CN103131685A - Marine-microbe-sourced low-temperature beta-galactosidase, and coding gene thereof and application thereof - Google Patents
Marine-microbe-sourced low-temperature beta-galactosidase, and coding gene thereof and application thereof Download PDFInfo
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Abstract
The invention relates to the field of marine biotechnology, and provides a marine-microbe-sourced low-temperature beta-galactosidase, which is obtained by fermentation culturing and purification of a marine halomonas sp. strain P6009-1 obtained by the applicant previously. The marine halomonas sp. strain P6009-1 is cultured and separated from seawater in East China Sea areas, and has a collection number of CCTCC No: M2011001. Under a temperature of 0 DEG C, a capacity of the enzyme in catalyzing lactose hydrolysis is 20% of a maximal activity, such that good low-temperature performance is shown. The invention further provides a coding gene of beta-galactosidase, and an application of beta-galactosidase in decomposing milk lactose.
Description
Technical field
The present invention relates to the marine biotechnology field, be specifically related to a kind of low temperature beta-galactosidase enzymes and encoding gene and application of made from ocean microorganism.
Background technology
(formal name used at school is β-D-galactoside galactohydrolase to beta-galactosidase enzymes, β-D-galactoside galatohydrolase, EC3.2.1.23) be a kind of can specific hydrolysis β-1, the lytic enzyme of 4-glycosidic link, can be glucose and semi-lactosi with lactose hydrolysis, also have the effect that galactoside shifts simultaneously.
Beta-galactosidase enzymes extensively is present in natural organism, in plant, bacterium, fungi (aspergillus oryzae, aspergillus niger, saccharomyces fragilis, saccharomyces lactis), actinomycetes and animal intestinal (particularly baby intestinal).
From the reaction of catalysis, beta-galactosidase enzymes belongs to glycosyl hydrolase (glycosyl hydrolase is abbreviated as GH), different according to its sequence and structure, and beta-galactosidase enzymes is divided into again four families, is respectively GH1, GH2, GH35 and GH42 family.
Lactose is a kind of distinctive disaccharides in Mammals milk, approximately contains 7% in human milk, approximately contains 5% in cow's milk.After milk enters in body, absorb after mainly will lactose wherein resolving into semi-lactosi and glucose by the beta-galactosidase enzymes that is present in human small intestine's endotheliocyte chorion brush border surface.If disappearance beta-galactosidase enzymes in body, or due to the growth at age, the activity decreased of beta-galactosidase enzymes, will make lactose too much in milk retain in enteron aisle is not absorbed, cause enteron aisle osmotic pressure uneven, unnecessary moisture can't be caused watery diarrhoea by large intestinal absorption, is called lactose intolerance.In adult yellow, the sickness rate of lactose intolerance is higher, has affected the absorption of nutritive ingredient in milk.
The main method that solves lactose intolerance is to add ectogenic beta-galactosidase enzymes when milk-producing, and lactose wherein is hydrolyzed partially or completely, can effectively alleviate lactose intolerance.The beta-galactosidase enzymes that only derives from present microorganism has industrial application value, produces beta-galactosidase enzymes because utilizing microbe fermentation method, and the enzyme source is abundant, and output is high, and production cost is low, and is with short production cycle, and is not subjected to the impact of the factors such as season, geographical position.Yeast and mould are the main sources of present suitability for industrialized production beta-galactosidase enzymes, and be also more deep to their research.The present beta-galactosidase enzymes that has been applied to the milk processing industry has the Kluyveromyces sp from Dutch DSM N. V., and this enzyme molecular weight 135kDa, optimal pH are pH6.8, and be close with the natural pH of fresh milk; But this enzyme has some significant shortcomings, and is as narrower in the pH stable range of this enzyme, at pH greater than 7.0 or less than the equal rapid inactivation of pH6.2; Optimum temperature is higher, is 45 ° of C, and the activity under low temperature is lower, and Ca
2+Its enzyme work is had remarkable restraining effect, and these shortcomings have all greatly limited its application in Dairy Industry.
The ecotope of ocean is different from land, has high salt, high pressure, low temperature and temperature variation characteristics slowly.The enzyme that derives from marine microorganism often has special zymologic property, and low and to metal ion tolerance etc. such as operative temperature, the beta-galactosidase enzymes of the type is exactly the enzyme of wherein a kind of worth exploitation.
The applicant will culture of isolated have obtained from the seawater of Area of The East China Sea strain ocean Halomonas (Halomonas sp.) bacterial strain P6009-1 be preserved in Chinese Typical Representative culture collection center (being called for short CCTCC) on January 5th, 2011, preserving number is CCTCC No:M2011001; And applied for Chinese invention patent CN201110029078.2 and obtained the authorization on January 27th, 2011, denomination of invention is " ocean Halomonas and application thereof that the low temperature beta-galactosidase enzymes is produced in a strain ", publication No. is CN102174439A.
Summary of the invention
The object of the present invention is to provide low temperature beta-galactosidase enzymes of a kind of made from ocean microorganism and preparation method thereof, another object of the present invention is to provide the encoding gene of this beta-galactosidase enzymes, and the application of this beta-galactosidase enzymes in the decomposing milk lactose.
A first aspect of the present invention, bacterial strain P6009-1 studies further to ocean Halomonas (Halomonas sp.), be to provide a kind of low temperature beta-galactosidase enzymes (P6009-BGalH) of made from ocean microorganism, obtain in order to the below legal system is standby, the method comprises the following steps:
(A) fermentation of Halomonas sp.P6009-1
Halomonas sp.P6009-1 bacterial classification (preserving number is CCTCC No:M2011001) is inoculated in Zobell 2216E liquid nutrient medium, 28 ℃, shaking table vibration (130rpm) is cultivated 24h and is activated, getting the bacterium liquid that 500 μ L activate is inoculated in the Zobell 2216E substratum that contains 2% lactose of 100mL, 28 ℃, 130rpm shaking culture 4d;
Described Zobell 2216E liquid culture based formulas is: peptone 5g, and yeast powder 1g, tertiary iron phosphate 0.1g is dissolved in the 1L artificial seawater, pH7.4; The formula of described artificial seawater is: NaCl25.0g, Na
2SO
44.0g, KCl0.7g, NaHCO
30.20g, KBr0.10g, H
3BO
30.03g, NaF0.003g, 53mL1.0mol/L MgCl
2Solution, 10mL1.0mol/l CaCl
2Solution, 0.90ml0.1mol/L SrCl
2Solution, distilled water 1000ml;
(B) preparation of crude enzyme liquid
After fermentation ends, fermented liquid centrifuging and taking thalline is resuspended in phosphoric acid buffer with thalline, uses the ultrasonic grinder smudge cells under condition of ice bath, and the bacterium liquid centrifuging and taking supernatant after broken bacterium is crude enzyme liquid;
(C) purifying of low temperature beta-galactosidase enzymes
With the resulting crude enzyme liquid 10ml of step (B), add (the NH of 2.2g
4)
2SO
4Saltout.The centrifuging and taking supernatant adds the (NH of 0.6g again
4)
2SO
4Carry out pickle change, the centrifuging and taking precipitation.The 0.1mol/L K that adds 3mL
2HPO
4-KH
2PO
4(contain 1mmol/L MgCl
2, pH7.2) resuspended precipitation, HiPrep
TM26/10 desalting column desalination is collected target protein; Further get 2mL and be splined on the PABTG affinity column, the 0.1mol/L K with 15%
2HPO
4-KH
2PO
4(contain 0.5mol/L KCl, 0.1mol/L lactose, 1mmol/LMgCl
2, buffer B buffer solution elution 60min pH7.2) follows the buffer B buffer solution elution 20min with 20%, and flow velocity is 1mL/min; With the active ingredient loading 2mL after concentrated after affinitive layer purification, displacement damping fluid in Capto DEAE weak anionic exchange column, the 0.1mol/LK with 0% ~ 100%
2HPO
4-KH
2PO
4(contain 1mol/L KCl, 1mmol/L MgCl
2, buffer C buffer solution for gradient elution 40min pH7.2), flow velocity is 1mL/min, detecting wavelength is 280nm and 214nm; Active ingredient is again after Capto Q reinforcing yin essence ion exchange column, Sepharcryl S200 molecular sieve further separate, obtain electrophoretically pure P6009-BGalH, carry out respectively SDS-PAGE and non-sex change-PAGE, find that its molecular weight is about 90kDa, the subunit size is the 48kDa left and right, illustrates that it is heterodimeric protein (Fig. 1).
The ability of this enzyme catalysis lactose hydrolysis in the time of 0 ℃ embodies good cold property for the most highly active 20%.
A second aspect of the present invention is that the low temperature beta-galactosidase enzymes of above-mentioned purifying is analyzed order-checking, and a kind of low temperature beta-galactosidase enzymes (P6009-rBGalH) is provided, and is following (a) or (b) or protein (c):
(a) protein that is formed by the aminoacid sequence shown in SEQ ID NO:2 in sequence table;
(b) with the aminoacid sequence shown in SEQ ID NO:2 through the replacement of one or several amino acid deformity and/or disappearance and/or interpolation and relevant to the beta-galactosidase enzymes protein that is derived by SEQ ID NO:2;
(c) aminoacid sequence shown in SEQ ID NO:2 is added the preceding paragraph sequence (as the affinity purification label) at N end or C end, and relevant to the beta-galactosidase enzymes sequence that is derived by SEQ ID NO:2.
SEQ ID NO:2 in sequence table is comprised of 389 amino-acid residues.
Described one or the number replacement of several amino-acid residues and/or disappearance and/or interpolation refer to be no more than replacement and/or disappearance and/or the interpolation of 10 amino-acid residues.
In order to make the P6009-rBGalH in (a) be convenient to purifying, adopt the pET-32a carrier to carry out amalgamation and expression.The amalgamation and expression protein sequence is compared with former sequence on the basis of the aminoacid sequence shown in SEQ ID NO:2 in by sequence table and is added sulphur hydrogen reduction albumen label (TrxTag) at the N of former sequence end, adds poly histidine-tagged (HisTag) at the C end.
Above-mentioned (b) but or the P6009-rBGalH synthetic (c), also can first synthesize its encoding gene, then carry out biological expression and obtain.The encoding gene of P6009-rBGalH in above-mentioned (b) can by will be in the DNA sequence dna shown in sequence in sequence table 2 codon of one or several amino-acid residue of disappearance, and/or the missense mutation of carrying out one or several base pair obtains.
The encoding gene of described beta-galactosidase enzymes (P6009-rBGalH) is following (ⅰ) or DNA molecular (ⅱ):
(ⅰ) its encoding sequence is the DNA molecular shown in SEQ ID NO:1 in sequence table;
(ⅱ) under stringent condition with the DNA sequence dna hybridization that (ⅰ) limits and the DNA molecular of the described beta-galactosidase enzymes of encoding;
SEQ ID NO:1 in sequence table is comprised of 1170 Nucleotide.
Described stringent condition can be at 0.1 * SSPE (or in the solution of 0.1 * SSC), 0.1%SDS, hybridizes under 65 ℃ and washes film.
The recombinant expression vector, expression cassette, transgenic cell line or the recombinant bacterium that contain described beta-galactosidase enzymes encoding gene all belong to protection scope of the present invention.
Described transgenosis recombinant bacterium specifically can be the recombination bacillus coli that contains described beta-galactosidase enzymes encoding gene, as contains intestinal bacteria Escherichia coli (E.coli) BL21 (DE3) pLysS of said gene.Described recombination bacillus coli is preferably E.coli BL21 (DE3) pLysS, produces restructuring beta-galactosidase enzymes (rBGalH).
A third aspect of the present invention is to provide the application of described low temperature beta-galactosidase enzymes in the decomposing milk lactose.Natural BGalH and the rBGalH of restructuring be (7 ° of C) at low temperatures all, with lactose reaction 12h, can obviously see the hydrolysis of lactose, and when 28 ° of C, after reaction 24h, lactose almost decomposes fully.
Description of drawings
Fig. 1 is SDS-PAGE (A) and non-sex change-PAGE (B) figure of the beta-galactosidase enzymes of bacterial strain P6009-1
(A) 1. protein molecular weight standard wherein; 2. sample after Sephacryl S200 molecular sieve purification; 3. the sample after Capto Q reinforcing yin essence ion Q column purification; 4. the sample after Capto DEAE weak anionic column purification; (B) 1. protein molecular weight standard; 2. sample after Sephacryl S200 molecular sieve purification.
Fig. 2 is the optimal reactive temperature of the beta-galactosidase enzymes of bacterial strain P6009-1.
Fig. 3 is the thermostability of the beta-galactosidase enzymes of bacterial strain P6009-1.
Fig. 4 is pH stability and the optimal pH of the beta-galactosidase enzymes of bacterial strain P6009-1.
Fig. 5 is beta-galactosidase enzymes and the reaction of natural pure milk of bacterial strain P6009-1
Wherein 1: natural pure milk; 2-6 is followed successively by: 7 ℃ of reaction 1h, 2h, 4h, 12h and 24h; 7:28 ℃ of reaction 24h.
Fig. 6 is SDS-PAGE and the non-sex change-PAGE of the beta-galactosidase enzymes (rBGalH) of restructuring
The SDS-PAGE of the beta-galactosidase enzymes (rBGalH) of a. restructuring wherein; Non-sex change-the PAGE of the beta-galactosidase enzymes (rBGalH) of b. recombinating.
Fig. 7 is the optimal reactive temperature of the beta-galactosidase enzymes (rBGalH) of restructuring.
Fig. 8 is the thermostability of the beta-galactosidase enzymes (rBGalH) of restructuring.
Fig. 9 is beta-galactosidase enzymes (rBGalH) pH stability and the optimal pH of restructuring.
Figure 10 is the beta-galactosidase enzymes (rBGalH) and natural pure milk reaction of restructuring
Wherein 1: natural pure milk; 2-6 is followed successively by: 7 ℃ of reaction 1h, 2h, 4h, 12h and 24h; 7:28 ℃ of reaction 24h.
Embodiment
Describe the present invention below in conjunction with embodiment and accompanying drawing.But the following example should not regarded limitation of the scope of the invention as.
Experimental technique in following embodiment if no special instructions, is ordinary method.
Biomaterial in following embodiment and main agents are available from Shanghai branch office of traditional Chinese medicines group.Capto Q anion-exchange column, Capto DEAE ion exchange column, Sepharcryl S200 gel chromatographic columns, PABTG affinity chromatography filler, HiPrep
TMGel chromatographic columns is all available from GE company.
Embodiment 1: a kind of preparation method of low temperature beta-galactosidase enzymes of made from ocean microorganism
One, the fermentation of Halomonas sp.P6009-1
Halomonas sp.P6009-1 bacterial classification is inoculated in the Zobell2216E liquid nutrient medium, and (tertiary iron phosphate 0.1g is dissolved in the 1L artificial seawater for peptone 5g, yeast powder 1g, and pH7.4), 28 ℃, shaking table vibration (130rpm) is cultivated 24h and activated.The formula of artificial seawater is: NaCl25.0g, Na
2SO
44.0g, KCl0.7g, NaHCO
30.20g, KBr0.10g, H
3BO
30.03g, NaF0.003g, 53mL1.0mol/L MgCl
2Solution, 10mL1.0mol/l CaCl
2Solution, 0.90ml0.1mol/L SrCl
2Solution, distilled water 1000ml.Get again the bacterium liquid that 500 μ L activate and be inoculated in the Zobell 2216E substratum that contains 2% lactose of 100mL, 28 ℃, 130rpm shaking culture 4d.
Two, the preparation of the beta-galactosidase enzymes crude enzyme liquid of Halomonas sp.P6009-1
After fermentation ends, fermented liquid is centrifugal, and supernatant is abandoned in 5000rpm/min * 15min * 4 ℃.To precipitate with the phosphoric acid buffer of 0.05mol/L pH7.2 resuspended, the washing thalline, centrifugal, supernatant is abandoned in 5000rpm/min * 15min * 4 ℃, repeated washing is once, and is centrifugal, supernatant is abandoned in 5000rpm/min * 15min * 4 ℃.Thalline is resuspended in the phosphoric acid buffer of 0.05mol/L pH7.2, uses the ultrasonic grinder smudge cells under condition of ice bath: each ultrasonic 1s, interval 1s, 5min altogether.Bacterium liquid after broken bacterium is centrifugal, 12000rpm/min * 20min * 4 ℃, and getting supernatant is crude enzyme liquid.
Three, the purifying of low temperature beta-galactosidase enzymes
With the resulting crude enzyme liquid 10ml of above-mentioned steps two, add (the NH of 2.2g while shaking
4)
2SO
4Fully vibration makes (NH
4)
2SO
4Fully dissolving is without obvious solid granular insolubles, standing 5min on ice.The centrifugal 20min of 12000rpm, careful sucking-off supernatant fast.(the NH that adds again 0.6g
4)
2SO
4, abundant mixing, the centrifugal 20min of 12000rpm gets precipitation and is the sample that contains thick enzyme.The 0.1mol/LK that adds 3mL
2HPO
4-KH
2PO
4(contain 1mmol/L MgCl
2, pH7.2) resuspended precipitation, be splined on HiPrep
TM26/10 desalting column is to remove (NH
4)
2SO
4, collect target protein.The thick enzyme sample of desalination 2mL is splined on the PABTG affinity column, the 0.1mol/L K with 15%
2HPO
4-KH
2PO
4(contain 0.5mol/L KCl, 0.1mol/L lactose, 1mmol/L MgCl
2, buffer B buffer solution elution 60min pH7.2) follows the buffer B buffer solution elution 20min with 20%, and flow velocity is 1mL/min, and detecting wavelength is 280nm and 214nm.With the active ingredient loading 2mL after concentrated after affinitive layer purification, displacement damping fluid in Capto DEAE weak anionic exchange column, the 0.1mol/L K with 0% ~ 100%
2HPO
4-KH
2PO
4(contain 1mol/L KCl, 1mmol/LMgCl
2, buffer C buffer solution for gradient elution 40min pH7.2), flow velocity is 1mL/min, detecting wavelength is 280nm and 214nm.Active ingredient after Capto Q reinforcing yin essence ion exchange column, Sepharcryl S200 molecular sieve further separate, obtains electrophoretically pure P6009-BGalH again, and purification is 1007.7 times, and active yield is 1.39%.Carry out respectively SDS-PAGE and Native-PAGE, find that its molecular weight is about 90kDa, the subunit size is the 48kDa left and right, illustrates that it is heterodimeric protein (Fig. 1).
Embodiment 2: thermostability and the optimal reactive temperature of the low temperature beta-galactosidase enzymes that embodiment 1 prepares
At 74 μ lK
2HPO
4-KH
2PO
4In (pH7.2,0.1mol/L) damping fluid, add 1 μ LP6009-BGalH, 40mmol/LONPG or lactose 25 μ l are placed in respectively 0-60 ℃ of reaction 10min, then add separately the Na of the 1mol/L of 100 μ L
2CO
3Termination reaction, 410nm measures at the place absorbance of ONP or the amount that HPLC measures lactose.In the thermostability experiment, first with the K of enzyme preincubate in differing temps
2HPO
4-KH
2PO
4In (pH7.2,0.1mol/L) damping fluid after 1h, then carry out above-mentioned reaction.Be determined at the ability that is hydrolyzed ONPG and lactose under optimum temperuture.Result shows (Fig. 2), and during take ONPG as substrate, the optimal reactive temperature of BGalH is 40 ℃; Optimal reactive temperature during take lactose as substrate is 35 ℃.In the time of 0 ℃, BGalH can keep 5% activity (take ONPG as substrate) or 20% activity (take lactose as substrate).P6009-BGalH is hatched the active beginning of 30min for 30 ℃ and is significantly descended, the rapid inactivation of BGalH when temperature surpasses 40 ℃ in 20 ℃ of thermostabilitys better (Fig. 3).
Embodiment 3: pH stability and the optimal pH of the low temperature beta-galactosidase enzymes that embodiment 1 prepares
(pH3.0-5.5 is the citric acid-sodium citrate damping fluid of 0.1mol/L to get the damping fluid of pH3.0-9.5 of P6009-BGalH, 74 μ L of 1 μ L; PH6.0-8.0 is the K of 0.1mol/L
2HPO
4-KH
2PO
4Damping fluid; PH8.5-9.5 is the Tris-HCl damping fluid of 0.1mol/L), respectively with the ONPG25 μ L of the 40mmol/L of above-mentioned each damping fluid dissolving, mixing, the reaction solution that will contain BGalH is incubated in 30 ℃, reaction 10min, then add separately the Na of the 1mol/L of 100 μ L
2CO
3Termination reaction, 410nm measures at the place absorbance of ONP or the amount that HPLC measures lactose.In the pH stability experiment, first with P6009-BGalH preincubate in the damping fluid of different pH 4 ℃, then 1h carries out above-mentioned decomposition reaction at 30 ℃.Experimental result shows (Fig. 4), and during take ONPG as substrate, the optimal reaction pH of BGalH is 8.0; During take lactose as substrate, the optimal reaction pH of BGalH is 7.5.Hatch 1h and can keep initial activity more than 80% in the damping fluid of pH7.0-9.5, the activity that is reduced to 6.5 P6009-BGalH when following as pH only has 50% of maximum activity.
Embodiment 4: the impact of the low temperature betagalactosidase activity that metal ion prepares embodiment 1
Get the P6009-BGalH of 1 μ L, the K of 73 μ L
2HPO
4-KH
2PO
4(pH7.2,0.1mol/L) damping fluid, 1 μ L concentration are the Ba of 0.5mol/L
2+, Li
+, Ca
2+, Mg
2+, Fe
2+, Sr
2+, Cu
2+, Zn
2+, Mn
2+, Fe
3+, Co
2+, 25 μ L K
2HPO
4-KH
2PO
4The ONPG of the 40mmol/L of (pH7.2,0.1mol/L) damping fluid preparation, the reaction mixture that will contain BGalH is incubated in 30 ℃ of reaction 10min, then adds separately the Na of the 1mol/L of 100 μ L
2CO
3Termination reaction, 410nm measures absorbance in the place, the impact of assessment metal ion on enzymic activity.With the enzymatic reaction that do not add other any metal ions in contrast.Result shows (table 1), and many kinds of metal ions does not suppress the activity of BGalH, Fe
2+(190.1%) and Mn
2+(129.77%) enzymic activity there is promoter action, Ca
2+, Mg
2+, Fe
3+, Co
2+And Ba
2+There is no obvious effect, Cu
2+Significant restraining effect (35.4%) is arranged, Zn
2+Partial inhibition (60.8%) is arranged.
Table 1: metal ion is on the impact of P6009-BGalH activity (take the enzyme work that do not add metal ion as 100%)
Embodiment 5: the apparent K of the low temperature beta-galactosidase enzymes that embodiment 1 prepares
mValue, V
maxValue and K
catValue
Measured take ONPG as substrate and the apparent K of P6009-BGalH during take lactose as substrate
mValue, V
maxValue and k
catValue.K with 0.1mol/L
2HPO
4-KH
2PO
4Damping fluid in the ONPG(pH7.0 of preparation different concns) and lactose (pH7.5), react at 40 ℃ (ONPG) and 35 ℃ (lactose), the Na of 1mol/L is used respectively in the reaction that is substrate to ONPG and lactose
2CO
3With heated and boiled method termination reaction, then adopt respectively the absorbance of 410nm place's detection ONP and the amount that the HPLC-differential refraction detector is measured lactose.Result shows, take ONPG as substrate, at K
2HPO
4-KH
2PO
4(pH8.0,0.1mol/L) damping fluid, record enzyme K 40 ℃ the time
mValue is 11.17mM, K
catValue is 1097.1s
-1Take lactose as substrate, at K
2HPO
4-KH
2PO
4In (pH7.5,0.1mol/L) damping fluid, record K in the time of 35 ℃
mValue is 204.6mM, K
catValue is 2.3s
-1
Embodiment 6: the low temperature beta-galactosidase enzymes decomposing milk experiment that embodiment 1 prepares
Get the 10 natural pure milks of μ L and mix with 20 μ LP6009-BGalH, respectively at 7 ℃ and 28 ℃ of reaction 1,2,4,12,24h, boiling water bath 5min termination reaction.8000rpm gets supernatant and is diluted to 100 μ L after 10min is centrifugal.Get again 1 μ L and carry out the TLC detection.Developping agent is propyl carbinol: pyridine: water=4:6:1, developer are 20% ethanol solution of sulfuric acid.(Fig. 5) can find out from figure, at 7 ℃, and reaction 12h, lactose begins to have obvious hydrolysis, can see the spot of semi-lactosi and glucose on thin layer plate; In the time of 28 ℃, after reaction 24h, lactose almost decomposes fully, cannot see obvious lactose spot on silica-gel plate.
Embodiment 7: the preparation of the low temperature beta-galactosidase enzymes rBGalH of restructuring
One, the acquisition of Halomonas sp.P6009-1 genomic dna
Halomonas sp.P6009-1 is inoculated in Zobell 2216E liquid nutrient medium, and (tertiary iron phosphate 0.1g is dissolved in the 1L artificial seawater for peptone 5g, yeast powder 1g, and pH7.4), 28 ℃, 48h is cultivated in shaking table vibration (130rpm).The formula of artificial seawater is: NaCl25.0g, Na
2SO
44.0g, KCl0.7g, NaHCO
30.20g, KBr0.10g, H
3BO
30.03g, NaF0.003g, 53mL1.0mol/L MgCl
2Solution, 10mL1.0mol/l CaCl
2Solution, 0.90ml0.1mol/L SrCl
2Solution, distilled water 1000ml.
The genome DNA extraction test kit of employing Axygen company extracts the genomic dna of bacterial strain Halomonas sp.P6009-1.Concrete operations are carried out according to the test kit specification sheets.
Two, the acquisition of the beta-galactosidase gene of Halomonas sp.P6009-1 (BGalH)
The genomic dna of the Halomonas that extracts in the embodiment 1 carries out pcr amplification as template with primer P6009-1-BGalHF and P6009-1-BGalHR.Wherein, upstream primer P6009-1-BGalHF sequence: 5 '-TT
GAATTCATGCATCAGCCGAGTCTG-3 ' (SEQ ID NO:3, underscore partly represent EcoR I recognition site), downstream primer P6009-1-BGalHR sequence: 5 '-TT
CTCGAGTTAAACCGGCAATTGCAA-3 ' (SEQ ID NO:4, underscore partly represent Xho I recognition site).
The PCR condition is: 94 ° of C5min → { 94 ° of C40s → 55 ° C45s → 72 ° of C1.2min, 30 circulations } → 72 ° of C10min → 4 ° C.
Detect through agarose gel electrophoresis, the band about 1170bp is carried out glue reclaim, products therefrom is the BGalH complete genome fragment of Halomonas sp.P6009-1.The BGalH full length gene 1170bp of Halomonas sp.P6009-1 is as shown in SEQ ID NO:1; GC content is 58.1%, and 389 amino acid of encoding are as shown in SEQ ID NO:2.
The BLAST result shows that the highest supposition function for from Pseudomonas stutzeri ATCC17588 of similarity is the gene (GenBankAccessionNo.CP002881.1) of carbohydrate lytic enzyme, and similarity is 67%.This enzyme gene and known beta-galactosidase gene sequence similarity are lower, with the similarity of LacZ gene be only 17.22%.
Three, the acquisition of recombinant plasmid pET-32a (+)-BGalH and conversion
With the resulting PCR product EcoRI/XhoI double digestion of embodiment 2, pET32a (+) plasmid vector also adopts same EcoR I/Xho I to carry out double digestion, enzyme cut product with the T4 ligase enzyme connect (16 ℃ 16h), connect product transformed competence colibacillus E.coli DH5 α cell.Choosing positive colony carries out EcoR I/Xho I double digestion evaluation and recombinant plasmid is delivered to Shanghai living work company check order.-20 ° of C after identifying a large amount of clones of correct recombinant plasmid are frozen stand-by.
Four, low temperature beta-galactosidase gene BGalH's is recombinant expressed
Enter in E.coli BL21 bacterial strain to express with building the correct pET32a (+) of sequence-BGalH recombinant plasmid transformed, it is 100 μ g/mL that the LB(that the E.coli BL21 bacterium liquid that contains pET32a (+)-BGalH of getting 500 μ L activation is inoculated in 100mL contains ammonia benzyl concentration) in nutrient solution, 37 ℃, 180r/min cultivates bacterial strain to OD
600=0.6, adding the IPTG(final concentration is 0.5mmol/L), be transferred to 28 ℃ and continue shaking culture 10h results.
Five, the purifying of low temperature beta-galactosidase enzymes rBGalH
With fermented liquid centrifugal (5000rpm, 20min), with the K of 0.1mol/L
2HPO
4-KH
2PO
4(pH7.2) after washing, resuspended precipitation, be interrupted ultrasonication on ice, again with the centrifugal 20min of 12000rpm, get supernatant and be the thick enzyme sample of recombinant protein.
Crude enzyme liquid is used Ni after 0.45 μ m filtering with microporous membrane
2+Affinity column Hitrap
TMObtained electrophoretically pure restructuring beta-galactosidase enzymes rBGalH after HP and Capto DEAE weak anionic exchange column purifying, purification is 146.24, and active yield is 53.23%, and enzyme is lived and is 118.45U/mg.Component after purifying is carried out SDS-PAGE analyze, electrophoresis showed is single band, and molecular weight is the 63kDa left and right, and non-sex change-PAGE shows that molecular weight is about 130kDa(Fig. 6), show that this enzyme is a homodimer albumen.
Embodiment 8: thermostability and the optimal reactive temperature of the restructuring low temperature beta-galactosidase enzymes rBGalH that embodiment 7 prepares
At K
2HPO
4-KH
2PO
4In (pH7.2,0.1mol/L) damping fluid, add 1 μ LrBGalH, the lactose of 10mmol/LONPG or 30mmol/L is placed in respectively 0-60 ℃ of 10min of reaction, then adds separately the Na of the 1mol/L of 100 μ L
2CO
3Termination reaction, 410nm measures at the place absorbance of ONP or the amount that HPLC measures lactose.In the thermostability experiment, first with the K of enzyme preincubate in differing temps
2HPO
4-KH
2PO
4In (pH7.2,0.1mol/L) damping fluid after 1h, then carry out above-mentioned reaction.Hydrolysis ONPG under being determined at and the ability of lactose.Result shows (Fig. 7), and during take ONPG as substrate, the optimal reactive temperature of rBGalH is 45 ℃; Optimal reactive temperature during take lactose as substrate is 35 ℃.In the time of 0 ℃, rBGalH can keep 50% activity (take ONPG as substrate) or 25% activity (take lactose as substrate).RBGalH has good thermostability (Fig. 8), and during take ONPG as substrate, rBGalH is hatched 1h at 50 ℃ can keep initial activity more than 55%, the rapid inactivation of rBGalH when temperature reaches 60 ℃.
Embodiment 9: pH stability and the optimal pH of the restructuring low temperature beta-galactosidase enzymes rBGalH that embodiment 7 prepares
(pH3.0-5.5 is the citric acid-sodium citrate damping fluid of 0.1mol/L to get the damping fluid of pH3.0-9.5 of P6009-BGalH, 74 μ L of 1 μ L; PH6.0-8.0 is the K of 0.1mol/L
2HPO
4-KH
2PO
4Damping fluid; PH8.5-9.5 is the Tris-HCl damping fluid of 0.1mol/L) 25 μ L, respectively with the ONPG of the 40mmol/L of above-mentioned each damping fluid dissolving, mixing, the reaction solution that will contain rBGalH is incubated in 40 ℃, reaction 10min, then add separately the Na of the 1mol/L of 100 μ L
2CO
3Termination reaction, 410nm measures at the place absorbance of ONP or the amount that HPLC measures lactose.In the pH stability experiment, first with rBGalH preincubate 1h in the damping fluid of different pH, and then carry out above-mentioned reaction.Experimental result shows (Fig. 9), and during take ONPG as substrate, the optimal reaction pH of rBGalH is 7.0; During take lactose as substrate, the optimal reaction pH of rBGalH is 7.5.Hatch 1h and can keep initial activity more than 80% in the damping fluid of pH5.5-9.5, when pH is reduced to the 5.5 rapid inactivations of rBGalH when following.
Embodiment 10: the impact of the restructuring low temperature beta-galactosidase enzymes rBGalH activity that metal ion prepares embodiment 7
Get the rBGalH of 1 μ L, the K of 73 μ L
2HPO
4-KH
2PO
4(pH7.2,0.1mol/L) damping fluid, 1 μ L concentration are the Ba of 0.5mol/L
2+, Li
+, Ca
2+, Mg
2+, Fe
2+, Sr
2+, Cu
2+, Zn
2+, Mn
2+, Fe
3+, Co
2+, 25 μ L K
2HPO
4-KH
2PO
4The ONPG of the 40mmol/L of (pH7.2,0.1mol/L) damping fluid preparation, the reaction mixture that will contain rBGalH is incubated in 40 ℃ of reaction 10min, then adds separately the Na of the 1mol/L of 100 μ L
2CO
3Termination reaction, 410nm measures absorbance in the place, the impact of assessment metal ion on enzymic activity.With the enzymatic reaction that do not add other any metal ions in contrast.Result shows (table 2), and many kinds of metal ions does not suppress the activity of rBGalH, Fe
2+(218.82%) and Mn
2+(124.36%) enzymic activity there is promoter action, Ba
2+, Li
+, Mg
2+, Co
2+There is no obvious effect, Cu
2+Significant restraining effect (50.84%) is arranged, Fe
3+Partial inhibition (79.91%) is arranged.
Table 2: metal ion is on the impact of P6009-BGalH activity (take the enzyme work that do not add metal ion as 100%)
Embodiment 11: the apparent K of the restructuring low temperature beta-galactosidase enzymes rBGalH that embodiment 7 prepares
mValue, V
maxValue and K
catValue
Measured take ONPG as substrate and the apparent K of rBGalH during take lactose as substrate
mValue, V
maxValue and K
catValue.K with 0.1mol/L
2HPO
4-KH
2PO
4Damping fluid in the ONPG(pH7.0 of preparation different concns) and lactose (pH7.5), react at 45 ℃ (ONPG) and 35 ℃ (lactose), the Na of 1mol/L is used respectively in the reaction that is substrate to ONPG and lactose
2CO
3With heated and boiled method termination reaction, then adopt respectively the absorbance of 410nm place's detection ONP and the amount that the HPLC-differential refraction detector is measured lactose.Result shows, take ONPG as substrate, at K
2HPO
4-KH
2PO
4(pH7.0,0.1mol/L) damping fluid, record K 45 ℃ the time
mValue is 2.9mM, K
catValue is 390.3s
-1Take lactose as substrate, at K
2HPO
4-KH
2PO
4In (pH7.5,0.1mol/L) damping fluid, record K during 35 ° of C
mValue is 32.06mM, K
catValue is 269.5s
-1
Embodiment 12: the restructuring low temperature beta-galactosidase enzymes rBGalH decomposing milk experiment that embodiment 7 prepares
Get the 10 natural pure milks of μ L and mix with 20 μ L rBGalH, respectively at 7 ℃ and 28 ℃ of reaction 1,2,4,12,24h, boiling water bath 5min termination reaction.8000rpm gets supernatant and is diluted to 100 μ L after 10min is centrifugal.Get again 1 μ L and carry out the TLC detection.Developping agent is propyl carbinol: pyridine: water=4:6:1, developer are 20% ethanol solution of sulfuric acid.(Figure 10) can find out from figure, at 7 ℃, and reaction 12h, lactose begins to have obvious hydrolysis, can see the spot of semi-lactosi and glucose on thin layer plate; In the time of 28 ℃, after reaction 24h, lactose almost decomposes fully, cannot see obvious lactose spot on silica-gel plate.
Above demonstration and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that describes in above-described embodiment and specification sheets just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (8)
1. the low temperature beta-galactosidase enzymes of a made from ocean microorganism, is characterized in that, this enzyme obtains in order to the below legal system is standby:
(A) fermentation of Halomonas sp.P6009-1
Halomonas sp.P6009-1 bacterial classification (preserving number is CCTCC No:M2011001) is inoculated in the Zobell2216E liquid nutrient medium, 28 ℃, 130rpm shaking table shaking culture 24h activates, getting the bacterium liquid that 500 μ L activate is inoculated in the Zobell2216E substratum that contains 2% lactose of 100mL, 28 ℃, 130rpm shaking culture 4d;
Described Zobell2216E liquid culture based formulas is: peptone 5g, and yeast powder 1g, tertiary iron phosphate 0.1g is dissolved in the 1L artificial seawater, pH7.4; The formula of described artificial seawater is: NaCl25.0g, Na
2SO
44.0g, KCl0.7g, NaHCO
30.20g, KBr0.10g, H
3BO
30.03g, NaF0.003g, 53mL1.0mol/L MgCl
2Solution, 10mL1.0mol/l CaCl
2Solution, 0.90ml0.1mol/L SrCl
2Solution, distilled water 1000ml;
(B) preparation of crude enzyme liquid
After fermentation ends, fermented liquid centrifuging and taking thalline is resuspended in phosphoric acid buffer with thalline, uses the ultrasonic grinder smudge cells under condition of ice bath, and the bacterium liquid centrifuging and taking supernatant after broken bacterium is crude enzyme liquid;
(C) purifying of low temperature beta-galactosidase enzymes
With the resulting crude enzyme liquid 10ml of step (B), add (the NH of 2.2g
4)
2SO
4Saltout.The centrifuging and taking supernatant adds the (NH of 0.6g again
4)
2SO
4Carry out pickle change, the centrifuging and taking precipitation.Add that 3mL's contain 1mmol/L MgCl
2, pH7.2 0.1mol/L K
2HPO
4-KH
2PO
4Resuspended precipitation, HiPrep
TM26/10 desalting column desalination is collected target protein; Further get 2mL and be splined on the PABTG affinity column, with 15% contain 0.5mol/L KCl, 0.1mol/L lactose, 1mmol/L MgCl
2, pH7.2 0.1mol/LK
2HPO
4-KH
2PO
4Buffer B buffer solution elution 60min, then with 20% buffer B buffer solution elution 20min, flow velocity is 1mL/min; With the active ingredient loading 2mL after concentrated after affinitive layer purification, displacement damping fluid in Capto DEAE weak anionic exchange column, the 1mol/LKCl that contains with 0% ~ 100%, 1mmol/L MgCl
2, the 0.1mol/LK of pH7.2
2HPO
4-KH
2PO
4Buffer C buffer solution for gradient elution 40min, flow velocity is 1mL/min, detecting wavelength is 280nm and 214nm; Active ingredient is again after Capto Q reinforcing yin essence ion exchange column, Sepharcryl S200 molecular sieve further separate, obtain electrophoretically pure P6009-BGalH, carry out respectively SDS-PAGE and non-sex change-PAGE, find that its molecular weight is about 90kDa, the subunit size is the 48kDa left and right.
2. the low temperature beta-galactosidase enzymes of a made from ocean microorganism, is characterized in that, this enzyme is following (a) or (b) or protein (c):
(a) protein that is formed by the aminoacid sequence shown in SEQ ID NO:2 in sequence table;
(b) with the aminoacid sequence shown in SEQ ID NO:2 through the replacement of one or several amino acid deformity and/or disappearance and/or interpolation and relevant to the beta-galactosidase enzymes protein that is derived by SEQ ID NO:2;
(c) aminoacid sequence shown in SEQ ID NO:2 is added the preceding paragraph sequence at N end or C end, and relevant to the beta-galactosidase enzymes sequence that is derived by SEQ ID NO:2.
3. the encoding gene of the low temperature beta-galactosidase enzymes of a made from ocean microorganism as claimed in claim 2, is characterized in that, encoding gene is following (ⅰ) or DNA molecular (ⅱ):
(ⅰ) its encoding sequence is the DNA molecular shown in SEQ ID NO:1 in sequence table;
(ⅱ) under stringent condition with the DNA sequence dna hybridization that (ⅰ) limits and the DNA molecular of the described beta-galactosidase enzymes of encoding.
4. the encoding gene of the low temperature beta-galactosidase enzymes of a kind of made from ocean microorganism according to claim 3, is characterized in that, described stringent condition is at 0.1 * SSPE or 0.1 * SSC, in the solution of 0.1%SDS, hybridizes under 65 ℃ and wash film.
5. recombinant expression vector, expression cassette, transgenic cell line or the recombinant bacterium of the encoding gene of a low temperature beta-galactosidase enzymes that contains made from ocean microorganism as claimed in claim 3.
6. the application of low temperature beta-galactosidase enzymes in the decomposing milk lactose of a made from ocean microorganism as claimed in claim 1.
7. the application of low temperature beta-galactosidase enzymes in the decomposing milk lactose of a made from ocean microorganism as claimed in claim 2.
8. the application of encoding gene in the decomposing milk lactose of the low temperature beta-galactosidase enzymes of a made from ocean microorganism as claimed in claim 3.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591262A (en) * | 2016-12-07 | 2017-04-26 | 江南大学 | Method for improving yield of cold-adapted beta-galactosidase |
| CN106754558A (en) * | 2017-01-25 | 2017-05-31 | 中国人民解放军第二军医大学 | Elongated lysine bacillus and its application that the polar region of one plant of product low temperature active beta galactosidase is originated |
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|---|---|---|---|---|
| CN102174439A (en) * | 2011-01-27 | 2011-09-07 | 中国人民解放军第二军医大学 | Marine halomonas for producing low-temperature beta-galactosidase and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174439A (en) * | 2011-01-27 | 2011-09-07 | 中国人民解放军第二军医大学 | Marine halomonas for producing low-temperature beta-galactosidase and application thereof |
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| 《中国优秀硕士学位论文全文数据库(电子期刊) 基础科学辑》 20121015 王国祥 海洋微生物低温beta-半乳糖苷酶的筛选、克隆及性质研究 图2.2，第16-32页 1-8 , * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591262A (en) * | 2016-12-07 | 2017-04-26 | 江南大学 | Method for improving yield of cold-adapted beta-galactosidase |
| CN106754558A (en) * | 2017-01-25 | 2017-05-31 | 中国人民解放军第二军医大学 | Elongated lysine bacillus and its application that the polar region of one plant of product low temperature active beta galactosidase is originated |
| CN106754558B (en) * | 2017-01-25 | 2020-12-08 | 中国人民解放军第二军医大学 | Polar-region-derived lysine bacillus for producing low-temperature active beta-galactosidase and application thereof |
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