CN101768578B - Eosinophilic lactase BGALA, gene and application thereof - Google Patents

Abstract

The invention relates to genetic engineering field, in particular to eosinophilic lactase BGALA, gene and application thereof. The invention provides a lactase BGALA of eosinophilic Bispora sp.; the amino acid sequence of the lactase BGALA is shown in SEQ ID NO.1; and the invention provides genome and cDNA coding gene bgalA for coding the lactase. The invention obtains an eosinophilic lactase with the most proper pH value of 1.5, which maintains high enzymatic activity in acid environment, has better pH stability and heat stability, and antiprotease hydrolysis. The eosinophilic lactase can effectively hydrolyze the lactose in milk in simulated digestive tract environment. The eosinophilic lactase BGALA with the properties can be used for treating lactose intolerance, diary industry no processing cow milk and whey.

Description

A kind of eosinophilic lactase BGALA and gene thereof and application

Technical field

The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of eosinophilic lactase BGALA and gene thereof, comprise recombinant vectors and the application of this gene.

Background technology

Beta-galactosidase enzymes (β-D galactoside galactohydrolase, β-D-galactosido-galatohydrolase, EC3.2.1.23) is commonly called Sumylact L (Lactase).This kind of enzyme can hydrolyze lactose into and be semi-lactosi and glucose, also has the transferance (Zhang Shuzheng etc., Enzymes Industry, Science Press, 1984, p818～819) of galactoside.Sumylact L is present in plant (especially at apricot, peach, apple), bacterium (milk-acid bacteria, intestinal bacteria etc.), fungi (aspergillus oryzae, aspergillus niger, sulphur ball aspergillus, saccharomyces fragilis, newborn combining yeast, saccharomyces lactis, candida tropicalis) and actinomycetes (sky blue streptomycete), the animal intestinal (particularly nursling).Lactose then is the main component in milk and the whey, and lactose accounts for 30% of milk dry-matter, but solubleness is low, solubleness only is 20% in 20 ℃ of water, and its sugariness only reaches 16% of sucrose, and the milk-product of storage are separated out owing to lactose for a long time, be the sand sample sensation, affect local flavor.In addition, many adults, particularly lack Sumylact L in baby's (different because of ethnic group, West Europe accounts for 2～8%, and Asia, Africa account for 60～90%) body, therefore they are difficult to pegnin, behind the milk drink, lactose has arrived in the intestines, by the intestinal bacteria decomposing and fermenting, produce great amount of carbon dioxide gas, enteron aisle is expanded, and excited intestines peristalsis, make to shrink and strengthen, cause borborygmus and diarrhoea, this disease is called lactose intolerance, and (G.G. uncle is strange etc., enzyme and food-processing, light industry press, 1991, p93～110).

Sumylact L is mainly used to treat lactose intolerance, processing cow's milk, whey etc., produces low-lactose milk and low-lactose dairy product and environmental contamination reduction.1. solve the problem (the G.G. uncle is strange etc., enzyme and food-processing, light industry press, 1991, p93～110) of lactose intolerance edible for patients milk-product.Owing to lack Sumylact L in the lactose intolerance patient body, they just can not take full advantage of the energy that lactose provides in the milk-product, lactose can not be absorbed in its body, just becomes the energy of enteric microorganism fungus strain, so just causes the formation of lactic acid and carbonic acid gas, they have hormesis to enteron aisle, and cause the body dehydration to enter colon, and diarrhoea finally can appear, intestinal obstruction and flatulence etc. occur simultaneously, cause intestines peristalsis to accelerate, also can reduce the dietetic alimentation of protein and inorganic salts.Diarrhoea also can be brought the problem of hygiology aspect and cause the chance of superinfection.Can directly be added to by oral Sumylact L or with Sumylact L and solve this problem in the milk-product.In industrial production, Sumylact L can be added to the milk-product of making low lactose in the milk-product, the impact that alleviates lactose intolerance on the one hand, the nutrition that can improve on the other hand milk-product.2. improvement concentrates the quality of breast and condensed whey.Because the solubleness of lactose is low, crystallization from concentrated breast and condensed whey very easily at low temperatures, the adding Sumylact L can prevent the lactose crystn in freezing breast and the condensed whey and cause solidifying of milk casein.In addition, whey is very high as the utility value of feed, but lactose has certain limitation to the growth of domestic animal, uses Sumylact L that lactose is decomposed into monose and has both improved digestibility, has also eliminated the operation inconvenience that lactose crystn produces simultaneously.3. improve ice-cream quality.In ice cream mix, during if any the skimming milk solid substance more than 12%, can produce lactose crystn in storage and the sale, this crystallization is the sand sample sense in mouth, reduced commodity value, be 16% such as the degreasing solid substance, after decomposing its lactose of 50% with enzyme, in refrigerator, place four months still stable, and, increased sweet taste, the granulated sugar in the ice cream mix can reduce by 1～2% consumption.In addition, hydrolyzed whey or lactose have very large purposes in sandwich, soft drink and textiles starching, use hydrolyzed whey can save the use albumin in the textiles starching, reduce production costs.

In recent years, utilizing beta-galactosidase enzymes to produce oligomeric galactose also comes into one's own.Therefore, the effect of Sumylact L in foodstuffs industry is produced is day by day remarkable.But Sumylact L is not used widely in foodstuffs industry at present, and major cause is: the output of Sumylact L is lower at present, and selling price is too high, and it is expensive to add cost; The Sumylact L of selling on the market is mainly derived from yeast, this kind enzyme poor heat resistance, and narrow application range can not satisfy the many-sided needs of foodstuffs industry.Along with the development of genetic engineering technique, it is low that cost is developed in searching, and output is high, and thermostability is strong, is more suitable for the Sumylact L that foodstuffs industry is produced, and become study hotspot.

At present, commercial Sumylact L is mainly derived from microorganism (bright work, enzyme application manual, Shanghai science tech publishing house, 1989,407～414 of waiting of phase chi filial piety).Take the variant of intestinal bacteria (Escherichia coli), aspergillus niger (Aspergillusniger), lactose-fermenting yeast (Kluyveromyces lactis) and smelly aspergillus (A.foetidus) as suitable.In and the relation that gene suppresses synthetic at Study on Protein, the intestinal bacteria beta-galactosidase enzymes of widespread use be first by the Sumylact L of crystallization, molecular weight is 850,000Da.From the Sumylact L that intestinal bacteria (E.coli) ML309 produces, infer that its molecular weight is 518,000Da.Colibacillary enzyme optimal pH is 7.0, but at Na ⁺Exist lower optimal pH to become 6.6.

In dairy products and other foodstuffs industry, obtain the Sumylact L of effective application mainly by obtaining in Crewe dimension Si Shi yeast (Kluyveromyces lactis) and the aspergillus niger (Aspergillus niger).Crewe dimension Si Shi yeast is separated to from cow's milk, and the natural pH (6.6～6.8) of the Sumylact L optimal pH of its generation and fresh milk is close, mainly is applicable to decompose the lactose in cow's milk and the skim-milk.This enzyme is stable in pH6.2～7.0,6.2～7.0 or more or the rapidly decline of living of following enzyme.Optimum temperuture is 35～40 ℃.Molecular weight is 135000Da.Process 1hr for 40 ℃, pH is stable between 6.5～8.5, even it is stable too to process 3hr, processing 24hr pH7～8 are stable.Just rapid inactivation more than 40 ℃.Ca ²⁺, Cu ²⁺, Fe ²⁺Can make enzyme deactivation, Mn ²⁺, Mg ²⁺Can recover Ca ²⁺To enzymeinhibition, K ⁺Individual existence just can improve Thermostability (Xie Yi etc., Fudan Journal (natural science edition), 1999, Vol.38, No.5:523-528) greatly.Take ONPG (o-NP-β-D galactoside) as substrate, 40 ℃ of reaction 3min, Km is 2.78mmol/L, and maximum reaction velocity is 0.1umol/min, and the hydrolysate semi-lactosi of enzyme has strong restraining effect to the activity of enzyme, it should be noted that, the restraining effect of ribose is also very strong, but its mechanism it be unclear that (Shen Weiqun etc., biotechnology journal, 1993,9 (4): 348～354; Dickson R C et al., Journal ofBacteriology, 1980,142 (3): 777～785).

Lower from the Sumylact L pH of aspergillus niger gained, and activity is at high temperature arranged.Molecular weight is 106000Da, 60 ℃ of optimum temperutures, and optimal pH is 3.5～4.0.Refining enzyme is stable in pH4～8, and thick enzyme is stable in pH2.2～8, and take ONPG and lactose as substrate, Km is respectively 7.2X10 ^-4M, 1.8X10 ^-2M, maximum reaction velocity are respectively 86.7umol/min/mg and 121.9umol/min/mg (LEE et.al., Archives of Biochemistry andBiophysics, 1970,138:264～271; AKASAKI M, et al., J.Biochem, 1976,80:1195～1200).Stable and the activation of enzyme does not all need metal ion.Mg2 ⁺Can suppress active.Sequestrant makes enzymatic inactivation, and the heavy metal ion of trace is not shown sensitivity yet.Nitrophenol dredge basic galactoside (galactonolactone) (TANAKA, etal., J.Biochem, 1975,77:241-247) and semi-lactosi be potent inhibitor.The maximum characteristics of the Sumylact L from mould are that thermostability is high, and optimum pH is low, just is not easy to be subject to like this microbiological contamination in foodstuffs industry, has more using value.

At present, the lactase gene that is cloned has multiple, and the lactase gene that is used for industrial bacterium, yeast and fungi all is cloned (Haijime SHIBUYA et al., Biotech.Biochem., 1995,59 (7): 1345-1348; Irma F.Den Herder, et al., Mol Gen Genet, 1992,238:404-410; Huo Keke.SCIENCE IN CHINA (Series B), 1995,38 (11): 1332-1340).Although these lactase gene sources are different, have higher homology between them.

Efficiently expressing of lactase gene mainly concentrates on the lactase gene that derives from aspergillus, because the Sumylact L Heat stability is good of aspergillus, pH is low, is more suitable for the needs of food-processing.

After food enters people's digestive tube, in digestive process, need from the wide variation of pH1.8-5.5, in stomach, also will stand pepsic Degradation through different pH processes.Therefore, in order more effectively to be hydrolyzed the lactose in the milk preparation in digestive tube, characteristic acidproof, protease inhibitor that the Sumylact L in the ideal should have also will have high reactivity at physiological temp and low pH.In addition, its optimal pH of Sumylact L that great majority are applied to foodstuffs industry is 2.5-5.5, and they all are the forms as tablet or capsule, and it is played a role in enteron aisle.These commercial zymins mostly casing are coated, and they are unsuitable for acting in whole digestive tube especially stomach.The coated mode of casing can increase treating processes and the cost of product.Therefore, screening has the Novel breast carbohydrase of more special good zymologic property, makes it adapt to whole gastral environment, as the zymin of direct interpolation, has better application prospect.

Summary of the invention

The purpose of this invention is to provide a kind of can efficient application have a liking for the yogurt carbohydrase.

A further object of the present invention provides the above-mentioned gene of having a liking for the yogurt carbohydrase of coding.

Another object of the present invention provides the recombinant vectors that comprises said gene.

Another object of the present invention provides the recombinant bacterial strain that comprises said gene.

Another object of the present invention provides a kind of above-mentioned gene engineering method of having a liking for the yogurt carbohydrase for preparing.

Another object of the present invention provides the above-mentioned application of having a liking for the yogurt carbohydrase.

The invention provides a kind of eosinophilic lactase BGALA, its aminoacid sequence is shown in SEQ ID NO.1.

The invention provides the above-mentioned yogurt carbohydrase gene bgalA that has a liking for that encodes.Particularly, the genome sequence of this gene is shown in SEQ ID NO.2.Its cDNA sequence is shown in SEQ ID NO.3.

The present invention also provides and has comprised the above-mentioned recombinant vectors of having a liking for yogurt carbohydrase gene bgalA, is preferably pPIC-bgalA.

The present invention also provides and has comprised the above-mentioned recombinant bacterial strain of having a liking for yogurt carbohydrase gene bgalA, and preferred described bacterial strain is intestinal bacteria, yeast, genus bacillus or lactobacillus.

The present invention also provides a kind of method for preparing eosinophilic lactase BGALA, may further comprise the steps:

1) with above-mentioned recombinant vectors transformed host cell, gets recombinant bacterial strain;

2) cultivate recombinant bacterial strain, induce the restructuring Sumylact L to express; And

3) reclaim the also expressed Sumylact L BGALA of purifying.

The present invention also provides the application of above-mentioned eosinophilic lactase BGALA.

The present invention obtains a kind of eosinophilic lactase BGALA, and its optimum pH is 1.5, all has high enzyme simultaneously and live stable in the scope of acidity.This enzyme has better protease resistant.In simulation artificial digestion road, keep stable and have higher lactose hydrolysis ratio.These character meet humans and animals digestive physiological characteristics, pH subject range, can directly apply to milk-product processing or be used for the treatment of lactose intolerance as oral preparation.

Description of drawings

The SDS-PAGE of the restructuring Sumylact L that Fig. 1 expresses in Pichia yeast analyzes, wherein, and 1: low molecular weight protein Marker; 2: the restructuring Sumylact L of purifying.3: the restructuring Sumylact L of de-glycosylation.

The recombinate optimal pH of Sumylact L BGALA of Fig. 2.

The recombinate pH stability of Sumylact L BGALA of Fig. 3.

The recombinate optimum temperuture of Sumylact L BGALA of Fig. 4.

The recombinate thermostability of Sumylact L BGALA of Fig. 5.

The recombinate protease resistant of Sumylact L BGALA of Fig. 6.

Fig. 7 stability of Sumylact L BGALA in simulated gastric fluid of recombinating.

Fig. 8 recombinates Sumylact L BGALA in the hydrolysis of simulation digestive tract environment to lactose.

Embodiment

Experiment condition:

1, bacterial strain and carrier:

Have a liking for sour fungi Bispora sp.MEY-1, be stored in (Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center on May 19th, 2008, Institute of Microorganism, Academia Sinica, 100101), its preserving number is: CGMCC No.2500.

2, enzyme and other biochemical reagents: restriction endonuclease is available from TaKaRa company, and ligase enzyme is available from Invitrogen company.Substrate is available from Sigma company, and other all is domestic reagent.

3. substratum: Bispora sp.MEY-1 substratum is the potato juice substratum: 1000mL potato juice, 10g glucose, 25g agar, pH2.5.Lactase activity inducing culture: 5g l ^-1Ammonium sulfate, 1g l ^-1Potassium primary phosphate, 0.5g l ^-1Bitter salt, 0.2g l ^-1Calcium chloride, 0.01g l ^-1Ferrous sulfate, 3g l ^-1Wheat bran, and 3g l ^-1Dregs of beans, 1.5% agarose, pH2.5.

The genetic recombination of using learns a skill and is routine techniques in this area among the present invention.The technology that does not describe in detail in following examples is all carried out according to the related Sections in following laboratory manual or the document or part, comprising: the people such as Sambrook, Molecular Cloning, A Laboratory Manual (the 3rd edition .2001); Kriegler, Gene Transfer and Expression:A Laboratory Manual (1990); With Current Protocols inMolecular Biology (people such as Ausubel compiles, 1994).

Embodiment 1 has a liking for sour fungi Bispora sp.MEY-1 and produces the enzyme characteristic

The uranium ore wastewater sample that derives from the ore deposit, Jiangxi after the potato juice culture medium culturing, is coated on the culture medium flat board, cultivated 5～6d for 30 ℃, produce at the dull and stereotyped visible transparent circle of culture medium.Prove that it has lactase activity.

Have a liking for sour fungi Bispora sp.MEY-1, be stored in (Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center on May 19th, 2008, Institute of Microorganism, Academia Sinica, 100101), its preserving number is: CGMCC No.2500.

Embodiment 2 has a liking for the clone of sour fungi Bispora sp.MEY-1 Sumylact L encoding gene bgalA

Sour fungi Bispora sp.MEY-1 genomic dna is had a liking in extraction:

3 days mycelium of liquid culture is put into mortar with the aseptic filter paper filtration, add the 2mL extracting solution, grind 5min, then lapping liquid is placed the 50mL centrifuge tube, 65 ℃ of water-bath cracking 20min, every the 10min mixing once, at 4 ℃ of centrifugal 5min of lower 10000rpm.Get supernatant extrct foreigh protein removing in phenol/chloroform, get again supernatant and add the equal-volume Virahol, after room temperature leaves standstill 5min, 4 ℃ of centrifugal 10min of lower 10000rpm.Abandon supernatant, precipitation is with 70% washing with alcohol twice, and vacuum-drying adds an amount of TE and dissolves, place-20 ℃ for subsequent use.

Degenerated primer BgalAP1:5 '-TWYGGNGGNACNAAYTGGGG-3 ' and BgalAP2:5 '-TCNCCNCCNAGRTTNCCNGT-3 ' have been synthesized in conserved sequence design according to lactase gene, W, and Y, N and R represent respectively A/T, C/T, A/C/T/G and A/G.Carry out pcr amplification to have a liking for the total DNA of sour fungi Bispora sp.MEY-1 as template.The PCR reaction parameter is: 94 ℃ of sex change 3min; Then 94 ℃ of sex change 30sec, 46 ℃ of annealing 30sec, 72 ℃ are extended 1min, 32 rear 72 ℃ of insulation 10min of circulation.Obtain an about 1494bp fragment, this fragment is reclaimed the rear order-checking that links to each other with the pEASY-T3 carrier.

According to the nucleotide sequence that order-checking obtains, each three TAIL-PCR Auele Specific Primer of design upstream and downstream: design direction is for needing the zone of ignorance direction of amplification, and the Position Design of sp2 is in the inboard of sp1, and sp3 is positioned at the inboard of sp2.Distance between per two primers does not have strict regulation, the general 22～30nt of primer length, and annealing temperature is at 60～65 ℃.And with they difference called after usp1, usp2, usp3 (upstream Auele Specific Primer), dsp1, dsp2, dsp3 (downstream Auele Specific Primer) sees Table 1.

Table 1. Sumylact L encoding gene bgalA TAIL-PCR Auele Specific Primer

Obtain the flanking sequence of known sequence by TAIL-PCR, amplification obtains sending after product reclaims the order-checking of three rich Bioisystech Co., Ltd.The result obtains the genome sequence of total length 3181bp.

The RT-PCR of embodiment 3 lactase genes analyzes

Extract total RNA of Bispora sp.MEY-1, the extracting method of total RNA is as follows: the thalline that will cultivate 54hr in bran mass is centrifugal, get weight in wet base 100mg, phosphoric acid buffer (PH7.0) with 1mmol/L is washed three times, grind into powder in liquid nitrogen, 600 μ L sex change liquid (the 26mM sodium-acetates that add the ice precooling, pH4.0, the acid of 0.5% lauryl creatine, 0.125M beta-mercaptoethanol, the 4M guanidine thiocyanate) in, then add successively 60 μ L 2mol/L sodium-acetates (PH4.0), mixing added 600 μ L phenol-chloroform-primary isoamyl alcohol (125: 24: 1, PH4.7), thermal agitation is placed 15min on ice, 4 ℃ of centrifugal 20min of lower 10000g, with the supernatant sucking-off, add isopyknic Virahol, place 30min, 4 ℃ of centrifugal 10min of lower 10000g for-20 ℃, supernatant discarded, after precipitation added 75% washing with alcohol of 1mL ice precooling, the precipitated rna seasoning was with for subsequent use after the Nuclease-free water dissolution.Utilize ThermoScript II to obtain the chain of cDNA, then design appropriate primer (P-BgalF:5 '-GTCTACGTAATGTTGTTGTCACGCTCTTTCGCGGCCGGT-3 ', P-BgalR:5 '-CGAGCGGCCGCTCAATACGCCCCAGGCCGTGGGCTATAC-3 ') amplification cDNA, obtain the cDNA sequence of coding Sumylact L, amplification obtains product and reclaims rear order-checking.

Find that this gene has 3 introns, cDNA total length 3009bp, 1002 amino acid of encoding after genome sequence by Sumylact L bgalA relatively and the cDNA sequence.The BLAST comparison result shows that the sequence similarity of the Sumylact L that itself and Aspergillus phoenicis originate is the highest.The highest similarity of amino acid is 55.5%.The gene of the proof coding Sumylact L that separating clone obtains from Bispora sp.MEY-1 is new gene.

The preparation of embodiment 4 restructuring Sumylact Ls

Expression vector pPIC9 is carried out double digestion (EcoRI+NotI), to encode the simultaneously gene bgalA double digestion (EcoRI+NotI) of Sumylact L, the gene fragment that cuts out the encoding mature Sumylact L is connected with expression vector pPIC9, acquisition contain Bispora sp.MEY-1 Sumylact L gene bgalA recombinant plasmid pPIC-bgalA and transform Pichia pastoris GS115, obtain recombinant pichia yeast strain GS115/bgalA.

Get the GS115 bacterial strain that contains recombinant plasmid, be inoculated in the 400mL BMGY nutrient solution, behind 30 ℃ of 250rpm shaking culture 48h, centrifugal collection thalline.Then resuspended in 200mL BMMY substratum, 30 ℃ of 250rpm shaking culture.After inducing 48h, centrifugal collection supernatant.Measure the lactose enzyme activity.The expression amount of restructuring Sumylact L is 0.08U/mL.SDS-PAGE result (Fig. 1) shows that the restructuring Sumylact L has obtained expression in pichia spp.

Nutrient solution 10000rpm was removed thalline in centrifugal 10 minutes, get supernatant liquor as crude enzyme liquid, crude enzyme liquid is placed ice bath, slowly add while stirring ammonium sulfate to 80%, the centrifugal 20min of 13000rpm gets precipitation, again dissolve with damping fluid, obtain concentrated enzyme liquid, further use HPLC (

FPLC, Pharmacia company) purifying.Upper HiTrap_Q_Sepharose_XL (Amersham Pharmacia Biotech prepacked column) anion column behind ammonium sulfate precipitation.Application of sample 2mL uses first pH8.0, the Tris-HCl buffered soln wash-out balance pillar of 0.02mol/L, and then with 10 post beds of 0～0.6mol/LNaCl gradient elution (about 50mL) of same buffer preparation, flow velocity is 5mL/min, fraction collection, every pipe 1mL.Then the solution in the collection tube being surveyed enzyme lives and the protein electrophoresis analysis.Fig. 1 demonstration has obtained electrophoresis pure lactose enzyme.Expressed Sumylact L is through after the purifying, and the content of its protein reaches more than 90% of total protein.The molecular weight of restructuring Sumylact L is about 160kD, and behind EndoH enzyme desugar base, its molecular weight becomes 110kD, conforms to its theoretical molecular.

The enzyme assay of embodiment 5 Sumylact Ls

Enzyme assay discharges the amount of nitrophenol and carries out by measuring hydrolysis substrate ONPG.Reaction mixture comprises 100 μ l enzyme diluents, 250 μ l ONPG (5mM) and 150 μ l glycine/HCl damping fluids (pH1.5).Mixture adds 1.5ml sodium carbonate solution (1M) termination reaction at 37 ℃ of incubation 5min.The nitrophenol that discharges is by measuring A ₄₂₀The method of absorbance value is quantitative.

The lactase activity unit definition: under certain condition, it is 1 activity unit (U) that per minute decomposition substrate generates the required enzyme amount of 1 μ mol nitrophenol.

The zymologic property analysis of embodiment 6 restructuring Sumylact Ls

Embodiment 4 purified Sumylact Ls are carried out enzymatic reaction to measure its optimal pH under different pH.Used damping fluid is the HCl-Gly damping fluid of pH1.0-3.5, the citric acid of pH2.2～8.0-Sodium phosphate dibasic series damping fluid and pH8.0～10.0 Tris-HCl series damping fluid.The Sumylact L of purifying is in the buffer system of different pH, and 37 ℃ of lower pH adaptive results (Fig. 2) that measure show: the optimal pH of BGALA is 1.5.Enzyme liquid is processed 30min in the damping fluid of different pH values, measure again enzymic activity with the pH stability of studying enzyme under 37 ℃.The result shows (Fig. 3), is keeping more than 80% of enzyme work under the optimal pH between pH0.5～10.0, and this illustrates that this enzyme has preferably pH stability.

Enzymatic reaction is carried out in being determined under citric acid-Sodium phosphate dibasic (pH5.0) buffer system and the differing temps of optimum temperuture.Temperature tolerance is determined as under differing temps and processes 30min, carries out enzyme activity determination again.Enzyme reaction optimum temperuture measurement result (Fig. 4) shows that the BGALA optimum temperuture is 60 ℃.The Thermostability test shows (Fig. 5), in 60 ℃ of lower insulation 60min, active not loss.At 70 ℃ of lower insulation 20min, the residual enzyme activity is about 80%.Illustrate that this enzyme has preferably thermotolerance.

In lactase soln, add 0.05mL trypsin 0.1mg/mL, with pH7.0, the preparation of Tris-HCl damping fluid), and stomach en-(0.1mg/mL is with pH2.0, glycine-HCl damping fluid preparation) in 37 ℃ of processing 60min, is measured enzymic activity again after the dilution.After processing 60min respectively with proteolytic enzyme, the enzymic activity (Fig. 6) of residue 100%.Illustrate that Sumylact L has the ability of preferably protease inhibitor hydrolysis.

The restructuring Sumylact L, detects its active method again and carries out by add the various chemical reagent of different concns in reaction system the resistant determination of metal ion and chemical reagent.The result shows (table 2), beta-mercaptoethanol, SDS, high density Mn ²⁺This enzymic activity of strongly inhibited; EDTA and high Pb concentration ²⁺To a certain degree suppress; Yet the various ions such as the Ca that exist in the milk ²⁺, Cr ³⁺, Cu ²⁺, Fe ³⁺Do not affect Deng on enzymic activity, or slight the activation.

The impact of each metal ion species counterweight group Sumylact L of table 2.

The stability of embodiment 7 restructuring Sumylact Ls in simulated gastric fluid

(SGF comprises 2.0mg ml to the restructuring Sumylact L among the 0.2M glycine/HCl at simulated gastric fluid ^-1NaCl and 3.2mg ml ^-1Stomach en-) stability in detects by the following method: restructuring Sumylact L (the 1U ml of purifying ^-1) 37 ℃ of incubations in the SGF of different pH (1.5,2.0,3.0,4.0, or 5.0, adjust with HCl or NaOH), 60min.Measure the residual lactose enzymic activity.Not adding pepsic simulated gastric fluid (SGF-NPEP) detects simultaneously.The result shows (Fig. 7), and restructuring Sumylact L BGALA is active not loss in the simulated gastric fluid of pH1-5, has showed stronger stability.Aspergillus oryzae in contrast (A.oryzae) Sumylact L does not have enzymic activity in the SGF of pH1.5-3.0.

Embodiment 8 restructuring Sumylact Ls in manual simulation's digestive tube to the degraded of lactose

Simulation digestive tube damping fluid is to add 2.0mg ml in 10ml milk ^-1NaCl, and 0.2M glycine and 3.2mg ml ^-1Stomach en-.Utilize NaOH or HCl to adjust the pH in each step.The pH environment of simulation and time change as follows: pH1.9,20 minutes; PH5.5,10 minutes; PH4.6,10 minutes; PH3.8,10 minutes; PH2.8,20 minutes; PH1.9,40 minutes.(pH1.9) adds respectively 50U and 100U restructuring Sumylact L in the initial soln, after each step places 37 ℃ of concussions (220rpm) incubation, gets 500 μ l reaction solutions and changes in the 50ml reagent bottle, adds 250 μ l, 20% plumbic acetate and 250 μ l K ₂C ₂O ₄/ Na ₂HPO ₄(3g potassium oxalate, the 7g Sodium phosphate dibasic is dissolved in 100ml water) placed dark surrounds 30 minutes again, made albumen precipitation.Dilute 20 times, its glucose content kit measurement.Lactose hydrolysis ratio represents with the glucose that generates and the ratio of lactose total amount.The result shows (Fig. 8), after adding the 100U Sumylact L and processing each step lactose hydrolysis ratio be respectively 46.0%, 36.8%, 42.6%, 46.1%, 58.8%85.8%.Commercial aspergillus oryzae Sumylact L in contrast, its final percent hydrolysis is 3.5%.Restructuring BGALA is up to 86% to the percent hydrolysis of milk lactose in the chyme digestive process, have preferably application prospect.

Sequence table

＜110〉Institute of Feeds,China Academy of Agriculture Sciences

＜120〉a kind of eosinophilic lactase BGALA and gene thereof and application

<160>3

<210>1

<211>1002

<212>PRT

＜213〉have a liking for sour fungi (Bispora sp.MEY-1)

<400>1

MLLSRSFAAG VVGCLTISSL AASIGPKVTN LKIREQDRLQ DIVTWDNYTL

LVRGERILFY 60

SGEFHPFRLP VASLYLDVFQ KIKALGYTGV SFYVDWALLE GTPGVYDDSG

IFNLQPFFDA 120

ASEAGIYLVA RPGPYINAEA SGGGFPGWLQ LVNGTLRALD APYLDATSLY

TAKVGEAIAK 180

NQITEGGPII LLQPENEYIP PNNVLTQTDR EYFAYVEKQF RDAGVVVPTI

INDASGKGIF 240

APGSGLGAVD IYGFDQYPLG FDCANPYIWP AGDLQTDYRE IHLDFSPTTP

QAIPEFQGGS 300

FDPWGGPGFN ACAILLNEEF ERVFYKNNFA AGLTIFNIYM TYGGTNWGNL

GHPGGYTSYD 360

YGAVIKEDRT VTREKYSEAK LEAVFVKTSL AYYTATPQNM SIAGQFVNTD

EISVTQSVGE 420

YGTNFYFIRH TNYSSLASTS YMLTVPTSRG NVTIPQLGGT LTLNGRDSKI

SVTDYKLGRI 480

NMLYSSSDIF TWQEFDGRTV VLLYGGAGET HELAFTGNLP PQPSCTGNPV

VKDKDGYTII 540

HWSVTPERQM VDFGYLTIYL LWRNDAYNYW SIEMPAAAPL GNFTSLTKTS

VIMKAGYLMR 600

TASVSGDILY LTGDVNATTP LEIISGASFE GTKAIYFNGK PLSFERTSYG

TFISTVEFSP 660

PSISVPNLSK LDWKSIDSLP EIQPDYSDAA WPSCDKPYTN DTYRALTTPT

SLYSSDYGFV 720

TGTLLYRGHF IATGKETTFF IETEGGFAFG HSVWLNSTYI GSWTGIAADQ

TYNQTFTLPS 780

LTAGKAYVFT IVIDTMGLSE NYNIPDDAMK QPRGILDYQL AGRPQSAITW

KMTGNLGGFN 840

YADKARGPLN EGGLYAERQG FYLPNPPSYS WPSSSPMTGF NSSGVRFYTA

SFTLDLPHGY 900

DIPLSFTFTN ASTTPYRAQL YVNGYQFGKY VNNIGPQKAF PVPEGILNYH

GTNWVALTLW 960

AQDEGGAKVD GLALTVDLVA LSGIETVVNS PMPGYSPRPG AY 1002

<210>2

<211>3181

<212>DNA

＜213〉have a liking for sour fungi (Bispora sp.MEY-1)

<400>2

atgttattgt cacgctcttt cgcggccggt gtagttggct gcctcacgat atccagcctt 60

gccgcctcca tcggacctaa agttacaaac ctcaagattc gagaacagga tcgtctacag 120

gacattgtaa gccgcagcct ccctcctcct accaaacccc tctttgatat ggcatctgaa 180

ttttctccag gtgacatggg acaactacac actcttggtg cgaggcgagc gcatcctctt 240

ctactctggt gaatttcacc ccttccgctt gcccgttgcc tcgctctatc tcgacgtgtt 300

ccagaaaatc aaggctttgg gctacaccgg cgtgtccttc tatgttgact gggctctcct 360

tgaaggcacg cccggcgtgt acgatgactc aggcatcttc aaccttcaac ccttcttcga 420

tgctgcatcg gaggctggaa tctacctcgt cgcgcgcccg ggcccgtata tcaacgccga 480

ggcttcggga ggtggctttc ctggatggct acagctcgtc aatggcaccc tgcgcgcctt 540

agatgctccc tacctcgacg ccacaagctt atacaccgca aaagtcggag aggccatcgc 600

caagaaccag atcacggagg gtggaccaat aattttgctc cagccagaaa acgaatatat 660

tccgcctaat aacgtgttga cacagactga tcgggagtac tttgcatacg ttgagaaaca 720

gttccgcgat gccggagttg tggtgccgac catcatcaac gacgcgagcg gcaaggggat 780

tttcgctccg ggcagcggct taggcgcagt agacatctac gggttcgatc aatatccgct 840

gggctttgat tgtgagagtt tctgcttttt atctatcatc aaaaatctgc taactcgcat 900

acaggcgcga atccctacat atggcccgct ggcgaccttc agacagatta ccgcgagatc 960

cacctggatt tcagtccgac aacgccgcaa gctattcctg agtttcaagg cggctcgttc 1020

gacccgtggg gagggccggg gttcaatgcg tgtgccatcc ttctaaacga ggagtttgag 1080

agggttttct ataagaacaa tttcgctgct ggactcacca ttttcaatat ctatatggta 1140

agtgctccgc tataaggtca tttacgaatc tatgctgatg atttgttgat agacttatgg 1200

cggaacaaac tggggcaatc ttggccatcc gggcggctat acatcgtacg actacggcgc 1260

cgtaatcaaa gaagaccgga ccgtgacgcg cgaaaagtac agcgaggcaa agctggaggc 1320

ggtttttgtg aagacatccc tagcgtacta cacggcgact cctcagaaca tgtcaattgc 1380

gggccagttc gtcaatacgg atgaaatatc ggtcacgcaa agcgtgggag agtacgggac 1440

caatttctac tttatccggc ataccaacta ctcctcatta gcatctacga gttacatgct 1500

caccgtgccg accagcagag gcaatgtgac aatccctcaa ctcggcggta ctcttacgct 1560

aaatgggcgg gattcgaaga tttccgttac ggactacaaa cttggccgga ttaacatgct 1620

ctactcctcc agtgacatct tcacgtggca agaatttgac ggtcggactg tcgtattact 1680

ttatggcggc gcgggggaaa cccacgagct cgcctttact ggtaatcttc caccacaacc 1740

ttcgtgcact ggaaacccag tcgtgaagga caaagatgga tacacgatca tccactggag 1800

tgtgacgccg gaacgacaaa tggtcgattt cggctacctc actatctacc tgctctggag 1860

gaatgatgca tataactact ggtcgattga aatgcccgcc gctgccccat taggcaactt 1920

cacctcgttg accaaaacgt cggtgatcat gaaggctggt tacctcatgc gcacggcaag 1980

tgtttctggg gacattttgt atctgacagg tgatgtcaat gcaacaaccc ctctggaaat 2040

cattagcgga gcttcctttg aggggactaa agctatatac ttcaacggca agcccctttc 2100

gttcgagcgg acgagttacg gcacttttat ctcgacggta gaattctcac caccttcaat 2160

atcggtgcca aatttgagca aattggattg gaaatccata gattcactac cagagattca 2220

gccggattac tctgatgcgg cttggccgtc ttgtgacaag ccgtatacca atgacacata 2280

tcgagccctg accacaccaa catcccttta ttcttccgat tatggtttcg tgacgggtac 2340

cctcttatac cgaggccact ttatcgcgac tggaaaagaa accacattct tcatcgagac 2400

agaaggtggt tttgcgttcg gccactccgt ctggctcaat agcacctata ttggctcctg 2460

gacgggtatc gctgctgatc agacatacaa ccaaacgttc accctacctt ctctcacggc 2520

cggcaaggcg tacgtcttca ccatcgtcat cgataccatg ggcctgagcg aaaattataa 2580

cattcccgac gacgccatga agcaaccccg tggcatcctc gactaccaac tcgcaggtcg 2640

tccccaatcc gcaattacat ggaagatgac tggcaatctt ggcggtttca actacgccga 2700

caaagcacgc ggccctctca atgaaggcgg cctatatgcc gaacgccaag ggttttatct 2760

ccccaatccg ccctcctact cttggccatc cagtagcccc atgacaggct tcaactcctc 2820

cggtgtgcgc ttttatacgg cttcttttac ccttgattta ccccacggct acgacattcc 2880

tctcagcttc acgtttacaa acgcttctac aaccccttac cgtgcacagt tgtacgtcaa 2940

cggctaccag ttcggaaagt acgtgaacaa tatcggtcca caaaaggctt tccccgttcc 3000

tgaaggaatt ctgaattatc atggtacgaa ttgggttgcg ttgacgttat gggcgcagga 3060

tgagggcggt gcaaaggtgg acgggttggc cttaacagtg gatttagtcg cgctgagtgg 3120

aattgagaca gtggtaaact cacctatgcc agggtatagc ccacggcctg gggcgtattg 3180

a 3181

<210>3

<211>3009

<212>DNA

＜213〉have a liking for sour fungi (Bispora sp.MEY-1)

<400>3

atgttattgt cacgctcttt cgcggccggt gtagttggct gcctcacgat atccagcctt 60

gccgcctcca tcggacctaa agttacaaac ctcaagattc gagaacagga tcgtctacag 120

gacattgtga catgggacaa ctacacactc ttggtgcgag gcgagcgcat cctcttctac 180

tctggtgaat ttcacccctt ccgcttgccc gttgcctcgc tctatctcga cgtgttccag 240

aaaatcaagg ctttgggcta caccggcgtg tccttctatg ttgactgggc tctccttgaa 300

ggcacgcccg gcgtgtacga tgactcaggc atcttcaacc ttcaaccctt cttcgatgct 360

gcatcggagg ctggaatcta cctcgtcgcg cgcccgggcc cgtatatcaa cgccgaggct 420

tcgggaggtg gctttcctgg atggctacag ctcgtcaatg gcaccctgcg cgccttagat 480

gctccctacc tcgacgccac aagcttatac accgcaaaag tcggagaggc catcgccaag 540

aaccagatca cggagggtgg accaataatt ttgctccagc cagaaaacga atatattccg 600

cctaataacg tgttgacaca gactgatcgg gagtactttg catacgttga gaaacagttc 660

cgcgatgccg gagttgtggt gccgaccatc atcaacgacg cgagcggcaa ggggattttc 720

gctccgggca gcggcttagg cgcagtagac atctacgggt tcgatcaata tccgctgggc 780

tttgattgcg cgaatcccta catatggccc gctggcgacc ttcagacaga ttaccgcgag 840

atccacctgg atttcagtcc gacaacgccg caagctattc ctgagtttca aggcggctcg 900

ttcgacccgt ggggagggcc ggggttcaat gcgtgtgcca tccttctaaa cgaggagttt 960

gagagggttt tctataagaa caatttcgct gctggactca ccattttcaa tatctatatg 1020

cttatggcg gaacaaactg gggcaatctt ggccatccgg gcggctatac atcgtacgac 1080

tacggcgccg taatcaaaga agaccggacc gtgacgcgcg aaaagtacag cgaggcaaag 1140

ctggaggcgg tttttgtgaa gacatcccta gcgtactaca cggcgactcc tcagaacatg 1200

tcaattgcgg gccagttcgt caatacggat gaaatatcgg tcacgcaaag cgtgggagag 1260

tacgggacca atttctactt tatccggcat accaactact cctcattagc atctacgagt 1320

tacatgctca ccgtgccgac cagcagaggc aatgtgacaa tccctcaact cggcggtact 1380

cttacgctaa atgggcggga ttcgaagatt tccgttacgg actacaaact tggccggatt 1440

aacatgctct actcctccag tgacatcttc acgtggcaag aatttgacgg tcggactgtc 1500

gtattacttt atggcggcgc gggggaaacc cacgagctcg cctttactgg taatcttcca 1560

ccacaacctt cgtgcactgg aaacccagtc gtgaaggaca aagatggata cacgatcatc 1620

cactggagtg tgacgccgga acgacaaatg gtcgatttcg gctacctcac tatctacctg 1680

ctctggagga atgatgcata taactactgg tcgattgaaa tgcccgccgc tgccccatta 1740

ggcaacttca cctcgttgac caaaacgtcg gtgatcatga aggctggtta cctcatgcgc 1800

acggcaagtg tttctgggga cattttgtat ctgacaggtg atgtcaatgc aacaacccct 1860

ctggaaatca ttagcggagc ttcctttgag gggactaaag ctatatactt caacggcaag 1920

cccctttcgt tcgagcggac gagttacggc acttttatct cgacggtaga attctcacca 1980

ccttcaatat cggtgccaaa tttgagcaaa ttggattgga aatccataga ttcactacca 2040

gagattcagc cggattactc tgatgcggct tggccgtctt gtgacaagcc gtataccaat 2100

gacacatatc gagccctgac cacaccaaca tccctttatt cttccgatta tggtttcgtg 2160

acgggtaccc tcttataccg aggccacttt atcgcgactg gaaaagaaac cacattcttc 2220

atcgagacag aaggtggttt tgcgttcggc cactccgtct ggctcaatag cacctatatt 2280

ggctcctgga cgggtatcgc tgctgatcag acatacaacc aaacgttcac cctaccttct 2340

ctcacggccg gcaaggcgta cgtcttcacc atcgtcatcg ataccatggg cctgagcgaa 2400

aattataaca ttcccgacga cgccatgaag caaccccgtg gcatcctcga ctaccaactc 2460

gcaggtcgtc cccaatccgc aattacatgg aagatgactg gcaatcttgg cggtttcaac 2520

tacgccgaca aagcacgcgg ccctctcaat gaaggcggcc tatatgccga acgccaaggg 2580

ttttatctcc ccaatccgcc ctcctactct tggccatcca gtagccccat gacaggcttc 2640

aactcctccg gtgtgcgctt ttatacggct tcttttaccc ttgatttacc ccacggctac 2700

gacattcctc tcagcttcac gtttacaaac gcttctacaa ccccttaccg tgcacagttg 2760

tacgtcaacg gctaccagtt cggaaagtac gtgaacaata tcggtccaca aaaggctttc 2820

cccgttcctg aaggaattct gaattatcat ggtacgaatt gggttgcgtt gacgttatgg 2880

gcgcaggatg agggcggtgc aaaggtggac gggttggcct taacagtgga tttagtcgcg 2940

ctgagtggaa ttgagacagt ggtaaactca cctatgccag ggtatagccc acggcctggg 3000

gcgtattga 3009

Claims (1)

1. the eosinophilic lactase BGALA of aminoacid sequence shown in the SEQ ID NO.1 application of lactose of under the sour environment of pH 1.0-5.0, degrading.

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