CN109897843A - A kind of recombination echinocandin B deacylase mutant and application - Google Patents

Abstract

It is to obtain the 287th, 527 progress single mutation of amino acid sequence shown in SEQ ID No.2 or double mutation the invention discloses a kind of recombination echinocandin B deacylase mutant and the application in biocatalysis echinocandin B synthesis echinocandin B parent nucleus, the deacylase mutant；The present invention changes the conformation of AuEBDA primary structure and enzyme, improves the catalytic performance of AuEBDA by mutation auebda gene order.Present system analyzes G287Q and R527V and replaces on the active influence of ECB deacylase.The enzyme activity for the double-mutant AuEBDA-G287Q/R527V that the present invention constructs improves 2.5 times compared with parent enzyme wild type AuEBDA enzyme activity, catalytic efficiency k_cat/K_mIncrease by 2.9 times.

Description

A kind of recombination echinocandin B deacylase mutant and application

Technical field

The present invention relates to the actinoplanes utahensis deacylases of an energy efficient catalytic echinocandin B amido bond hydrolysis AuEBDA and its encoding gene auebda.Half is carried out to AuEBDA by bioinformatics, homologous modeling and molecular docking method The AuEBDA mutant enzyme for having robustness using Protocols in Molecular Biology, catalysis are realized in design and rational and molecular modification for the first time The enzyme activity of echinocandin B improves 2.5 times compared with wild type AuEBDA, catalytic efficiency k_cat/K_mImprove 2.9 times.

Technical background

In recent years, fungal infection seriously threatens the life and health of the mankind, especially deep fungal infection.According to statistics, in Europe The 70% of continent fungal infection death is related with Aspergillus, monilial infection.By the death of aggressive monilial infection Rate is up to 30%, and in the case where cannot get timely and effective treatment, lethality is close to 100%.

The echinocandin class drugs such as anidulafungin (anidulafungin, Fig. 1), Caspofungin (caspofungin) are new Generation antifungal drug, belongs to Cyclic lipopeptide antibiotic, is made of cyclic hexapeptide parent nucleus and fatty acid side chain structure two parts.Spine The action target spot of white bacteriums drug is β -1, and 3- glucan synthase hinders fungi thin by the Noncompetition inhibition enzymatic activity The synthesis of cell wall inhibits fungal cell's proliferation.Echinocandin B (echinocandin B, ECB) is Eurotium The secondary metabolite of the fungies such as Aspergilusnidulans, Aspergilusrugulosus, hydrolysis ECB amido bond removal The ECB parent nucleus (echinocandin B nucleus, ECBN) obtained after side-chain structure is synthesis anidulafungin, Caspofungin etc. The critical materials of semisynthetic antibiotics.

Anidulafungin is mainly used for treating candida infection disease, and antifungal activity is than amphotericin B, Fluconazole etc. By force, and its action target spot is single-minded, toxic side effect is low.Anidulafungin synthesis technology is divided into three phases: 1. aspergillus fermentation preparation Echinocandin B raw material；2. the hydrolysis of echinocandin B amido bond obtains ECBN, this step reaction is catalyzed by ECB deacylase to be completed；③ ECBN again with

1-({[4″-(pentyloxy)-1,1′:4′,1″-terphenyl-4-yl]carbonyl}oxy)-1H-1,2,3- Benzotriazole is acylated in dimethylformamide (DMF) to generate product anidulafungin.Wherein, ECB deacylase is catalyzed ECB hydrolysis is the key that entire technique rate-determining steps, and therefore, implementing molecular modification to ECB deacylase has important science Meaning and industrial value.

ECB deacylase is a kind of embrane-associated protein of amido bond between specific for hydrolysis ECBN and side chain in the present invention, By the inactive Precursor Peptide of structural gene coding through post translational processings such as excision signal peptide, spacer peptides at α, β subunit, and just Really it is folded into heterodimer α β.

In early-stage study, we are successful from actinoplanes utahensis (Actinoplanes utahensis) ZJB- The deacylase AuEBDA that can be catalyzed the hydrolysis of ECB acyl side-chain is excavated to one in 08196, its encoding gene auebda is inserted into Then pSET152 plasmid adds erythromycin strong promoter PermE* in gene front end, constructs constitutive expression plasmid pSET152- PermE*-auebda realizes the auebda composing type in muta lead mycillin (Streptomyces lividans) TK24 point Secrete expression.But the ECB deacylase of the muta lead mycillin secreting, expressing of building haves the defects that enzyme activity is low, it is still necessary to pass through egg The catalytic performance of white matter engineering technology raising enzyme.

The present invention determines the space structure of AuEBDA using technological means such as bioinformatics, homologous modeling and molecular simulations As constructing Streptomyces lividansTK24 mutant and analyzing the space structure of AuEBDA, near enzymatic activity pocket Amino acid residue and the entrance of possible substrate, product release channel etc., pass through protein engineering and improve ECB deacylation base Catalysis activity and substrate tolerance of the enzyme to echinocandin B.

The present invention is using the molecular docking of ECB and parent AuEBDA as a result, choosing the amino acid position near enzymatic activity pocket Point, and rite-directed mutagenesis is carried out to it, screening obtains two plants of direct mutation sites, respectively G287Q and R527V.The present invention will G287Q, R527V are combined mutation, obtain the deacylase gene that enzymatic activity is further promoted.

The present invention provides a kind of ECB deacylase renovation technique, building mutated gene, muta lead mycillin expression vector and Engineering bacteria is simultaneously applied to the synthesis of ECBN biocatalysis.

Summary of the invention

Object of the present invention is to the enzyme activity of the ECB deacylase for existing maternal muta lead mycillin secreting, expressing it is low this Defect provides a kind of recombination echinocandin B deacylase mutant for the first time, utilizes the gene recombination bacterium of the deacylase mutant Or enzyme solution synthesizes ECBN as biocatalyst.

The technical solution adopted by the present invention:

The present invention provides a kind of recombination echinocandin B deacylase mutant, and the deacylase mutant is by SEQ The 287th, 527 progress single mutation of amino acid sequence shown in ID No.2 or double mutation obtain；Shown in the SEQ ID No.2 The nucleotides sequence of amino acid AuEBDA encoding gene auebda is classified as shown in SEQ IDNo.1.

Further, the preferably described recombination echinocandin B deacylase mutant is one of following: (1) AuEBDA-G287Q, The 287th glycine mutation of amino acid sequence shown in SEQ ID No.2 is glutamine, and encoding gene is labeled as auebda- G287Q；(2) the 527th arginine of amino acid sequence shown in AuEBDA-R527V, SEQ ID No.2 sports valine, coding Genetic marker is auebda-R527V；(3) amino acid sequence the 287th shown in AuEBDA-G287Q/R527V, SEQ ID No.2 Glycine mutation is glutamine, and the 527th arginine sports valine, and encoding gene is labeled as auebda-G287Q/ R527V。

Deacylase parent AuEBDA and deacylase mutant AuEBDA-G287Q, AuEBDA-R527V of the invention and AuEBDA-G287Q/R527V base sequence overall length is 2364bp, is stopped from the 1st base to the 2364th base, is originated close Numeral is GTG, and terminator codon TGA, G+C content is 72.1%.

The invention further relates to encoding gene, recombinant vector and the engineering bacteria structures of recombination echinocandin B deacylase mutant It builds.It is preferred that recombinant expression carrier pSET152 (BioVector NTCC Inc.Beijing)；The preferred lead-changing penicillium strepto- of host cell Bacterium (Streptomyces lividans) TK24 (building and application study of State of Zhao's tinkling of pieces of jade echinocandin deacylase genetic engineering bacterium [J] Chinese biological engineering magazine 2015,35 (1): 67-74.).Engineering bacteria construction method are as follows: by deacylase mutant code Gene is connect with strong constitutive promoter PermE*, and is connected to expression plasmid pSET152, and conversion enters E.coli JM109 and feels By state, deacylase mutant Escherichia coli cloning vector is obtained, extracts plasmid；The recombinant plasmid transformed of acquisition is entered E.coli ET12567 competence obtains engagement transfer E. coli cloning vector；Engagement transfer imports muta lead mycillin TK24 spore suspension is coated on containing 30 mMs of every liter of MgCl₂MS solid medium on, deacylase mutant gene is whole It is bonded on muta lead mycillin TK24 genome, obtains the engineering bacteria of the mutant gene containing deacylase.

It is white in biocatalysis ECB synthesis spine that the present invention also provides a kind of recombination echinocandin B deacylase mutant Application in rhzomorph B parent nucleus (echinocandin B nucleus, ECBN), the specific application method are as follows: recombination will be contained The deacylase mutant that the supernatant that the fermented culture of the engineering bacteria of echinocandin B deacylase mutant gene obtains extracts Pure enzyme is constituted reaction system by cosolvent of methanol using ECB as substrate for catalyst, in 35 degrees Celsius, 600 rpms of items It is reacted under part, reaction terminates, and reaction solution isolates and purifies, and obtains ECBN.

The catalyst is prepared as follows: the fermented culture of the engineering bacteria of the mutant gene containing deacylase is obtained Supernatant with 1 mole of every liter of KH₂PO₄Adjust pH to 7.5, as crude enzyme liquid；Crude enzyme liquid is successively used microfiltration membranes, ultrafiltration membrane and Nanofiltration membrane is filtered, and takes the filtrate of nanofiltration membrane dialysed overnight in 7.5,20 mMs of every liter of kaliumphosphate buffers of pH； Bio-Rad HighS strong cat ion exchange column (first being balanced with pH7.5,20 mMs of every liter of kaliumphosphate buffers) in permeate, Foreign protein is eluted with 0.1 mole of every liter of KCl aqueous solution again, is finally eluted with 0.2 mole of every liter of KCl aqueous solution, flow velocity is 1 milliliter Per minute, when destination protein peak occurs in Protein Detection device, destination protein is collected, until the collection of destination protein peak is completely, Dialysed overnight in pH7.5,20 mMs of every liter of kaliumphosphate buffers takes permeate, obtains the pure enzyme solution of deacylase, is as catalyzed Agent.

Further, in the reaction system, final concentration of 0.1-1.5 mMs every liter of ECB, 1.00~5.02U/ of enzyme dosage ML, methanol volumetric concentration are 5% (v/v).

Further, the fermentation liquid is prepared as follows: the engineering bacteria of the mutant gene containing deacylase is inoculated into In the seed culture medium of the 25 micrograms per millilitre Pristinamycin containing final concentration, 28 degrees Celsius, 200 rpms are cultivated 3 days, as one Grade seed culture fluid；Be inoculated into the inoculum concentration of volumetric concentration 10% in fresh seed culture medium, 28 degrees Celsius, 200 turns it is every Minute culture 2 days, as secondary seed culture solution；Fresh fermentation medium is inoculated into the inoculum concentration of volumetric concentration 10% In, 30 degrees Celsius, 200 rpms are cultivated 2.5 days, and 4 degrees Celsius, 8000 rpms are centrifuged 10 minutes, obtain supernatant；Institute State seed culture medium composition are as follows: peptone 10g/L, yeast extract 10g/L, NaCl 0.5g/L, KH₂PO₄ 1g/L、 K₂HPO₄·3H₂O1.5g/L uses H₃PO₄PH to 7.0 is adjusted with KOH, solvent is deionized water；The fermentation medium composition: egg White peptone 10g/L, yeast extract 10g/L, glucose 10g/L, NaCl 0.5g/L, KH₂PO₄ 1g/L、K₂HPO₄·3H₂O 1.5g/L uses H₃PO₄PH to 7.0 is adjusted with KOH, solvent is deionized water.The molecular modification of ECB deacylase reported in the literature Mainly in promoter screening, signal peptide screening and increase gene copy number, it is intended to increase the expression quantity and secretory volume of albumen, but imitate Fruit is general.

Compared with prior art, beneficial effect of the present invention is mainly reflected in: the present invention passes through mutation auebda gene order, The conformation for changing AuEBDA primary structure and enzyme, improves the catalytic performance of AuEBDA.Present system analyze G287Q and R527V replaces on the active influence of ECB deacylase.The enzyme activity for the double-mutant AuEBDA-G287Q/R527V that the present invention constructs is 2.5 times of parent enzyme wild type AuEBDA enzyme activity, catalytic efficiency k_cat/K_mImprove 2.9 times.

Detailed description of the invention

Fig. 1 is ECBN and anidulafungin synthesis technology.

Fig. 2 is Streptomyces lividansTK24/pSET152-PermE*-gene expression vector plasmid map. The restriction enzyme site at ECB deacylase mutant gene sequence both ends is EcoR I/Xba I, and plasmid designations are respectively pSET152-PermE*-auebda、pSET152-PermE*-auebda-G287Q、pSET152-PermE*-auebda-R527V And pSET152-PermE*-auebda-G287Q/R527V.

Fig. 3 is positive colony PCR verifying.M: standard nucleic acid molecules amount；Swimming lane 1: host Streptomyceslividans The 16SrRNA gene of TK24；Swimming lane 2:Streptomyces lividans TK24-auebda gene；Swimming lane 3: Streptomyces lividans TK24/pSET152-PermE*-auebda 16SrRNA gene；Swimming lane 4: Streptomyces lividans TK24/pSET152-PermE*-auebda gene；Swimming lane 5:Streptomyces Lividans TK24/pSET152-PermE*-auebda-G287Q mutant 16S rRNA gene；Swimming lane 6: Streptomyces lividans TK24/pSET152-PermE*-auebda-G287Q mutant auebda gene.Swimming lane 7: Streptomyces lividans TK24/pSET152-PermE*-auebda-R527V mutant 16S rRNA gene；Swimming lane 8:Streptomyces lividans TK24/pSET152-PermE*-auebda-R527V mutant auebda gene.Swimming lane 9:Streptomyceslividans TK24/pSET152-PermE*-auebda-G287Q/R527V mutant 16SrRNA base Cause；Swimming lane 10:Streptomyces lividans TK24/pSET152-PermE*-auebda-G287Q/R527V mutant Auebda gene.

Fig. 4 is that WT-AuEBDA, AuEBDA-G287Q, AuEBDA-R527V and AuEBDA-G287Q/R527V catalysis ECB are de- Acyl group efficiency.

Influence of Fig. 5 AuEBDA-G287Q/R527V enzyme dosage to ECB deacylation base synthesis ECBN.

Influence of Fig. 6 substrate additive amount to AuEBDA-G287Q/R527V catalysis ECB deacylation base synthesis ECBN.

Fig. 7 is pH value on the active influence of deacylase mutant AuEBDA-G287Q/R527V, and experimental material is using pure Change enzyme.

Fig. 8 is deacylase mutant AuEBDA-G287Q/R527V pH stability, and experimental material is using purifying enzyme.

Fig. 9 is temperature on the active influence of deacylase mutant AuEBDA-G287Q/R527V, and experimental material is using pure Change enzyme.

Figure 10 is deacylase mutant AuEBDA-G287Q/R527V thermal stability, and experimental material is using purifying enzyme.

Specific embodiment

Involved culture medium prescription is as follows in example:

LB liquid medium: peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, solvent are deionized water, pH 7.0。

LB solid medium: peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar powder 20g/L, solvent are Deionized water uses H₃PO₄PH to 7.0 is adjusted with KOH.

Synthesize V solid medium: soluble starch 20g/L, beef extract 1g/L, K₂HPO₄·3H₂O 0.5g/L、NaCl 0.5g/L、FeSO₄ 0.01g/L、KNO₃ 1.0g/L、MgSO₄0.5g/L, agar 24.0g/L, use H₃PO₄With KOH adjust pH to 7.0, solvent is deionized water.

MS culture medium: soybean powder 20.0g/L, mannitol 20.0g/L, agar 20.0g/L, solvent are deionized water, pH value It is natural.

Seed culture medium: peptone 10g/L, yeast extract 10g/L, NaCl 0.5g/L, KH₂PO₄ 1g/L、 K₂HPO₄·3H₂O 1.5g/L, uses H₃PO₄PH to 7.0 is adjusted with KOH, solvent is deionized water.

Fermentation medium: peptone 10g/L, yeast extract 10g/L, glucose 10g/L, NaCl0.5g/L, KH₂PO₄ 1g/L、K₂HPO₄·3H₂O 1.5g/L, uses H₃PO₄PH to 7.0 is adjusted with KOH, solvent is deionized water.

Embodiment 1: homologous modeling and molecular docking

To the deacylation base that sets out in SWISS-MODEL database (https: //www.swissmodel.expasy.org/) (shown in SEQ ID No.2, encoding gene auadea nucleotides sequence is classified as SEQ ID No.1 institute to the amino acid sequence of enzyme AuEBDA Show) it is compared.The homologous highest model protein of degree is derived from the antibiotic acyltransferase of Acidovoraxsp.MR-S7 MacQ (PDB ID:5C9i_1), amino acid sequence and deacylase AuEBDA homology of setting out are 40.77%.

Program coding is carried out with 9.14 software of Modeller, using MacQ protein sequence as template, constructs AuEBDA egg The three-dimensional space model of Bai Xulie obtains best model.It is subsequent to use ChemDraw Software on Drawing and convert substrate ECB three-dimensional mould Type carries out molecular docking using 4.2 software of AutoDock.Using Pymol1.6.0.0 carry out analysis albumen and substrate molecule it Between three-dimensional structure relationship.

By sequence alignment, Gln259 corresponding to the site Gly287 and the MacQ albumen in AuEBDA albumen is found not Together.Molecular docking is carried out using AutoDock software, it is found that the R527 site side chains near enzyme activity pocket are larger, there are space bits Inhibition effect, therefore, R527 may have more consequence to enzymatic structure.The G287 of AuEBDA albumen is mutated into Q (AuEBDA-G287Q), R527 is mutated into V (AuEBDA-R527V), the G287 of AuEBDA albumen is mutated into Q and is mutated R527 At V (AuEBDA-G287Q/R527V).With 9.14 software of Modeller using MacQ protein sequence as template, building The three-dimensional space model of AuEBDA mutain.

The results show that AuEBDA mutant protein molecules are larger, and original AuEBDA enzyme active center catalytic pocket is smaller, phase For G, the Q in 287 sites has biggish side chain after mutation, has a certain impact to the structure tool of enzyme, so that the knot of enzyme pocket Structure becomes larger, so that reducing substrate enters the space resistance of holoenzyme molecular activity pocket or the space resistance of product release.This Outside, compared to parent's AuEBDA albumen, the valine of R527V mutant is hydrophobic amino acid, to the affine enzyme activity pocket of substrate Positive effect is played, furthermore forms hydrogen bond on S525 and substrate, the increase of hydrogen bond is so that the combination of substrate and enzyme produces actively It influences.

Embodiment 2:PCR method constructs E. coli cloning vector

1, pSET152-auebda plasmid

Will travelling Utah actinomyces Actinoplanes utahensis ZJB-08196 (Wang Y J, Liu L L, Feng Z H,et al.Optimization of media composition and culture conditions for acarbose production by Actinoplanes utahensis ZJB-08196[J].World Journal of Microbiology&Biotechnology, 2011,27 (12): 2759-2766.) in the synthesis V solid medium of non-resistant On carry out scribing line activation, picking single bacterium is fallen in the seed culture medium of non-resistant, 28 degrees Celsius, 200 rpms cultivate 3 days, Travelling Utah actinomyces ZJB-08196 thallus is obtained, genome is extracted.Using the genome of extraction as amplification template, in gene EcoR I and Xba I restriction enzyme site are added in the both ends of sequence, and PCR amplification is carried out using auebda-F and auebda-R as primer (amplification system and condition are shown in Tables 1 and 2), amplified production are the gene auebda that sets out.

Primer sequence is as follows:

Auebda-F:5 '-GAATTCGTGACGTCCTCG-3’

Auebda-R:5 '-TCTAGATCAGCGTCCCCG-3’

Double digestion is carried out to pSET152 plasmid simultaneously, then by the digestion products for gene auebda and the pSET152 plasmid that sets out Connection, conversion enter E.coli JM109, are coated on LB plate (containing 25 micrograms per millilitre Pristinamycins), 37 degrees Celsius Overnight incubation carries out bacterium colony PCR to positive clone molecule, and extracts plasmid order-checking verifying, obtains pSET152-auebda plasmid.

2, plasmid pSET152-PermE*-auebda

EcoR I restriction enzyme site is added at the both ends of strong constitutive promoter PermE* sequence, expands PermE*.Amplification is anti- Condition such as table 3 is answered, primer sequence is as follows:

PermE*-F:5 '-GAATTCGTGCACGCGGTCG-3’

PermE*-R:5 '-GAATTCCGCTGGATCCTAC-3’

Simultaneously to pSET152-auebda plasmid carry out EcoR I single endonuclease digestion, 37 degrees Celsius digestion 6 hours, 65 degrees Celsius guarantor Temperature 10 minutes inactivates EcoR I enzyme.The digestion products of PermE* and pSET152-auebda plasmid T4 ligase is connected again It connects, overnight, conversion imports E.coli JM109 for 37 degrees Celsius of connections, enterprising in LB plate (containing 25 micrograms per millilitre Pristinamycins) Row coating, 37 degrees Celsius of overnight incubations carry out bacterium colony PCR to positive clone molecule, and extract plasmid pSET152-PermE*- Auebda sequence verification chooses gene bacterial strain in the right direction.Plasmid map is shown graphically in the attached figures 2.

3, rite-directed mutagenesis

Using plasmid pSET152-PermE*-auebda as template, using the method for rite-directed mutagenesis respectively by Parent Protease AuEBDA (shown in SEQ ID NO.2) the 287th site glycine rite-directed mutagenesis is at glutamine, the 527th site arginine fixed point It is mutated into valine, and carries out combinatorial mutagenesis, obtains mutant pSET152-PermE*-auebda-G287Q, pSET152- The amplified production of PermE*-auebda-R527V and pSET152-PermE*-auebda-G287Q/R527V, the same table of amplification system 1, amplification condition such as table 4.Primer sequence is as follows:

G287Q-F:5 '-GCGACCCGATCATCCAGATCGGGCACAAC-3 '

G287Q-R:5 '-GTTGTGCCCGATCTGGATGATCGGGT-3 '

R527V-F:5 '-CGCGCAGCCTGGTCACCCGGCTC-3 '

R527V-R:5 '-GAGCCGGGTGACCAGGCTGCGCG-3 '

Amplified production is first subjected to digestion original template with DpnI enzyme, then is converted into E.coli JM109, then micro- containing 25 Gram every milliliter of Pristinamycin resistance LB plate is coated, 37 degrees Celsius of overnight incubations.Bacterium colony PCR is carried out to positive clone molecule, And extract plasmid order-checking verifying.Plasmid pSET152-PermE*-auebda-G287Q, pSET152-PermE*- are obtained respectively Auebda-R527V and pSET152-PermE*-auebda-G287Q/R527V.

PCR praises Phanta Max kit using Novi, and amplification reaction system (μ L) is as follows:

1 amplification reaction system of table

Table 2 expands auebda segment reaction condition

Table 3 expands PermE* segment reaction condition

4 rite-directed mutagenesis amplification reaction condition of table

Embodiment 3: muta lead mycillin expression vector establishment

1, the plasmid conversion in embodiment 2 is entered into E.coli ET12567/pUZ8002, picking positive clone molecule respectively Single bacterium is fallen within containing 10 milliliters of LB culture mediums (containing 50 micrograms per millilitre kanamycins, 25 micrograms per millilitre chloramphenicol, the every milli of 25 micrograms Rise Pristinamycin) in test tube, 37 degrees Celsius, 200 rpms of overnight incubations.Bacterium solution is overnight with the inoculum concentration of volumetric concentration 1% It is transferred in 50 milliliters of LB liquid mediums containing corresponding antibiotic, 37 degrees Celsius, 200 rpms are cultivated 6 to 8 hours, are made OD₆₀₀Reach 0.4~0.6.It is washed 2 times with isometric sterile LB medium, then with 0.5 milliliter of fresh sterile LB medium It suspends, as E.coli suspension, measures E.coli concentration with blood counting chamber.

2, muta lead mycillin spore suspension is prepared.To host's muta lead mycillin Streptomyces LividansTK24 crosses on synthesis V solid medium, 28 degrees Celsius, cultivates 7 days or so, when spore growth is plentiful, It performs the following operation:

(1) 10 milliliters of sterile waters are added on inclined-plane；

(2) spore is gently scraped with spreading rod, makes spore suspension in sterile water；

(3) spore suspension is poured into 10 milliliters of centrifuge tubes containing bead, puts to vibrate 1 minute or so on the oscillator and fills Divide and breaks up spore；

(4) suspension is filtered with the funnel equipped with sterile absorbent cotton, removes mycelium and solid culture based draff；

(5) 6000 rpms of centrifugations precipitate spore in 10 minutes；

(6) supernatant is outwelled, adds a little sterile water that spore is resuspended；

(7) it is 30% glycerine water solution that sterile volumetric concentration, which is added, in the ratio with volume ratio for 1:1, as Streptomyces lividans TK24 spore suspension, final glycerol concentration 15% measure spore concentration with blood counting chamber Afterwards, -80 DEG C of freezings.

3, engagement transfer.500 microlitres of LB of Streptomyces lividans TK24 spore suspension prepared by step 2 Fluid nutrient medium washs 2 times, by spore suspension in 500 microlitres of LB liquid mediums, then 50 C water bath's Heat thermostability 10 minutes, room temperature natural cooling.By the Streptomyces lividans TK24 spore of step 1 escherichia coli suspension and step 2 Sub- suspension is 10 by concentration ratio⁸:10⁶Ratio mixing, 6000 rpms of centrifugations suck most of supernatant after five minutes, to remain Again suspension thalline precipitates liquid, and is coated on MS plate and (contains 30 mMs of every liter of MgCl₂).30 degrees Celsius of cultures 16 to 20 Hour, by 1 milliliter of sterile water uniform fold containing 50 microgram nalidixic acids and 50 microgram Pristinamycins in planar surface.To dry Afterwards, it is cultivated in 28 degrees Celsius of inversions.After 3 to 5 days, screening positive clone.Need to carry out multiple single colonie purifying when necessary.Point Not Huo get the mutant gene of deacylase containing ECB recombination muta lead mycillin: Streptomyces lividans TK24/ pSET152-PermE*-auebda、Streptomyces lividans TK24/pSET152-PermE*-auebda-G287Q、 Streptomyces lividans TK24/pSET152-PermE*-auebda-R527V and Streptomyces lividans TK24/pSET152-PermE*-auebda-G287Q/R527V。

Embodiment 4: the recombination muta lead mycillin positive strain verifying of the mutant gene of deacylase containing ECB

The weight of 4 plants of mutant genes of deacylase containing ECB of acquisition is constructed in picking muta lead mycillin TK24 and example 3 Group muta lead mycillin positive clone molecule single colonie (totally 5 plants), until boiling after twenty minutes in 20 microlitres of sterile waters as template. Using target gene auebda both ends gene order as primer, amplifying target genes.It is lead-changing penicillium strepto- to verify the bacterial strain Bacterium is expanded, segment using the 16SrRNA genetic fragment of Streptomyces lividans TK24 as template design primer For 500bp.In addition to it can not amplify target gene fragment in control group Streptomyces lividans TK24, if remaining Target gene and 16S rRNA fragment gene can be amplified respectively, then are otherwise false positive to construct successfully.Primer sequence is such as Under:

Primer-F:ACGTCCTCGTACATGCGCCTGAAAGC

Primer-R:TCAGCGTCCCCGCTGTGCCACCCGG

S-16S-rRNA-F:TGGCAACACGGGACAAGGGT

S-16S-rRNA-R:CCGTCACTTTCGCTTCTTCC

PCR praises Phanta Max kit using Novi, and amplification reaction system is as follows:

It is as follows to expand auebda segment reaction condition:

It is as follows to expand 500bp segment reaction condition in 16SrRNA:

Then agarose nucleic acid electrophoresis is carried out, electrophoresis result is as shown in Figure 3.And sequence verification is carried out, host is obtained respectively to be become Lead blueness streptomycete and recombination muta lead mycillin expression vector positive strain: Streptomyces lividansTK24/ pSET152-PermE*-auebda、Streptomyces lividans TK24/pSET152-PermE*-auebda-G287Q、 Streptomyces lividans TK24/pSET152-PermE*-auebda-R527V and Streptomyces lividans TK24/pSET152-PermE*-auebda-G287Q/R527V。

Embodiment 5: recombination muta lead mycillin producing enzyme culture

The recombination muta lead mycillin bacterium colony constructed in about 1 square centimeter of size embodiment 4 is selected respectively in 30 milliliters of kinds (contain 25 micrograms per millilitre Pristinamycins) in sub- culture medium, 28 degrees Celsius, 200 rpms are cultivated 3 days, as first order seed Liquid.Be forwarded in 30 milliliters of antibiotic seed culture mediums with the inoculum concentration of volumetric concentration 10%, 28 degrees Celsius, 200 turns it is every Minute culture 2 days, as secondary seed solution.30 milliliters of antibiotic fermentation trainings are forwarded to the inoculum concentration of volumetric concentration 10% It supports in base, 30 degrees Celsius, 200 rpms are cultivated 2.5 days.Fermentation liquid is collected, 8000 rpms of centrifugations must be sent out after ten minutes Ferment stoste, with 1 mole of every liter of KH₂PO₄PH to 7.5 is adjusted, obtains deacylase AuEBDA crude enzyme liquid, deacylase mutant respectively AuEBDA-G287Q crude enzyme liquid, deacylase mutant AuEBDA-R527V crude enzyme liquid, deacylase mutant AuEBDA- G287Q/R527V crude enzyme liquid.

Embodiment 6: recombination muta lead mycillin deacylase purifying

Crossing aperture to deacylase crude enzyme liquid in embodiment 5 respectively is 0.2 micron, the micropore filtering film that diameter is 50 millimeters Mycelium and impurity in fermentation liquid are removed, collects micro-filtrate, micro-filtrate volume is identical with crude enzyme liquid volume at this time.By micro-filtrate mistake 10kDa ultrafiltration membrane is concentrated by ultrafiltration, and as 1/10th of enzyme solution volume remaining in ultrafiltration system to initial volume, is stopped Ultrafiltration, enzyme solution is ignored in ultrafiltration system pipeline, collects ultrafiltrate, ultrafiltrate at this time, which is equivalent to, concentrates 10 times.Ultrafiltration Liquid carry out nanofiltration, nanofiltration specification be 10kDa, 4000 rpms be centrifuged 30 minutes, collect nanofiltration pipe in enzyme solution, with pH7.5, 20 mMs of every liter of kaliumphosphate buffer polishing enzyme solution volumes are to 0.1 times of ultrafiltrate volume, and nanofiltration liquid is equivalent to concentration at this time General times are 100 times, obtain nanofiltration liquid.All steps operate in ice bath.By nanofiltration liquid at 7.5,20 mMs every liter of pH Dialysed overnight, the Bio- balanced in permeate through pH7.5,20 mMs of every liter of kaliumphosphate buffers are carried out in kaliumphosphate buffer Rad HighS strong cation exchange gel column elutes foreign protein with 0.1 mole of every liter of KCl aqueous solution, with 0.2 mole of every liter of KCl Aqueous solution elutes destination protein.Flow velocity is 1 milliliter per minute, when destination protein peak occurs in Protein Detection device, collects purpose egg It is white, until destination protein peak terminates.By elution under destination protein in 7.5,20 mMs of every liter of kaliumphosphate buffers of pH into Row dialysed overnight takes permeate, obtains the pure enzyme solution of deacylase AuEBDA, the pure enzyme of deacylase mutant AuEBDA-G287Q respectively Liquid, the pure enzyme solution of deacylase mutant AuEBDA-R527V, the pure enzyme solution of deacylase mutant AuEBDA-G287Q/R527V.Root According to the concentration of standard Bradford measuring method measurement protein purification.

Embodiment 7: deacylase enzyme activity determination

Enzyme-activity unit definition: under the conditions of 35 degrees Celsius, 7.5 pH, every liter of product ECBN institute of 1 micromole is generated per minute The enzyme amount needed is defined as 1 enzyme-activity unit (U).

The pure enzyme solution of deacylase in Example 6 is catalyst respectively, is 5% with volumetric concentration using ECB as substrate Methanol constitutes reaction system as cosolvent.It takes 4.75 milliliters of pure enzyme solutions to be placed under 35 degrees celsius and keeps the temperature 5 minutes, be added ECB makes final concentration of 1.0 gram per liters, and 250 microlitres of methanol are cosolvent.It is carried out under the conditions of 35 degrees Celsius, 600 rpms anti- It answers, reaction takes 200 microlitres of reaction solutions after ten minutes, and 200 microlitres of anhydrous methanols are added immediately and terminate and react, 12000 rpms Centrifugation 5 minutes, takes supernatant.Using HPLC analysis detection enzyme activity, analysis is repeated 3 times.As a result as shown in Fig. 4, AuEBDA's Specific enzyme activity is 152.3U/mg, the specific enzyme activity of mutant AuEBDA-G287Q is 212.1U/mg, mutant AuEBDA-R527V Specific enzyme activity is 310.1U/mg, the specific enzyme activity of mutant AuEBDA-G287Q/R527V is 380.8U/mg, mutant AuEBDA- The specific enzyme activity of G287Q/R527V improves 2.5 times compared with parent enzyme AuEBDA.

In addition, the present invention also constructs other 5 plant mutant body using molecular docking, be negative mutant, specific building side Method such as Examples 1 to 66, specific enzyme activity is as follows: mutant AuEBDA-G340S is 136.9U/mg, mutant AuEBDA-G340I For 148.3U/mg, mutant AuEBDA-F254G be 136.9U/mg, mutant AuEBDA-F254W is 140.5U/mg and mutation Body AuEBDA-F254Y is 128.2U/mg.Enzyme activity calculation formula is as follows:

Wherein c_ECBNIt is the concentration of ECBN in the reaction system, g/L；React total system, 5mL；N is extension rate；Reaction Time, 10min；4.75mL enzyme preparation additive amount；808 be the molal weight of ECBN, g/mol.

2, ECB and ECBN detection method

ECB and ECBN is detected with the method for HPLC.HPLC model selects Agilent 1260infinity II, USA, purple External detector model 1260DAD WR.Chromatographic column is C18 column (4.6 × 250mm, 5 μm, Welchrom, Shanghai).Column temperature is kept At 40 degrees Celsius.By solvent A (2 gram per liters of ammonium acetate solutions) and solvent B, (2 gram per liters of ammonium acetate solutions, solvent are body to mobile phase Product 60% acetonitrile solution of concentration) composition.C₁₈Column is balanced each other by the flowing that 92% solvent A and 8% solvent B are formed, elution program As follows: from 0 minute to 15 minute, linear gradient is from 92%A → 8%B to 2%A → 98%B；From 15 minutes to 26 minute, linearly Gradient is from 2%A → 98%B to 8%A → 92%B；From 25 minutes to 33 minute, constant ratio is 92%A → 8%B.Mobile phase With 0.8 milliliter of flow velocity operation per minute.The retention time of ECBN and ECB is respectively 6.3 minutes and 26.1 minutes.Using identical Method measurement ECB concentration and draw standard curve, calibration curve equation are as follows: y=4917.0x-33.36, R²=0.999.

Embodiment 8: deacylase mutant AuEBDA-G287Q/R527V catalytic condition optimization

This example determines deacylase mutant AuEBDA-G287Q/R527V in different enzyme amount, different concentration of substrate Under the conditions of, catalysis ECB generates the catalytic efficiency of ECBN.It is reacted under the conditions of 35 degrees Celsius, pH7.5, reaction total system is 5 Milliliter, final concentration of 1.0 gram per liters of ECB, methanol volumetric concentration is 5% (v/v).

Enzyme amount range is 1.00~5.02U/mL, uses 7.5,20 mMs of every liter of kaliumphosphate buffer polishing enzymes of pH respectively Liquid product is to 4.75 milliliters.It is sampled respectively when reacting the different time points between 5 minutes to 12 hours.As a result such as attached drawing 5, when When enzyme additive amount is 5.02U/mL, reaction reaches balance after 7 hours, and the conversion ratio after reaction 12 hours is 80%.Enzyme additive amount When respectively 4.01U/mL and 3.01U/mL, reach balance after reacting 8 hours and 10 hours respectively.When enzyme additive amount is respectively When 2.00U/mL and 1.00U/mL, after reaction 12 hours, conversion ratio is respectively 72% and 56%.

Concentration of substrate selects 0.50,0.75,1.00,1.25 and 1.50 mM every liter respectively, and enzyme additive amount is 5.02U/mL.It is sampled respectively when reacting the different time points between 5 minutes to 12 hours.As a result as shown in fig. 6, when substrate is dense When degree is respectively 0.50 and 0.75 mM every liter, reaction reaches balance behind 1 hour and 7 hours respectively, and conversion ratio is 100%.When ECB concentration is 1.00 mMs every liter or more, reaction reaches balance, the production quantity of ECBN after 8 hours At 0.76 mM or so, and difference is unobvious within 12 hours.

Embodiment 9: deacylase mutant AuEBDA-G287Q/R527V kinetics

This example determines the reactive kinetics parameters of parent's deacylase AuEBDA and its mutant.35 degrees Celsius, It is reacted under the conditions of pH 7.5.The dynamics ginseng of ECB is measured at the ECB (0.1~0.5 mM every liter) of various concentration Number.And Michaelis constant K is estimated by the nonlinear fitting of general initial reaction rate_mWith maximum reaction rate v_maxValue.Rice Family name's equation is as follows:

As a result it is shown in table 5, at optimum conditions, the catalytic efficiency k of parent's deacylase AuEBDA_cat/K_mFor 11.26s^-1·mM^-1, mutant AuEBDA-G287Q catalytic efficiency is 18.38s^-1·mM^-1, mutant AuEBDA-R527V catalytic efficiency is 25.05s^-1·mM^-1, mutant AuEBDA-G287Q/R527V catalytic efficiency is 44.13s^-1·mM^-1.With parent's AuEBDA phase Than the K of each mutant_mReduce, catalytic efficiency k_cat/K_mIncrease separately 0.6 times, 1.2 times and 2.9 times.

The comparison of the stability kinetics parameter of 5 WT-AuEBDA of table and its mutant

Embodiment 10: deacylase mutant AuEBDA-G287Q/R527V reacts pH optimization and pH stability

The pure enzyme solution of the AuEBDA-G287Q/R527V that embodiment 6 is prepared, it is every with 1 mole of every liter of phosphoric acid and 1 mole It rises potassium hydroxide and adjusts pH value to 5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0.The 4.75 of different pH value are taken respectively The pure enzyme solution of milliliter, which is placed under 35 degrees Celsius, keeps the temperature 10 minutes, and ECB, which is added, makes final concentration of 1.0 gram per liters, and 250 microlitres of methanol are to help Solvent, reaction total system are 5 milliliters, are reacted under the conditions of 35 degrees Celsius, 600 rpms, carry out enzyme activity determination (with real Apply example 7).As a result as shown in fig. 7, in pH 7.5, specific enzyme activity highest reaches 380.8U/mg.

The pure enzyme solution of AuEBDA-G287Q/R527V is kept the temperature 0~12 hour under the conditions of pH5.5~9.0 respectively, Mei Geyi The section time is measured enzyme activity.Fig. 8 the results show that deacylase mutant AuEBDA-G287Q/R527V in pH 7.0~9.0 It is with good stability in range.

Embodiment 11: the optimization of deacylase mutant AuEBDA-G287Q/R527V reaction temperature and temperature stability

Pipette the pure enzyme solution of AuEBDA-G287Q/R527V that 4.75mL embodiment 6 is prepared, respectively 20,25,30, 35,40,45 degrees Celsius shaking table reactor inside holding 10 minutes, ECB, which is added, makes final concentration of 1.0 gram per liters, and 250 microlitres of methanol are Cosolvent, reaction total system are 5 milliliters, and 600 rpms are reacted 10 minutes, and enzyme activity is measured by sampling.As a result as shown in figure 9, AuEBDA-G287Q/R527V enzyme activity highest at 35 degrees Celsius, specific enzyme activity reach 376.2U/mg.

Enzyme solution under the conditions of each temperature is kept the temperature 0~12 hour respectively, is measured enzyme activity at regular intervals.Mutant enzyme Temperature stability be shown in Figure 10, AuEBDA-G287Q/R527V has very strong stability at low temperature.Its 4 degrees Celsius, Remain to remain above 95% relative activity under the conditions of pH 7.5 after saving 15 days；It is saved under the conditions of 25 degrees Celsius, 7.5 pH 90% relative activity is still remained above after 12 hours；It saves 12 hours, remains above under the conditions of 35 degrees Celsius, 7.5 pH 80% relative activity.In addition, half-life period difference of the AuEBDA-G287Q/R527V under 45 degrees Celsius and 55 degrees celsius For 28.1 hours and 4.3 hours.

Embodiment 12:EDTA and metal ion are on the active influence of deacylase mutant AuEBDA-G287Q/R527V

The pure enzyme solution of AuEBDA-G287Q/R527V that 4.75mL embodiment 6 is prepared is pipetted, final concentration is successively separately added For 5 mMs of every liter of EDTA, heavy metal ion, such as Li⁺、Zn⁺、Mn²⁺、Mg²⁺、Fe³⁺、Ni²⁺And Cu²⁺, it is placed on 35 degrees Celsius of heat preservations 10 minutes, ECB, which is added, made final concentration of 1.0 gram per liters, and 5% (v/v) methanol is cosolvent, and reaction total system is 5 milliliters, and 600 It is reacted under the conditions of rpm, enzyme activity is measured by sampling after ten minutes.The results are shown in Table 6, AuEBDA-G287Q/R527V Activity do not influenced by EDTA, show that AuEBDA-G287Q/R527V is not metalloenzyme.In addition, AuEBDA-G287Q/R527V Activity do not influenced by metal ion.

The influence of 6 metal ion of table and EDTA to AuEBDA-G287Q/R527V enzyme activity

Sequence table

<110>Zhejiang Polytechnical University

<120>a kind of recombination echinocandin B deacylase mutant and application

<160> 2

<170> SIPOSequenceListing 1.0

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<211> 2364

<212> DNA

<213>actinoplanes utahensis (Actinoplanes utahensis)

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accgcagccg tgccgtcacc cgcttccggc agggaacatg acggcggcta tgcggccctg 120

atccgccggg cctcgtacgg cgtcccgcac atcaccgccg acgacttcgg gagcctcggt 180

ttcggcgtcg ggtacgtgca ggccgaggac aacatctgcg tcatcgccga gagcgtagtg 240

acggccaacg gtgagcggtc gcggtggttc ggtgcgaccg ggccggacga cgccgatgtg 300

cgcagcgacc tcttccaccg caaggcgatc gacgaccgcg tcgccgagcg gctcctcgaa 360

gggccccgcg acggcgtgcg ggcgccgtcg gacgacgtcc gggaccagat gcgcggcttc 420

gtcgccggct acaaccactt cctacgccgc accggcgtgc accgcctgac cgacccggcg 480

tgccgcggca aggcctgggt gcgcccgctc tccgagatcg atctctggcg tacgtcgtgg 540

gacagcatgg tccgggccgg ttccggggcg ctgctcgacg gcatcgtcgc cgcgacgcca 600

cctacagccg ccgggcccgc gtcagccccg gaggcacccg acgccgccgc gatcgccgcc 660

gccctcgacg ggacgagcgc gggcatcggc agcaacgcgt acggcctcgg cgcgcaggcc 720

accgtgaacg gcagcgggat ggtgctggcc aacccgcact tcccgtggca gggcgccgca 780

cgcttctacc ggatgcacct caaggtgccc ggccgctacg acgtcgaggg cgcggcgctg 840

atcggcgacc cgatcatcgg gatcgggcac aaccgcacgg tcgcctggag ccacaccgtc 900

tccaccgccc gccggttcgt gtggcaccgc ctgagcctcg tgcccggcga ccccacctcc 960

tattacgtcg acggccggcc cgagcggatg cgcgcccgca cggtcacggt ccagaccggc 1020

agcggcccgg tcagccgcac cttccacgac acccgctacg gcccggtggc cgtgatgccg 1080

ggcaccttcg actggacgcc ggccaccgcg tacgccatca ccgacgtcaa cgcgggcaac 1140

aaccgcgcct tcgacgggtg gctgcggatg ggccaggcca aggacgtccg ggcgctcaag 1200

gcggtcctcg accggcacca gttcctgccc tgggtcaacg tgatcgccgc cgacgcgcgg 1260

ggcgaggccc tctacggcga tcattcggtc gtcccccggg tgaccggcgc gctcgctgcc 1320

gcctgcatcc cggcgccgtt ccagccgctc tacgcctcca gcggccaggc ggtcctggac 1380

ggttcccggt cggactgcgc gctcggcgcc gaccccgacg ccgcggtccc gggcattctc 1440

ggcccggcga gcctgccggt gcggttccgc gacgactacg tcaccaactc caacgacagt 1500

cactggctgg ccagcccggc cgccccgctg gaaggcttcc cgcggatcct cggcaacgaa 1560

cgcaccccgc gcagcctgcg cacccggctc gggctggacc agatccagca gcgcctcgcc 1620

ggcacggacg gtctgcccgg caagggcttc accaccgccc ggctctggca ggtcatgttc 1680

ggcaaccgga tgcacggcgc cgaactcgcc cgcgacgacc tggtcgcgct ctgccgccgc 1740

cagccgaccg cgaccgcctc gaacggcgcg atcgtcgacc tcaccgcggc ctgcacggcg 1800

ctgtcccgct tcgatgagcg tgccgacctg gacagccggg gcgcgcacct gttcaccgag 1860

ttcgccctcg cgggcggaat caggttcgcc gacaccttcg aggtgaccga tccggtacgc 1920

accccgcgcc gtctgaacac cacggatccg cgggtacgga cggcgctcgc cgacgccgtg 1980

caacggctcg ccggcatccc cctcgacgcg aagctgggag acatccacac cgacagccgc 2040

ggcgaacggc gcatccccat ccacggtggc cgcggggaag caggcacctt caacgtgatc 2100

accaacccgc tcgtgccggg cgtgggatac ccgcaggtcg tccacggaac atcgttcgtg 2160

atggccgtcg aactcggccc gcacggcccg tcgggacggc agatcctcac ctatgcgcag 2220

tcgacgaacc cgaactcacc ctggtacgcc gaccagaccg tgctctactc gcggaagggc 2280

tgggacacca tcaagtacac cgaggcgcag atcgcggccg acccgaacct gcgcgtctac 2340

cgggtggcac agcggggacg ctga 2364

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Val Thr Ser Ser Tyr Met Arg Leu Lys Ala Ala Ala Ile Ala Phe Gly

1 5 10 15

Val Ile Val Ala Thr Ala Ala Val Pro Ser Pro Ala Ser Gly Arg Glu

20 25 30

His Asp Gly Gly Tyr Ala Ala Leu Ile Arg Arg Ala Ser Tyr Gly Val

35 40 45

Pro His Ile Thr Ala Asp Asp Phe Gly Ser Leu Gly Phe Gly Val Gly

50 55 60

Tyr Val Gln Ala Glu Asp Asn Ile Cys Val Ile Ala Glu Ser Val Val

65 70 75 80

Thr Ala Asn Gly Glu Arg Ser Arg Trp Phe Gly Ala Thr Gly Pro Asp

85 90 95

Asp Ala Asp Val Arg Ser Asp Leu Phe His Arg Lys Ala Ile Asp Asp

100 105 110

Arg Val Ala Glu Arg Leu Leu Glu Gly Pro Arg Asp Gly Val Arg Ala

115 120 125

Pro Ser Asp Asp Val Arg Asp Gln Met Arg Gly Phe Val Ala Gly Tyr

130 135 140

Asn His Phe Leu Arg Arg Thr Gly Val His Arg Leu Thr Asp Pro Ala

145 150 155 160

Cys Arg Gly Lys Ala Trp Val Arg Pro Leu Ser Glu Ile Asp Leu Trp

165 170 175

Arg Thr Ser Trp Asp Ser Met Val Arg Ala Gly Ser Gly Ala Leu Leu

180 185 190

Asp Gly Ile Val Ala Ala Thr Pro Pro Thr Ala Ala Gly Pro Ala Ser

195 200 205

Ala Pro Glu Ala Pro Asp Ala Ala Ala Ile Ala Ala Ala Leu Asp Gly

210 215 220

Thr Ser Ala Gly Ile Gly Ser Asn Ala Tyr Gly Leu Gly Ala Gln Ala

225 230 235 240

Thr Val Asn Gly Ser Gly Met Val Leu Ala Asn Pro His Phe Pro Trp

245 250 255

Gln Gly Ala Ala Arg Phe Tyr Arg Met His Leu Lys Val Pro Gly Arg

260 265 270

Tyr Asp Val Glu Gly Ala Ala Leu Ile Gly Asp Pro Ile Ile Gly Ile

275 280 285

Gly His Asn Arg Thr Val Ala Trp Ser His Thr Val Ser Thr Ala Arg

290 295 300

Arg Phe Val Trp His Arg Leu Ser Leu Val Pro Gly Asp Pro Thr Ser

305 310 315 320

Tyr Tyr Val Asp Gly Arg Pro Glu Arg Met Arg Ala Arg Thr Val Thr

325 330 335

Val Gln Thr Gly Ser Gly Pro Val Ser Arg Thr Phe His Asp Thr Arg

340 345 350

Tyr Gly Pro Val Ala Val Met Pro Gly Thr Phe Asp Trp Thr Pro Ala

355 360 365

Thr Ala Tyr Ala Ile Thr Asp Val Asn Ala Gly Asn Asn Arg Ala Phe

370 375 380

Asp Gly Trp Leu Arg Met Gly Gln Ala Lys Asp Val Arg Ala Leu Lys

385 390 395 400

Ala Val Leu Asp Arg His Gln Phe Leu Pro Trp Val Asn Val Ile Ala

405 410 415

Ala Asp Ala Arg Gly Glu Ala Leu Tyr Gly Asp His Ser Val Val Pro

420 425 430

Arg Val Thr Gly Ala Leu Ala Ala Ala Cys Ile Pro Ala Pro Phe Gln

435 440 445

Pro Leu Tyr Ala Ser Ser Gly Gln Ala Val Leu Asp Gly Ser Arg Ser

450 455 460

Asp Cys Ala Leu Gly Ala Asp Pro Asp Ala Ala Val Pro Gly Ile Leu

465 470 475 480

Gly Pro Ala Ser Leu Pro Val Arg Phe Arg Asp Asp Tyr Val Thr Asn

485 490 495

Ser Asn Asp Ser His Trp Leu Ala Ser Pro Ala Ala Pro Leu Glu Gly

500 505 510

Phe Pro Arg Ile Leu Gly Asn Glu Arg Thr Pro Arg Ser Leu Arg Thr

515 520 525

Arg Leu Gly Leu Asp Gln Ile Gln Gln Arg Leu Ala Gly Thr Asp Gly

530 535 540

Leu Pro Gly Lys Gly Phe Thr Thr Ala Arg Leu Trp Gln Val Met Phe

545 550 555 560

Gly Asn Arg Met His Gly Ala Glu Leu Ala Arg Asp Asp Leu Val Ala

565 570 575

Leu Cys Arg Arg Gln Pro Thr Ala Thr Ala Ser Asn Gly Ala Ile Val

580 585 590

Asp Leu Thr Ala Ala Cys Thr Ala Leu Ser Arg Phe Asp Glu Arg Ala

595 600 605

Asp Leu Asp Ser Arg Gly Ala His Leu Phe Thr Glu Phe Ala Leu Ala

610 615 620

Gly Gly Ile Arg Phe Ala Asp Thr Phe Glu Val Thr Asp Pro Val Arg

625 630 635 640

Thr Pro Arg Arg Leu Asn Thr Thr Asp Pro Arg Val Arg Thr Ala Leu

645 650 655

Ala Asp Ala Val Gln Arg Leu Ala Gly Ile Pro Leu Asp Ala Lys Leu

660 665 670

Gly Asp Ile His Thr Asp Ser Arg Gly Glu Arg Arg Ile Pro Ile His

675 680 685

Gly Gly Arg Gly Glu Ala Gly Thr Phe Asn Val Ile Thr Asn Pro Leu

690 695 700

Val Pro Gly Val Gly Tyr Pro Gln Val Val His Gly Thr Ser Phe Val

705 710 715 720

Met Ala Val Glu Leu Gly Pro His Gly Pro Ser Gly Arg Gln Ile Leu

725 730 735

Thr Tyr Ala Gln Ser Thr Asn Pro Asn Ser Pro Trp Tyr Ala Asp Gln

740 745 750

Thr Val Leu Tyr Ser Arg Lys Gly Trp Asp Thr Ile Lys Tyr Thr Glu

755 760 765

Ala Gln Ile Ala Ala Asp Pro Asn Leu Arg Val Tyr Arg Val Ala Gln

770 775 780

Arg Gly Arg

785

Claims (9)

1. a kind of recombination echinocandin B deacylase mutant, it is characterised in that the deacylase mutant is by SEQ ID The 287th, 527 progress single mutation of amino acid sequence shown in No.2 or double mutation obtain.

2. as described in claim 1 recombination echinocandin B deacylase mutant, it is characterised in that the mutant be it is following it One: (1) the 287th glycine mutation of amino acid sequence shown in SEQ ID No.2 is glutamine；(2) shown in SEQ ID No.2 The 527th arginine of amino acid sequence sports valine；(3) the 287th glycine of amino acid sequence shown in SEQ ID No.2 Glutamine is sported, and the 527th arginine sports valine.

3. recombinating the recombination engineering bacteria of echinocandin B deacylase mutation construction described in a kind of claim 1.

4. engineering bacteria as claimed in claim 3, it is characterised in that the engineering bacteria constructs as follows: deacylase is dashed forward Variant encoding gene is connect with strong constitutive promoter PermE*, and is connected to expression plasmid pSET152, and conversion enters E.coli JM109 competence obtains deacylase mutant Escherichia coli cloning vector, extracts plasmid；By the recombinant plasmid transformed of acquisition Into E.coli ET12567 competence, engagement transfer E. coli cloning vector is obtained；Engagement transfer imports lead-changing penicillium chain Mould (Streptomyces lividans) TK24 screens positive recombinant, obtains the engineering of the mutant gene containing deacylase Bacterium.

5. recombination echinocandin B deacylase mutant described in a kind of claim 1 is white in biocatalysis echinocandin B synthesis spine Application in rhzomorph B parent nucleus.

6. application as claimed in claim 5, it is characterised in that the application method are as follows: recombination echinocandin B deacylation will be contained The pure enzyme of deacylase mutant that the supernatant that the fermented culture of the engineering bacteria of base enzyme mutant gene obtains extracts is catalyst, Using echinocandin B as substrate, reaction system is constituted by cosolvent of methanol, is carried out under the conditions of 35 degrees Celsius, 600 rpms Reaction, reaction terminate, and reaction solution isolates and purifies, and obtain echinocandin B parent nucleus.

7. application as claimed in claim 6, it is characterised in that in the reaction system, echinocandin B final concentration of 0.1~ 1.5 mMs every liter, catalyst amount is 1.00~5.02U/mL, and methanol volumetric concentration is 5%.

8. application as claimed in claim 6, it is characterised in that the catalyst is prepared as follows: will be contained deacylase and be dashed forward 1 mole of every liter of KH of the supernatant that the fermented culture of the engineering bacteria of variant gene obtains₂PO₄Adjust pH to 7.5, as crude enzyme liquid； Crude enzyme liquid is successively filtered with microfiltration membranes, ultrafiltration membrane and nanofiltration membrane, takes the filtrate of nanofiltration membrane in pH 7.5,20 mmoles Dialysed overnight in your every liter of kaliumphosphate buffer；In permeate after pH7.5,20 mMs of every liter of kaliumphosphate buffer balances Bio-Rad HighS strong cat ion exchange column, then foreign protein is eluted with 0.1 mole of every liter of KCl aqueous solution, finally with 0.2 mole Every liter of KCl aqueous solution elution, flow velocity are 1 milliliter per minute, destination protein are collected, in pH7.5,20 mMs of every liter of potassium phosphates Dialysed overnight in buffer takes permeate, obtains the pure enzyme solution of deacylase, as catalyst.

9. the use as claimed in claim 7, it is characterised in that the fermentation liquid is prepared as follows: will be contained deacylase and be dashed forward The engineering bacteria of variant gene is inoculated into the seed culture medium of the 25 micrograms per millilitre Pristinamycin containing final concentration, 28 degrees Celsius, 200 Rpm culture 3 days, as first order seed culture solution；Fresh seed culture is inoculated into the inoculum concentration of volumetric concentration 10% In base, 28 degrees Celsius, 200 rpms are cultivated 2 days, as secondary seed culture solution；It is connect with the inoculum concentration of volumetric concentration 10% Kind is into fresh fermentation medium, and 30 degrees Celsius, 200 rpms are cultivated 2.5 days, 4 degrees Celsius, 8000 rpms of centrifugations 10 minutes, obtain supernatant；The seed culture medium composition are as follows: peptone 10g/L, yeast extract 10g/L, NaCl 0.5g/ L、KH₂PO₄ 1g/L、K₂HPO₄·3H₂O 1.5g/L, uses H₃PO₄PH to 7.0 is adjusted with KOH, solvent is deionized water；The hair Ferment culture medium composition: peptone 10g/L, yeast extract 10g/L, glucose 10g/L, NaCl 0.5g/L, KH₂PO₄ 1g/L、 K₂HPO₄·3H₂O 1.5g/L, uses H₃PO₄PH to 7.0 is adjusted with KOH, solvent is deionized water.