CN106957832A - A kind of bacteria beta-galactosidase and its encoding gene and application - Google Patents
A kind of bacteria beta-galactosidase and its encoding gene and application Download PDFInfo
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- 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)
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Abstract
The invention discloses a kind of bacteria beta-galactosidase and its encoding gene and application.Protein provided by the present invention is following a1) or a2) or a3) or a4)：A1) amino acid sequence is the protein shown in sequence 2 in sequence table；A2) the fused protein that the N-terminal of the protein in sequence table shown in sequence 2 or/and C-terminal connection label are obtained；A3) amino acid sequence is the protein shown in sequence 4 in sequence table；A4) by a1) or a2) or a3) shown in substitution and/or missing and/or addition obtained protein with betagalactosidase activity of the protein by one or several amino acid residues.It is demonstrated experimentally that the protein that provides of the present invention both can effectively hydrolyzing lactose, galactooligosaccharide can be efficiently synthesized again, for the production of low-lactose dairy product in food industry and galactooligosaccharide, with important economic worth and realistic meaning.
Description
Technical field
The invention belongs to microorganism field, and in particular to a kind of bacteria beta-galactosidase and its encoding gene and application.
Background technology
Dairy products are rich in nutritional ingredients such as protein and fat, also containing about 4.5% lactose.Most of Asians suffer from
Lactose intolerance, limits the consumption of dairy products.Beta galactosidase (β-galactosidase, EC 3.2.1.23)
Scientific name be β-D- galactohydrolases, trade name lactase, it can hydrolyze the lactose in dairy products, make lactose not
Resistance to crowd can consume.Part beta galactosidase also has the effect for turning galactosyl, i.e., with lactose hydrolysate or
Lactose is glycosyl acceptor, synthesis of oligonucleotides galactolipin.Galactooligosaccharide is a kind of important prebiotics, can promote enteron aisle
The propagation of middle beneficial bacterium, so that regulating intestinal canal flora.
The beta galactosidase of microorganism has that yield of enzyme is big, property is various, low cost and other advantages：
Pawlak-Szukalska etc. reports an arthrobacterium (Arthrobacter sp.32cB) and fits cold beta galactosidase,
Optimum pH and temperature are respectively 8.0 and 28 DEG C, and 90% lactose is (enzyme-added in 24h hydrolyzables milk at 10 DEG C
Measure 2U/mL), and can carry out turning glucosides reaction by acceptor of a variety of substrates；Liu etc. is from Lactobacillus
Fermentum K4 have cloned a GH2 families beta-galactosidase gene and the heterogenous expression in Escherichia coli, weight
The optimal pH and temperature of group enzyme are respectively 7.0 and 40 DEG C (substrate is oNPG), and galactooligosaccharide yield is 37%；Iqbal
Deng the Lactobacillus sakei Lb790 beta-galactosidase gene in expression in escherichia coli, recombinase
Optimal pH and temperature are respectively 6.5 and 55 DEG C (substrate is oNPG), and galactooligosaccharide yield is the 41% of total reducing sugar；Woods
Lian Bing etc. has cloned a thermostable beta-galactosidase gene from the grand genome of hot spring, and the optimum temperature of recombinase is
55℃；Zheng Zhaojuan etc. is by the beta-galactosidase gene of bacillus coagulans (Bacillus coagulans) in large intestine
Expressed in bacillus, the optimal pH of its recombinase is 6.0, and optimum temperature is 60 DEG C, can effectively hydrolyze lactose；
Liu Bin etc. has cloned a beta-galactosidase gene from hot spring anaerobic bacteria flora, and optimum temperature is 75 DEG C, to decontamination
The tolerance of agent, inhibitor and metal ion is strong；Liu Yuhuan etc. is obtained by screening the grand genomic library of ocean mud
One beta galactosidase, its optimal pH is 8.0, and optimum temperature is 55 DEG C, low when lactose concn is 30%
Poly- galactolipin synthesis yield is 47%.The beta galactosidase of these reports can only be closed in lactose hydrolysis or galactooligosaccharide
There is preferable effect into aspect, and lactose hydrolysis and galactooligosaccharide synthesis performance more excellent beta galactose glycosides
Enzyme report is seldom.Therefore, excavate both can effectively hydrolyzing lactose, β-gala of oligosaccharide galactolipin can be efficiently synthesized again
Glycosidase, realizes that an enzyme is dual-purpose, for the production of low-lactose dairy product in food industry and galactooligosaccharide, with weight
The economic worth and realistic meaning wanted.
The content of the invention
The technical problems to be solved by the invention be to provide not only hydrolyzable lactose but also be capable of the β of oligosaccharide synthesis galactolipin-
Galactosidase.
In order to solve the above technical problems, present invention firstly provides a kind of protein.
Protein provided by the present invention, from balun Pueraria lobota hereby series bacillus (Paenibacillus
Barengoltzii) CAU904, is following a1) or a2) or a3) or a4)：
A1) amino acid sequence is the protein shown in sequence 2 in sequence table；
A2) the fused protein that the N-terminal of the protein in sequence table shown in sequence 2 or/and C-terminal connection label are obtained；
A3) amino acid sequence is the protein shown in sequence 4 in sequence table；
A4) by a1) or a2) or a3) shown in protein by one or several amino acid residues substitution and/or
The protein with betagalactosidase activity that missing and/or addition are obtained.
Wherein, sequence 2 is made up of 1014 amino acid residues in sequence table；Sequence 4 is by 1037 ammonia in sequence table
Base acid residue composition.
In order that a1) in protein be easy to purifying, the amino of protein that can be in sequence table shown in sequence 2
End or the upper label as shown in table 1 of carboxyl terminal connection.
The sequence of table 1, label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being usually 5) | RRRRR |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned a4) in protein, the substitution of one or several amino acid residues and/or missing and/or be added to
No more than the substitution and/or missing and/or addition of 10 amino acid residues.
Above-mentioned a4) in protein can be artificial synthesized, also can first synthesize its encoding gene, then carry out biological expression and obtain.
Above-mentioned a4) in the encoding gene of protein can be by by sequence in sequence table 1 or sequence table shown in sequence 3
DNA sequence dna in lack the codons of one or several amino acid residues, and/or carry out the mistake of one or several base-pairs
Justice mutation, and/or obtained in the coded sequence that its 5 ' end and/or 3 ' ends connect the label shown in table 1.
The nucleic acid molecules of code for said proteins fall within protection scope of the present invention.
The nucleic acid molecules of the code for said proteins can be following b1) or b2) or b3) or b4) shown in DNA
Molecule：
B1) nucleotide sequence is the DNA molecular shown in sequence 1 in sequence table；
B2) nucleotide sequence is the DNA molecular shown in sequence 3 in sequence table；
B3) and b1) or b2) nucleotide sequence that limits has 75% or more than 75% homogeneity, and encodes the egg
The DNA molecular of white matter；
B4) under strict conditions with b1) or the b2) nucleotide sequence hybridization that limits, and code for said proteins
DNA molecular.
Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid point
Son can also be RNA, such as mRNA or hnRNA.
Wherein, sequence 1 is made up of 3045 nucleotides in sequence table, the nucleotide coding sequence of sequence 1 in sequence table
Amino acid sequence in table shown in sequence 2；Sequence 3 is made up of 3114 nucleotides in sequence table, sequence in sequence table
Amino acid sequence in 3 nucleotide coding sequence table shown in sequence 4.
Those of ordinary skill in the art can be easily using known method, such as side of orthogenesis and point mutation
Method, is mutated to the nucleotide sequence of the code for said proteins of the present invention.Those have by manually modified
The nucleotide sequence 75% or the nucleotides of higher homogeneity of the isolated protein with the present invention, as long as compiling
The code protein and with betagalactosidase activity, is the nucleotide sequence derived from the present invention and equivalent
In the sequence of the present invention.
Term " homogeneity " used herein refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this
The nucleosides of the protein of amino acid sequence composition shown in the sequence 2 of the polynucleotide of invention or the sequence 4 of sequence table
Acid sequence has 75% or higher, or 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher
The nucleotide sequence of homogeneity.Homogeneity can with the naked eye or computer software is evaluated.Using computer software,
Homogeneity between two or more sequences can represent with percentage (%), and it can be used to evaluate between correlated series
Homogeneity.
Expression cassette, recombinant vector, recombinant microorganism or the transgenosis of nucleic acid molecules containing the code for said proteins
Cell line falls within protection scope of the present invention.
The recombinant vector can be shown in the sequence 1 in carrier pET-28a (+) multiple cloning sites insetion sequence table
The recombinant plasmid pET-gal that DNA molecular is obtained.
The recombinant vector concretely carrier pET-28a (+) multiple cloning sites insetion sequence table the institute of sequence 1
The recombinant plasmid that the DNA molecular shown is obtained.
The recombinant vector concretely replaces carrier pET-28a (+) with the DNA molecular shown in sequence 1 in sequence table
NheI and XhoI recognition sequences between fragment (carrier pET-28a (+) by restriction endonuclease NheI and
XhoI is cut into a large fragment and a small fragment, and the DNA is the small fragment), obtain recombinant plasmid pET-gal.Weight
The fusion protein with His-tag labels shown in the sequence 4 of group plasmid pET-gal expressed sequence tables.
The recombinant microorganism can be obtained by the way that the recombinant vector is imported into the microorganism that sets out.
The microorganism that sets out can be yeast, bacterium, algae or fungi.The bacterium can for gram-positive bacterium or
Gramnegative bacterium.The gramnegative bacterium can be Escherichia bacteria.The Escherichia bacteria can
For Escherichia coli (Escherichia coli).The Escherichia coli (Escherichia coli) can be Escherichia coli
Rosetta(DE3)。
The transgenic plant cells system does not include propagating materials.The genetically modified plants are interpreted as not only including and incited somebody to action
The first generation genetically modified plants that the encoding gene transformation receptor plant of the protein obtains, also including its filial generation.It is right
In genetically modified plants, the gene can be bred in the species, it is also possible to which traditional breeding method is by the gene transfer
Into other kinds of same species, particularly including in commercial variety.The genetically modified plants include seed, callus
Tissue, intact plant and cell.
The present invention also protects the application of the protein, can be following c1) or c2)：
C1) as the application of beta galactosidase；
C2) the application in beta galactosidase product is prepared.
The present invention also protects the application of the protein, can d1 as follows) or d2)：
D1) the application in hydrolysis lactose；
D2) application in being used to hydrolyze the product of lactose is being prepared.
The present invention also protects the application of the protein, can be following e1) or e2)：
E1) the application in production galactooligosaccharide；
E2) application in being used to produce the product of galactooligosaccharide is being prepared.
In above-mentioned application, " the production galactooligosaccharide " is to be used as substrate using lactose.
It is demonstrated experimentally that protein provided by the present invention has betagalactosidase activity, it both can effectively hydrolyzing breast
Sugar, can efficiently synthesize oligosaccharide galactolipin again：By the lactose complete hydrolysis in commercially available milk into glucose and half in 2h
By the lactose complete hydrolysis in whey powder into glucose and galactolipin in lactose, 1h, a variety of degree of polymerization can be synthesized different
Galactooligosaccharide, reaction 8h after conversion lactose generation galactooligosaccharide maximum conversion rate be 47.9%.It can be seen that,
Protein provided by the present invention for low-lactose dairy product in food industry and galactooligosaccharide production, with important
Economic worth and realistic meaning.
Brief description of the drawings
Fig. 1 is the SDS-PAGE results of crude enzyme liquid and beta galactosidase solution.
Fig. 2 is the optimum pH and pH stability of restructuring beta galactosidase.
Fig. 3 is the optimum temperature and temperature stability of restructuring beta galactosidase.
Fig. 4 is the result of lactose in TLC and HPLC detection beta galactosidase hydrolysed milks.
Fig. 5 is the result of lactose in TLC and HPLC detection beta galactosidase hydrolysed whey powder solution
Fig. 6 is the result that TLC and HPLC detection beta galactosidases catalyze and synthesize galactooligosaccharide.
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided only for
The present invention is illustrated, the scope being not intended to be limiting of the invention.
Experimental method in following embodiments, unless otherwise specified, is conventional method.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
Hereby series bacillus (Paenibacillus barengoltzii) CAU904 is documented in following document to balun Pueraria lobota
In：Fu X, Yan Q, Yang S, Yang X, Guo Y, Jiang Z.An acidic, thermostable
exochitinase withβ-N-acetylglucosaminidase activity from Paenibacillus
barengoltzii converting chitin to N-acetyl glucosamine.Biotechnology for
Biofuels,2014,7(1):The 1-11. public can obtain from China Agricultural University, to repeat this experiment.Balun
Pueraria lobota hereby series bacillus (Paenibacillus barengoltzii) CAU904 hereinafter abbreviation balun Pueraria lobota hereby classes
Bacillus CAU904.
Agarose Ni-IDA affinity columns are that catalog number is 17-0575-01 from U.S.'s GE Products.
Determine beta galactosidase vigor method with reference to Katrolia (Katrolia P, Yan Q, Jia H,
Li Y, Jiang Z, Song C.Molecular cloning and high-level expression of a β
-galactosidase gene from Paecilomyces aerugineus in Pichia pastoris.
Journal of Molecular Catalysis B:Enzymatic,2011,69(3):Side 112-119.)
Method, specific determination step is following (this method is named as universal method)：(1) 75 μ L 15mM adjacent nitro β is taken
- D- galactopyranosides (oNPG) solution (solvent is water), the MOPS for adding 150 μ L 50mM, pH7.5 delays
In fliud flushing, 3min is preheated in 40 DEG C of waters bath with thermostatic control；(2) complete after step (1), add 25 μ L testing samples
Liquid (dilution of enzyme liquid to be measured or enzyme liquid to be measured), which is placed in 45 DEG C of waters bath with thermostatic control, reacts 10min；(3) step is completed
Suddenly after (2), 750 μ L 2M Na are added2CO3The aqueous solution, terminating reaction, using spectrophotometer, passes through
Detect that light absorption value is (by suitably diluting enzyme liquid by OD at 410nm410nmControl is in the range of 0.200-0.800) come
The vigor of beta galactosidase is determined, the definition of per unit beta galactosidase vigor is under these conditions, often
Enzyme amount needed for minute catalysis 1 μm of ol o-nitrophenol of generation.
The computing formula of the vigor of beta galactosidase is：Beta galactosidase vigor (U/mL)=
(0.326×OD410nm- 0.006) extension rate × 4 of × analyte sample fluid
Using bovine serum albumin as standard protein, using Lowry methods (Lowry O H, Rosebrough N J, Farr A
L,Randall R J.Protein measurement with the Folin phenol reagent.The Journal
Of Biological Chemistry, 1951,193 (1):265-275.) determine protein content.
Whey powder solution：The MOPS buffer solutions of pH7.5,50mmol/L containing 5% (weight/mass percentage composition) whey powder；
Wherein whey powder is U.S.'s Bio-Nutrition International Products.
Buffer A：Phosphate buffer (pH8.0, di(2-ethylhexyl)phosphate containing 20mM containing 10% (mass percent) glycerine
Hydrogen sodium-disodium hydrogen phosphate buffer solution).
Buffer B：Phosphate buffer containing 10% (mass percent) glycerine, 300mM NaCl and 20mM imidazoles
(pH8.0, sodium dihydrogen phosphate containing 20mM-disodium hydrogen phosphate buffer solution).
Buffer solution C：Phosphate buffer containing 10% (mass percent) glycerine, 300mM NaCl and 50mM imidazoles
(pH8.0, sodium dihydrogen phosphate containing 20mM-disodium hydrogen phosphate buffer solution).
Buffer solution D：Phosphate-buffered containing 10% (mass percent) glycerine, 300mM NaCl and 100mM imidazoles
Liquid (pH8.0, sodium dihydrogen phosphate containing 20mM-disodium hydrogen phosphate buffer solution).
Protein shown in the sequence 2 of sequence table is named as protein first, the sequence of its encoding gene such as sequence table
Shown in 1.
Embodiment 1, the preparation for recombinating beta galactosidase
First, the structure of beta galactosidase encoding gene expression plasmid is recombinated
1st, balun Pueraria lobota hereby series bacillus CAU904 genomic DNA and using it as template is extracted, with artificial synthesized
PbBgal2ANheF：5’-ctcagGCTAGC(underscore is restricted interior to GTGGAACTGAACCGTGAATGG-3 '
Enzyme cutting NheI restriction enzyme site) and PbBgal2AXhoR：5’
-gtcatCTCGAG(underscore is restriction enzyme XhoI enzyme to CTATTCTTCTTCCTTAAATATCGGCTG-3 '
Enzyme site) it is primer, enter performing PCR amplification, obtain DNA fragmentation.
Response procedures are：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 3min,
35 circulations；72 DEG C of extension 5min.
2nd, the DNA fragmentation obtained with restriction enzyme NheI and XhoI double digestion step 1, reclaims digestion products.
3rd, with restriction enzyme NheI and XhoI double digestion carrier pET-28a (+) (Novagen Products,
Catalog number is 69864-3CN), reclaim about 5300bp carrier framework.
4th, digestion products are connected with carrier framework, obtain recombinant plasmid pET-gal.
According to sequencing result, structure is carried out to recombinant plasmid pET-gal and is described as follows：By carrier pET-28a's (+)
DNA small fragments between NheI recognition sequences and XhoI recognition sequences replace with the sequence 1 that nucleotide sequence is sequence table
Shown DNA molecular.In recombinant plasmid pET-gal, DNA molecular and carrier framework shown in the sequence 1 of sequence table
On His-tag labels (being made up of 6 histidine residues) coded sequence fusion, the institute of sequence 3 of formation sequence table
The fusion shown, the fusion protein with His-tag labels shown in the sequence 4 of expressed sequence table.
2nd, the expression of beta galactosidase is recombinated
1st, recombinant plasmid pET-gal is imported into Escherichia coli Rosetta (DE3), recombinant bacterium is obtained, by the recombinant bacterium
It is named as Rosetta (DE3)-pET-gal.
2nd, by Rosetta (DE3)-pET-gal be inoculated in 200mL LB fluid nutrient mediums (containing 50 μ g/mL cards that
Mycin), 37 DEG C, 200rpm shaken cultivations to OD600Value 0.6~0.8, adds IPTG and makes its concentration be 1mM,
Then 20 DEG C, 200rpm shaken cultivation 2h, are collected by centrifugation thalline.
3rd, the thalline that step 2 is obtained is taken, with 1：10 (volume ratios) add buffer A, and resuspended rear ultrasonication is (super
Acoustic power 200W, cyclic program is：Broken 3s, stops 4s, totally 180 circulations), then 4 DEG C, 10000rpm
10min is centrifuged, supernatant, as crude enzyme liquid is collected.
3rd, the purifying of beta galactosidase is recombinated
Use agarose Ni-IDA for chromatography column packing, purified using 1 × 5cm chromatographic column, it is specific as follows：
Chromatographic column (flow velocity is 1mL/min) first is balanced with the buffer B of 5~10 column volumes, then loading step two is obtained
Crude enzyme liquid (flow velocity is 0.5mL/min), then with buffer B rinse chromatographic column (flow velocity is 1mL/min) until
OD280nmIt is straight that ＜ 0.05 rinses chromatographic column (flow velocity is 1mL/min) to wash away uncombined albumen, then with buffer solution C
To OD280nm＜ 0.05, finally rinses chromatographic column (flow velocity is 1mL/min) with buffer solution D and collects OD280>=1.0
Cross solution after post, as beta galactosidase solution.
The SDS-PAGE results of crude enzyme liquid and beta galactosidase solution are shown in Fig. 1.In Fig. 1, M is HMW mark
Quasi- protein, 1 is crude enzyme liquid, and 2 be beta galactosidase solution.As a result show, beta galactosidase solution shows
Show unimodal molecular weight band, correspondence molecular weight is 124.2KDa.
Detect that crude enzyme liquid and protein concentration and beta galactosidase vigor in beta galactosidase solution (are used
Universal method is detected).The protein concentration and beta galactosidase vigor of crude enzyme liquid and beta galactosidase solution are shown in
Table 1.
The protein concentration of table 1. and beta galactosidase vigor result
Embodiment 2, the property for recombinating beta galactosidase
First, the optimum pH and pH stability of beta galactosidase are recombinated
Buffer solution to be measured (concentration is 50mmol/L)：PH4.0 acetic acid-sodium acetate buffer solution, pH4.5 acetic acid-
Sodium acetate buffer, pH5.0 acetic acid-sodium acetate buffer solution, pH5.5 acetic acid-sodium acetate buffer solution, pH6.0
Acetic acid-sodium acetate buffer solution, pH5.5 MES buffer solutions, pH6.0 MES buffer solutions, pH6.5 MES bufferings
Liquid, pH6.0 MOPS buffer solutions, pH6.5 MOPS buffer solutions, pH7.0 MOPS buffer solutions, pH7.5
MOPS buffer solutions, pH8.0 MOPS buffer solutions, pH6.0 sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, pH6.5
Sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, pH7.0 sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, pH7.5
Sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, pH8.0 sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, pH8.5
Sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, pH8.0 CHES buffer solutions, pH8.5 CHES buffer solutions, pH9.0
CHES buffer solutions, pH9.5 CHES buffer solutions, pH10.0 CHES buffer solutions, pH10.0 CAPS buffering
The CAPS buffer solutions of liquid, pH10.5 CAPS buffer solutions or pH11.0.
Beta galactosidase solution prepared by embodiment 1 is lived as analyte sample fluid, detection beta galactosidase
Power, method is as follows：(1) 75 μ L 15mM adjacent nitro β-D- galactopyranosides solution (solvent is water) is taken,
Add in 150 μ L buffer solutions to be measured, 3min is preheated in 40 DEG C of waters bath with thermostatic control；Other same universal methods.With enzyme activity
The peak of power is used as 100%.The relative enzyme activity of beta galactosidase is shown in Fig. 2 (A) under each pH.Fig. 2
In in (A), open diamonds represent acetic acid-sodium acetate buffer solution, and filled square represents MES buffer solutions, open circles
Shape represents MOPS buffer solutions, and black triangle represents sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, hollow square generation
Table CHES buffer solutions, solid circles represent CAPS buffer solutions.As a result show, recombinate the most suitable of beta galactosidase
PH is 7.5.
Beta galactosidase solution prepared by embodiment 1 is diluted with buffer solution to be measured, then 40 DEG C of water-bath 30min,
It is immediately placed in again in frozen water and cools down 30min, then as analyte sample fluid, beta galactose is detected using universal method
Glycosides enzyme activity.Will (above-mentioned processing refers to 40 DEG C of water-bath 30min, then is immediately placed in cold in frozen water without above-mentioned processing
But 30min) beta galactosidase solution dilution be used as control.100% is used as using the enzyme activity of control.Using
Relative enzyme activity after each pH processing is shown in Fig. 2 (B).As a result show, restructuring beta galactosidase pH 6.0~
Keep stable in the range of 8.0, residual enzyme activity is more than 80% after processing 30min.
2nd, the optimum temperature and temperature stability of beta galactosidase are recombinated
Beta galactosidase solution prepared by embodiment 1 is lived as analyte sample fluid, detection beta galactosidase
Power, method is as follows：By 45 DEG C in step (2) replace with 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C,
50 DEG C, 55 DEG C or 60 DEG C, other same universal methods.Using enzyme activity peak as 100%.Under each reaction temperature
Enzyme activity see in Fig. 3 (A).As a result show, the optimum temperature of restructuring beta galactosidase is 45 DEG C.
The beta galactosidase solution prepared with pH7.5,50mmol/L MOPS buffer solutions dilution embodiment 1, so
Afterwards water-bath 30min (bath temperature be 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C or
60 DEG C), then be immediately placed in frozen water and cool down 30min, then as analyte sample fluid, using universal method detect β-
Galactoside enzyme activity.Will (above-mentioned processing refers to water-bath 30min, then is immediately placed in frozen water without above-mentioned processing
Cooling 30min) beta galactosidase solution dilution be used as control.100% is used as using the enzyme activity of control.Adopt
Seen with the relative enzyme activity after each Temperature Treatment in Fig. 3 (B).As a result show, recombinate 35~45 DEG C of beta galactosidase
In the range of keep stable, residual enzyme activity is maintained at more than 90%.
Embodiment 3, the application for recombinating beta galactosidase
TLC detection methods：By sample point sample in opening up layer twice in TLC analysis plates, drying is soaked completely with nitrite ion,
100 DEG C of colour developings.Developing agent is n-butanol/ethanol/water=5:3:2 (v/v/v), developer is 5% methanolic solution,
Standard control is used as using the melange of the standard items of glucose, galactolipin and lactose.
HPLC detection methods one：Contain for determining the lactose in glucose content, galactose content and lactose hydrolysis experiment
Amount, chromatographic column used is cation-exchange chromatography post BP-800Pb2+(Benson Polymeric, Reno, NE, USA),
Column temperature is 80 DEG C, and flow velocity is 0.6mL/min, and mobile phase is ultra-pure water, and sample size is 20 μ L.Computing formula is as follows：
Glucose content (g/L)=[glucose peaks area (mAu*s) -21376]/458.8/1000.
Galactose content (g/L)=[galactolipin peak area (mAu*s)+1101]/491.1/1000.
Lactose content (g/L)=[lactose peak area (mAu*s) -22151]/501.7/1000.
HPLC detection methods two：For determining the content of the lactose in the experiment of synthesis of oligonucleotides galactolipin, chromatographic column used is
Sugar-D (250 × 4.6mm) nh 2 column, column temperature is 40 DEG C, and flow velocity is 1.0mL/min, and mobile phase is 75% second
The nitrile aqueous solution, sample size is 20 μ L.Computing formula is as follows：
Lactose content (g/L)=[lactose peak area (mAu*s) -2858]/391.7/1000.
It is pure that glucose, galactolipin and lactose standard are commercially available analysis.
Lactose hydrolysis ratio (%, w/w)=[lactose content (%, w/v) during initial lactose content (%, w/v)-sampling]/
Initial lactose content (%, w/v).
Galactooligosaccharide conversion ratio (%, w/w)=[lactose during initial lactose content (%, w/v)-sampling, glucose and
Galactolipin sugared content sum (%, w/v)]/initial lactose content (%, w/v).
First, lactose hydrolysis
Reaction system (cumulative volume is 1mL)：Beta galactosidase solution prepared by substrate and embodiment 1 is mixed,
Obtain reaction system.In reaction system, the initial concentration of beta galactosidase is 5U/mL.Substrate is that commercially available ternary is pure
Milk or whey powder solution.
Reaction condition：40 DEG C of water-baths.
Interval time is sampled, and enzyme activity of going out is heated in boiling water bath, is then detected lactose content and is calculated lactose hydrolysis ratio.
When substrate is milk, TLC testing results are shown in Fig. 4 (A) (M is standard control), and HPLC testing results are shown in figure
In 4 (B).As a result show, restructuring beta galactosidase is in 2h by the lactose complete hydrolysis in commercially available milk into Portugal
Grape sugar and galactolipin.
When substrate is whey powder, TLC testing results are shown in Fig. 5 (A) (M is standard control), and HPLC testing results are shown in
In Fig. 5 (B).As a result show, restructuring beta galactosidase is in 1h by the lactose complete hydrolysis in whey powder into Portugal
Grape sugar and galactolipin.
In Fig. 4 in (B) and Fig. 5 in (B), solid diamond represents lactose, and solid circles represent glucose, hollow
Triangle represents galactolipin.
2nd, synthesis of oligonucleotides galactolipin (Galactooligosaccharides, GOS)
Reaction system (cumulative volume is 1mL)：By pH7.5,50mmol/L MOPS buffer solutions, lactose and embodiment 1
The beta galactosidase solution mixing of preparation, obtains reaction system.In reaction system, beta galactosidase it is initial
Concentration is 5U/mL, and the concentration of lactose is 35% (weight/mass percentage composition).
Reaction condition：40 DEG C of water-baths.
Interval time is sampled, and enzyme activity of going out is heated in boiling water bath, is then detected lactose content and is calculated galactooligosaccharide turn
Rate.
As a result Fig. 6 is seen, wherein (A) is TLC testing results (M is standard control), (B) is HPLC testing results.
As a result show, restructuring beta galactosidase turns after can synthesizing the different galactooligosaccharide of a variety of degree of polymerization, reaction 8h
The maximum conversion rate for changing lactose generation galactooligosaccharide is 47.9%.In Fig. 6 in (B), solid diamond represents lactose,
Solid circles represent glucose, and open triangles represent galactolipin, and hollow square represents galactooligosaccharide.
Claims (10)
1. protein, is following a1) or a2) or a3) or a4)：
A1) amino acid sequence is the protein shown in sequence 2 in sequence table；
A2) the fused protein that the N-terminal of the protein in sequence table shown in sequence 2 or/and C-terminal connection label are obtained；
A3) amino acid sequence is the protein shown in sequence 4 in sequence table；
A4) by a1) or a2) or a3) shown in protein by one or several amino acid residues substitution and/or
The protein with betagalactosidase activity that missing and/or addition are obtained.
2. encode the nucleic acid molecules of protein described in claim 1.
3. nucleic acid molecules as claimed in claim 2, it is characterised in that：The nucleic acid molecules are following b1) or b2)
Or b3) or b4) shown in DNA molecular：
B1) nucleotide sequence is the DNA molecular shown in sequence 1 in sequence table；
B2) nucleotide sequence is the DNA molecular shown in sequence 3 in sequence table；
B3) and b1) or b2) nucleotide sequence that limits has 75% or more than 75% homogeneity, and coding right will
Seek protein DNA molecule described in 1；
B4) under strict conditions with b1) or b2) limit nucleotide sequence hybridization, and coding claim 1 described in
Protein DNA molecule.
4. expression cassette, recombinant vector, recombinant microorganism or transgenosis containing nucleic acid molecules described in Claims 2 or 3
Cell line.
5. recombinant vector as claimed in claim 4, it is characterised in that：The recombinant vector is in carrier pET-28a
The recombinant plasmid pET-gal that DNA molecular shown in the sequence 1 of the multiple cloning sites insetion sequence table of (+) is obtained.
6. recombinant microorganism as claimed in claim 4, it is characterised in that：The recombinant microorganism is by claim
Recombinant plasmid pET-gal described in 5 imports the recombinant bacterium that the microorganism that sets out obtains.
7. the application of protein described in claim 1, is following c1) or c2)：
C1) as the application of beta galactosidase；
C2) the application in beta galactosidase product is prepared.
8. the application of protein described in claim 1, is following d1) or d2)：
D1) the application in hydrolysis lactose；
D2) application in being used to hydrolyze the product of lactose is being prepared.
9. the application of protein described in claim 1, is following e1) or e2)：
E1) the application in production galactooligosaccharide；
E2) application in being used to produce the product of galactooligosaccharide is being prepared.
10. application as claimed in claim 9, it is characterised in that：" the production galactooligosaccharide " is with lactose
It is used as substrate.
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| CN111154740A (en) * | 2020-02-07 | 2020-05-15 | 中国农业大学 | Arenicola microvesicle bacterium β -galactosidase, and coding gene and application thereof |
| CN112351990A (en) * | 2018-04-05 | 2021-02-09 | 合同酒精株式会社 | Enzyme capable of producing galactooligosaccharide from Paenibacillus bifidus (Paenibacillus pabuli) and method for producing galactooligosaccharide |
| WO2024021455A1 (en) * | 2022-07-26 | 2024-02-01 | 江南大学 | β-GALACTOSIDASE GENE AND USE THEREOF IN ENCODING OF ENZYME |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112351990A (en) * | 2018-04-05 | 2021-02-09 | 合同酒精株式会社 | Enzyme capable of producing galactooligosaccharide from Paenibacillus bifidus (Paenibacillus pabuli) and method for producing galactooligosaccharide |
| JPWO2019194062A1 (en) * | 2018-04-05 | 2021-07-01 | 合同酒精株式会社 | An enzyme capable of producing a galactooligosaccharide derived from Paenibacillus pavuli, and a method for producing a galactooligosaccharide. |
| EP3778625A4 (en) * | 2018-04-05 | 2022-02-09 | Godo Shusei Co., Ltd. | Enzyme derived from paenibacillus pabuli and capable of producing galactooligosaccharide and method for producing galactooligosaccharide |
| JP7344195B2 (en) | 2018-04-05 | 2023-09-13 | 合同酒精株式会社 | Enzyme capable of producing galactooligosaccharide derived from Paenibacillus pabuli, and method for producing galactooligosaccharide |
| CN112351990B (en) * | 2018-04-05 | 2024-04-26 | 合同酒精株式会社 | Enzyme capable of producing galactooligosaccharides from paenibacillus feed (Paenibacillus pabuli) and method for producing galactooligosaccharides |
| CN111154740A (en) * | 2020-02-07 | 2020-05-15 | 中国农业大学 | Arenicola microvesicle bacterium β -galactosidase, and coding gene and application thereof |
| WO2024021455A1 (en) * | 2022-07-26 | 2024-02-01 | 江南大学 | β-GALACTOSIDASE GENE AND USE THEREOF IN ENCODING OF ENZYME |
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