CN114958893B - Construction method of lactase required by preparation of suckling pig high-temperature creep feed - Google Patents

Construction method of lactase required by preparation of suckling pig high-temperature creep feed Download PDF

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CN114958893B
CN114958893B CN202210671673.4A CN202210671673A CN114958893B CN 114958893 B CN114958893 B CN 114958893B CN 202210671673 A CN202210671673 A CN 202210671673A CN 114958893 B CN114958893 B CN 114958893B
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CN114958893A (en
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游锡火
王玉万
姜本荣
胡燕
梁大明
曾徐浩
夏胜
薛栋升
蒋慧
田美华
齐义清
沈力
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Zhongnong Huawei Biopharmaceutical Hubei Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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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)
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    • C12Y—ENZYMES
    • 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)
    • C12Y302/01108—Lactase (3.2.1.108)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

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Abstract

The invention discloses a construction method of lactase required for preparing a high-temperature creep feed for a suckling pig, belonging to the technical field of genetic engineering. The invention discloses a construction method of lactase required by preparing a suckling pig high-temperature creep feed, which comprises target gene amplification; enzyme cutting, connecting and converting; inducing the expression of a target gene by using IPTG, and obtaining a crude protein enzyme solution through ultrasonic disruption; and purifying the crude protein enzyme solution by adopting a Ni-NTA purifying medium. The invention utilizes the directed evolution technology to obtain lactase with better catalytic activity and thermal stability at higher temperature, the specific enzyme activity is 141.3U/mg (pH 7.0, 90 ℃), and the half life is 268.3min at 100 ℃. The enzyme is more suitable for a high-temperature granulating process or a high-temperature treatment process, and has wide application prospect and great potential economic benefit.

Description

Construction method of lactase required by preparation of suckling pig high-temperature creep feed
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a construction method of lactase required by preparing a high-temperature creep feed for suckling pigs.
Background
Pig raising has a huge market share in China, and along with the development of economic level, the living standard of people is improved, and the demand for meat is gradually increased. The space for raising pigs is huge in China, and the total amount of the pig feed industry is gradually increased.
The suckling pigs are lack of lactase because the bodies are not developed completely, and various organs are not fully mature. Lactose, if not digested, can cause malabsorption of nutrients and nutritional diarrhea. Therefore, lactase is added into the feed of the suckling pigs, so that nutrition absorption can be promoted, factors such as nutrition diarrhea and the like are avoided, and the benefit of suckling pig cultivation is improved.
Due to the influence of African swine fever, high-temperature granulation or high-temperature virus inactivation is required in the processing process of the feed. During the processing of feeds at high temperatures, enzymes that are not thermostable may be inactivated, thereby reducing the performance of the enzymes. The general methods for improving the stability of enzymes are as follows: enzyme immobilization, enzyme chemical modification, enzyme molecular fusion and the like. Molecular engineering of enzymes or fusion of enzyme molecules is considered an economic way. The molecular transformation of the enzyme has the advantages of greenness, low cost, simple industry and the like compared with other approaches. Based on the method, the heat resistance of lactase is improved by using a genetic engineering method, so that the lactase is more suitable for a high-temperature granulating or high-temperature feed process, the maximum catalytic capability is exerted, and the raising cost of the suckling pigs is reduced.
Therefore, providing a construction method of lactase needed for preparing a high-temperature creep feed for suckling pigs is a problem to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a construction method of lactase required for preparing a high-temperature creep feed for suckling pigs.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a construction method of lactase required by the preparation of a suckling pig high-temperature creep feed comprises the following specific steps:
(1) Amplification of target Gene
PCR amplification is carried out by taking Primer-F and Primer-R as primers and taking synthesized lactase gene sequence SEQ ID NO.1 as a template to obtain a target gene;
(2) Cleavage, ligation and transformation
Respectively carrying out double digestion on the target gene obtained in the step (1) and pRSFduet-1 expression vector, and connecting to obtain plasmid pRSFduet-1-SQ, so as to transform E.coli DH5 alpha competent cells;
(3) Inducing the expression of a target gene by using IPTG, and obtaining a crude protein enzyme solution through ultrasonic disruption;
(4) And purifying the crude protein enzyme solution by adopting a Ni-NTA purification medium, and analyzing the enzyme activity.
Further, the Primer sequences of Primer-F and Primer-R in the step (1) are as follows:
Primer-F：5’-GGGATCCATGTTCCCAGAAAA-3’；SEQ ID NO.3；
Primer-R：5’-CGAATTCTTAGTGATGGTGGTG-3’；SEQ ID NO.4。
further, the enzymes used for the double cleavage in step (2) are BamHI and EcoRI.
Compared with the prior art, the invention discloses a construction method of lactase required by the preparation of the suckling pig high-temperature creep feed, and the lactase with better catalytic activity and thermal stability at higher temperature is obtained by utilizing a directed evolution technology, wherein the specific enzyme activity is 141.3U/mg (pH 7.0, 90 ℃), and the half-life period is 268.3min at 100 ℃. The enzyme is more suitable for a high-temperature granulating process or a high-temperature treatment process, and has wide application prospect and great potential economic benefit.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
E, purchasing the color DH5 alpha in a commercial way and preserving in a laboratory; restriction enzymes, takaRa; plasmid extraction kit, tiangen Biochemical technology (Beijing) Co., ltd; the ready-to-use error-prone PCR kit is available from Beijing Tianen technologies Co., ltd; BCA protein concentration assay kit, fozhou omnirange laboratories, inc; plasmid miniprep extraction kit, gel recovery kit, tiangen Biochemical technology (Beijing) limited; plasmid pRSFduet-1 was purchased from Coley Biotech Co.Ltd; the sequence is composed of Optimus synthesized by limited company.
LB medium: 10g/L tryptone, 10g/L sodium chloride and 5g/L yeast extract.
Washing buffer: 10mM imidazole, 50mM K 2 HPO 4 -KH 2 PO 4 300mM NaCl,10% glycerol, pH 8.0.
Elution buffer: 200mM imidazole, 50mM K 2 HPO 4 -KH 2 PO 4 300mM NaCl,10% glycerol, pH 8.0.
Preservation buffer: 50mM K 2 HPO4-KH 2 PO4, 300mM NaCl,10% glycerol, pH 8.0.
EXAMPLE 1 error-prone PCR
(1) Amplification of target Gene
Error-prone PCR is performed by using the synthesized Primer-F and Primer-R as primers and the synthesized lactase gene sequence SEQ ID NO.1 (comprising restriction enzyme cleavage site, terminator TAA and His tag) as templates to obtain the target gene.
GGGATCCATGTTCCCAGAAAAATTCCTGTGGGGTGTTGCACAGTCTGGCTTCCAGTTCGAAATGGGTGACAAACTGCGTCGTAACATCGACACCAACACCGATTGGTGGCATTGGGTTCGCGATAAAACGAACATTGAGAAAGGTCTGGTTTCTGGCGACCTGCCGGAAGAGGGCATTAACAACTACGAGCTGTACGAAAAAGACCACGAGATCGCACGTAAACTGGGTCTGAACGCGTATCGTATCGGTATTGAATGGTCCCGTATCTTCCCGTGGCCGACGACCTTCATTGACGTGGACTACAGCTACAACGAGTCCTATAACCTGATCGAAGACGTTAAAATTACCAAGGACACCCTGGAAGAACTGGATGAAATCGCAAACAAACGTGAAGTTGCTTATTATCGTAGCGTTATTAACTCCCTGCGCTCCAAAGGCTTCAAAGTTATTGTAAACCTGAACCACTTCACGCTGCCATACTGGCTGCACGACCCGATCGAAGCGCGTGAACGTGCACTGACCAACAAACGTAACGGCTGGGTGAACCCGCGCACGGTGATTGAGTTCGCTAAATATGCGGCCTACATCGCGTATAAATTCGGTGACATCGTAGACATGTGGAGCACCTTCAACGAACCGATGGTAGTTGTTGAACTGGGTTACCTGGCGCCGTACAGCGGTTTCCCGCCGGGTGTACTGAACCCAGAAGCAGCCAAGCTGGCAATTCTGCACATGATTAACGCTCATGCTCTGGCCTATCGTCAGATTAAAAAATTCGACACCGAAAAAGCGGACAAAGACAGCAAGGAACCTGCGGAGGTAGGCATCATCTACAACAACATCGGCGTGGCCTACCCGAAAGATCCGAACGACAGCAAGGACGTGAAAGCGGCTGAGAACGATAACTTCTTTCACTCTGGTCTGTTCTTCGAAGCTATCCATAAAGGTAAACTGAACATCGAGTTCGATGGTGAAACTTTCATCGATGCTCCGTACCTGAAAGGCAACGACTGGATCGGTGTTAACTATTATACCCGCGAAGTTGTTACTTACCAGGAACCGATGTTCCCTTCTATTCCGCTGATCACCTTCAAAGGTGTTCAAGGCTACGGCTATGCGTGTCGCCCGGGCACCCTGTCCAAAGACGACCGTCCGGTTAGCGACATCGGCTGGGAGCTGTATCCGGAGGGTATGTACGATTCTATCGTGGAAGCGCACAAATATGGTGTGCCGGTGTACGTCACCGAAAACGGTATTGCAGATTCTAAAGACATTCTGCGTCCTTATTACATCGCGTCTCACATCAAGATGATTGAAAAAGCGTTCGAAGATGGTTACGAAGTTAAAGGTTATTTCCATTGGGCGCTGACGGACAACTTTGAATGGGCCCTGGGTTTCCGCATGCGTTTCGGCCTGTATGAAGTGAACCTGATCACGAAAGAACGCATCCCACGTGAAAAATCTGTTTCTATTTTCCGTGAAATTGTTGCTAACAACGGCGTTACCAAGAAAATTGAAGAGGAACTGCTGCGTGGCCACCATCACCACCATCACTAAGAATTCG；SEQ ID NO.1。
The amino acid sequence of lactase is shown in SEQ ID NO. 2:
MFPEKFLWGVAQSGFQFEMGDKLRRNIDTNTDWWHWVRDKTNIEKGLVSGDLPEEGINNYELYEKDHEIARKLGLNAYRIGIEWSRIFPWPTTFIDVDYSYNESYNLIEDVKITKDTLEELDEIANKREVAYYRSVINSLRSKGFKVIVNLNHFTLPYWLHDPIEARERALTNKRNGWVNPRTVIEFAKYAAYIAYKFGDIVDMWSTFNEPMVVVELGYLAPYSGFPPGVLNPEAAKLAILHMINAHALAYRQIKKFDTEKADKDSKEPAEVGIIYNNIGVAYPKDPNDSKDVKAAENDNFFHSGLFFEAIHKGKLNIEFDGETFIDAPYLKGNDWIGVNYYTREVVTYQEPMFPSIPLITFKGVQGYGYACRPGTLSKDDRPVSDIGWELYPEGMYDSIVEAHKYGVPVYVTENGIADSKDILRPYYIASHIKMIEKAFEDGYEVKGYFHWALTDNFEWALGFRMRFGLYEVNLITKERIPREKSVSIFREIVANNGVTKKIEEELLRGHHHHHH；SEQ ID NO.2。
Primer-F and Primer-R Primer sequences were as follows:
Primer-F：5’-GGGATCCATGTTCCCAGAAAA-3’；SEQ ID NO.3；
Primer-R：5’-CGAATTCTTAGTGATGGTGGTG-3’；SEQ ID NO.4。
the error-prone PCR reaction system is as follows: error-prone PCR Mix 3.0. Mu.L, 10. Mu.g/. Mu.L DNA template 1. Mu.L, 10. Mu.M PCR upstream and downstream primers 1. Mu.L each, sterilized double distilled water 24. Mu.L.
Error-prone PCR procedure was as follows: pre-denaturation at 95℃for 3min; denaturation at 94℃for 1min, annealing at 45℃for 1min, extension at 72℃for 4min, 32 cycles total; extending at 72℃for 10min.
(2) Cleavage, ligation and transformation
(1) Adding double distilled water, an endonuclease buffer solution, an enzyme cutting substrate and a restriction endonuclease into a PCR small tube respectively to carry out double enzyme cutting reaction, wherein the adding sequence is from more to less. The target gene and pRSFduet-1 are subjected to double digestion at 37 ℃, and the reaction system is as follows:
target gene double enzyme digestion system:
11. Mu.L of ultrapure water, 1. Mu.L of BamH I, 1. Mu.L of EcoR I, 3. Mu.L of Buffer and 14. Mu.L of the target gene.
pRSFduet-1 double cleavage system:
pRSFduet-1 plasmid 43. Mu.L, bamH I1. Mu.L, ecoR I1. Mu.L, buffer 5. Mu.L.
After the double digestion reaction for 3 hours at 37℃using BamH I and EcoR I restriction enzymes, the reaction was terminated by adding Loading Buffer, and the double digested product was purified and recovered according to the gel recovery kit instructions.
(2) After cleavage with the same restriction enzyme, the double digested products have the same cohesive ends and can be ligated into a complete plasmid by DNA ligase. The target gene sequence containing the same cohesive end and pRSFduet-1 double enzyme digestion product are placed in a same PCR small tube, a 10 mu L system is adopted for the ligation reaction, and the target gene and plasmid enzyme digestion product are subjected to the following steps of 3:1, and 1. Mu. L T4 DNA ligase, and ligating overnight at 16 ℃. The ligated plasmid was designated pRSFduet-1-SQ.
(3) The conversion step:
the ligation product was transformed into E.coli DH 5. Alpha. Competent cells as follows:
1) The temperature of the thermostatic water bath was first adjusted to 42 ℃.
2) A tube (100. Mu.l) of competent bacteria was removed from an ultra-low temperature freezer at-80℃and immediately thawed using a finger, and then inserted into ice for 10min ice bath.
3) Mu.l of ligation product pRSFduet-1-SQ was added and left on ice for 20min after gentle shaking.
4) After shaking gently, the mixture was inserted into a water bath at 42℃and subjected to heat shock for 90s, and then rapidly returned to ice and allowed to stand for 5min.
5) 900. Mu.L of LB medium without antibiotics was added to the above tube and gently mixed, then fixed on a spring holder of a shaker, and shaken at 37℃for 50min.
6) To a solid LB plate culture dish containing 30. Mu.g/mL kanamycin, 300. Mu.l of the above-mentioned transformation mixture taken out of the ultra-clean bench was dropped, and the mixture was uniformly coated by using a glass coating rod burned by an alcohol burner and cooled.
7) The coated culture dish is marked, firstly placed in a constant temperature incubator at 37 ℃ for 30-60 min, and then placed in the constant temperature incubator at 37 ℃ for overnight in a reverse way after the surface liquid permeates into the culture medium.
(4) Induction of expression
A. Single colonies growing on the LB solid medium are picked up, inoculated into 5mL of LB medium containing kanamycin, cultured overnight at 37 ℃ under the condition of 200r/min, partial bacterial liquid is taken to store strains by a glycerol tube, and the strains are sequentially numbered.
B. The transformed strain was inoculated in 6mL of LB medium (containing 50mg/L kanamycin sulfate) and cultured overnight at 37℃at 220rpm for 14 hours;
C. according to the following steps of 1:100 transferring the seeds to 500mL LB culture medium, culturing at 37 ℃ and 150rpm until reaching OD 600 =0.5; induction of gene expression with 0.7mM IPTG; culturing at 18 ℃ for 24 hours at 150rpm overnight after induction;
D. 4000g of the cells were collected by centrifugation at 4℃for 5min, resuspended in 30mL of preservation buffer, and then disrupted by an ultrasonic disrupter (working for 3s, at 7s intervals, for 10min, with an ultrasonic power of about 200W);
E. the crushed cell suspension is centrifuged for 30min at 12,000 rpm4deg.C, and the obtained supernatant is crude protease solution.
EXAMPLE 2 purification
The protein was purified using a Ni-NTA purification medium (south kyi gold sri biotechnology limited) from Shanghai:
in step 1, 6mL of Ni-NTA purification medium was added to a 50mL empty column, the medium allowed to settle freely, and the stock solution was drained.
Step 2, adding 4 times of column volume of washing buffer solution to balance the chromatography medium.
And 3, loading the crude protein enzyme solution into a column, wherein the flow rate is controlled to be 0.5mL/min.
Step 4, washing the column with washing buffer solution with a flow rate of 1mL/min, and removing the impurity protein by a flow rate of 10 times of the volume of the column.
And step 5, eluting with 5 times of column volume eluting buffer solution at the flow rate of 0.5-1 mL/min, and collecting the eluent.
Step 6, collecting target protein eluent, desalting and concentrating the purified protein by a Millipore ultrafiltration tube (15 mL of 100 kDa), and replacing the imidazole-containing elution buffer by a preservation buffer.
EXAMPLE 3 analysis of enzymatic Activity
Half-life and specific enzyme activity were measured as described in (Barkat, ali, zhuolin, yi, yang, fang, lanchai, chen, kaize, he, characterization of a fungal thermostable endoglucanase from Chinese Nong-flavor daqu by metatranscriptomic method, internationaljournal ofbiological macromolecules, 2019).
The purified protein concentration was determined using BCA protein concentration assay kit.
Mutant enzymes were selected based on half-life and specific enzyme activity, and the specific enzyme activity was determined to be 141.3U/mg (pH 7.0, 90 ℃) for an evolved enzyme, and at 100℃half-life was 268.3min (pH 7.0).
The amino acid sequence of lactase with high heat stability obtained by directed evolution is shown as SEQ ID NO. 5.
MFPEKFLWGVAQSSFQFEMGDKLRRLIDTNTDWWHVVRDKTNIEKGLVSGDLPEEGINNYELYEKDHEIARKLGLNAYRIGIEWSRIFPWPTTFIDVDYSYNESYNLIEDVKITKDTLEELDEIANKREVAYYRSVINSLRSKGFKVIVNLNHFTLPYWLHDPIEARERALTNKRNGWVNPRTVIEFAKYAAYIAYKFGDIVDMWSTFNEPMVVVELGYLAPYSGFPPGVLNPEAAKLAILHMINAHALAYRQIKKFDTEKADKDSKEPAEVGIIYNNIGVAYPKDPNDSKDVKAAENDNFFHSGLFFEAIHKGKLNIEFDGETFIDAPYLKGNDWIGVNYYTREVVTYQEPMFPSIPLITFKGVQGYGYACRPGTLSKDDRPVSDIGWELYPEGMYDSIVEAHKYGVPVYVTENGIADSKDILRPYYIASHIKMIEKAFEDGYEVKGYFHWALTDNFEWALGFRMRFGLYMVNLITKERIPREMSVSIFREIVHNNGVTKKIEEELLRGHHHHHH；SEQ ID NO.5。
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> North China biological pharmacy (Hubei) Co., ltd
<120> method for constructing lactase required for preparing suckling pig high-temperature creep feed
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1565
<212> DNA
<213> Artificial Sequence
<400> 1
gggatccatg ttcccagaaa aattcctgtg gggtgttgca cagtctggct tccagttcga 60
aatgggtgac aaactgcgtc gtaacatcga caccaacacc gattggtggc attgggttcg 120
cgataaaacg aacattgaga aaggtctggt ttctggcgac ctgccggaag agggcattaa 180
caactacgag ctgtacgaaa aagaccacga gatcgcacgt aaactgggtc tgaacgcgta 240
tcgtatcggt attgaatggt cccgtatctt cccgtggccg acgaccttca ttgacgtgga 300
ctacagctac aacgagtcct ataacctgat cgaagacgtt aaaattacca aggacaccct 360
ggaagaactg gatgaaatcg caaacaaacg tgaagttgct tattatcgta gcgttattaa 420
ctccctgcgc tccaaaggct tcaaagttat tgtaaacctg aaccacttca cgctgccata 480
ctggctgcac gacccgatcg aagcgcgtga acgtgcactg accaacaaac gtaacggctg 540
ggtgaacccg cgcacggtga ttgagttcgc taaatatgcg gcctacatcg cgtataaatt 600
cggtgacatc gtagacatgt ggagcacctt caacgaaccg atggtagttg ttgaactggg 660
ttacctggcg ccgtacagcg gtttcccgcc gggtgtactg aacccagaag cagccaagct 720
ggcaattctg cacatgatta acgctcatgc tctggcctat cgtcagatta aaaaattcga 780
caccgaaaaa gcggacaaag acagcaagga acctgcggag gtaggcatca tctacaacaa 840
catcggcgtg gcctacccga aagatccgaa cgacagcaag gacgtgaaag cggctgagaa 900
cgataacttc tttcactctg gtctgttctt cgaagctatc cataaaggta aactgaacat 960
cgagttcgat ggtgaaactt tcatcgatgc tccgtacctg aaaggcaacg actggatcgg 1020
tgttaactat tatacccgcg aagttgttac ttaccaggaa ccgatgttcc cttctattcc 1080
gctgatcacc ttcaaaggtg ttcaaggcta cggctatgcg tgtcgcccgg gcaccctgtc 1140
caaagacgac cgtccggtta gcgacatcgg ctgggagctg tatccggagg gtatgtacga 1200
ttctatcgtg gaagcgcaca aatatggtgt gccggtgtac gtcaccgaaa acggtattgc 1260
agattctaaa gacattctgc gtccttatta catcgcgtct cacatcaaga tgattgaaaa 1320
agcgttcgaa gatggttacg aagttaaagg ttatttccat tgggcgctga cggacaactt 1380
tgaatgggcc ctgggtttcc gcatgcgttt cggcctgtat gaagtgaacc tgatcacgaa 1440
agaacgcatc ccacgtgaaa aatctgtttc tattttccgt gaaattgttg ctaacaacgg 1500
cgttaccaag aaaattgaag aggaactgct gcgtggccac catcaccacc atcactaaga 1560
attcg 1565
<210> 2
<211> 516
<212> PRT
<213> Artificial Sequence
<400> 2
Met Phe Pro Glu Lys Phe Leu Trp Gly Val Ala Gln Ser Gly Phe Gln
1 5 10 15
Phe Glu Met Gly Asp Lys Leu Arg Arg Asn Ile Asp Thr Asn Thr Asp
20 25 30
Trp Trp His Trp Val Arg Asp Lys Thr Asn Ile Glu Lys Gly Leu Val
35 40 45
Ser Gly Asp Leu Pro Glu Glu Gly Ile Asn Asn Tyr Glu Leu Tyr Glu
50 55 60
Lys Asp His Glu Ile Ala Arg Lys Leu Gly Leu Asn Ala Tyr Arg Ile
65 70 75 80
Gly Ile Glu Trp Ser Arg Ile Phe Pro Trp Pro Thr Thr Phe Ile Asp
85 90 95
Val Asp Tyr Ser Tyr Asn Glu Ser Tyr Asn Leu Ile Glu Asp Val Lys
100 105 110
Ile Thr Lys Asp Thr Leu Glu Glu Leu Asp Glu Ile Ala Asn Lys Arg
115 120 125
Glu Val Ala Tyr Tyr Arg Ser Val Ile Asn Ser Leu Arg Ser Lys Gly
130 135 140
Phe Lys Val Ile Val Asn Leu Asn His Phe Thr Leu Pro Tyr Trp Leu
145 150 155 160
His Asp Pro Ile Glu Ala Arg Glu Arg Ala Leu Thr Asn Lys Arg Asn
165 170 175
Gly Trp Val Asn Pro Arg Thr Val Ile Glu Phe Ala Lys Tyr Ala Ala
180 185 190
Tyr Ile Ala Tyr Lys Phe Gly Asp Ile Val Asp Met Trp Ser Thr Phe
195 200 205
Asn Glu Pro Met Val Val Val Glu Leu Gly Tyr Leu Ala Pro Tyr Ser
210 215 220
Gly Phe Pro Pro Gly Val Leu Asn Pro Glu Ala Ala Lys Leu Ala Ile
225 230 235 240
Leu His Met Ile Asn Ala His Ala Leu Ala Tyr Arg Gln Ile Lys Lys
245 250 255
Phe Asp Thr Glu Lys Ala Asp Lys Asp Ser Lys Glu Pro Ala Glu Val
260 265 270
Gly Ile Ile Tyr Asn Asn Ile Gly Val Ala Tyr Pro Lys Asp Pro Asn
275 280 285
Asp Ser Lys Asp Val Lys Ala Ala Glu Asn Asp Asn Phe Phe His Ser
290 295 300
Gly Leu Phe Phe Glu Ala Ile His Lys Gly Lys Leu Asn Ile Glu Phe
305 310 315 320
Asp Gly Glu Thr Phe Ile Asp Ala Pro Tyr Leu Lys Gly Asn Asp Trp
325 330 335
Ile Gly Val Asn Tyr Tyr Thr Arg Glu Val Val Thr Tyr Gln Glu Pro
340 345 350
Met Phe Pro Ser Ile Pro Leu Ile Thr Phe Lys Gly Val Gln Gly Tyr
355 360 365
Gly Tyr Ala Cys Arg Pro Gly Thr Leu Ser Lys Asp Asp Arg Pro Val
370 375 380
Ser Asp Ile Gly Trp Glu Leu Tyr Pro Glu Gly Met Tyr Asp Ser Ile
385 390 395 400
Val Glu Ala His Lys Tyr Gly Val Pro Val Tyr Val Thr Glu Asn Gly
405 410 415
Ile Ala Asp Ser Lys Asp Ile Leu Arg Pro Tyr Tyr Ile Ala Ser His
420 425 430
Ile Lys Met Ile Glu Lys Ala Phe Glu Asp Gly Tyr Glu Val Lys Gly
435 440 445
Tyr Phe His Trp Ala Leu Thr Asp Asn Phe Glu Trp Ala Leu Gly Phe
450 455 460
Arg Met Arg Phe Gly Leu Tyr Glu Val Asn Leu Ile Thr Lys Glu Arg
465 470 475 480
Ile Pro Arg Glu Lys Ser Val Ser Ile Phe Arg Glu Ile Val Ala Asn
485 490 495
Asn Gly Val Thr Lys Lys Ile Glu Glu Glu Leu Leu Arg Gly His His
500 505 510
His His His His
515
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 3
gggatccatg ttcccagaaa a 21
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 4
cgaattctta gtgatggtgg tg 22
<210> 5
<211> 516
<212> PRT
<213> Artificial Sequence
<400> 5
Met Phe Pro Glu Lys Phe Leu Trp Gly Val Ala Gln Ser Ser Phe Gln
1 5 10 15
Phe Glu Met Gly Asp Lys Leu Arg Arg Leu Ile Asp Thr Asn Thr Asp
20 25 30
Trp Trp His Val Val Arg Asp Lys Thr Asn Ile Glu Lys Gly Leu Val
35 40 45
Ser Gly Asp Leu Pro Glu Glu Gly Ile Asn Asn Tyr Glu Leu Tyr Glu
50 55 60
Lys Asp His Glu Ile Ala Arg Lys Leu Gly Leu Asn Ala Tyr Arg Ile
65 70 75 80
Gly Ile Glu Trp Ser Arg Ile Phe Pro Trp Pro Thr Thr Phe Ile Asp
85 90 95
Val Asp Tyr Ser Tyr Asn Glu Ser Tyr Asn Leu Ile Glu Asp Val Lys
100 105 110
Ile Thr Lys Asp Thr Leu Glu Glu Leu Asp Glu Ile Ala Asn Lys Arg
115 120 125
Glu Val Ala Tyr Tyr Arg Ser Val Ile Asn Ser Leu Arg Ser Lys Gly
130 135 140
Phe Lys Val Ile Val Asn Leu Asn His Phe Thr Leu Pro Tyr Trp Leu
145 150 155 160
His Asp Pro Ile Glu Ala Arg Glu Arg Ala Leu Thr Asn Lys Arg Asn
165 170 175
Gly Trp Val Asn Pro Arg Thr Val Ile Glu Phe Ala Lys Tyr Ala Ala
180 185 190
Tyr Ile Ala Tyr Lys Phe Gly Asp Ile Val Asp Met Trp Ser Thr Phe
195 200 205
Asn Glu Pro Met Val Val Val Glu Leu Gly Tyr Leu Ala Pro Tyr Ser
210 215 220
Gly Phe Pro Pro Gly Val Leu Asn Pro Glu Ala Ala Lys Leu Ala Ile
225 230 235 240
Leu His Met Ile Asn Ala His Ala Leu Ala Tyr Arg Gln Ile Lys Lys
245 250 255
Phe Asp Thr Glu Lys Ala Asp Lys Asp Ser Lys Glu Pro Ala Glu Val
260 265 270
Gly Ile Ile Tyr Asn Asn Ile Gly Val Ala Tyr Pro Lys Asp Pro Asn
275 280 285
Asp Ser Lys Asp Val Lys Ala Ala Glu Asn Asp Asn Phe Phe His Ser
290 295 300
Gly Leu Phe Phe Glu Ala Ile His Lys Gly Lys Leu Asn Ile Glu Phe
305 310 315 320
Asp Gly Glu Thr Phe Ile Asp Ala Pro Tyr Leu Lys Gly Asn Asp Trp
325 330 335
Ile Gly Val Asn Tyr Tyr Thr Arg Glu Val Val Thr Tyr Gln Glu Pro
340 345 350
Met Phe Pro Ser Ile Pro Leu Ile Thr Phe Lys Gly Val Gln Gly Tyr
355 360 365
Gly Tyr Ala Cys Arg Pro Gly Thr Leu Ser Lys Asp Asp Arg Pro Val
370 375 380
Ser Asp Ile Gly Trp Glu Leu Tyr Pro Glu Gly Met Tyr Asp Ser Ile
385 390 395 400
Val Glu Ala His Lys Tyr Gly Val Pro Val Tyr Val Thr Glu Asn Gly
405 410 415
Ile Ala Asp Ser Lys Asp Ile Leu Arg Pro Tyr Tyr Ile Ala Ser His
420 425 430
Ile Lys Met Ile Glu Lys Ala Phe Glu Asp Gly Tyr Glu Val Lys Gly
435 440 445
Tyr Phe His Trp Ala Leu Thr Asp Asn Phe Glu Trp Ala Leu Gly Phe
450 455 460
Arg Met Arg Phe Gly Leu Tyr Met Val Asn Leu Ile Thr Lys Glu Arg
465 470 475 480
Ile Pro Arg Glu Met Ser Val Ser Ile Phe Arg Glu Ile Val His Asn
485 490 495
Asn Gly Val Thr Lys Lys Ile Glu Glu Glu Leu Leu Arg Gly His His
500 505 510
His His His His
515

Claims (1)

1. A lactase with high heat stability is characterized in that the amino acid sequence is shown as SEQ ID NO. 5.
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CN114958809A (en) * 2022-06-14 2022-08-30 中农华威生物制药(湖北)有限公司 Construction method of endo-beta-glucanase suitable for high-temperature granulation of feed

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JPH11137247A (en) * 1997-11-10 1999-05-25 Toyobo Co Ltd Production of beta 1,4-galactose transferase
JPH11225768A (en) * 1998-02-20 1999-08-24 Tadayuki Imanaka Hyper-thermostable beta-glycosidase
CN1610746A (en) * 2000-07-22 2005-04-27 辛根塔参与股份公司 Process for pyrohydrolyzing oligose containing galactose in complex mixture
WO2009000063A1 (en) * 2007-06-25 2008-12-31 National Research Council Of Canada Inducible/regulated gene expression systεm in e coli
CN113234743A (en) * 2021-06-07 2021-08-10 广东溢多利生物科技股份有限公司 Heat-resistant alpha-galactosidase gene and application thereof
CN113817763A (en) * 2021-10-26 2021-12-21 中国水产科学研究院黄海水产研究所 Beta-galactosidase family gene directed evolution method, mutant and application thereof
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JP2022524312A (en) * 2019-02-28 2022-05-02 デュポン ニュートリション バイオサイエンシス エーピーエス How to reduce lactose at high temperatures
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JPH11137247A (en) * 1997-11-10 1999-05-25 Toyobo Co Ltd Production of beta 1,4-galactose transferase
JPH11225768A (en) * 1998-02-20 1999-08-24 Tadayuki Imanaka Hyper-thermostable beta-glycosidase
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WO2009000063A1 (en) * 2007-06-25 2008-12-31 National Research Council Of Canada Inducible/regulated gene expression systεm in e coli
CN113234743A (en) * 2021-06-07 2021-08-10 广东溢多利生物科技股份有限公司 Heat-resistant alpha-galactosidase gene and application thereof
CN113817763A (en) * 2021-10-26 2021-12-21 中国水产科学研究院黄海水产研究所 Beta-galactosidase family gene directed evolution method, mutant and application thereof
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