CN102115718B - Recombinant strain for expressing beta-galactosidase and construction method and application thereof - Google Patents
Recombinant strain for expressing beta-galactosidase and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant strain for expressing beta-galactosidase and a construction method and application thereof. The galactosidase gene bgal of Thermus thermophilus HB27 is obtained by cloning, and is inserted into a eukaryotic expression vector pPIC9K, and transformed into a Pichia pastoris strain GS 115, so that the expression quantity of beta-galactosidase can reach 14000U/ml. The molecular weight of beta-galactosidase gene bgal expression is 58Dk, the optimum temperature is 70 ℃, and the optimum temperature is stable. The lactase has the highest hydrolytic activity and better stability at pH6.5. The beta-galactosidase with high expression can be directly used for pasteurization of dairy products, can degrade lactose while sterilizing, and avoids secondary pollution caused by adding enzyme after sterilization. At 60 degrees, 30 minutes, HB27 galactosidase was able to degrade 90% of lactose in milk.
Description
Technical field
The present invention relates to a kind of recombinant bacterial strain and construction process and application of expressing beta-galactosidase enzymes, belong to biological technical field.
Background technology
Beta-galactosidase enzymes (EC 3.2.1.23) formal name used at school is β-D-galactoside galactohydrolase (hereinafter to be referred as tilactase), Sumylact L by name commonly used, and this enzyme can hydrolyze lactose into and be semi-lactosi and glucose.
Tilactase is used for the milk-product industry the earliest; Because milk occupies very big proportion in westerner's dietary structure; So abroad to the research of tilactase early, many research work mainly concentrate in the diagnoses and treatment of low lactose milk processing and lactose intolerance (Lactose Intolerance).Lactose is a distinctive carbohydrate in the Mammals milk, and lactose-content is different because of kind in the Mammals milk, and lactose-content is 6.7%. in the human milk, and the Lactose in Milk average content is 4.5-5%.Although cow's milk and milk-product thereof have the good reputation of " being close to perfect food ", in China, the development of milk-product still is in the junior stage, and wherein lactose intolerance is the one of the main reasons that limits its development.Because lactose need pass through by mucous membrane of small intestine epithelial cell excretory tilactase and be hydrolyzed into utilizations that just can be absorbed by the body behind semi-lactosi and the glucose, so the cause of disease of lactose intolerance mainly is on the mucous membrane of small intestine galactoside enzyme activity than due to hanging down.Lactose in Milk can not get hydrolysis, and lactose concn improves in the small intestine, and osmotic pressure is increased, thereby the moisture content that causes getting in the enteric cavity improves, and the symptom of its generation is that abdominal pressure increases, inflatable, stomachache and diarrhoea.The mucous membrane of small intestine lactase activity is higher after most Mammals births; Can digest and assimilate lactose from breast milk or other milk-product; After the ablactation; Lactase activity descends with age growth gradually, and galactosidase activity is merely the 5-10% of normal infant level to being grown up afterwards, and develops into lactose intolerance gradually.Lactose intolerance affects the absorption of the people (especially Asia and African) in the whole world about 70% to breast according to statistics.
Producing low lactose milk, is the best method that overcomes lactose intolerance at present.Classical way be with fresh milk behind pasteurization, be cooled to 40-45 ℃, add tilactase and insulation, make lactose hydrolysis.Discover when lactose hydrolysis ratio is 70-80%, can solve the lactose intolerance problem, can produce sapid product again.Commercialization already outside the producing country of low lactose milk, domestic then ground zero.The mode of production of low lactose milk has multiple, a kind of in above-mentioned just wherein many working methods.Theoretically, the low lactose milk working method has physical removal method, chemical acid-hydrolysis method, enzyme hydrolysis method and genetic engineering technique method.Wherein enzyme hydrolysis method has obtained promoting widely because reaction conditions is gentle, and product is simple, does not destroy other nutritive ingredients in the cow's milk, and the side reaction product of generation is less, and mouthfeel is better.Up to now, enzyme hydrolysis method is to produce low the most safe, practical, the effective means of lactose cow's milk.Enzyme hydrolysis method is produced in the process of low lactose milk, enzyme be added with 2 kinds of modes.A kind of is with the optimum temps of raw dairy through pasteurize postcooling to tilactase effect, adds enzyme and under this temperature, is incubated for some time, makes it reach desired lactose hydrolysis degree, then product is sterilized, and carries out sterile filling.This method is simple to operate; Product is not easy to be polluted; But the Maillard reaction that takes place during sterilising treatment (reducing sugar in the food and amine substance at high temperature produce a kind of reaction of chocolate material) causes the brown stain of product easily, thereby influences the aesthetic quality of product, and consuming time longer.Another kind method is with after raw dairy sterilization, the cooling, before the product can, by a certain percentage tilactase is joined in the product, and in storage process, tilactase is decomposed into glucose and semi-lactosi with the lactose in the cow's milk.This method required equipment investment is low, and the color and luster of product changes little, and when still adding enzyme, the cow's milk after the sterilization receives microbiological contamination easily, and the difficult control action time of enzyme, possibly need extra sterilisation step, for example UHT or UV etc. after the enzyme reaction.
Thermus thermophilus HB27 is a very thermophilic widely bacterial classification of a kind of research, and genetic resources wherein is very abundant.Because its living environment temperature is high, nutrition is poor, causes the character of the enzyme system in its pathways metabolism and special.So far, there have been many genes to be cloned and in different expression systems, express, comprised many glycoside hydrolases, for example glycase and glucuroide etc. from Thermus thermophilus HB27.The thermotolerance of these enzymes is all very high, and optimum temperuture is between the 60-95 degree.Also has no at present the report of being cloned and expressing from the beta-galactosidase gene of Thermus thermophilus HB27; And the protein sequence of this enzyme and other species difference are bigger, even also be not more than 30% with the homology of the Thermus thermophilus A4 bacterial classification of same subspecies.Utilize the resistant to elevated temperatures character of the beta-galactosidase enzymes of Thermusthermophilus HB27, can set up and be applicable to milk-product pasteurization technology.
Summary of the invention
Technical problem to be solved by this invention provides a kind of recombinant bacterial strain of expressing beta-galactosidase enzymes, and the expressed high temperature resistant beta-galactosidase enzymes of this bacterial strain is applicable to the milk-product pasteurization.
The technical problem that the present invention also will solve provides the construction process of above-mentioned recombinant bacterial strain.
The technical problem that the present invention will solve at last provides the application of above-mentioned recombinant bacterial strain in producing high temperature resistant beta-galactosidase enzymes.
A kind of recombinant bacterial strain of expressing beta-galactosidase enzymes, this bacterial strain are the pichia spp of having integrated the beta-galactosidase gene bgal that derives from thermus thermophilus (Thermus thermophilus) HB27 on the genomic dna.
The construction process of the recombinant bacterial strain of above-mentioned expression beta-galactosidase enzymes; To be cloned among the yeast expression vector pPIC9K from the beta-galactosidase gene bgal of thermus thermophilus (Thermusthermophilus) HB27; After utilizing SacI restriction enzyme linearized vector; It is converted in the pichia spp, utilizes the G418 microbiotic to carry out the multiple copied transformant screening and obtain.
The recombinant bacterial strain of above-mentioned expression beta-galactosidase enzymes efficiently expresses the method for beta-galactosidase enzymes, it is characterized in that with methyl alcohol being that inductor inducing culture recombinant bacterial strain produces enzyme.With 3L fermented liquid culture bacteria bulk-growth 35h, OD
600After reaching 450, add form with stream and add methyl alcohol, the adding total amount is 1.0～1.5L, and induction time is 40～48 hours.
The application of the recombinant bacterial strain of above-mentioned expression beta-galactosidase enzymes in the fermentative prodn beta-galactosidase enzymes.The condition of the beta-galactosidase enzymes hydrolyzes lactose that recombinant bacterial strain of the present invention produced is: ph optimum is 6.5, reaches half that maximum enzyme is lived at pH5.3 and this beta-galactosidase enzymes of 7.6 places, and this pH scope meets the soda acid scope of most of food; Optimum temperuture is 70 ℃, and when temperature continued to increase, the Denaturation of zymoprotein made the strong decline of enzyme living drama.No matter whether add halfcystine, the optimum temperuture of this enzyme does not all have considerable change.
The used pichia spp of the present invention is a kind of eukaryotic expression system efficiently; Efficiently express tilactase with it many-sided advantage is arranged: the widespread use of (1) pichia spp Ceng Zuowei single cell protein; Itself just has good security; And, do not contain Toxic matter and pyrogen in the yeast culture base.(2) pichia spp has PE system efficiently.We adopt zymic a factor signal skin sequence with the tilactase direct secretion of expressing in substratum, will simplify the purifying process of enzyme so greatly, reduce the production cost of enzyme.(3) its high-cell density, low-cost fermentation process are generally adopted, and employed carbon source, nitrogenous source, salt, trace element etc. all are the general industry raw material in the fermention medium, cheaply are easy to get.Utilize pichia spp to lay a good foundation for large-scale industrialization, low-cost fermentative prodn tilactase as the bio-reactor of galactoside expression of enzymes.The foreign protein of (4) expressing content in total secretory protein is high.Galactosidase gene bgal secreting, expressing in yeast among the present invention, foreign protein seldom.So just simplify the separation purifying technique of target protein, reduced cost.
Beneficial effect: the beta-galactosidase enzymes that recombinant bacterial strain produced that the present invention makes up has higher thermostability; By immobilized thermophilic beta-galactosidase enzymes; Degraded lactose when can accomplish to sterilize; Avoided the sterilization back to add the secondary pollution that enzyme caused, do not needed extra sterilisation step, and be not easy to take place the brown stain that Maillard reaction causes product.Do like this and can improve product appearance, reduce production costs.
Description of drawings
Fig. 1 recombinant plasmid pPIC9K-bgal makes up synoptic diagram.
Thalli growth and dissolved oxygen in Fig. 2 beta-galactosidase enzymes expression process.
The mensuration of Fig. 3 beta-galactosidase enzymes ph optimum.
The mensuration of Fig. 4 beta-galactosidase enzymes optimum temperuture.
The thermostability of Fig. 5 beta-galactosidase enzymes.
Fig. 6 lactose standard substance HPLC detects collection of illustrative plates.
Lactose degradation curve in the commercially available milk of Fig. 7, the i.e. relation in lactose residual content and reaction times.
Embodiment
According to following embodiment, can understand the present invention better.Yet, those skilled in the art will readily understand that embodiment is described only to be used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
Embodiment 1:
The Biometra PCR of company appearance is adopted in the clone of bgal gene: PCR reaction.Basi gene operative techniquies such as the DNA enzyme is cut, connected, conversion and the preparation of bacterium competence are carried out with reference to the operational manual that relevant company provides.Adopt the test kit of Shanghai China Shun biotechnology ltd, extract T.thermophilus HB27 genomic dna.With it is template, and primer is G-sense and G-anti, carries out pcr amplification.PCR carries out in 50 μ L systems, and reaction conditions is: 94 ℃ of sex change 5min, circulate 34 times by following parameter: 94 ℃ of sex change 1min, 55 ℃ of annealing 50s, 72 ℃ are extended 1.5min.Last 72 ℃ are extended 10min.The PCR reaction product is with 0.8% agarose gel electrophoresis analytical results.Behind a large amount of amplifying target genes of PCR, gel electrophoresis separation and purification PCR product behind the recovery goal gene, is connected to goal gene on the T-vector from gel.In the positive recombinant of confirming, select two and serve the order-checking of Hai Boya company.Sequencing result and the sequence of being reported are carried out the sequence alignment analysis with ClustalW software.
Bacterial classification and carrier: thermus thermophilus Thermus thermophilus HB27, available from Chinese microbiological industry DSMZ.Bgal gene clone carrier pMD18-T Vector (hereinafter to be referred as T-Vector) is available from Takara company.Yeast expression vector pPIC9K is available from Invitrogen company; Pichiapastoris expression strain GSl15 is available from Invitrogen company;
Culture medium preparation:
10x YNB (the no amino acid yeast nitrogen of 134g/L): the 134gYNB solid is dissolved in the 1L deionized water, filtration sterilization, 4 ℃ of preservations;
500x B solution (0.2g/L vitamin H): vitamin H 20mg is dissolved in the 100mL deionized water, filtration sterilization, 4 ℃ of preservations;
10x D solution (200g/L glucose): 200g D-glucose is dissolved in 1000mL water, filtration sterilization, 4 ℃ of preservations;
10x GY (glycerine of 100g/L): 100mL glycerine is dissolved in 900mL water, filtration sterilization, 4 ℃ of preservations;
100x AA (containing the every seed amino acid of 0.5g/L): take by weighing L-L-glutamic acid, L-methionine(Met), L-leucine, L-Isoleucine, each 500g of L-Methionin and be dissolved in 100mL water, filtration sterilization, 4 ℃ of preservations;
The phosphoric acid buffer seal (pH6.0) of 1mol/L: the potassium hydrogenphosphate of 132mL1mol/L and the potassium primary phosphate of 868ml 1mol/L are mixed, transfer pH to 6.0 with phosphoric acid and potassium hydroxide solution then, filtration sterilization, normal temperature is preserved;
The Sorbitol Solution USP of 1mol/L: the D-sorbyl alcohol of 182.1g is dissolved in the water of 1L filtration sterilization, 4 ℃ of preservations;
YPD substratum: 10g/L yeast extract, 20g/L peptone, 20g/L glucose;
MM solid medium: 13.4g/LYNB, 0.0004g/L vitamin H, 2% glucose, 1.5% agar; 0.005% L-glutamic acid, 0.005% methionine(Met), 0.005% Methionin, 0.005% leucine; 0.005% Isoleucine, 2% agar powder, all amino acid are the L type;
MD solid medium: 13.4g/L YNB, 0.0004g/L vitamin H, 2% glucose, 1.5% agar, the G418 vitriol of 0.5～4mg/L (adopting different concentration) according to screening process;
Fermention medium: 26.7mL/L 85%H
3PO
4, 0.93g/L CaSO
42H
2O, 18.2g/LK
2SO
4, 14.9g/LMgSO
47H
2O, 4.13g/L KOH, 40g/L glycerine, yeast extract paste 10g/L, peptone 20g/L, ammoniacal liquor adjust pH to 5.0 with 25%.
PTM1 nutritive salt: CuSO
45H
2O 6g/L, NaI 0.09g/L, MnCl
24H
2O 3g/L, H
3BO
30.02g/L, Na
2MoO
42H
2O 0.2g/L, CoCl
20.5g/L, ZnCl
242.2g/L, FeSO
4H
2O 65g/L, biotin 0.2g/L, H
2SO
45.0mL/L.(adding 4.35mLPTM1 nutritive salt in the 1L fermention medium)
The stream liquid feeding: 25% ammoniacal liquor: 500mL, keep fermentation pH 5.0.50% glycerine: 500mL glycerine adds deionized water to 1L, sterilization back Ensure Liquid salt 12mL.100% methyl alcohol: 1L methyl alcohol adds 12mL nutritive salt.
Primer: according to bgal gene order information, with Prime premier software design pcr amplification primer.
(9K-sense EcoRI) is: 5 '-cggaattcatgcggctggaccccaaccacc-3 ' upstream primer;
(9K-anti NotI) is: 5 '-gccaccttgcggccgcctactccgcgagaa-3 ' downstream primer.
All primers can betting office synthesize by the Shen, Shanghai.
The structure of pichia spp recombinant expression plasmid: recombinant plasmid T-bgal is carried out pcr amplification with 9K-sense and 9K-anti primer; PCR carries out in 50 μ L systems; Reaction conditions is: 94 ℃ of sex change 5min; Circulate 34 times by following parameter: 94 ℃ of sex change 1min, 55 ℃ of annealing 50s, 72 ℃ are extended 1.5min.Last 72 ℃ are extended 10min.Reclaim the fragment (bgal gene) of about 1.8kb, with EcoRI, NotI double digestion yeast expression vector pPIC9K and recovery.16 ℃ of connections, CaCl
2Method transformed into escherichia coli DH5 α is applied to LB (Amp
+) flat board, plasmid, double digestion evaluation and screening pichia spp recombinant expression plasmid, called after pPIC9K-bgal are cultivated and extracted to picking list bacterium colony.The building process of expression vector sees Fig. 1 for details.
The preparation of pichia spp competent cell: picking pichia spp recipient bacterium GS115 (His
-, Mut
+) single colony inoculation in the 10mLYPD liquid nutrient medium, 30 ℃ of shaking table overnight cultures are transferred in the 100mLYPD substratum with the inoculum size of 1% (v/v) again, 30 ℃ are cultured to OD600=1.3-1.5.4 ℃ of centrifugal 5min of 5000rpm, supernatant discarded, with the sterilized water of 100mL ice precooling that thalline is resuspended; 4 ℃ of centrifugal 10min of 5000m once more, supernatant discarded, with the sterilized water of 50mL ice precooling that thalline is resuspended; 4 ℃ of centrifugal 10mln of 5000rpm; With 20mL 1mol/L sorbyl alcohol washing 1 time, be dissolved in the sorbyl alcohol of 20mL 1mol/L ice precooling, again in order to transforming.
The electric shock of pichia spp transforms: with SacI restriction endonuclease single endonuclease digestion recombinant expression plasmid pPIC9K-bgal, make plasmid linearization.The unit point polygene inserts and can spontaneously form really in the cell, though ratio is low, accounts for the 1-10% of all HIS+ transformants, but can survey.What multiple copied inserted incident is because gene can insert in AOX1, aox1::AGR4 site or his4 site.The result produces the Mut+ phenotype in GS115, in KM71, produce the Muts phenotype.Transfer to after getting 80 μ L competent cells and linearization plasmid (1～5 μ g) mixing in the electric shock cup of prior 0.2cm with the ice bath precooling; Electric shock transformed yeast competent cell. (2.5KV; 5ms), in the electric shock cup, add the Sorbitol Solution USP of the ice-cold 1mol/L of 1mL immediately, behind the mixing; Be applied on the MM solid plate with every flat board 200 μ L bacterium liquid, 28 ℃ of cultivations are up to growing transformant.The His+ that grows on the MM flat board is transformed bacterium colony distinguish substratum MD flat board is selected in dibbling in yeast corresponding position sequentially.
Pichia spp efficiently expresses the screening and the expression of son: picking is inoculated in respectively after the well-grown transformant numbering on the MM flat board that (concentration is 0.5mg/L, 1.0mg/L, 1.5mg/L in the MD solid plate that contains different concns G418 vitriol; 2.0mg/L, 2.5mg/L, 3.0mg/L; 3.5mg/L); Cultivate 24～72h (the dull and stereotyped growth time of different G418 concentration is different) in 28 ℃ of insulation cans, distinguish the single bacterium colony in the picking different concns G418 flat board, carry out next step abduction delivering.Whether effective for detecting expression condition, with the GS115 transformant that imports blank pPIC9K plasmid blank as secreting, expressing.
The picking mono-clonal is inoculated among the seed culture medium YPD, behind the shake-flask culture 24h, transfers in the fermentor tank of 5L mixing speed 1000rpm; Air flow 4VVM cultivates 23.5 hours DO% and rises to 80% in jar, begin to flow glycerol adding; When being cultured to 35h, OD600 reaches 450, begins stream and adds methyl alcohol; Through improving dissolved oxygen and the strategy that adds organic nitrogen source, recombinant yeast pichia pastoris GS115 in the NBS of 5L fermentor tank, OD
600Reach 700, enzymic activity can reach 14000U/L in the fermented liquid.
Embodiment 2:
The preparation of crude enzyme liquid: get bacterium liquid behind the abduction delivering in 8000rpm, 4 ℃ of centrifugal 10min abandon supernatant.Deposition is with phosphoric acid buffer (100mM, pH 6.0) washed twice, and the deposition after the washing is resuspended in the lysis buffer (the phosphoric acid buffer 1.0mM PMSF of 100mM), puts ultrasonication in the ice bath.With the 10mm probe, in 200w ultrasonication on ice, ultrasonic time 3s, interval 5s, ultrasonic altogether 8min.Bacterium liquid after supersound process is in 12000rpm, 4 ℃ of centrifugal 15min.Get supernatant and be crude enzyme liquid.
Expression product is analyzed: using and concentrating colloid amount mark is 5%, and the separation gel massfraction is that 10% sds polyacrylamide gel electrophoresis is investigated proteic expression.
Tilactase enzyme activity determination method: take by weighing 280mg ortho-nitrophenyl β-D-gala pyranoside (ONPG), be dissolved in earlier in the 1mL methyl alcohol, the phosphoric acid buffer with 100mM pH6.0 is settled to 100mL again, is made into the ONPG mother liquor of 20mM.Obtain the ONPG aqueous solution of different concns through the phosphoric acid buffer that adds different ONPG mother liquors of measuring and 100mM pH6.0 respectively.Before measuring enzyme and living, ONPG solution can be under corresponding temperature water-bath for some time, to guarantee the accuracy of temperature.Add crude enzyme liquid to be measured during measurement, making TV is 200 μ l, insulation for some time, measure its absorbance value then at 420nm, and system is through adding 1800 μ l 100mM dilute sulphuric acid stopped reactions.1 unit enzyme (U) alive is defined as PM hydrolysis 1nmONPG.
The ph optimum of this beta-galactosidase enzymes hydrolysis ortho-nitrophenyl β-D-gala pyranoside (ONPG) is 6.5 as a result.This result and some are similar from the tilactase of other yeast and bacterium, and the ph optimum of most of beta-galactosidase from yeast and bacterium is all between 6.5 to 7.5.Reach half that maximum enzyme is lived at pH5.3 and this beta-galactosidase enzymes of 7.6 places, this pH scope meets the soda acid scope of most of food, and this this enzyme of explanation is useful on the potentiality of lactose in hydrolysed milk or the sweetened condensed milk.Optimum temperuture from the beta-galactosidase enzymes of T.thermophilus is 70 ℃, and is very stable under its optimum temperuture.
Embodiment 3: lactose degradation experiment in the milk.
Commercially available milk detects through performance liquid chromatography, and lactose-content is 42g/L.Adopt Ultimate 3000 chromatographic systems, differential refraction detector (RID), differential temperature 35 degree, chromatographic column is the NH of Chinese nation
2Post, moving phase are 80% acetonitrile, flow velocity 1ml/min, detection time 30min, appearance time 18min.
The lactose standard curve determination:
Commercially available lactose is made into the lactose solution of 0g/L-50g/L concentration with volumetric flask, detects the differential value with above-mentioned performance liquid.The gained typical curve, y=0.609x+0.0452, R
2=0.9991.Fig. 6 is that lactose standard substance HPLC detects collection of illustrative plates.
Lactose degradation experiment in the commercially available milk
With milk heating in water bath to 60 ℃, add Sumylact L (be embodiment 1 with embodiment 2 in the crude enzyme liquid that obtains), its unit vigor reaches 30000U/L, sampling in per 5 minutes, the content of liquid chromatogram measuring lactose.After 30 minutes, lactose degradation reaches more than 85% in the milk.Fig. 7 is the lactose degradation curve, i.e. the relation in lactose residual content and reaction times.
Claims (1)
1. utilize the recombinant bacterial strain of expressing beta-galactosidase enzymes to efficiently express the method for beta-galactosidase enzymes, it is characterized in that with methyl alcohol being that inductor inducing culture recombinant bacterial strain produces enzyme, every 3L fermented liquid culture bacteria bulk-growth 35h, OD
600After reaching 450, add form with stream and add methyl alcohol, adding total amount is the methyl alcohol of 1.0～1.5L, and induction time is 40～48h;
Wherein, the recombinant bacterial strain of described expression beta-galactosidase enzymes is for having integrated the pichia spp of the beta-galactosidase gene bgal that derives from thermus thermophilus (Thermus thermophilus) HB27 on the genomic dna.
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| CN103937767B (en) * | 2014-04-08 | 2016-03-30 | 昆明理工大学 | The polynucleotide of a kind of tilactase and this enzyme of coding |
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| CN110628792B (en) * | 2019-10-22 | 2021-04-20 | 怀化学院 | Cold-tolerant lactase gene and expression vector and protein thereof |
| CN111440782B (en) * | 2020-04-22 | 2021-09-17 | 青岛大学 | Beta-galactosidase GalA and application thereof |
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| CN1258452A (en) * | 1998-12-25 | 2000-07-05 | 浙江医科大学 | Method of producing low-lactose milk with solid thermophilic bacteria lactase |
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