVPNHTGLGF WAFQDVVKRG PRSPYWNWYF 420
IKRWPFVPGD GSAYEGWWGL GSLPKLNTAN PGVKRYLIEV TKYWVRFGFD GVRVDMPGDV 480
LNPHAFFKEM RAELKAIKPD AYLVAEIWQR DPSWLRGDEF DSLMNYAIGR DILLRFAKGG 540
SLALYNARRA LADLARVYAL YPEAVAGMGF NLITSHDTAR LLTELGGGGL KDVPSPEARA 600
RQRLAAAMLY ALPGLPVTFQ GDECGFTGER PADPPHELNR YPFQWEKCHG ETLAFYQELA 660
GLRRELAALR SAVFRTYFGE GHLLAFFRGE PGEGEVLAAF NNGVEAVTLP LPPGGWRDPL 720
EGRTYRKEVS LPPLGFRYLV HLGR 744
<212> Type : PRT
<211> Length : 744
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 AGGTGGTGGT 60
GGTTCCGGTG GTGGTGGTTC CGGTGGTGGT GGTAGCGGAA GAATCCTGGC GGTGTGGGTA 120
TGGCTTAGCC TGGCCCTGGC GGTCCCGGTG ACCTTCCGCT ACACACCCCC TTCGGGCCTC 180
GAGGTGCGCT CGGTAAGCCT CCGGGGCTCC TTCAACAGCT GGGGGGAAAC CCCCATGCAG 240
AAGGAGGACG GGTCCTGGGC GGTAACCGTG GACCTGGATC CAGGGGAGCA CCAGTACAAG 300
TTCTTCATCA ACGGCCAGTG GCCCAGGGAC ATGTGCAACG ATCCCACCTT CGGCACGCCC 360
ATGGTGGACC CGAAGGCGGC AGGGTGTGTG GACGATGGCT TTGGGGGTCA GAACGCCGTG 420
ATCGTGGTCC AGGCCCCGGT AGCCCCCACC CCTCCTGCGG GGCCCGTGGC CCTGGACTTC 480
ACCCATGATC CGTTGGACGC CCAGTATGTG TCCCATGCCG ACGGCAAGCT TTCCGTGCGC 540
TTCCGGGCAG GGGAGGGGGC GGTGGCGGCC GCCTGGGTCG AGGTGCAGGG AAAGAGGCTC 600
CCCATGCACC TGCAGCTGAG TTTTCCGGGA AGCGAGGTTT GGCGTGGGAC CTTACCTGGA 660
GGCGTGGGAG CCTACCGCAT CCTGGTGCGG ACCCAGGATG GCAAGGAGGA GGTGTTCGGC 720
CCCTTTAACC CTCCCGAAAG GCCCTTCGCC GAGGTGGCAT GGGTGGGCGA GGGGGTGGGT 780
TATCAGATCT TCCCCGAGCG CTTCTACAAC GGGGATTCCA GCAACGACGC CTTGGCCCTG 840
GAAACCGACG AGTACCGCTT TAACCAGGTG TGGCAGCGCT CCTCTGGGCC CAAGCCCCAT 900
CTTTCCCGCT GGGGCGATCC CCCCTCGCCC CTGCACTGCT GCCACCAGTA CTTCGGGGGG 960
GATCTTGCCG GGGTGCTGGC CAAGCTTCCT TACCTGAAGG CCCTGGGGGT TAGCGTCCTC 1020
TACCTGAATC CCATCTTTGA TTCCGGGTCG GCCCACGGCT ACGACACCCA CGACTACCTC 1080
AAGGTTTCCC CCAAGTTCGG CGACAAACCC CTCTTGCGCA AGCTGCTGGA CGAGGCCCAC 1140
CGCCTCGGCA TGCGGGTGAT CTTTGACTTC GTCCCCAACC ACACTGGCCT GGGCTTTTGG 1200
GCTTTTCAGG ATGTGGTAAA GAGGGGTCCC CGTTCCCCTT ACTGGAACTG GTACTTCATC 1260
AAGCGGTGGC CCTTTGTGCC GGGTGACGGA TCGGCCTACG AGGGATGGTG GGGGTTAGGG 1320
AGCCTGCCCA AGCTGAACAC CGCAAACCCC GGGGTGAAGC GCTACCTGAT CGAGGTGACC 1380
AAGTACTGGG TACGCTTCGG CTTTGACGGG GTGCGGGTGG ATATGCCCGG GGATGTGCTA 1440
AATCCTCACG CTTTCTTTAA GGAAATGCGG GCCGAACTGA AGGCCATCAA GCCCGACGCC 1500
TACCTGGTGG CGGAGATCTG GCAGAGGGAT CCTAGCTGGC TTCGGGGGGA TGAGTTTGAC 1560
TCCCTGATGA ACTACGCCAT CGGCCGGGAT ATCCTCCTCC GCTTTGCTAA GGGGGGAAGC 1620
CTGGCCCTGT ACAACGCCCG CCGAGCCTTG GCGGACCTAG CCCGGGTTTA CGCCCTTTAC 1680
CCGGAGGCGG TGGCCGGGAT GGGCTTCAAC TTGATCACCT CCCACGATAC GGCCCGCCTC 1740
CTTACCGAGC TTGGGGGCGG GGGCCTGAAG GACGTTCCCA GCCCGGAAGC CAGGGCCCGG 1800
CAGCGGCTTG CGGCGGCCAT GCTCTACGCC CTTCCCGGCC TCCCCGTAAC CTTCCAGGGG 1860
GATGAGTGCG GTTTCACCGG GGAAAGGCCG GCCGACCCCC CTCACGAGCT CAACCGGTAT 1920
CCCTTCCAGT GGGAGAAATG CCATGGGGAA ACCCTAGCCT TTTACCAGGA GCTGGCGGGG 1980
CTGCGCCGGG AGCTTGCGGC CCTCAGGAGC GCCGTGTTCC GGACCTACTT CGGAGAGGGC 2040
CATCTTCTGG CCTTCTTCCG GGGTGAGCCG GGAGAAGGGG AGGTGCTTGC CGCCTTCAAT 2100
AACGGGGTGG AGGCCGTCAC CTTGCCCTTG CCTCCTGGGG GCTGGCGGGA TCCCCTCGAG 2160
GGGCGCACCT ACCGGAAGGA AGTGAGCCTG CCCCCCCTGG GCTTCCGGTA CCTGGTCCAC 2220
CTGGGGCGGC ATCATCATCA TCATCATTAG 2250
<212> Type : PRT
<211> Length : 2250
SequenceName : SEQ ID NO:3
SequenceDescription :
Claims (7)
1. An artificially engineered β -galactosidase GaLT1, characterized by:
the beta-galactosidase GaLT1 is formed by connecting an artificially-modified amphiphilic short peptide sequence to the N-terminal amino acid sequence of a wild beta-galactosidase GaLT0 derived from T.scotoductus, wherein the amphiphilic short peptide sequence is shown as SEQ ID NO. 2.
2. The β -galactosidase GaLT1 according to claim 1, wherein:
the amino acid sequence of the beta-galactosidase GaLT1 is shown in SEQ ID NO. 1.
3. The gene encoding the beta-galactosidase GaLT1 according to claim 2, wherein the nucleotide sequence is represented by SEQ ID NO. 3.
4. A recombinant plasmid for expressing the β -galactosidase GaLT1 of claim 2, characterized in that the recombinant plasmid comprises at least the amino acid sequence shown in SEQ ID No. 1.
5. A recombinant strain for expressing the β -galactosidase GaLT1 of claim 2, characterized in that the recombinant strain comprises the recombinant plasmid of claim 4.
6. Use of the β -galactosidase GaLT1 according to claim 1 or 2, wherein:
the beta-galactosidase GaLT1 is used for hydrolyzing lactose, and has high thermal stability and lactose hydrolysis rate.
7. Use according to claim 6, characterized in that:
the lactose hydrolysis condition parameters are as follows: the enzyme content of the natural milk is 20U/mL, the reaction temperature is 55 ℃, and the reaction time is 2 h.
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| CN202111483822.6A CN114149987B (en) | 2021-12-07 | 2021-12-07 | Artificially modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis |
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Cited By (1)
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| CN114958893A (en) * | 2022-06-14 | 2022-08-30 | 中农华威生物制药(湖北)有限公司 | Construction method of lactase required by preparation of high-temperature creep feed for porkets |
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| CN114958893A (en) * | 2022-06-14 | 2022-08-30 | 中农华威生物制药(湖北)有限公司 | Construction method of lactase required by preparation of high-temperature creep feed for porkets |
| CN114958893B (en) * | 2022-06-14 | 2023-08-29 | 中农华威生物制药(湖北)有限公司 | Construction method of lactase required by preparation of suckling pig high-temperature creep feed |
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| CN114149987B (en) | 2024-02-13 |
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