CN108220308B - Beta-galactosidase, gene and carrier thereof, bacterial strain, product containing beta-galactosidase and method for converting lactose and fructose into lactulose - Google Patents
Beta-galactosidase, gene and carrier thereof, bacterial strain, product containing beta-galactosidase and method for converting lactose and fructose into lactulose Download PDFInfo
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- 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)
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Abstract
The invention relates to the field of microorganisms, in particular to beta-galactosidase, a gene, a carrier and a strain thereof, a product containing the beta-galactosidase and a method for converting lactose and fructose into lactulose. The beta-galactosidase coding gene has: SEQ ID NO: 1; SEQ ID NO: 1, and the nucleotide sequence still has beta-galactosidase activity after being substituted by one or more of the nucleotide sequences shown in the specification; beta-galactosidase has enzymatic activity that catalyzes the conversion of lactose and fructose to lactulose. The method comprises the following steps: the beta-galactosidase as described above is contacted with lactose and fructose to catalyze the conversion of said lactose and fructose into lactulose. The method is environment-friendly, can efficiently convert lactose and fructose into lactulose, and does not generate harmful byproducts. The beta-galactosidase coded by the gene can be catalyzed at 70 ℃, and has low requirement on pH and good thermal stability.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a gene for coding beta-galactosidase, a recombinant vector containing the gene for coding beta-galactosidase, a strain containing the gene for coding beta-galactosidase or the recombinant vector, beta-galactosidase coded by the gene for coding beta-galactosidase, a product containing the beta-galactosidase and a method for converting lactose and fructose into lactulose.
Background
Lactulose (Lactulose) is an important functional disaccharide, has moderate sweet taste, is difficult to be digested and absorbed by human intestinal tracts, is a safe and effective medicament for promoting intestinal tract movement and treating constipation as a medicament, is a good sweet substitute as a food additive, and is suitable for being used as an additive in foods with special requirements (such as foods of diabetics). Because of its high safety, its production and application are receiving more and more attention from people. However, the synthesis cost of lactulose is high, the catalytic efficiency is slow, and the large-scale production of lactulose is limited.
Lactulose is less distributed and less abundant in natural products and is therefore difficult to extract from animals and plants. Lactulose is obtained by condensing a molecule of galactose and a molecule of fructose, and has a structure which is slightly different from lactose, so that lactose can be converted into lactulose by a chemical catalysis method, and the lactose can be converted into lactulose by an electrochemical method. However, the two methods for converting lactulose have problems such as high reaction cost, difficult catalyst removal, difficult separation of by-products and generation of by-products harmful to human body. The chemical catalyst and the mode of catalyzing lactose isomerization by electrochemistry to generate lactulose greatly limit the application of the lactulose in pharmacy and food.
Therefore, a method for producing lactulose at low cost and without toxic and side effects to human body is needed.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides beta-galactosidase which is low in cost and does not produce toxic or side products in the production process and can be used for efficiently producing lactulose, a coding gene of the beta-galactosidase, a recombinant vector containing the gene for coding the beta-galactosidase, a strain containing the gene for coding the beta-galactosidase or the recombinant vector, a product containing the beta-galactosidase and a method for converting lactose and fructose into lactulose.
In order to achieve the above objects, in one aspect, the present invention provides a gene encoding β -galactosidase, wherein the gene has any one of the following nucleotide sequences:
(a) SEQ ID NO: 1;
(b) SEQ ID NO: 1, wherein one or more of the nucleotides at positions 214-216, 256, 341, 427, 466, 609, 613, 863, 891, 910, 989, 1092, 1270 and 1298 in the nucleotide sequence shown in the sequence 1 is/are substituted to still have beta-galactosidase activity;
(c) SEQ ID NO: 1, wherein one or more of nucleotides at positions 38, 107, 108, 170, 197, 245, 290, 314, 449, 457, 470, 476, 548, 559, 578, 608, 668, 764, 776, 803, 914, 986, 1053, 1058, 1100, 1220, 1283, 1286, 1355, 1368, 1403 and 1436 in the nucleotide sequence shown in 1 is/are substituted to still have beta-galactosidase activity;
wherein the beta-galactosidase has an enzymatic activity which catalyzes the conversion of lactose and fructose to lactulose.
In a second aspect, the present invention provides a recombinant vector, wherein the recombinant vector contains the gene encoding β -galactosidase as described above.
In a third aspect, the present invention provides a strain, wherein the strain contains the gene encoding beta-galactosidase or the recombinant vector as described above.
In a fourth aspect, the present invention provides a β -galactosidase encoded by the gene encoding β -galactosidase as described above, wherein said β -galactosidase has an enzymatic activity which catalyzes the conversion of lactose and fructose into lactulose.
In a fifth aspect, the invention provides a product comprising a β -galactosidase as described above.
In a sixth aspect, the present invention provides a process for converting lactose and fructose to lactulose, the process comprising: the beta-galactosidase as described above is contacted with lactose and fructose to catalyze the conversion of said lactose and fructose into lactulose.
The invention has the following advantages: the method is environment-friendly, can efficiently convert lactose and fructose into lactulose, does not generate any harmful by-product, and is beneficial to the expanded production of the lactulose as a medicament and food additive. The beta-galactosidase coded by the gene sequence can be catalyzed at high temperature of 70 ℃, has low requirement on the pH value, and has the characteristics of good stability, low industrial production cost and the like.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Detailed Description
The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In a first aspect, the invention discloses a gene encoding beta-galactosidase, wherein the gene has any one of the following nucleotide sequences:
(a) SEQ ID NO: 1;
(b) SEQ ID NO: 1, wherein one or more of the nucleotides at positions 214-216, 256, 341, 427, 466, 609, 613, 863, 891, 910, 989, 1092, 1270 and 1298 in the nucleotide sequence shown in the sequence 1 is/are substituted to still have beta-galactosidase activity;
(c) SEQ ID NO: 1, wherein one or more of nucleotides at positions 38, 107, 108, 170, 197, 245, 290, 314, 449, 457, 470, 476, 548, 559, 578, 608, 668, 764, 776, 803, 914, 986, 1053, 1058, 1100, 1220, 1283, 1286, 1355, 1368, 1403 and 1436 in the nucleotide sequence shown in 1 is/are substituted to still have beta-galactosidase activity;
wherein the beta-galactosidase has an enzymatic activity which catalyzes the conversion of lactose and fructose to lactulose.
The inventors of the present invention found in their studies that substitutions at the sites as listed above have a smaller effect on the activity of the encoded beta-galactosidase. Therefore, the object of the present invention can be achieved by any of the above nucleotide sequences.
In addition, the present inventors have also found that, in the case where the mutation site as in (b) above is satisfied, the mutation site of SEQ ID NO: 1, substitution of one or more nucleotides selected from the group consisting of nucleotides 190, 230, 247, 316, 332, 366, 399, 400, 496, 526, 677, 859, 928, 968, 1040, 1073, 1120, 1222 and 1308 among the nucleotide sequences shown in FIG. 1 is also effective for the purpose of the present invention.
Furthermore, it is also to be understood in the art that 18 amino acids, of the 20 different amino acids that make up a protein, are each encoded by 2 to 6 codons, except for Met (ATG) or Trp (TGG), which are each encoded by a single codon (Sambrook et al, molecular cloning, Cold spring harbor laboratory Press, New York, USA, second edition, 1989, see appendix D page 950). That is, due to the degeneracy of genetic code, there is usually more than one codon determining one amino acid, and the substitution of the third nucleotide in the triplet codon will not change the composition of the amino acid, so that the nucleotide sequences of genes encoding the same protein may differ. From the amino acid sequences encoded by the nucleotide sequences disclosed in the present invention, the nucleotide sequences of other genes capable of encoding these amino acid sequences can be completely deduced by those skilled in the art from well-known codon tables, and the nucleotide sequences are obtained by biological methods (e.g., PCR method, mutation method) or chemical synthesis methods, so that nucleotide sequences having a portion different from the nucleotide sequences of the present invention due to the degeneracy of genetic code are also included in the scope of the present invention.
Specific examples after such sequence changes include, but are not limited to, SEQ ID NOs: 2. SEQ ID NO: 3 or SEQ ID NO: 4.
According to the present invention, to facilitate purification of the encoded β -galactosidase, a nucleotide sequence encoding a tag common in the art (e.g., at least one of Poly-Arg, Poly-His, FLAG, Strep-tag II, and c-myc, which may also be present in the attached plasmid) may also be added to the above nucleotide sequence to add modifications to (a) or (b). The tag does not influence the enzymatic activity of the encoded beta-galactosidase, and whether the tag is added or not can be selected according to requirements in the actual application process.
According to the present invention, a cleavage site, a promoter, an enhancer, a terminator and the like, which are conventionally used in the art, may be added to the above nucleotide sequence, and a nucleotide sequence containing the cleavage site, the promoter, the enhancer and the terminator should be included in the scope of the present invention.
The nucleotide sequence provided by the invention can be obtained by a Polymerase Chain Reaction (PCR) amplification method, a recombination method or an artificial synthesis method. For example, one skilled in the art can easily obtain templates and primers based on the nucleotide sequences provided by the present invention, and obtain the relevant sequences by PCR amplification.
Once the nucleotide sequence of interest has been obtained, the protein (enzyme) of interest can be obtained in large quantities by recombinant methods. The nucleotide sequence obtained is usually cloned into a vector and transferred into a host, and the relevant protein (enzyme) is isolated from the propagated host cells by conventional methods.
In a second aspect, the present invention also provides a recombinant vector, wherein the recombinant vector contains the gene encoding β -galactosidase.
As the "vector" used in the recombinant vector of the present invention, various vectors known in the art, such as various commercially available plasmids, cosmids, phages, retroviruses, and the like, can be used, and the pET-28a vector is preferably used in the present invention. The recombinant vector can be constructed by digesting a linear plasmid with various endonucleases having cleavage sites at the multiple cloning sites of the vector (e.g., Sal I, BamH I, EcoR I for pUC 18; Nde I, Nhe I, EcoR I, BamH, HindIII for pPICZ alpha A) and ligating the linear plasmid with a gene fragment cleaved with the same endonuclease to obtain a recombinant plasmid.
In a third aspect, the present invention also provides a strain, wherein the strain contains the gene encoding β -galactosidase or the recombinant vector.
In the present invention, the strain may be a recombinant strain obtained by transforming, transducing or transfecting the above recombinant vector into a host by a method conventional in the art, such as calcium chloride method, chemical transformation or shock transformation, preferably shock transformation, and the present invention will not be described in detail herein.
According to the invention, the strain can be prokaryotic or eukaryotic. The prokaryotic strain may be various prokaryotic strains conventionally used in the art, for example, Escherichia coli. The eukaryotic strain may be various fungal strains conventionally used in the art, for example, yeast. Preferably, the strain is a prokaryotic strain, more preferably E.coli.
In a fourth aspect, the present invention also provides a β -galactosidase encoded by the gene encoding β -galactosidase as described above, wherein said β -galactosidase has an enzymatic activity which catalyzes the conversion of lactose and fructose into lactulose.
As mentioned above, conventional modifications may also be made to the enzymes provided by the present invention, and modifications (which do not generally alter the primary structure, i.e.do not alter the amino acid sequence) may include: chemically derivatized forms of proteins such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those proteins that result from glycosylation modifications during synthesis and processing of the protein or during further processing steps. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Modifications may also be included to improve its resistance to proteolysis, to optimize proteolytic performance, or to improve protein thermostability, and the like. These modifications are conventional in the art and are not described herein in detail. However, it should be noted that the protein obtained by these conventional modifications is also included in the scope of the present invention.
In a fifth aspect, the invention provides a product comprising a β -galactosidase as described above.
The product may be a pharmaceutical and/or a food additive. The amount of the beta-galactosidase contained in the medicine and/or food additive can be referred to the content of the beta-galactosidase in the conventional medicine and/or food additive, and the detailed description of the invention is omitted.
In a sixth aspect, the present invention provides a process for converting lactose and fructose to lactulose, the process comprising: the beta-galactosidase as described above is contacted with lactose and fructose to catalyze the conversion of said lactose and fructose into lactulose.
The "beta-galactosidase" used in the "contacting beta-galactosidase with lactose and fructose" described above may be purified beta-galactosidase or a lysate containing beta-galactosidase obtained by directly cleaving the expression strain. The present invention is not particularly limited thereto as long as it contains the beta-galactosidase.
Compared with the existing beta-galactosidase, the beta-galactosidase provided by the invention has the advantages of obviously improved heat resistance and thermal stability, and can perform catalytic reaction under acidic and alkaline pH values.
According to the invention, the beta-galactosidase can catalyze the polymerization reaction of lactose and fructose at a temperature of up to 70 ℃ or even higher. Preferably, the temperature of the catalysis is 30-70 ℃, more preferably, the temperature of the catalysis is 40-60 ℃, and most preferably, the temperature of the catalysis is 50 ℃.
According to the present invention, the beta-galactosidase can catalyze the polymerization of lactose and fructose at a pH of 9 or higher and a pH of 4 or lower. Preferably, the catalytic pH is 4 to 9, more preferably, the catalytic pH is 5 to 8, and most preferably, the catalytic pH is 6.
The present invention will be described in detail below by way of examples.
In the following test examples:
the method for measuring the content of lactulose comprises the following steps: the content of lactulose is determined by TLC method, and the spot area formed on thin layer chromatography silica gel plate by sample amount of lactulose with concentration of 1 μ L of 0.1-0.5g/L is determined by external standard method. The area determination method is shown in reference literature (thin-layer chromatography spot area method for determining emodin content in radix et rhizoma Rhei, Curibanjiang, A.kikai; proceedings of Isui academy of teachers and academy of sciences (Nature version); stage 1 of 3 months in 2008).
The method for measuring the enzyme activity comprises the following steps: measurement of beta-galactosidase Activity the amount of glucose produced using lactose as a substrate was measured according to the Sigma glucose (HK) colorimetric kit. Per unit of enzyme activity is defined as: the amount of enzyme required to catalyze the production of each 1. mu. mol of glucose per minute. The reaction system was 50mM lactose and 50mM sodium phosphate buffer.
Examples 1 to 4
This example illustrates the obtention of beta-galactosidase provided by the present invention
(1) Obtaining of genes
According to SEQ ID NO: 1-4 (named as: gal1, gal2, gal3, gal4, respectively) were synthesized by artificial chemical synthesis (Shinbao bioengineering, Inc., Kyowa, Inc.) to obtain the corresponding gene encoding beta-galactosidase.
(2) Amplification of genes
The primers (forward primer F: CGCGGATCCATGTCATCC, SEQ ID NO: 5; reverse primer R: CCCAAGCTTTCAGTCCTCC, SEQ ID NO: 6) were used to amplify the DNA fragment of SEQ ID NO: 1-4, performing amplification.
The system for amplification was as follows:
template: 2uL
10x PCR buffer：5uL
dNTP：2uL
And (3) primer F: 0.5uL
And (3) primer R: 0.5uL
Taq enzyme: 0.5uL
Water: 39.5uL
Amplification conditions:
94℃4min；
1min at 94 ℃; 40s at 55 ℃; 2min at 72 ℃; 30 cycles;
10min at 72 ℃; storing at 16 ℃.
The amplified products were subjected to electrophoresis detection (Biowest, agarose), and the target fragments were recovered using a gel recovery kit from Beijing Bomeide according to the instructions to obtain amplification products of gal1, gal2, gal3, and gal 4.
(3) After the amplification products of gal1, gal2, gal3 and gal4 were digested with BamHI and HindIII (restriction enzymes from NEB) (the digestion sites are BamHI and HindIII (NEB are provided by SEQ ID NO: 5-6) and ligated to the similarly digested pET-28a plasmid (commercial plasmid, pET-28a-c (+)) obtained from Novogene according to the instructions contained in the plasmid), the ligation products were transformed into e.coli (top 10 competent cells from Beijing Mebo de company and transformed according to the instructions) as competent to obtain recombinant strains containing gal1, gal2, gal3 and gal4, which were designated as EG1, EG2, EG3 and EG4, respectively.
(4) Obtaining beta-galactosidase
The above 4 recombinant strains were expressed in M9 medium containing kanamycin (20. mu.g/ml) and IPTG (0.5mM) at 32 ℃ and 200 rpm. And (4) after 12h of culture, centrifuging the thalli, washing for 3 times by using PBS buffer solution, and then carrying out ultrasonic lysis to obtain a lysate.
(5) The lysate obtained in step (4) was subjected to PCR amplification according to the method of step (2), and the amplified product was sent to Beijing Bomeide for sequencing, which showed that the genes GAL1, GAL2, GAL3, and GAL4 were successfully transformed into E.coli, thereby indicating that lysates containing GAL1, GAL2, GAL3, and GAL4 were obtained in step (3), respectively.
Comparative example 1
Preparation of lysates was carried out as in examples 1-4, except that empty pET-28a plasmid was transformed into competent E.coli.
Comparative example 2
This comparative example serves to illustrate the acquisition of a reference beta-galactosidase
Preparation of lysates was performed as in examples 1-4, except that the gene encoding β -galactosidase ligated into the pET-28a plasmid was as set forth in SEQ ID NO: shown at 7.
Test example 1
(1) Detection of enzymatic Activity
The disrupted solutions obtained in example 1 and comparative examples 1-2 were added to PBS (pH6.0) buffer containing 1g/L lactose and 1g/L fructose, reacted at 40 ℃ for 30min, and the lactulose content and enzyme activity were measured and calculated.
As a result of the measurement, the enzyme specific activities of GAL1, GAL2, GAL3 and GAL4 were 4.0U/mg, 3.6U/mg and 3.6U/mg, respectively, lactulose was not detected in comparative example 1, and the enzyme specific activity was 1.2U/mg in comparative example 2.
(2) Heat resistance test
The determination of the enzyme specific activity was carried out in the same manner as in the above step (1) except that the temperatures for the catalysis were 30 ℃, 40 ℃, 50 ℃, 60 ℃ and 70 ℃ respectively, and the results are shown in Table 1.
(3) Thermal stability test
The enzyme specific activity was measured as in the above step (1) except that the catalytic temperature was 50 ℃ and 70 ℃ and the reaction time was 0.5h, 1h, 2h, 4h, 8h, 16h, 32h and 64h, respectively, and the enzyme specific activities at different temperatures and time points were measured. The reaction substrates were in excess at each time point, and the results are shown in Table 2.
(4) Acid and alkali resistance test
The determination of the specific activity of the enzyme was carried out in the same manner as in the above step (1) except that the catalytic pH was 4, 5, 6, 7, 8 and 9, respectively, and the results are shown in Table 3.
TABLE 1
TABLE 2
TABLE 3
From the above, the biotransformation method provided by the invention can efficiently convert lactose and fructose into lactulose, and no raw material harmful to human body is used in the process, and no by-product harmful to human body is generated, so that the method is beneficial to the expanded production of lactulose as a medicament and food additive. The beta-galactosidase coded by the gene sequence can be catalyzed at high temperature of 70 ℃, has low requirement on pH value (can perform catalysis in acidic and alkaline environments), and has the characteristics of good thermal stability, low industrial production cost and the like.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
SEQUENCE LISTING
<110> Zhongliang group Co., Ltd
COFCO NUTRITION AND HEALTH RESEARCH INSTITUTE Co.,Ltd.
<120> beta-galactosidase, gene and vector thereof, strain, product containing beta-galactosidase and method for converting lactose and fructose
Process for preparing lactulose
<130> I42021COF
<160> 7
<170> PatentIn version 3.3
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tatctggtta gccatgtgta tcaggtgcat cgcgccatta atagcggtgc agatgtgcgc 1260
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tttggcctgc tgaaagtgga ttataatacc aaacgtctgt attggcgtcc gagcgcactg 1380
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ggtaattatg aaacctttca tgataatgcc caggcgatgg gtctgaaaaa tgcccgtctg 240
aatgttcaat ggagtagtat ttttccgaat ccgctgccgc gtccgcagaa ttttgatgaa 300
agtaaacagg atgtttccga agttgaaatt attgaaaatg tactgaaacg cctggatgaa 360
tatgccaata aagatgcgct gaatcattat cgcgaaatgg ttaaagatct gaaatcacgc 420
ggcctgaatt ttattctgaa tatgtatcat tggccgctgc cgctgaggct gcatgatccg 480
attcgcgtgc gccgccgtga ttttaccggc ccgtctggct ggctgactac ccgcaccgtg 540
tatgaatttg cgcgcttttc agcgtatatt gcgtggaaat ttgatgatct ggtggatgaa 600
tattcaacga tgcatgaacc gaatgttgtt ggtggtctgg gttatgttgg tgttaaaagc 660
ggttttccgc cgggttgtct gagttttgaa ctgagtcgtc gtgccatgta taatattatt 720
caggcccatg cccgtgccta tgatggtatt aaaagtgtta gtaaaaaacc ggtgggcatt 780
atttatgcga attcatcatt tcagccgctg accgataaag atatggaagc tgtggaaatg 840
gctgaaaatg ataatcgccg gtagtttttt gatgctatta ttcgcggtga tattacccgt 900
ggtaatgaag aaattgttcg tgatgatatg aaaggtcgtc tggattggat tggtgttaat 960
tattatagcc gtaccgtggt gaaacgcagc gaaaaaggct atgtgtcact gggcggctat 1020
ggccatggct gcgaacgcag ttctgtgtct ctggccggtc tgccgaccag cggttttggt 1080
tgggaatttt tcccggaagg tctgtatgat gttctgaccg aatattggaa tcgttatcat 1140
ctgtatatgt atgttaccga aaatggtatt gcggatgatg ctgattatca gcgtccgtat 1200
tatctggtta gccatgtgta taaggtgcat cgcgccatta atagcggtgc agatgtgcgc 1260
ggctatctgg attggagtct ggcagataat tatgaatcgg catcaggatt ttcaatgcgt 1320
tttggcctgc tgaaagtgga ttataatacc aaacgtctgt attggcgtcc gagcgcactg 1380
gtttatcgtg aaattgcaac caatggcgca attaccgatg aaattgaaca tctgaatagc 1440
gttccgccgg ttaaaccgct gcgtcattaa 1470
<210> 3
<211> 1470
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 3
atgtatagct ttccgaattc ttttcgcttt ggctggtctc aggctggctt tcagtctgaa 60
atgggcaccc cgggctctga agatccgaat accgattggt ataaatgggt gcatgatccg 120
gaaaatatgg ctgctggcct ggtgagtggt gatctgccgg aaaatggtcc gggttattgg 180
ggtaattata aaacctttca tgataatgcc caggcgatgg gtctgaaaat tgcccgtctg 240
aatgttgaat ggagtagtat ttttccgaat ccgctgccgc gtccgcagaa ttttgatgaa 300
agtaaacagg atgttaccga agttgaaatt aatgaaaatg tactgaaacg cctggatgaa 360
tatgcgaata aagatgcgct gaatcattat cgcgaaattt ttaaagatct gaaatcacgc 420
ggcctgaatt ttattctgaa tatgtatcat tggccgctgc cgctgaggct gcatgatccg 480
attcgcgtgc gccgcggtga ttttaccggc ccgtctggct ggctgtctac ccgcaccgtg 540
tatgaatttg cgcgcttttc agcgtatatt gcgtggaaat ttgatgatct ggtggatgaa 600
tattcaacga tgcatgaacc gaatgttgtt ggtggtctgg gttatgttgg tgttaaaagc 660
ggttttccgc cgggttatct gagttttgaa ctgagtcgtc gtgccatgta taatattatt 720
caggcccatg cccgtgccta tgatggtatt aaaagtgtta gtaaaaaacc ggtgggcatt 780
atttatgcga attcatcatt tcagccgctg accgataaag atatggaagc tgtggaaatg 840
gctgaaaatg ataatcgctg gtagtttttt gatgctatta ttcgcggtga tattacccgt 900
ggtaatgaag aaattgttcg tgatgatctg aaaggtcgtc tggattggat tggtgttaat 960
tattataccc gtaccgtggt gaaacgcagc gaaaaaggct atgtgtcact gggcggctat 1020
ggccatggct gcgaacgcaa ttctgtgtct ctggccggtc tgccgaccag cgattttggt 1080
tgggaatttt tcccggaagg tctgtatgat gttctgacca aatattggaa tcgttatcat 1140
ctgtatatgt atgttaccga aaatggtatt gcggatgatg ctgattatca gcgtccgtat 1200
tatctggtta gccatgtgta tcaggtgcat cgcgccatta atagcggtgc agatgtgcgc 1260
ggctatctgg attggagtct ggcagataat tatgaatcgg catcaggctt ttcaatgcgt 1320
tttggcctgc tgaaagtgga ttataatacc aaacgtctgt attggcgtcc gagcgcactg 1380
gtttatcgtg aaattgcaac caatggcgca attaccgatg aaattgaaca tctgaatagc 1440
gttccgccgg ttaaaccgct gcgtcattaa 1470
<210> 4
<211> 1470
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 4
atgtatagct ttccgaattc ttttcgcttt ggctggtatc aggctggctt tcagtctgaa 60
atgggcaccc cgggctctga agatccgaat accgattggt ataaatcagt gcatgatccg 120
gaaaatatgg ctgctggcct ggtgagtggt gatctgccgg aaaatggtca gggttattgg 180
ggtaattata aaacctatca tgataatgcc cagaaaatgg gtctgaaaat tgcccgtctg 240
aatgctgaat ggagtcgtat ttttccgaat ccgctgccgc gtccgcagag ttttgatgaa 300
agtaaacagg atgctaccga agttgaaatt aatgaaaatg aactgaaacg cctggatgaa 360
tatgcgaata aagatgcgct gaatcattat cgcgaaattt ttaaagatct gaaatcacgc 420
ggcctgtatt ttattctgaa tatgtatcgt tggccggtgc cgctgtggca gcatgctccg 480
attcgcgtgc gccgcggtga ttttaccggc ccgtctggct ggctgtctac ccgcaccgtg 540
tatgaatgtg cgcgctttac agcgtatatt gcgtggatat ttgatgatct ggtggatgaa 600
tattcaagca tgaatgaacc gaatgttgtt ggtggtctgg gttatgttgg tgttaaaagc 660
ggttttcagc cgggttatct gagttttgaa ctgagtcgtc gtgccatgta taatattatt 720
caggcccatg cccgtgccta tgatggtatt aaaagtgtta gtataaaacc ggtggacatt 780
atttatgcga attcatcatt tccgccgctg accgataaag atatggaagc tgtggaaatg 840
gctgaaaatg ataatcgctg gtggtttttt gatgctatta ttcgcggtga aattacccgt 900
ggtaatgaaa aaagtgttcg tgatgatctg aaaggtcgtc tggattggat tggtgttaat 960
tattataccc gtaccgtggt gaaacccacc gaaaaaggct atgtgtcact gggcggctat 1020
ggccatggct gcgaacgcaa ttctgtgtct cttgccgatc tgccgaccag cgattttggt 1080
tgggaatttt ttccggaagt tctgtatgat gttctgacca aatattggaa tcgttatcat 1140
ctgtatatgt atgttaccga aaatggtatt gcggatgatg ctgattatca gcgtccgtat 1200
tatctggtta gccatgtgtt tcaggtgcat cgcgccatta atagcggtgc agatgtgcgc 1260
ggctatctgc attggagtct gggaggtaat tatgaatggg catcaggctt ttcaatgcgt 1320
tttggcctgc tgaaagtgga ttataatacc aaacatctgt attggcctcc gagcgcactg 1380
gtttatcgtg aaattgcaac cattggcgca attaccgatg aaattgaaca tctgagtagc 1440
gttccgccgg ttaaaccgct gcgtcattaa 1470
<210> 5
<211> 18
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 5
cgcggatcca tgtcatcc 18
<210> 6
<211> 19
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 6
cccaagcttt cagtcctcc 19
<210> 7
<211> 1470
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 7
atgtatacct ttccgaattc ttatcgcttt ggctggtctc aggctggctt tcagtctgta 60
atgggcaccc caggctctga agatccgaat accgattggt ataaatgggt gcaggatccg 120
gaagctatgg ctgctggcct ggtgtgtggt gatctgccgg aaaatggtcc gggttattgg 180
ggtaattata aaacctgtca tgataatgcc cagaaaatgg gtctgaaaat tgctcgtctg 240
aatgttgaat ggagtcgtat ttttccgaat ccgctgccgc gtccgcagaa ttttgatgaa 300
agtaaacagg atgttaccga agttgaaatt aatgaaaatg aactgaaacg cctggaagaa 360
tatgcgaata aagatgcgct gaatcattat cgcgaaattt ttaaagatct gaaatcacgc 420
ggcctgtatt ttattctgaa tatgtatcat tggccgctgc cgctgtggct gcatgacccg 480
attcgcgtgc gccgcggtga ttttaccggc ccgtctggct ggctgtctac ccgcaccgtg 540
tatgaatttg cgcgcttttc agcgtatatt gcgtggaaat ttgatgatct ggtggattaa 600
tattcaacca tgaatgaacc gaatgttgtt ggtggtctgg gttatgttgg tgttagaagc 660
ggttttccgc cgggttatct gagttttgaa ctgagtcgtc gtgccatgta taatattatt 720
caggcccatg cccgtgccta tgatggtatt aaaagtgtta gtaaaaaacc ggtgggcatt 780
atttatgcga attcatcatt tcagccgctg accgataaag atatggaagc tgtggaaatg 840
gctgaaaatg ataatcgctg gtggtttttt gatgctatta ttcgcggtga aattacccgt 900
ggtaatgaaa aaattgttcg tgatgatctg aaaggtcgtc tggattggat tggtgttaat 960
tattataccc gtaccgtggt gaaacgcacc gaaaaaggct atgtgtcact gggcggttat 1020
ggccatggct gcgaacgcaa ttctgtgtct ctggccggtc tgccgaccag cgattttggt 1080
tgggaattgt ttccggaagg tctgtatgat gttctgacca aatattggaa tcgttatcat 1140
ctgtatatgt atgttaccga aaatggtatt gcggatgatg ctgattatca gcgtccgtat 1200
tatctggtta gccatgtgta tcaggtgcat cgcgccatta atagcggtgc agatgagcgc 1260
ggctatctgc attggagtct ggcagataat tatgaatggg catcaggctt ttcaatgcgt 1320
tttggcttgc tgaaagtgga ttataatacc aaacgtctgt attggcgtcc gagcgcactg 1380
gtttatcgtg aaattgcaac caatggcgca attaccgatg aaattgaaca tcagaatagc 1440
gttccgccgg ttaaaccgct gcgtcattaa 1470
Claims (3)
1. A gene encoding β -galactosidase, wherein the gene is SEQ ID NO: 1;
wherein the beta-galactosidase has an enzymatic activity which catalyzes the conversion of lactose and fructose to lactulose.
2. A recombinant vector comprising the gene encoding β -galactosidase of claim 1.
3. A recombinant Escherichia coli comprising the gene encoding β -galactosidase of claim 1 or the recombinant vector of claim 2.
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| CN201611130148.2A CN108220308B (en) | 2016-12-09 | 2016-12-09 | Beta-galactosidase, gene and carrier thereof, bacterial strain, product containing beta-galactosidase and method for converting lactose and fructose into lactulose |
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| CN201611130148.2A CN108220308B (en) | 2016-12-09 | 2016-12-09 | Beta-galactosidase, gene and carrier thereof, bacterial strain, product containing beta-galactosidase and method for converting lactose and fructose into lactulose |
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