CN107345225A - A kind of beta galactosidase combination mutant with high transglycosylation and its preparation method and application - Google Patents

A kind of beta galactosidase combination mutant with high transglycosylation and its preparation method and application Download PDF

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CN107345225A
CN107345225A CN201710423347.0A CN201710423347A CN107345225A CN 107345225 A CN107345225 A CN 107345225A CN 201710423347 A CN201710423347 A CN 201710423347A CN 107345225 A CN107345225 A CN 107345225A
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张伟
张宇宏
刘波
孙宁
张佳琳
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Biotechnology Research Institute of CAAS
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Abstract

The invention belongs to genetic engineering and field of genetic engineering, disclose a kind of beta galactosidase combination mutant with high transglycosylation, it is on the basis of the aspergillus candidus and the amino acid sequence of the beta galactosidase of aspergillus oryzae for eliminating native signal peptide, obtained by the fixed point saturation mutation of double site, in the case where oligosaccharide growing amount keeps constant, the glucosides reaction rate that turns of the mutant improves more than 100% than wild type.Meanwhile the invention also discloses the host cell of the DNA molecular of coding combinations thereof mutant, the recombinant expression carrier containing above-mentioned DNA molecular and the above-mentioned DNA molecular of expression.In addition, present invention also offers the application of the method and combinations thereof mutant, DNA molecular, recombinant expression carrier and host cell of the beta galactosidase with high transglycosylation in beta galactosidase is prepared is prepared using above-mentioned recombinant expression carrier.

Description

A kind of beta galactosidase combination mutant and its preparation with high transglycosylation Methods and applications
The application is Application No. 201410514519.1, and the applying date is September in 2014 29, and entitled " one kind has The division Shen of the application for a patent for invention of beta galactosidase combination mutant of high transglycosylation and its preparation method and application " Please.
Technical field
The present invention relates to genetic engineering and field of genetic engineering, and in particular to a kind of β-gala with high transglycosylation Glycosidase combination mutant and its preparation method and application.
Background technology
Galactooligosaccharide (GOS) is a kind of absorption can not to be digested in human gastrointestinal tract and to be directly entered large intestine various The oligosaccharide with special biological function that Bifidobacterium well utilizes.It can improve micro-ecological environment in human body, favorably In Bifidobacterium and the propagation of other beneficial bacteriums, immune function of human body is improved.Meanwhile GOS makes enteral through metabolism generation organic acid PH value reduces, and suppresses the growth of enteral salmonella and spoilage organisms, reduces the generation of poisonous tunning and harmful bacteria enzyme, adjusts Gastrointestinal function is saved, alleviates the burden that liver decomposes toxin.Due to galactooligosaccharide have it is more excellent than other functional oligoses Good property, so that galactooligosaccharide is more convenient easy as additive large-scale application, it can adapt to more food species Class and the wider array of consumer group, there are huge application value and market potential.
GOS preparation methods generally have five kinds：Extraction, the synthesis of the sour water solution of natural polysaccharide, chemical method, hair from natural material Ferment method and enzymatic clarification.Due to GOS, content is low in nature, colourless neutral thus be difficult to extraction separation；Natural polysaccharide turns It is low to change product yield, product component is complicated, it is difficult to purifies；Chemical method toxicity great Yi is remained, environmental pollution weight；Fermentation method produces GOS research is seldom, still in laboratory scale, can not largely be produced.The industrialized production of galactooligosaccharide is at present Completed by beta galactosidase.Beta galactosidase (β-D-galactoside galatohydrolase, EC 3.2.1.23 lactase (Lactase)) is also known as, there is hydrolysis and turn the double action of glucosides.Previously for beta galactosidase Research be concentrated mainly on and produce low-lactose dairy product using its hydrolysis function, to release lactose-intolerant person because edible breast is made A variety of adverse reactions such as diarrhoea, abdominal distension caused by product.With the determination of the special healthcare function of galactooligosaccharide, β-half is utilized The transglucosidation production galactooligosaccharide of lactoside enzyme turns into study hotspot.It is concentrated mainly on following three aspects：
1st, the beta-galactosidase bacteria of high transglycosylation is screened
Have in the multiple-microorganisms such as yeast, bacillus, aspergillus, mould, Bifidobacterium tool transglycosylase activity β- Galactosidase.Research shows, the beta galactosidase of separate sources is because its zymologic property is different, during synthesis of oligonucleotides galactolipin Reaction condition differs widely.Beta galactosidase can be divided into acid and neutral two kinds according to optimal pH.The β in usual mould source- Galactosidase is acidicenzym, and it is higher (50~60 DEG C) in pH2.5~5.5, optimal reactive temperature that it acts on optimal pH；Yeast and The beta galactosidase that bacterium is produced is neutral enzymatic, and its optimal pH is between pH 6~7.5, optimal reactive temperature relatively low (30~40 ℃).The substrate type of the beta galactosidase effect of separate sources is also not quite similar, oligosaccharides in the galactooligosaccharide thus generated Species and ratio also vary, this just makes the newcomer of galactooligosaccharide family emerge in an endless stream.Nevertheless, screen The transglycosylation of natural beta galactosidase is generally relatively low, the maximum output generally only 5~30% of galactooligosaccharide, can not Meet the requirement of industrialized production.
2nd, optimize reaction condition, improve production technology
Having scholar to attempt working condition by optimizing galactooligosaccharide and technique, its transglycosylation is low to be lacked to make up Fall into, improve the yield of galactooligosaccharide, achieved certain effect.Main method has：The concentration of increase starting lactose, use Organic solvent controls water activity and using immobilization technology.Due to beta galactosidase hydrolysis and turn glucosides reaction be reversible 's.When concentration of substrate is relatively low, hydrolysate galactose concentration is relatively low, and its inhibitory action to hydrolysis is smaller, now enzyme It is higher to show hydrolysis vigor, and it is relatively low to turn glucosides vigor, therefore contents of monosaccharides is higher in product.When lactose concn is higher, Hydrolysate galactose concentration is higher, and just inhibitory action is produced to hydrolyzing activity when it reaches certain value.Galactolipin is to turn glucosides Substrate, concentration height is advantageous to the synthesis of galactooligosaccharicomposition, therefore when concentration of substrate is higher, oligosaccharide content is higher in product.Make The synthesis for being advantageous to oligosaccharide with organic solvent is because organic solvent can reduce the activity of reaction system reclaimed water to influence enzyme Avtive spot and reaction mechanism, the reverse transgalactosidation reaction of guiding hydrolysis enzymatic, make reaction balance be offset to by hydrolysis Oligosaccharide synthesizes.The pH of resolvase can be then greatly increased using immobilization technology and heat endurance and can be recycled, reduces life Produce cost.Beta galactosidase is adsorbed in phenolic resin and with after glutaraldehyde cross-linking by Mozaffar, and the yield of oligosaccharide improves 20%；But also studies have found that, when immobilised enzymes acts on the lactose solution of higher concentration, caused oligosaccharide ratio is used free Enzyme is also few.It can not be tackled the problem at its root all the time by optimal conditions merely as can be seen here.
3rd, improve the expression of beta galactosidase using genetic engineering means and property improves
Between wild beta galactosidase GOS yield typically all maintains 20~45% in nature, yield is relatively low, difficult To meet production requirement, therefore turn the excellent mutant enzyme of glycosidic nature by molecular modification screening to turn into study hotspot.Hansen O. the discovery such as (2001) derives from the beta galactosidase BIF3 in Bifidobacterium, misses out enzyme after 580 amino acid of C-terminal Albumen is changed into an efficient transglycosylase, can utilize almost 90% lactose generation GOS, and hydrolysing component accounts for 10%, and 9 can be kept all the time under 10% to 40% lactose concn:1 ratio turns glucosides vigor and hydrolysis vigor；Placier G. In 2009 to being oriented evolution from Geobacillus stearothermophilus KVE39 beta galactosidase, glucosides work will be turned Power, which improves, then hydrolyzes vigor reduction as screening foundation, three plant mutant body R109W, R109V, R109K is successfully screened, 18% (w/v) oligomeric candy output is respectively 23%, 11.5%, 21% in lactose, and wild enzyme only has 2%；Wu Y. (2013) etc. are to sulphur The beta galactosidase for changing leaf bacterium source carries out molecular modification, studies its most suitable GOS formation condition, is dashed forward under respective optimum condition Variant F441Y GOS yield is 61.7%, F359Q 58.3%, and wild enzyme is 50.9%.
Then, there is scholar to propose a kind of method of iteration combinatorial mutagenesis (Ji J, Fan K.et al, 2012), i.e., will be each The mutant for taking turns highest vigor in the selection result carries out next round combinatorial mutagenesis as template, until the mutation vitality of subject of generation is equal It can not surmount when setting out template, iteration combination terminates.Screened by this method from four-wheel combinatorial mutagenesis totally 24 plant mutant bodies The mutant that 11 plants of vigor improve, final vigor highest mutant (C155Y Y184H V275I C281Y) is than original wild enzyme 1874% is improved, but the method for the combinatorial mutagenesis is not yet had been reported that in the molecular modification applied to beta galactosidase.
Up to the present, either the screening separation of native enzyme, process optimization still by genetic engineering means improve β- The expression of galactosidase and property improvement, it is all low with the present situation that yields poorly without beta galactosidase transglycosylation is changed, Low so as to also result in galactooligosaccharide synthetic yield, reaction time length, production cost is too high, and this seriously constrains oligomeric gala The cheap production and popularization and application of sugar.
Therefore, the new beta galactosidase of high transglycosylation is formulated, and it is current research and life it is inexpensively produced One of subject matter of urgent need to resolve in production.
The content of the invention
For drawbacks described above present in prior art, one aspect of the present invention provides a kind of with high transglycosylation Beta galactosidase combination mutant, it is on the basis of aspergillus candidus or aspergillus oryzae beta galactosidase, passes through double site Fixed point saturation mutation and obtain, preferably on the basis of the amino acid sequence shown in sequence 2 or sequence 4, pass through dibit What the fixed point saturation mutation of point was obtained, the mutant turns glucosides reaction in the case where oligosaccharide growing amount keeps constant Speed ratio wild type improves more than 100%, preferably improves more than 150%, more preferably improves more than 200%.
In a preferred embodiment of the present invention, mutational site is respectively the amino acid of the 219th and the 785th.
In further preferred embodiment of the present invention, the fixed point saturation mutation of double site is respectively to use glycine residue Substitute the serine residue (S219G) of the 219th and substitute the glutaminic acid residue (E785V) of the 785th with valine residue, use Alanine residue substitutes the serine residue (S219A) of the 219th and substitutes the glutaminic acid residue of the 785th with valine residue (E785V), substitute the serine residue (S219N) of the 219th with asparagine residue and substitute the 785th with valine residue Glutaminic acid residue (E785V) or substitute the serine residue (S219V) of the 219th and residual with valine with valine residue Base substitutes the glutaminic acid residue (E785V) of the 785th.
Another aspect provides the DNA molecular of coding combinations thereof mutant.
Another aspect of the invention provides the recombinant expression carrier containing above-mentioned DNA molecular, preferably recombination yeast table Up to carrier.
Another aspect of the invention provides the host cell for expressing above-mentioned DNA molecular, preferably saccharomyces, Klu Saccharomyces, Schizasaccharomyces and methylotrophic yeast strain, methylotrophic yeast strain are more preferably to finish Chi Shi ferment Mother's category bacterial strain.
Another aspect of the invention provides a kind of method for preparing the beta galactosidase with high transglycosylation, bag Include following steps：
1st, host cell is converted with above-mentioned recombinant expression carrier, obtains recombinant bacterial strain；
2nd, above-mentioned recombinant bacterial strain, the expression of induction restructuring beta galactosidase are cultivated；
3rd, reclaim and purify the beta galactosidase of expressed high transglycosylation.
Last aspect of the present invention provide combination mutant of the present invention, DNA molecular, recombinant expression carrier with And application of the host cell in beta galactosidase is prepared.
The present invention pinpoints β-gala of the saturation mutation technology to the aspergillus candidus of removal native signal peptide using double site The beta-galactosidase gene laco ˊ of the aspergillus oryzae of glycosidase genes lacb ˊ and removal native signal peptide have carried out fixed point saturation Mutation, obtains the beta galactosidase combination mutant with high transglycosylation, so that it turns glucosides reaction rate ratio Wild type improves more than 100%, or even improves more than 200%, makes β-gala of preparation high efficiency and high transglycosylation Glycosidase becomes a reality, and is had laid a good foundation for the application of beta galactosidase.
Brief description of the drawings
Fig. 1：The molecular docking process of the beta galactosidase and substrate of aspergillus candidus and aspergillus oryzae.
Fig. 2：Mutational site and lactose molecule spatial relation in the beta galactosidase of aspergillus candidus and aspergillus oryzae.
Fig. 3：The building process of the recombinant expression carrier of the beta-galactosidase gene containing mutant.
Fig. 4：S219 sites typical case's mutant galactooligosaccharide growing amount.
Fig. 5：F245 site mutants storehouse galactooligosaccharide growing amount trend.
Fig. 6：F245 sites typical case's mutant galactooligosaccharide growing amount.
Fig. 7：E785 site mutants storehouse galactooligosaccharide growing amount trend.
Fig. 8：S219/E785 double-site mutant body galactooligosaccharide growing amounts.
Embodiment
Below by embodiment, the present invention is described in further detail, it is intended to limits this for illustrating rather than Invention.It should be pointed out that to those skilled in the art, under the premise without departing from the principles of the invention, can also be to this hair Bright to carry out some improvement and modification, these are improved and modification is similarly fallen under the scope of the present invention.
Embodiment 1：Beta galactosidase tertiary structure and the prediction in mutational site
The beta-galactosidase gene lacb ˊ of native signal peptide are removed, are this laboratory from aspergillus candidus Clone obtains in (Aspergillus candidus), removes the gene of its own signal peptide sequence by 2958 nucleotides groups Into as shown in sequence 1, the protein of the gene code is made up of particular sequence 986 amino acid, the particular sequence such as institute of sequence 2 Show.
The beta-galactosidase gene laco ˊ of native signal peptide are removed, are this laboratory from aspergillus oryzae (Aspergillus Oryze clone obtains in), and it is also made up of 2958 nucleotides, and particular sequence is as shown in sequence 3, the egg of the gene code White matter is also made up of 986 amino acid, and particular sequence is as shown in sequence 4.The albumen of its amino acid sequence and lacb ˊ gene codes Matter only has the difference of three amino acid：Respectively at the 231st：Lacb ˊ (Gly), laco ˊ (Ser)；401st：lacbˊ (Met), laco ˊ (Ile)；970th：Lacb ˊ (Asp), laco ˊ (Asn).
Using the beta galactosidase of aspergillus candidus and aspergillus oryzae as research material.To obtain the Penicillium β of crystal structure-half Lactoside enzyme (PDB accession number：1TG7), aspergillus oryzae beta galactosidase (PDB accession number：4IUG) and trichoderma reesei beta galactose Glycosides enzyme (PDB accession number：Protein structures 3OG2) are homology model, to the 3D structures and and substrate of beta galactosidase With reference to region be predicted.The structure of prediction is highly similar (referring to The crystal to the prediction result of document report structure of acidicβ-galactosidase from Aspergillus oryzae,Mirko M.Maksimainen, International Journal of Biological Macromolecules, 2013,109- 115).Zymoprotein is made up of 5 domains：The close N-terminal of domain 1 (1~394 amino acid), it is the activated centre place of the enzyme, Activated centre is TIM barrel shape structures；Domain 2 (395~573 amino acid) by 16 antiparallel beta-pleated sheets and 1 α- Spiral forms, and contains the subdomain of a similar immunoglobulin；Domain 3 (574~661 amino acid) is reverse by 8 Parallel beta-pleated sheet forms the β-interlayer and 1 alpha-helix composition of one " Greece's key "；(662~857 amino of domain 4 Acid) and domain 5 (858~1005 amino acid) be mainly made up of the topological structure of " spring roll " type.To activated centre selective analysis It was found that Glu160, Glu258 on TIM buckets activated centre the 4th and the 7th article of beta-pleated sheet be probably participate in catalytic reaction must Indispensable amino acid, and Asn140 and Tyr96 may be used for fixing lactose molecule.
According to the three-dimensional structure of obtained beta galactosidase, enzyme-to-substrate is carried out by Discovery Studio softwares Molecular docking simulate (referring to accompanying drawing 1), according to docking result parsing, would know that with substrate exist interaction amino acid (referring to accompanying drawing 2).Assess the evolution entropy of these amino acid one by one using calculation biology software, final screening determines that six are entered Change amino acid sites S219, D239, S240, Y241, F245 and E785 (referring to table 1) that entropy changes greatly to dash forward for pinpointing saturation Become.
The evolution entropy of the beta galactosidase typical case's amino acid of table 1
Embodiment 2：The structure of Pichia pastoris single-point saturated mutant library
1st, materials and methods
(1) strain and carrier
Wild type gene derives from the beta-galactosidase gene lacb ˊ for the aspergillus candidus for removing native signal peptide, and From the beta-galactosidase gene laco ˊ of aspergillus oryzae, clone to obtain early stage for this laboratory, the particular sequence such as He of sequence 1 Shown in sequence 3, it is connected on pPIC9 expression vectors, and expressed in Pichia pastoris GS115；Escherichia coli Trans1-T1 competent cells are purchased from TransGen companies；PPIC9 expression vectors, Pichia pastoris GS115 are purchased from Invitrogen Company.
(2) preparation of culture medium and related solution
Pichia pastoris conversion, culture and screening conventional medium and the specification of reagent reference Invitrogen companies.
PTM trace salts：0.6%CuSO4, 0.008%NaI2, 0.3%MnSO4, 0.02%Na2MoO4, 0.002%H3BO3, 0.05%CoCl2, 2%ZnCl2, 6.5%FeSO4, 0.5% sulfuric acid (v/v).
Yeast fermentation basal salt media (FBSM)：0.5%KH2PO4, 5%NH4H2PO4, 1.485%MgSO4, 1.82% K2SO4, 0.093%CaSO4, 0.15%KOH, 0.00011%Biotin, 0.44%PTM trace salts, 2% glucose.
The basic Salt treatment culture medium (FBIM) of yeast fermentation：0.5%KH2PO4, 5%NH4H2PO4, 1.485%MgSO4, 1.82%K2SO4, 0.093%CaSO4, 0.15%KOH, 0.00011%Biotin, 0.44%PTM trace salts, 0.5% methanol.
Na2HPO4- citrate buffer solution (0.1mol/L pH5.2)：0.2mol/L disodium hydrogen phosphates 536ml, 0.1mol/L Citric acid 464ml, pH to 5.2 is adjusted after mixing.
(3) Oligonucleolide primers
Specific primer sequence employed in gene mutation is as shown in table 2.
The primer table in the gene mutation of table 2
2nd, over-lap PCR amplification mutational site
Saturation mutation is carried out to the Single locus of gene using the method for over-lap PCR (Overlap PCR).That is two fragments Expand through PCR, then merged by overlap-extension PCR respectively.A pair of degenerate primers are designed, it is had one near target site Determine overlapping (referring to B, C primer in accompanying drawing 3) of degree, hold primer (to draw referring to A, D in accompanying drawing 3 with gene 5 ' and 3 ' respectively Thing) combination, expand containing the fragment upstream and segments downstream for being mutated target site, because these primers are complementary thus caused PCR primer chain will be overlapped, and upstream and downstream fragment is interlocked at target site, and template overlap-extension PCR is into full-length gene each other.Take 1 μ L pPIC9-lacb ' plasmids are template, respectively with primer pair A and C and B and D, use TransStart FastPfu DNA Polymerase, which is expanded, is detected PCR primer with agarose gel electrophoresis, and (method is with reference to day for the correct fragment of recovery size Root biochemical reagents (Beijing) company, Ago-Gel DNA QIAquick Gel Extraction Kits).
3rd, external homologous recombination construction expression vector
By two sections of PCR fragments with homology arm, equimolar amounts mixing, add homologous recombination enzyme and carry out vitro recombination.Will Mixture reacts 30min at 25 DEG C, places 5min on ice afterwards.It can immediately convert or -20 DEG C preserve.By homologous recombination product Take 10ul to be transferred to using chemical method in 100ul Escherichia coli Trans1-T1 competent cells respectively, and be coated on containing Amp's On LB flat boards, 37 DEG C of inversion overnight incubations.
3~5 times of theoretical mutations quantum counts (Mutated codons MNN, the theoretical value 4 in variant storehouse are grown on LB flat boards × 4 × 2=32, single-point saturation mutation, which selects 32 clones, can cover whole mutation) all mutational sites of can covering. Determined dna sequence is carried out from 6~8 monoclonals of picking on the LB flat boards of each mutant library at random, Beijing is entrusted in examining order Complete Mai Aodeen bio tech ltd.Each mutant library is different by mutational site, be named as successively S219 storehouses, D239 storehouses, S240 storehouses, Y241 storehouses, F245 storehouses and E785 storehouses.
4th, expression and the screening technique of high transglycosylation bacterial strain of the beta galactosidase mutant in Pichia pastoris
(1) expression of the recombinant plasmid in Pichia pastoris
Extraction mixing plasmid (about 200-230 μ g), is entered with enough Bgl II enzymes from the Escherichia coli of each mutant library After the digestion of row complete degestion, isopropanol precipitating, it is dissolved in after the washing of 70% ethanol in deionized water and converts Pichia pastoris.Ferment will be converted Mother is applied on the MM flat boards containing x-gal, becomes blue bacterial strain on MM flat boards to there is positive gram of betagalactosidase activity It is grand, the bacterium colony on its corresponding MD flat board is chosen into 48 well culture plates, different mutants are first cultivated in 48 orifice plates through FBSM After base culture makes its fast-growth 48h, then take after FBIM culture medium Fiber differentiations 48h β-half of supernatant measure Positive mutants strain Gal activity.
(2) beta galactosidase is with the activity determination method of oNPG substrates
ONPG substrate 0.1g accurately are weighed, are dissolved in 40mL Na2HPO4- citrate buffer solution (pH 5.2,0.1mol/L), i.e., For the oNPG solution that concentration is 0.25% (W/V).Crude enzyme liquid to be measured uses pH 5.2,0.1mol/L Na2HPO4- citric acid is dilute Release to suitable multiple, to take in the substrate solution addition test tube for inhale 800 μ l and 2min is preheated in 60 DEG C of water-baths, it is dilute to add 200 μ l Release rear enzyme liquid to mix, 1mL 10%TCA terminations, 2ml 1mol/L Na are sequentially added after reacting 15min2CO3Colour developing, measure Absorbance value (OD at 420nm420).To add Na2HPO4- citrate buffer solution (concentration 0.1mol/L, pH5.2) is as empty White control, using standard curve, the oNP of reaction generation amount is calculated, and then calculate the enzyme activity of beta galactosidase.Enzyme activity Unit of force defines：The betagalactosidase activity of one unit (1U) refers under the conditions of 60 DEG C, pH 5.2, catalysis substrate per minute O-nitrophenol-β-D- galactopyranosides (oNPG) generate the enzyme amount needed for 1 μm of ol ortho-nitrophenol (oNP).
According to the result of beta galactosidase standard curve, enzyme activity calculation formula is：
Enzymatic activity (U/mL)=5*N* (0.9472X+0.0046)/15
x：Absorbance value at 420nm；N：The extension rate of enzyme liquid；15：React 15min；5：200 μ L are diluted in enzyme liquid The enzymatic activity that enzyme activity conversion is 1mL.
(3) mutant strain turns glucosides vitality test fundamental reaction system and reaction condition
By the crude enzyme liquid of each mutant through pH 5.2,0.1mol/L Na2HPO4- citrate buffer solution such as is diluted at the albumen Concentration, i.e., contain 5 μ g albumen (concentration about 0.08mg/mL) in every 60 μ L enzyme liquids.Enzyme liquid is placed in EP pipes after drawing 60 μ L dilutions, Add 440 μ L 30% (w/v) lactose substrate, substrate mixes as far as possible quick with enzyme liquid, ensures each sample initial action time Interval is minimum.Each reactant is placed in 50 DEG C, reacts different time in 200rpm constant-temperature tables.Reaction terminate rear 12000rpm from Heart 10min, it is placed in 100 DEG C of water-baths and boils 10min terminating reactions.
12000rpm centrifugations 10min, takes 700 μ L to carry out HPLC detections after reaction product dilutes 16 times using ultra-pure water.
High performance liquid chromatograph (HPLC) testing conditions：
HPLC draws glucose, galactolipin, the standard curve of lactose before quantitatively detecting.The inspection of glucose, galactolipin, lactose It is 0-25.6mg/mL to survey section, and testing conditions are：Waters e2695Separations Moule, mobile phase：50mM Ethylenediaminetetraacetic Acid Calcium Salt salt；Column temperature：85℃；Flow velocity, time：0.5ml/min, 12min/sample.
Oligosaccharide GOS yield (mg/mL)=starting lactose yield (mg/mL)-remaining lactose yield (mg/mL)-glucose amount (mg/mL)-gala sugar amount (mg/mL) (Jorgensen F et al, 2001)；
GOS conversion ratios=oligomeric sugar amount (mg/mL)/starting lactose yield (mg/mL)；
Consume yield=oligomeric sugar amount (mg/mL)/[starting lactose yield (mg/mL)-remaining lactose that Lactose conversion is GOS Measure (mg/mL)].
Embodiment 3：The screening of S219 saturated mutant libraries and the synthesis of oligosaccharide
200 Pichia pastoris positive colonies for choosing S219 mutant libraries determine its transglycosylation (oligosaccharide production Amount), and determine nucleotide sequence.Sequencing shows that these mutant sport 8 kinds of different amino acid respectively can improve mutation The transglycosylation (referring to table 3) of enzyme, especially with the amino acid with smaller side chain such as Gly, Ala, Val and negatively charged pole Acidic amino acid Glu is more prominent, wherein sporting, Gly is the most obvious (referring to accompanying drawing 4), and oligosaccharide growing amount can improve 26.6%. Oligosaccharide growing amount improves 25.7% after sporting small side chain and electronegative Glu, oligosaccharide after S219 sports Ala, Val 15.0% and 15.5% is respectively increased in growing amount, sports Asp, Arg, Leu oligosaccharide growing amount and is respectively increased 10.4%, 7.9% and 8.2%, sporting bulky side chain aromatic amino acid Phe, it turns glucosides and is also substantially improved, and improves 16.4%.And dash forward Being changed into Pro and Trp, it turns glucosides ability and is decreased obviously, and declines 16.7% and 28.7% respectively.As can be seen here, S219 sports it He turn after amino acid glucosides ability present it is high have low situation, show that the site is one and turns glucosides energy with beta galactosidase The related important site of power.The site is spatially positioned in-TIM the buckets of betagalactosidase activity center, passes through electric charge and pole Property effect produce certain effect and be the nonconserved amino acid in the activated centre with lactose substrate.
Table 3S219 different aminoacids mutant oligosaccharide generates situation
Note：WT represents wild enzyme.
Embodiment 4：The screening of F245 saturated mutant libraries and the synthesis of oligosaccharide
The measure that 200 Positive mutants bodies carry out transglycosylation has been screened from F245 storehouses.Surveyed with reference to transglycosylation To determine result and sequencing result shows, all mutant present height on oligosaccharide growing amount low situation (referring to accompanying drawing 5), Fractional mutant oligosaccharide growing amount declines much than wild enzyme, and fractional mutant oligosaccharide growing amount is higher by 30% than wild enzyme Left and right.As can be seen here, F245 sites are also an important site related to the generation of beta galactosidase oligosaccharide.
Specifically, oligosaccharide growing amount improves at most after F245 site mutations are Arg, about 35%, next to that Lys and Gly, It is respectively increased about 30% and 24.7%.After sporting other amino acid such as Ser, Glu, Ala, Thr and Met, oligomeric candy output Also it is substantially improved (referring to accompanying drawing 6).
Embodiment 5：The screening of E785 saturated mutant libraries and the synthesis of oligosaccharide
Compared with wild type, in E785 saturated mutant libraries, the oligosaccharide synthetic quantity of only a small amount of mutant improves, and carries High-amplitude is not more than 20% (referring to accompanying drawing 7).In the mutant that E785 mutant library oligosaccharide growing amount improves, Glu is sported 15% is improved after Val, remaining most of mutant oligosaccharide yield is less than wild type or little with wild enzyme difference.
The construction and screening in the combination mutant storehouse of embodiment 6
1st, materials and methods
(1) strain and carrier
Initiation mutant is the beta-galactosidase gene lacb ˊ of aspergillus candidus mutant E785V, is connected to pPIC9 On expression vector, and expressed in Pichia pastoris GS115；Escherichia coli Trans1-T1 competent cells are purchased from TransGen companies；PPIC9 expression vectors and Pichia pastoris GS115 are purchased from Invitrogen companies.
(2) preparation of culture medium and related solution
Pichia pastoris conversion, culture and screening conventional medium and the specification of reagent reference Invitrogen companies.
PTM trace salts：0.6%CuSO4, 0.008%NaI2, 0.3%MnSO4, 0.02%Na2MoO4, 0.002%H3BO3, 0.05%CoCl2, 2%ZnCl2, 6.5%FeSO4, 0.5% sulfuric acid (v/v).
Yeast fermentation basal salt media (FBSM)：0.5%KH2PO4, 5%NH4H2PO4, 1.485%MgSO4, 1.82% K2SO4, 0.093%CaSO4, 0.15%KOH, 0.00011%Biotin, 0.44%PTM trace salts, 2% glucose.
The basic Salt treatment culture medium (FBIM) of yeast fermentation：0.5%KH2PO4, 5%NH4H2PO4, 1.485%MgSO4, 1.82%K2SO4, 0.093%CaSO4, 0.15%KOH, 0.00011%Biotin, 0.44%PTM trace salts, 0.5% methanol.
Na2HPO4- citrate buffer solution (0.1mol/L pH5.2)：0.2mol/L disodium hydrogen phosphates 536ml, 0.1mol/L Citric acid 464ml, pH to 5.2 is adjusted after mixing.
(3) mutation Oligonucleolide primers
Specific primer sequence employed in combinatorial mutagenesis is as shown in table 4.
The primer table in the combinatorial mutagenesis of table 4
Note：Overstriking font is mutating alkali yl.
2nd, the structure in combination mutant storehouse
Based on the above-mentioned analysis to different loci saturated mutant library, two sites of S219 and E785 are selected as combination The site of mutation, and determine according to the sequencing result to each site mutant the purpose mutating acid in each site..Really Primer is designed after determining mutational site and purpose mutating acid, the structure in combination mutant storehouse uses the method being mutated by several times, The mutant plasmid in one of site is first built, another one point is mutated again on this basis, is dashed forward until completing purpose Become.For S219/E785 two point combinatorial mutagenesises, using E785 single-points are first mutated, E785 simple point mutation plasmids are recycled to enter The mutation in row S219 sites.
The structure of combinatorial mutagenesis recombinant plasmid is then established on the basis of above-mentioned simple point mutation, i.e., with the mixing of simple point mutation Plasmid is that template continues through above-mentioned PCR amplifications and recombinant technique obtains double mutant.
By combinatorial mutagenesis recombinant plasmid transformed into Pichia pastoris GS115, expressed in Pichia pastoris GS115, and Determine the betagalactosidase activity of Positive mutants strain.
3rd, the screening in combination mutant storehouse
500 mutant of random picking, determine its betagalactosidase activity from S219/E785 double mutants storehouse. As shown in figure 8, the oligosaccharide growing amount of most of mutant is all higher than wild enzyme in S219/E785 double mutants storehouse.It is heavier Want, the GOS generating rates of part combination mutant are greatly speeded up compared with wild type, reach the time needed for highest GOS yield Also greatly shorten, the property that different mutants turn glucosides is listed in Table 5 below respectively.
The different mutants of table 5 turn glycosidic nature
From the data of table 5, the height filtered out turns the generation speed of glucosides mutant enzyme oligosaccharide in identical lactose substrate Rate is substantially accelerated, namely means to turn the quickening of glucosides reaction rate, the most obvious with S219G/E785V mutant.S219G/ For the yield of E785V oligosaccharide when reacting 2h with regard to high, most of mutant has just reached oligosaccharide synthesis most in 5-6h or so Largely, that is, hydrolyze and turn glucosides reaction speed and balance each other the stage, only E785V mutant just reaches maximum oligomeric in 36h or so Sugared growing amount, and wild enzyme just reaches the equilibrium stage for turning glucosides and hydrolysis in 48h or so, reaction time is very long.It is most of prominent Variant adds GOS yield while reaction rate is accelerated, highest such as S219V/E785V conversion ratios by 23.3% to 27.1% improves 16%.It is visible to calculate the generating rate of each mutant oligosaccharide to react the yield of 2h oligosaccharide, remove E785V mutant improves 22% or so than wild enzyme, and remaining mutant raising amount is all more than 100%, wherein reaction rate Most fast S219G/E785V mutant raising amounts are wild enzymes more than 3 times 211% or so.
Sequence table
<110>Biological Technology institute, Chinese Academy of Agricultural Sciences
<120>A kind of beta galactosidase combination mutant with high transglycosylation and its preparation method and application
<160> 24
<170> PatentIn version 3.3
<210> 1
<211> 2958
<212> DNA
<213>Artificial sequence
<400> 1
tccatcaagc atcgtctcaa tggcttcacg atcctggaac atccggatcc ggcgaaaaga 60
gacttgctgc aagacattgt tacatgggat gacaaatctc tgttcatcaa tggagagagg 120
attatgttat tcagcggaga agtgcatcct ttcagattgc cagtaccttc gctttggctt 180
gatatcttcc acaagatcag agctcttggt ttcaactgtg tatctttcta tattgattgg 240
gctcttctgg agggaaagcc tggcgactac agagcagaag gcatctttgc tctggaaccc 300
ttcttcgatg cagccaagga agcaggcatt tatctgatcg cccgccccgg ttcgtacatc 360
aatgccgagg tctcaggcgg tggcttccct ggatggttgc agagggtcaa tggcactctt 420
cgctcgtctg atgagccatt ccttaaagct actgataact atatcgccaa tgccgctgct 480
gccgtggcga aggctcaaat cacgaatgga gggccagtaa ttctctacca gcccgaaaac 540
gaatacagcg gtggctgctg cggtgtcaaa taccccgatg cagactacat gcagtatgtt 600
atggatcagg cccggaaggc tgacattgtt gtacctttca tcagcaacga tgcctcacct 660
tctgggcaca atgctcctgg aagtggaacg ggcgctgttg atatttatgg tcacgatagc 720
tatccccttg gctttgattg cgcaaaccca tccgtatggc ccgagggtaa actgcccgac 780
aacttccgca cgctccatct tgagcagagc ccatcaactc cgtattcact tcttgagttc 840
caagcgggtg ctttcgaccc atggggtgga cccggctttg aaaaatgcta tgccctcgtt 900
aaccacgaat tctcgagagt tttctatagg aacgacttga gtttcggagt ttctaccttt 960
aacttataca tgactttcgg cggaacaaac tggggtaacc tcggacatcc cggtggatat 1020
acatcctacg actacggctc gcctataact gaaacgcgaa acgttacacg ggagaagtac 1080
agcgacataa agctccttgc caactttgtc aaagcatcgc catcctatct caccgctact 1140
cccagaaacc tgactactgg tgtttacaca gacacatctg acctggctgt caccccgtta 1200
atgggtgata gtccaggctc attcttcgtg gtcagacata cggactattc cagccaagag 1260
tcaacctcgt acaaacttaa gcttcctacc agtgctggta acctgactat tccccagctg 1320
gagggcactc taagtctcaa cggacgtgac tcaaaaattc atgttgttga ttataatgtg 1380
tctggaacga acattatcta ttcgacagct gaagtcttca cctggaagaa gtttgacggt 1440
aacaaggtcc tggtgttata cggcggaccg aaggaacacc atgaattggc cattgcctcc 1500
aagtcaaatg tgaccatcat cgaaggttcg gactctggaa ttgtctcaac gaggaagggc 1560
agctctgtta tcattggctg ggatgtctct tctactcgtc gcatcgttca agtcggtgac 1620
ttgagagtgt tcctgcttga tagaaactct gcttacaact actgggtccc cgaactcccc 1680
acagaaggta cttctcccgg gttcagcact tcgaagacga ccgcctcctc cattattgtg 1740
aaggccggct acctcctccg aggggctcac ctggatggtg ctgatcttca tcttactgct 1800
gatttcaatg ccaccacccc gattgaagtg atcggtgctc caacaggcgc caagaatctg 1860
ttcgtgaatg gtgaaaaggc tagccacaca gtcgacaaaa acggcatctg gagtagtgag 1920
gtcaagtacg cggctccaga gatcaagctc cccggtttga aggatttgga ctggaagtat 1980
ctggacacgc ttcccgaaat taagtcttcc tatgatgact cggcctgggt ttcggcagac 2040
cttccaaaga caaagaacac tcaccgtcct cttgacacac caacatcgct atactcctct 2100
gactatggct tccacactgg ctacctgatc tacaggggtc acttcgttgc caacggcaag 2160
gaaagcgaat tttttattcg cacacaaggc ggtagcgcat tcggaagttc cgtatggctg 2220
aacgagacgt atctgggctc ttggactggt gccgattatg cgatggacgg taactctacc 2280
tacaagctat ctcagctgga gtcgggcaag aattacgtca tcactgtggt tattgataac 2340
ctgggtctcg acgagaattg gacggtcggc gaggaaacca tgaagaatcc tcgtggtatt 2400
cttagctaca agctgagcgg acaagacgcc agcgcaatca cctggaagct cactggtaac 2460
ctcggaggag aagactacca ggataaggtt agaggacctc tcaacgaagg tggactgtac 2520
gcagagcgcc agggcttcca tcagcctcag cctccaagcg aatcctggga gtcgggcagt 2580
ccccttgaag gcctgtcgaa gccgggtatc ggattctaca ctgcccagtt cgaccttgac 2640
ctcccgaagg gctgggatgt gccgctgtac ttcaactttg gcaacaacac ccaggcggct 2700
cgggcccagc tctacgtcaa cggttaccag tatggcaagt tcactggaaa cgttgggcca 2760
cagaccagct tccctgttcc cgaagggatc ctgaactacc gcggaaccaa ctatgtggca 2820
ctgagtcttt gggcattgga gtcggacggt gctaagctgg gtagcttcga actgtcctac 2880
accaccccag tgctgaccgg atacggggat gttgagtcac ctgagcagcc caagtatgag 2940
cagcggaagg gagcatac 2958
<210> 2
<211> 986
<212> PRT
<213>Artificial sequence
<400> 2
Ser Ile Lys His Arg Leu Asn Gly Phe Thr Ile Leu Glu His Pro Asp
1 5 10 15
Pro Ala Lys Arg Asp Leu Leu Gln Asp Ile Val Thr Trp Asp Asp Lys
20 25 30
Ser Leu Phe Ile Asn Gly Glu Arg Ile Met Leu Phe Ser Gly Glu Val
35 40 45
His Pro Phe Arg Leu Pro Val Pro Ser Leu Trp Leu Asp Ile Phe His
50 55 60
Lys Ile Arg Ala Leu Gly Phe Asn Cys Val Ser Phe Tyr Ile Asp Trp
65 70 75 80
Ala Leu Leu Glu Gly Lys Pro Gly Asp Tyr Arg Ala Glu Gly Ile Phe
85 90 95
Ala Leu Glu Pro Phe Phe Asp Ala Ala Lys Glu Ala Gly Ile Tyr Leu
100 105 110
Ile Ala Arg Pro Gly Ser Tyr Ile Asn Ala Glu Val Ser Gly Gly Gly
115 120 125
Phe Pro Gly Trp Leu Gln Arg Val Asn Gly Thr Leu Arg Ser Ser Asp
130 135 140
Glu Pro Phe Leu Lys Ala Thr Asp Asn Tyr Ile Ala Asn Ala Ala Ala
145 150 155 160
Ala Val Ala Lys Ala Gln Ile Thr Asn Gly Gly Pro Val Ile Leu Tyr
165 170 175
Gln Pro Glu Asn Glu Tyr Ser Gly Gly Cys Cys Gly Val Lys Tyr Pro
180 185 190
Asp Ala Asp Tyr Met Gln Tyr Val Met Asp Gln Ala Arg Lys Ala Asp
195 200 205
Ile Val Val Pro Phe Ile Ser Asn Asp Ala Ser Pro Ser Gly His Asn
210 215 220
Ala Pro Gly Ser Gly Thr Gly Ala Val Asp Ile Tyr Gly His Asp Ser
225 230 235 240
Tyr Pro Leu Gly Phe Asp Cys Ala Asn Pro Ser Val Trp Pro Glu Gly
245 250 255
Lys Leu Pro Asp Asn Phe Arg Thr Leu His Leu Glu Gln Ser Pro Ser
260 265 270
Thr Pro Tyr Ser Leu Leu Glu Phe Gln Ala Gly Ala Phe Asp Pro Trp
275 280 285
Gly Gly Pro Gly Phe Glu Lys Cys Tyr Ala Leu Val Asn His Glu Phe
290 295 300
Ser Arg Val Phe Tyr Arg Asn Asp Leu Ser Phe Gly Val Ser Thr Phe
305 310 315 320
Asn Leu Tyr Met Thr Phe Gly Gly Thr Asn Trp Gly Asn Leu Gly His
325 330 335
Pro Gly Gly Tyr Thr Ser Tyr Asp Tyr Gly Ser Pro Ile Thr Glu Thr
340 345 350
Arg Asn Val Thr Arg Glu Lys Tyr Ser Asp Ile Lys Leu Leu Ala Asn
355 360 365
Phe Val Lys Ala Ser Pro Ser Tyr Leu Thr Ala Thr Pro Arg Asn Leu
370 375 380
Thr Thr Gly Val Tyr Thr Asp Thr Ser Asp Leu Ala Val Thr Pro Leu
385 390 395 400
Met Gly Asp Ser Pro Gly Ser Phe Phe Val Val Arg His Thr Asp Tyr
405 410 415
Ser Ser Gln Glu Ser Thr Ser Tyr Lys Leu Lys Leu Pro Thr Ser Ala
420 425 430
Gly Asn Leu Thr Ile Pro Gln Leu Glu Gly Thr Leu Ser Leu Asn Gly
435 440 445
Arg Asp Ser Lys Ile His Val Val Asp Tyr Asn Val Ser Gly Thr Asn
450 455 460
Ile Ile Tyr Ser Thr Ala Glu Val Phe Thr Trp Lys Lys Phe Asp Gly
465 470 475 480
Asn Lys Val Leu Val Leu Tyr Gly Gly Pro Lys Glu His His Glu Leu
485 490 495
Ala Ile Ala Ser Lys Ser Asn Val Thr Ile Ile Glu Gly Ser Asp Ser
500 505 510
Gly Ile Val Ser Thr Arg Lys Gly Ser Ser Val Ile Ile Gly Trp Asp
515 520 525
Val Ser Ser Thr Arg Arg Ile Val Gln Val Gly Asp Leu Arg Val Phe
530 535 540
Leu Leu Asp Arg Asn Ser Ala Tyr Asn Tyr Trp Val Pro Glu Leu Pro
545 550 555 560
Thr Glu Gly Thr Ser Pro Gly Phe Ser Thr Ser Lys Thr Thr Ala Ser
565 570 575
Ser Ile Ile Val Lys Ala Gly Tyr Leu Leu Arg Gly Ala His Leu Asp
580 585 590
Gly Ala Asp Leu His Leu Thr Ala Asp Phe Asn Ala Thr Thr Pro Ile
595 600 605
Glu Val Ile Gly Ala Pro Thr Gly Ala Lys Asn Leu Phe Val Asn Gly
610 615 620
Glu Lys Ala Ser His Thr Val Asp Lys Asn Gly Ile Trp Ser Ser Glu
625 630 635 640
Val Lys Tyr Ala Ala Pro Glu Ile Lys Leu Pro Gly Leu Lys Asp Leu
645 650 655
Asp Trp Lys Tyr Leu Asp Thr Leu Pro Glu Ile Lys Ser Ser Tyr Asp
660 665 670
Asp Ser Ala Trp Val Ser Ala Asp Leu Pro Lys Thr Lys Asn Thr His
675 680 685
Arg Pro Leu Asp Thr Pro Thr Ser Leu Tyr Ser Ser Asp Tyr Gly Phe
690 695 700
His Thr Gly Tyr Leu Ile Tyr Arg Gly His Phe Val Ala Asn Gly Lys
705 710 715 720
Glu Ser Glu Phe Phe Ile Arg Thr Gln Gly Gly Ser Ala Phe Gly Ser
725 730 735
Ser Val Trp Leu Asn Glu Thr Tyr Leu Gly Ser Trp Thr Gly Ala Asp
740 745 750
Tyr Ala Met Asp Gly Asn Ser Thr Tyr Lys Leu Ser Gln Leu Glu Ser
755 760 765
Gly Lys Asn Tyr Val Ile Thr Val Val Ile Asp Asn Leu Gly Leu Asp
770 775 780
Glu Asn Trp Thr Val Gly Glu Glu Thr Met Lys Asn Pro Arg Gly Ile
785 790 795 800
Leu Ser Tyr Lys Leu Ser Gly Gln Asp Ala Ser Ala Ile Thr Trp Lys
805 810 815
Leu Thr Gly Asn Leu Gly Gly Glu Asp Tyr Gln Asp Lys Val Arg Gly
820 825 830
Pro Leu Asn Glu Gly Gly Leu Tyr Ala Glu Arg Gln Gly Phe His Gln
835 840 845
Pro Gln Pro Pro Ser Glu Ser Trp Glu Ser Gly Ser Pro Leu Glu Gly
850 855 860
Leu Ser Lys Pro Gly Ile Gly Phe Tyr Thr Ala Gln Phe Asp Leu Asp
865 870 875 880
Leu Pro Lys Gly Trp Asp Val Pro Leu Tyr Phe Asn Phe Gly Asn Asn
885 890 895
Thr Gln Ala Ala Arg Ala Gln Leu Tyr Val Asn Gly Tyr Gln Tyr Gly
900 905 910
Lys Phe Thr Gly Asn Val Gly Pro Gln Thr Ser Phe Pro Val Pro Glu
915 920 925
Gly Ile Leu Asn Tyr Arg Gly Thr Asn Tyr Val Ala Leu Ser Leu Trp
930 935 940
Ala Leu Glu Ser Asp Gly Ala Lys Leu Gly Ser Phe Glu Leu Ser Tyr
945 950 955 960
Thr Thr Pro Val Leu Thr Gly Tyr Gly Asp Val Glu Ser Pro Glu Gln
965 970 975
Pro Lys Tyr Glu Gln Arg Lys Gly Ala Tyr
980 985
<210> 3
<211> 2958
<212> DNA
<213>Artificial sequence
<400> 3
tccatcaagc atcgtctcaa tggcttcacg atcctggaac atccggatcc ggcgaaaaga 60
gacttgctgc aagacattgt tacatgggat gacaaatctc tgttcatcaa tggagagagg 120
attatgttat tcagcggaga agtgcatcct ttcagattgc cagtaccttc gctttggctt 180
gatatcttcc acaagatcag agctcttggt ttcaactgtg tatctttcta tattgattgg 240
gctcttctgg agggaaagcc tggcgactac agagcagaag gcatctttgc tctggaaccc 300
ttctttgatg cagccaagga agcaggcatt tatctgatcg cccgccccgg ttcgtacatc 360
aatgccgagg tctcaggcgg tggcttccct ggatggttgc agagggtcaa tggcactctt 420
cgctcgtctg atgagccatt ccttaaagct actgataact atatcgccaa tgccgctgct 480
gccgtggcga aggctcaaat cacgaatgga gggccagtaa ttctctacca gcccgaaaac 540
gaatacagcg gtggctgctg cggtgtcaaa taccccgatg cagactacat gcagtatgtt 600
atggatcagg cccggaaggc tgacattgtt gtacctttca tcagcaacga tgcctcacct 660
tctgggcaca atgctcctgg aagtggaacg agcgctgttg atatttatgg tcacgatagc 720
tatcccctcg gctttgattg cgcaaaccca tccgtatggc ccgagggtaa actgcccgac 780
aacttccgca cgctccatct tgagcagagc ccatcaactc cgtattcact tcttgagttc 840
caagcgggtg ctttcgaccc atggggtgga cccggctttg aaaaatgcta tgccctcgtt 900
aaccacgaat tctcgagagt tttctatagg aacgacttga gtttcggagt ttctaccttt 960
aacttataca tgactttcgg cggaacaaac tggggtaacc tcggacatcc cggtggatat 1020
acatcctacg actacggatc gcctataact gaaacgcgaa acgttacgcg ggagaagtac 1080
agcgacataa agctccttgc caactttgtc aaagcatcgc catcctatct caccgctact 1140
cccagaaacc tgactactgg tgtttacaca gacacatctg acctggctgt caccccgtta 1200
attggtgata gtccaggctc attcttcgtg gtcagacata cggactattc cagccaagag 1260
tcaacctcgt acaaacttaa gcttcctacc agtgctggta acctgactat tccccagctg 1320
gagggcactc taagtctcaa cggacgtgac tcaaaaattc atgttgttga ttataatgtg 1380
tctggaacga acattatcta ttcgacagct gaagtcttca cctggaagaa gtttgacggt 1440
aacaaggtcc tggtgttata cggcggaccg aaggaacacc atgaattggc cattgcctcc 1500
aagtcaaatg tgaccatcat cgaaggttcg gactctggaa ttgtctcaac gaggaagggc 1560
agctctgtta tcattggctg ggatgtctct tctactcgtc gcatcgttca agtcggtgac 1620
ttgagagtgt tcctgcttga taggaactct gcttacaact actgggtccc cgaactcccc 1680
acagaaggta cttctcccgg gttcagcact tcgaagacga ccgcctcctc cattattgtg 1740
aaggctggct acctcctccg aggcgctcac cttgatggtg ctgatcttca tcttactgct 1800
gatttcaatg ccaccacccc gattgaagtg atcggtgctc caacaggcgc taagaatctg 1860
ttcgtgaatg gtgaaaaggc tagccacaca gtcgacaaga acggcatctg gagcagtgag 1920
gtcaagtacg cggctccaga gatcaagctc cccggtttga aggatttgga ctggaagtat 1980
ctggacacgc ttcccgaaat taagtcttcc tatgatgact cggcctgggt ttcggcagac 2040
cttccaaaga caaagaacac tcaccgtcct cttgacacac caacatcgct atactcctct 2100
gactatggct tccacactgg ctacctgatc tacaggggtc acttcgttgc caacggcaag 2160
gaaagcgaat tttttattcg cacacaaggc ggtagcgcat tcggaagttc cgtatggctg 2220
aacgagacgt atctgggctc ttggactggt gccgattatg cgatggacgg taactctacc 2280
tacaagctat ctcagctgga gtcgggcaag aattacgtca tcactgtggt tattgataac 2340
ctgggtctcg acgagaattg gacggtcggc gaggaaacca tgaagaatcc tcgtggtatt 2400
cttagctaca agctgagcgg acaagacgcc agcgcaatca cctggaagct cactggtaac 2460
ctcggaggag aagactacca ggataaggtt agaggacctc tcaacgaagg tggactgtac 2520
gcagagcgcc agggcttcca tcagcctcag cctccaagcg aatcctggga gtcgggcagt 2580
ccccttgaag gcctgtcgaa gccgggtatc ggattctaca ctgcccagtt cgaccttgac 2640
ctcccgaagg gctgggatgt gccgctgtac ttcaactttg gcaacaacac ccaggcggct 2700
cgggcccagc tctacgtcaa cggttaccag tatggcaagt tcactggaaa cgttgggcca 2760
cagaccagct tccctgttcc cgaaggtatc ctgaactacc gcggaaccaa ctatgtggca 2820
ctgagtcttt gggcattgga gtcggacggt gctaagctgg gtagcttcga actgtcctac 2880
accaccccag tgctgaccgg atacgggaat gttgagtcac ctgagcagcc caagtatgag 2940
cagcggaagg gagcatac 2958
<210> 4
<211> 986
<212> PRT
<213>Artificial sequence
<400> 4
Ser Ile Lys His Arg Leu Asn Gly Phe Thr Ile Leu Glu His Pro Asp
1 5 10 15
Pro Ala Lys Arg Asp Leu Leu Gln Asp Ile Val Thr Trp Asp Asp Lys
20 25 30
Ser Leu Phe Ile Asn Gly Glu Arg Ile Met Leu Phe Ser Gly Glu Val
35 40 45
His Pro Phe Arg Leu Pro Val Pro Ser Leu Trp Leu Asp Ile Phe His
50 55 60
Lys Ile Arg Ala Leu Gly Phe Asn Cys Val Ser Phe Tyr Ile Asp Trp
65 70 75 80
Ala Leu Leu Glu Gly Lys Pro Gly Asp Tyr Arg Ala Glu Gly Ile Phe
85 90 95
Ala Leu Glu Pro Phe Phe Asp Ala Ala Lys Glu Ala Gly Ile Tyr Leu
100 105 110
Ile Ala Arg Pro Gly Ser Tyr Ile Asn Ala Glu Val Ser Gly Gly Gly
115 120 125
Phe Pro Gly Trp Leu Gln Arg Val Asn Gly Thr Leu Arg Ser Ser Asp
130 135 140
Glu Pro Phe Leu Lys Ala Thr Asp Asn Tyr Ile Ala Asn Ala Ala Ala
145 150 155 160
Ala Val Ala Lys Ala Gln Ile Thr Asn Gly Gly Pro Val Ile Leu Tyr
165 170 175
Gln Pro Glu Asn Glu Tyr Ser Gly Gly Cys Cys Gly Val Lys Tyr Pro
180 185 190
Asp Ala Asp Tyr Met Gln Tyr Val Met Asp Gln Ala Arg Lys Ala Asp
195 200 205
Ile Val Val Pro Phe Ile Ser Asn Asp Ala Ser Pro Ser Gly His Asn
210 215 220
Ala Pro Gly Ser Gly Thr Ser Ala Val Asp Ile Tyr Gly His Asp Ser
225 230 235 240
Tyr Pro Leu Gly Phe Asp Cys Ala Asn Pro Ser Val Trp Pro Glu Gly
245 250 255
Lys Leu Pro Asp Asn Phe Arg Thr Leu His Leu Glu Gln Ser Pro Ser
260 265 270
Thr Pro Tyr Ser Leu Leu Glu Phe Gln Ala Gly Ala Phe Asp Pro Trp
275 280 285
Gly Gly Pro Gly Phe Glu Lys Cys Tyr Ala Leu Val Asn His Glu Phe
290 295 300
Ser Arg Val Phe Tyr Arg Asn Asp Leu Ser Phe Gly Val Ser Thr Phe
305 310 315 320
Asn Leu Tyr Met Thr Phe Gly Gly Thr Asn Trp Gly Asn Leu Gly His
325 330 335
Pro Gly Gly Tyr Thr Ser Tyr Asp Tyr Gly Ser Pro Ile Thr Glu Thr
340 345 350
Arg Asn Val Thr Arg Glu Lys Tyr Ser Asp Ile Lys Leu Leu Ala Asn
355 360 365
Phe Val Lys Ala Ser Pro Ser Tyr Leu Thr Ala Thr Pro Arg Asn Leu
370 375 380
Thr Thr Gly Val Tyr Thr Asp Thr Ser Asp Leu Ala Val Thr Pro Leu
385 390 395 400
Ile Gly Asp Ser Pro Gly Ser Phe Phe Val Val Arg His Thr Asp Tyr
405 410 415
Ser Ser Gln Glu Ser Thr Ser Tyr Lys Leu Lys Leu Pro Thr Ser Ala
420 425 430
Gly Asn Leu Thr Ile Pro Gln Leu Glu Gly Thr Leu Ser Leu Asn Gly
435 440 445
Arg Asp Ser Lys Ile His Val Val Asp Tyr Asn Val Ser Gly Thr Asn
450 455 460
Ile Ile Tyr Ser Thr Ala Glu Val Phe Thr Trp Lys Lys Phe Asp Gly
465 470 475 480
Asn Lys Val Leu Val Leu Tyr Gly Gly Pro Lys Glu His His Glu Leu
485 490 495
Ala Ile Ala Ser Lys Ser Asn Val Thr Ile Ile Glu Gly Ser Asp Ser
500 505 510
Gly Ile Val Ser Thr Arg Lys Gly Ser Ser Val Ile Ile Gly Trp Asp
515 520 525
Val Ser Ser Thr Arg Arg Ile Val Gln Val Gly Asp Leu Arg Val Phe
530 535 540
Leu Leu Asp Arg Asn Ser Ala Tyr Asn Tyr Trp Val Pro Glu Leu Pro
545 550 555 560
Thr Glu Gly Thr Ser Pro Gly Phe Ser Thr Ser Lys Thr Thr Ala Ser
565 570 575
Ser Ile Ile Val Lys Ala Gly Tyr Leu Leu Arg Gly Ala His Leu Asp
580 585 590
Gly Ala Asp Leu His Leu Thr Ala Asp Phe Asn Ala Thr Thr Pro Ile
595 600 605
Glu Val Ile Gly Ala Pro Thr Gly Ala Lys Asn Leu Phe Val Asn Gly
610 615 620
Glu Lys Ala Ser His Thr Val Asp Lys Asn Gly Ile Trp Ser Ser Glu
625 630 635 640
Val Lys Tyr Ala Ala Pro Glu Ile Lys Leu Pro Gly Leu Lys Asp Leu
645 650 655
Asp Trp Lys Tyr Leu Asp Thr Leu Pro Glu Ile Lys Ser Ser Tyr Asp
660 665 670
Asp Ser Ala Trp Val Ser Ala Asp Leu Pro Lys Thr Lys Asn Thr His
675 680 685
Arg Pro Leu Asp Thr Pro Thr Ser Leu Tyr Ser Ser Asp Tyr Gly Phe
690 695 700
His Thr Gly Tyr Leu Ile Tyr Arg Gly His Phe Val Ala Asn Gly Lys
705 710 715 720
Glu Ser Glu Phe Phe Ile Arg Thr Gln Gly Gly Ser Ala Phe Gly Ser
725 730 735
Ser Val Trp Leu Asn Glu Thr Tyr Leu Gly Ser Trp Thr Gly Ala Asp
740 745 750
Tyr Ala Met Asp Gly Asn Ser Thr Tyr Lys Leu Ser Gln Leu Glu Ser
755 760 765
Gly Lys Asn Tyr Val Ile Thr Val Val Ile Asp Asn Leu Gly Leu Asp
770 775 780
Glu Asn Trp Thr Val Gly Glu Glu Thr Met Lys Asn Pro Arg Gly Ile
785 790 795 800
Leu Ser Tyr Lys Leu Ser Gly Gln Asp Ala Ser Ala Ile Thr Trp Lys
805 810 815
Leu Thr Gly Asn Leu Gly Gly Glu Asp Tyr Gln Asp Lys Val Arg Gly
820 825 830
Pro Leu Asn Glu Gly Gly Leu Tyr Ala Glu Arg Gln Gly Phe His Gln
835 840 845
Pro Gln Pro Pro Ser Glu Ser Trp Glu Ser Gly Ser Pro Leu Glu Gly
850 855 860
Leu Ser Lys Pro Gly Ile Gly Phe Tyr Thr Ala Gln Phe Asp Leu Asp
865 870 875 880
Leu Pro Lys Gly Trp Asp Val Pro Leu Tyr Phe Asn Phe Gly Asn Asn
885 890 895
Thr Gln Ala Ala Arg Ala Gln Leu Tyr Val Asn Gly Tyr Gln Tyr Gly
900 905 910
Lys Phe Thr Gly Asn Val Gly Pro Gln Thr Ser Phe Pro Val Pro Glu
915 920 925
Gly Ile Leu Asn Tyr Arg Gly Thr Asn Tyr Val Ala Leu Ser Leu Trp
930 935 940
Ala Leu Glu Ser Asp Gly Ala Lys Leu Gly Ser Phe Glu Leu Ser Tyr
945 950 955 960
Thr Thr Pro Val Leu Thr Gly Tyr Gly Asn Val Glu Ser Pro Glu Gln
965 970 975
Pro Lys Tyr Glu Gln Arg Lys Gly Ala Tyr
980 985
<210> 5
<211> 32
<212> DNA
<213>Artificial sequence
<400> 5
cgcgaggcag agatcttgag ataaatttca cg 32
<210> 6
<211> 33
<212> DNA
<213>Artificial sequence
<400> 6
acgtgaaatt tatctcaaga tctctgcctc gcg 33
<210> 7
<211> 40
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (29)..(30)
<223> n is a, c, g, or t
<400> 7
acttccagga gcattgtgcc cagaaggmnn ggcatcgttg 40
<210> 8
<211> 31
<212> DNA
<213>Artificial sequence
<400> 8
ttctgggcac aatgctcctg gaagtggaac g 31
<210> 9
<211> 36
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (29)..(30)
<223> n is a, c, g, or t
<400> 9
tgcgcaatca aagccaaggg gatagctmnn gtgacc 36
<210> 10
<211> 27
<212> DNA
<213>Artificial sequence
<400> 10
tccccttggc tttgattgcg caaaccc 27
<210> 11
<211> 36
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (26)..(27)
<223> n is a, c, g, or t
<400> 11
tgcgcaatca aagccaaggg gatamnnatc gtgacc 36
<210> 12
<211> 32
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (25)..(26)
<223> n is a, c, g, or t
<400> 12
tttgcgcaat caaagccaag gggmnngcta tc 32
<210> 13
<211> 30
<212> DNA
<213>Artificial sequence
<400> 13
tggctttgat tgcgcaaacc catccgtatg 30
<210> 14
<211> 31
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (24)..(25)
<223> n is a, c, g, or t
<400> 14
atacggatgg gtttgcgcaa tcmnngccaa g 31
<210> 15
<211> 24
<212> DNA
<213>Artificial sequence
<400> 15
tgcgcaaacc catccgtatg gccc 24
<210> 16
<211> 29
<212> DNA
<213>Artificial sequence
<400> 16
tttcctcgcc gaccgtccaa ttaacgtcg 29
<210> 17
<211> 27
<212> DNA
<213>Artificial sequence
<400> 17
tggacggtcg gcgaggaaac catgaag 27
<210> 18
<211> 32
<212> DNA
<213>Artificial sequence
<400> 18
cgcgaggcag agatcttgag ataaatttca cg 32
<210> 19
<211> 33
<212> DNA
<213>Artificial sequence
<400> 19
acgtgaaatt tatctcaaga tctctgcctc gcg 33
<210> 20
<211> 40
<212> DNA
<213>Artificial sequence
<400> 20
acttccagga gcattgtgcc cagaaggaak ggcatcgttg 40
<210> 21
<211> 40
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (29)
<223> n is a, c, g, or t
<400> 21
acttccagga gcattgtgcc cagaaggmnc ggcatcgttg 40
<210> 22
<211> 31
<212> DNA
<213>Artificial sequence
<400> 22
ttctgggcac aatgctcctg gaagtggaac g 31
<210> 23
<211> 29
<212> DNA
<213>Artificial sequence
<400> 23
tttcctcgcc gaccgtccaa ttaacgtcg 29
<210> 24
<211> 27
<212> DNA
<213>Artificial sequence
<400> 24
tggacggtcg gcgaggaaac catgaag 27

Claims (9)

1. a kind of beta galactosidase combination mutant with high transglycosylation, it is bright shown in sequence 2 or sequence 4 On the basis of aspergillus albicans or aspergillus oryzae beta galactosidase amino acid sequence, obtained by the fixed point saturation mutation of double site , wherein the fixed point saturation mutation of double site is to substitute the serine residue (S219A) of the 219th with alanine residue and use figured silk fabrics Histidine residue substitutes the glutaminic acid residue (E785V) of the 785th.
2. encode the DNA molecular of combination mutant described in claim 1.
3. the recombinant expression carrier containing DNA molecular described in claim 2.
4. recombinant expression carrier according to claim 3, it is recombinant yeast expression vector.
5. express the host cell of DNA molecular described in claim 2.
6. host cell according to claim 5, selected from saccharomyces, genus Kluyveromyces, Schizasaccharomyces and Methylotrophic yeast strain.
7. host cell according to claim 6, wherein methylotrophic yeast strain are pichia category bacterial strain.
8. a kind of method for preparing the beta galactosidase with high transglycosylation, comprises the following steps：
1) with the recombinant expression carrier conversion host cell described in claim 3 or 4, recombinant bacterial strain is obtained；
2) above-mentioned recombinant bacterial strain, the expression of induction restructuring beta galactosidase are cultivated；
3) reclaim and purify the beta galactosidase of expressed high transglycosylation.
9. the DNA molecular described in combination mutant, claim 2 described in claim 1, the restructuring described in claim 3 or 4 Application of the host cell in beta galactosidase is prepared any one of expression vector, claim 5 to 7.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113493799A (en) * 2020-04-02 2021-10-12 青岛蔚蓝生物股份有限公司 Aspergillus niger strain for high yield of acid lactase
CN114214304A (en) * 2021-10-20 2022-03-22 江南大学 Beta-galactosidase mutant with improved GOS conversion rate and application thereof

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106682452A (en) * 2017-01-17 2017-05-17 新疆大学 Method for analyzing biological evolution law through entropy change
CN107937365B (en) * 2018-01-15 2020-03-24 江南大学 β -galactosidase mutant and preparation method and application thereof
CN110938614A (en) * 2019-12-06 2020-03-31 宁波希诺亚海洋生物科技有限公司 High-activity β -galactosidase, plasmid for high-throughput screening of same and preparation method thereof
CN111154741B (en) * 2020-02-27 2021-10-29 武汉轻工大学 Thermostable acidic lactase, gene, strain and application
CN112608914A (en) * 2021-01-13 2021-04-06 马金佑 Preparation of high activity beta-galactosidase
CN113484512B (en) * 2021-06-01 2023-06-27 中国农业科学院油料作物研究所 Aflatoxin risk early warning intelligent sensing card and application thereof
CN114395547B (en) * 2022-02-16 2023-06-13 江南大学 Mutant of beta-galactosidase and application of mutant in milk
CN116926047A (en) * 2022-03-31 2023-10-24 北京锦篮基因科技有限公司 Functional beta-galactosidase variant, AAV-mediated human beta-galactosidase expression vector and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102250856A (en) * 2011-06-23 2011-11-23 江南大学 Construction of heat-resistant beta-galactosidase mutant
CN103881994A (en) * 2014-04-14 2014-06-25 中国农业科学院生物技术研究所 Beta-galactosidase mutant with high transglycosylation activity and preparation method and application thereof

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1197872C (en) * 2000-08-11 2005-04-20 中国人民解放军军事医学科学院野战输血研究所 Polypeptide with alpha-galactosidase activity and its coding nucleic acid
CN102776215B (en) * 2012-06-13 2014-08-27 中国农业科学院生物技术研究所 Optimized lactase gene, and secretory expression method and application thereof
CN103031289A (en) * 2012-11-30 2013-04-10 青岛蔚蓝生物集团有限公司 Lactase and recombinant expression engineering bacterium thereof
CN103525793B (en) * 2013-10-29 2015-09-30 中国农业科学院饲料研究所 The alkalescent xylanase of a kind of thermostability improvement and encoding gene thereof and application
CN103627685B (en) * 2013-11-20 2015-07-01 华南理工大学 Higher-activity partial glyceride lipase mutant and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102250856A (en) * 2011-06-23 2011-11-23 江南大学 Construction of heat-resistant beta-galactosidase mutant
CN103881994A (en) * 2014-04-14 2014-06-25 中国农业科学院生物技术研究所 Beta-galactosidase mutant with high transglycosylation activity and preparation method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
许牡丹等: "米曲霉β-半乳糖苷酶催化合成低聚半乳糖的工艺研究", 《食品工业科技》 *
邢肖肖等: "β半乳糖苷酶酶学性质及其在低聚半乳糖合成中的应用", 《生物加工过程》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113493799A (en) * 2020-04-02 2021-10-12 青岛蔚蓝生物股份有限公司 Aspergillus niger strain for high yield of acid lactase
CN114214304A (en) * 2021-10-20 2022-03-22 江南大学 Beta-galactosidase mutant with improved GOS conversion rate and application thereof
CN114214304B (en) * 2021-10-20 2023-07-18 江南大学 Beta-galactosidase mutant with improved GOS conversion rate and application thereof

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