CN105602916B - A kind of DBAT mutant enzymes R363H and its application - Google Patents

Abstract

The present invention discloses a kind of DBAT mutant enzymes R363H and its application.The mutant enzyme is that amino acid sequence is SEQ ID NO：363rd amino acids of 2 DBAT enzymes change into His by Arg；Its amino acid sequence is SEQ ID NO：14.The present invention successfully constructs mutant enzyme R363H.The mutant enzyme improves 12 times on the catalytic efficiency using acetyl coenzyme A than wild-type enzyme, substantially increases the yield of the utilization rate and product Bakating III of acetyl coenzyme A.Meanwhile to reduce production cost, the present invention has found 7 kinds of novel acry radical donors, and mutant enzyme can efficiently utilize these acry radical donors, wherein improving 100 times to the utilization rate of vinyl acetate, can be used to prepare Bakating III as expensive acetyl coenzyme A is substituted.So far, in the research of biosynthesis Bakating III, and find no using non-plant coenzyme class as acry radical donor and embody the research of high usage.

Description

A kind of DBAT mutant enzymes R363H and its application

Technical field

The invention belongs to biomolecule catalytic field, more particularly to a kind of DBAT enzymes (10- deacetylates baccatin III -10 β acetyltransferases) mutant enzyme R363H and its application.

Background technology

Customization enzyme is always the target of biochemist struggle.With the gradual maturation of Protocols in Molecular Biology, to albumen Sequence carries out arbitrary modification, can understand the catalytic mechanism of enzyme and continue design and rational enzyme.This design and rational can be increased Have the property of enzyme, even redesigns a protein.

High catalytic activity enzyme is the key point of important potential to be played in biological industry, and modern protein engineering is ground The main direction studied carefully.The design and rational of new catalyst often expands our understanding to albumen biochemical characteristic, especially that A little mistakes less, the time use rational protein rationality transformation process.Utilize design and rational mutational site technology to day at present The catalytic activity of right zymoprotein, inoxidizability, substrate specificity, thermal stability and improve enzyme allosteric effect etc. carried out at The transformation (Kries et al., 2013) of work(.However, Rational design method be only applicable to three-dimensional structure understand, structure and function Correlation also more clear enzyme.

The thinking of design and rational still mainly gave albumen by Hellinga and Richards in proposition in 1991 at present Increase the viewpoint of new binding site.In practical operation, the first step introduces new functional domain in protein molecular, second step again into Row optimization (Feldmeier et al., 2013；Koga et al.,2012).

Enzyme active center is the core position of enzymatic, and specific conformation not only facilitates Binding Capacity, moreover it is possible to promote Efficient catalytic action.Therefore the transformation for whole protein being compared to enzyme active sites transformation can be more direct, significantly more Promote the catalytic activity (Kiss et al., 2013) of enzyme.But the activated centre of enzyme needs conformation adjusting with high accuracy, it Both it had needed certain flexibility to remove identification substrate, and catalysis reaction had been completed, and certain rigidity was needed to maintain rational conformation, to keep Good biological activity；The activated centre of enzyme remains the delicate balance between flexible and rigidity, arbitrarily dashes forward to activated centre Become, the rapid unstability or inactivation (Hilvert, 2013) of enzyme can be caused.

As scholars are to the gradual perfection of enzymatic structure and functional relationship cognition, the fast development of calculation biology utilizes The method of CAD evolution zymologic property also reaches its maturity.It, can be with Rapid identification using the method for computer simulation The amino acid that can not be mutated and the target site that is possible to promotion enzymatic activity directly related with catalysis, is transformed for protein engineering Quickly guidance is provided.

The present invention studies 10- and the substrate of acetyl Bakating III acetyltransferase is gone to open up by the means of synthetic biology Exhibition and the new enzyme of design and rational establish the new method of synthesis Bakating III.The present invention can be by efficiently preparing Bakating III to pole The big improvement taxol present situation that supply falls short of demand.

Invention content

In order to overcome the disadvantages and deficiencies of the prior art, the purpose of the present invention is primarily to provide a kind of DBAT mutant enzymes R363H.The DBAT mutant enzymes R363H has higher catalytic efficiency than wild type DBAT enzymes.

Another object of the present invention is to provide the applications of above-mentioned DBAT mutant enzymes R363H.

The purpose of the invention is achieved by the following technical solution：

It is SEQ ID NO that a kind of DBAT mutant enzymes R363H of present invention offer, which is amino acid sequence,：The of 2 DBAT enzymes 363 amino acids become histidine (His) from arginine (Arg).

The amino acid sequence of above-mentioned DBAT mutant enzymes R363H is SEQ ID NO：14, the nucleotide sequence of encoding gene For SEQ ID NO：13.

Another aspect of the present invention, which is related to carrying, has coded sequence for SEQ ID NO：13 DBAT mutant enzyme R363H genes Recombinant plasmid, the recombinant plasmid be pET-R363H.

The invention further relates to a kind of engineered strains, carry above-mentioned recombinant plasmid.

The engineered strain is recombinant strains TBL21-R363H, i.e. recombinant plasmid transformed into host e. coli Obtained by (Escherichia coli) BL21.

Applications of the DBAT mutant enzymes R363H in catalysis 10-DAB synthesis Bakating IIIs.Using acetyl coenzyme A as acyl When base donor, the catalytic efficiency of DBAT mutant enzymes R363H ratio DBAT enzymes improves 12 times.

It is supplied with acry radical donor 1, acry radical donor 2, acry radical donor 3, acry radical donor 4, acry radical donor 5, acry radical donor 6 or acyl group When body 7 is acry radical donor, the DBAT mutant enzymes R363H ratio DBAT enzymes have higher catalytic efficiency.

The acry radical donor 1 is vinyl acetate；The acry radical donor 2 is butyl acetate；The acry radical donor 3 For isobutyl acetate；The acry radical donor 4 is sec-butyl acetate；The acry radical donor 5 is pentyl acetate；The acyl group Donor 6 is isoamyl acetate；The acry radical donor 7 is methylvinyl acetate.

The present invention compared with the existing technology, has the following advantages and effect：

The present invention uses homologous modeling, the computer aided technique of molecular docking to carry out design and rational, success to DBAT enzymes Construct mutant enzyme R363H.The mutant enzyme improves 12 times on the catalytic efficiency using acetyl coenzyme A than wild-type enzyme, greatly The yield of the big utilization rate and product Bakating III for improving acetyl coenzyme A.Meanwhile to reduce production cost, the present invention is found 7 kinds of novel acry radical donors, mutant enzyme can efficiently utilize these acry radical donors, wherein the utilization rate to vinyl acetate improves 100 times, it can be used as to substitute acetyl coenzyme A and become synthesis substrate and be used to prepare Bakating III.So far, in biosynthesis bar In the research of card pavilion III, and find no using non-plant coenzyme class as acry radical donor and embody the research of high usage.

Description of the drawings

Fig. 1 is the SDS-PAGE results of each mutant enzyme.

Fig. 2 is the SDS-PAGE results of each mutant enzyme after purification.

Fig. 3 is the result that mutant enzyme D166H catalysis various concentrations acry radical donor 1,5,7 generates Bakating III.

Fig. 4 is the result that mutant enzyme R363H catalysis various concentrations acry radical donor 1,7 generates Bakating III.

Fig. 5 is that mutant enzyme D166HR363H (abbreviation DHRH) catalysis various concentrations acry radical donor 1,6,7 generates Bakating III Result.

Fig. 6 is DHRH, R363H and D166H difference versus wild type DBAT using on acry radical donor 1,2,3,4,5,6 Efficiency comparative schemes.

Specific implementation mode

Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited In this.

In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition.

The acquisition of 1 wild type DBAT of embodiment

The gene order SEQ ID NO for the DBAT enzymes announced on NCBI：1(GenBank：JQ029678.1), rise at it Increase GGAATTC (I restriction enzyme sites of EcoR) before beginning codon ATG, increases GCGGCCGCTTTA after its terminator codon TGA (I restriction enzyme sites of Not) obtains the gene order of the DBAT enzymes with restriction enzyme site.

Double digestion is carried out to the gene of DBAT enzymes with restriction enzyme EcoR I and Not I (Invitrogen companies)；Together When, double digestion is carried out to pET-32a (+) carrier (Invitrogen companies) with restriction enzyme EcoR I and Not I.Using solidifying Gel purification kit purifies digestion products, is used in combination T4DNA ligases (Invitrogen companies) by above-mentioned two digestion products Connection.Connection product is transformed into E.coli DH5 α competent cells (Invitrogen companies), small amount plasmid extraction, enzyme Identification is cut, send sequencing to determine expression vector establishment success after PCR (primer is dbat-F and dbat-R) identifications, is named as pET- DBAT.Recombinant plasmid transformed E.coli BL21 (Invitrogen companies) obtain recombinant strains TBL21-DBAT.It is described The amino acid sequence of DBAT is SEQ ID NO：2(GenBank：AFD32413.1).

dbat-F：5′-GGAATTCATGGCAGGCTCAACAGAAT-3′；(underscore is I restriction enzyme sites of EcoR)

dbat-R：5′-ATTTGCGGCCGCTCAAGGTTTAGTTA-3′；(underscore is I restriction enzyme sites of Not)

The acquisition of embodiment 2DBAT mutant enzymes

(1) full plasmid PCR method structure mutation zymophore is utilized

1, the small amount plasmid extraction of recombinant plasmid pET-DBAT；

2, it using plasmid pET-DBAT as template, amplifies and contains using overlap extension pcr in conjunction with both ends mutant primer There are the dbat plasmids in mutational site, PCR system as follows：

The Primer-F and Primer-R needs are changed accordingly according to different mutant enzymes, specific such as table 1 It is shown.

Primer pair D166AF and D166AR are SEQ ID NO for obtaining DBAT mutant enzyme D166A, amino acid sequence： 4, coding nucleotide sequence is SEQ ID NO：3.

Primer pair D166HF and D166HR are for obtaining DBAT mutant enzymes D166H or D166HR363H；DBAT mutant enzymes The amino acid sequence of D166H is SEQ ID NO：6, coding nucleotide sequence is SEQ ID NO：5；DBAT mutant enzymes The amino acid sequence of D166HR363H is SEQ ID NO：8, coding nucleotide sequence is SEQ ID NO：7.

Primer pair H162AF and H162AR are for obtaining DBAT mutant enzymes H162A or H162AR363H；DBAT mutant enzymes The amino acid sequence of H162A is SEQ ID NO：10, coding nucleotide sequence is SEQ ID NO：9；DBAT mutant enzymes The amino acid sequence of H162AR363H is SEQ ID NO：12, coding nucleotide sequence is SEQ ID NO：11.

Primer pair R363HF and R363HR are for obtaining DBAT mutant enzymes R363H or D166HR363H；DBAT mutant enzymes The amino acid sequence of R363H is SEQ ID NO：14, coding nucleotide sequence is SEQ ID NO：13；DBAT mutant enzymes The amino acid sequence of D166HR363H is SEQ ID NO：8, coding nucleotide sequence is SEQ ID NO：7.

PCR amplification program：98 DEG C of 3min of pre-degeneration；Cycle setting：98 DEG C of 15s are denaturalized, anneal 56 DEG C of 15s, extends 72 DEG C 8min, 20 cycles；Finally extend：72℃10min；After the completion of reaction, utilizeCycle Pure Kit purifying, It measures spare after recycling concentration.

Respectively in negative control and PCR recovery products, I restriction enzymes of Dpn are added, and (10U/ μ L, can be not added with Buffer), 1h is digested in 37 DEG C of metal baths.Utilize DNA gel electrophoresis detection digestion situation.

(2) sequence verification mutant enzyme vector construction success

Postdigestive PCR product is converted into E.coli DH5 α competent cells, coated plate overnight incubation, the quasi- sun of picking Property clone carry out sequence verification, determination successfully obtain mutant enzyme expression vector.

(3) mutant strain is obtained

After digestion is identified and identification is sequenced, recombinant plasmid transformed E.coli BL21 bacterial strains obtain the recombination of DBAT mutant enzymes Express bacterial strain.

Table 1 obtains the primer used in DBAT mutant enzymes

Interpretation of result：

(1) activated centre positive tetrahedron

Activated centre is the core position of enzymatic, and specific conformation not only facilitates Binding Capacity, moreover it is possible to promote enzyme Efficient catalytic.However, the subtle variation of enzyme active center may also can bring the prodigious variation of the property of enzyme (or even to influence enzyme Normal biological function), therefore, transformation active site (Schmid, 2011) is generally avoided as far as possible.

It is studied to vigor of a large amount of different type enzymes in degenerative process, conformation change, scholars early 20th century It was found that the inactivation of enzyme is frequently experienced in before detectable protein entirety conformation change, i.e., enzyme is first to inactivate, rear to be denaturalized.This table It is bright, the conformation fragility of enzyme active center may be cause the deactivated basic reason of enzyme (Benkovic et al., 2003； C.-L.Tsou,1998；C.Tsou,1993).It is therefore believed that under the premise of not destroying the conformation in activated centre, to enzyme Appropriate design and rational, not only may not destructive enzyme activity, it is also possible to change the activity of enzyme.

Wherein, it has been found that after D166 and R363 are mutated into Ala, enzyme activity declines；Molecular docking the result shows that, D166 and R363 forms hydrophobic effect with acetyl coenzyme A.Further rite-directed mutagenesis experiment shows the mutation in the two sites Still it can retain a part of vigor, therefore, this hydrophobic effect not fully determines that can entire enzymatic reaction process carry out.

In addition, it has been found that in raw type DBAT out of office, H162, D166 and R363 phase at a distance from acetyl coenzyme A thioester bond When, and the distance of these three amino acid residues is also close, constitutes positive tetrahedron structure.It is presumed that the two sites can be with It is modified.The acyl group transfer that especially DBAT is catalyzed acetyl coenzyme A is initiated by single His, these are to introduce new catalytic sites Point provides theoretical foundation.

(2) sites D166

(1)D166A

We obtain the three-dimensional structure of D166A mutant first with homologous modeling.Again with acetyl coenzyme A be substrate into Row molecular docking.Ala166 and acetyl coenzyme A form active force after mutation, and it is related that this with Ala belongs to hydrophobic amino acid.The open country and Raw type is compared, and the amino acid in D166A in pocket is slightly changed.Speculate that this is related with active decline.

From distance, the distance of the S atom of His162 and acetyl coenzyme A isThis is than wild type closer to theoretically It says, this is more conducive to His162 and starts nucleophillic attack.And the distance of Ala166 equally becomes close, increases space steric effect, thus it is speculated that These are related with active decline.

(2)D166H

According to the docking of wild type DBAT and D166A as a result, the site D166 (or A166) is closer apart from acetyl coenzyme A, in It is, it is contemplated that, after D166 is mutated into His, two active sites may be formed, it is theoretically anti-it is also possible to participate in It answers.

First with homologous modeling, the three-dimensional structure of D166H is obtained.The skeleton of D166H and WT (wild type DBAT enzymes) RMSD values areShow that mutation does not cause the acute variation of protein structure.Again molecule pair is carried out with acetyl coenzyme A It connects.His166 and acetyl coenzyme A form hydrophobic forces after mutation, and it is related that this with His belongs to hydrophobic amino acid, thus it is speculated that this pole It is possible that changing original zymologic property.Compared with wild type, in D166H, formed pocket amino acid residue numbers and type simultaneously There is no larger change.

From the point of view of space length, in D166H, the distance of His162 and acetyl coenzyme A is caused to beThis and wild type DBAT is compared, and closer distance is more conducive to offensive attack.In addition, the amino acid residue His166 newly introduced is equally auxiliary apart from acetyl Enzyme A is closer, and in other words, the carbonyl of acetyl coenzyme A is possible to simultaneously by the nucleophillic attack of two histidines, this may lead Cause the raising of enzyme activity.In addition, aspartic acid and histidine difference are negatively charged and on schedule, it means that the optimal pH of enzyme may be sent out Changing.

(3) sites R363

(1)R363H

In wild type DBAT, R363 is in solvent channel and acetyl coenzyme A forms hydrophobic forces.And it is dashed forward After becoming Ala, enzyme activity is caused to decline, shows that R363 does not determine that can reaction carry out.It is contemplated that after being mutated into His, Zymologic property may be changed.Carry out homologous modeling first, the RMSD values of the skeleton of R363H and WT areShow to be mutated Do not cause the acute variation of protein structure, then docked with acetyl coenzyme A, His363 is same and acetyl coenzyme A formed it is hydrophobic Active force.It is compared with wild type DBAT, the amino acid in pocket is slightly changed.In addition arginic pKa=12.5, and histidine PKa=6.1, thus it is speculated that mutation may cause enzyme optimal pH variation.

Further investigated His162 and His363 to acetyl coenzyme A distance, respectivelyWithThis shows Acetyl coenzyme A can may result in zymology simultaneously by the attack of two His (His363 and original His162 that newly introduce) The change of matter.

(2)H162AR363H

His162 is the Key residues of DBAT, and enzyme activity is lost completely after being mutated.And the imidazole radicals on His is to start to attack The direct participant hit.We guess, cause enzyme activity to be lost original His162 mutation, then will be in pocket and acetyl coenzyme A Closer R363 residue mutations may result in the recovery of enzyme activity at His.

Equally, first with homologous modeling, the three-dimensional structure of H162AR363H is built, the skeleton of H162AR363H and WT RMSD values areShow that mutation does not cause the acute variation of protein structure.It is docked again with acetyl coenzyme A.Newly Active force is formed between the His363 and acetyl coenzyme A of introducing, shows that this new active site may play catalytic action.And Although the sites Asp162 are mutated into Ala, but the Ala162 newly introduced is also involved in the cohesive process with substrate.

Distances of the His363 apart from acetyl coenzyme A beThis distance enables the imidazole group of His effectively to touch The carbonyl carbon of acetyl coenzyme A further demonstrates that this site may participate in acyl group transfer process.

(3)D166HR363H

From the point of view of the docking result of acetyl coenzyme A and above-mentioned each mutant enzyme, single mutation can't be to entire albumen Three-dimensional structure has big influence, these sites may be the optimal candidate position of rationality transformation enzyme instead.It is contemplated that in DBAT Solvent channel in, while introducing 3 His, and this 3 His distance away from substrate is sufficiently small, is increased by acetyl coenzyme A By the possibility of any one His residue nucleophillic attack, to promote enzyme activity.

Therefore, we construct the bis- mutant enzymes of D166HR363H using homologous model, the skeleton of D166HR363H and WT RMSD values areShow that mutation does not cause the acute variation of protein structure.Itself and acetyl coenzyme A are carried out pair again It connects, tri- amino acid residues of His162, His166 and His363 are and acetyl coenzyme A forms active force.

The distance of His162, His166 and His363 and acetyl coenzyme A is respectivelyWithThis is enough Allow acetyl coenzyme A by the nucleophillic attack of these three amino acid residues.It is therefore believed that for theoretically, D166HR363H The activity of double mutant enzymes can equally increase.

The fermentation verification of 3 mutant enzyme engineered strain TBL-R363H of embodiment and enzyme activity analysis

(1) the fermenting of engineered strain, protein induced and purifying

1, protein induced expression

With recombinant plasmid transformed expressive host E.coli BL21, the recombinant strains TBL21-R363H of acquisition.According to Positive colony bacterium solution is inoculated into 100mL LB liquid mediums (ampicillin that 100 μ g/mL are added) by 1% inoculum concentration, 37 DEG C, 220rpm is cultivated to OD600=0.6~0.8 or so, and final concentration of 1mM derivants IPTG, 28 DEG C of induction 3h is added.

At 4 DEG C, 8000rpm centrifuges 10min and collects thalline, and per 100mL, 20mL phosphate buffers are added in original zymotic fluid (PBS, pH 7.4) is resuspended.It is crushed using ultrasonic cell disintegration instrument to thalline is resuspended, total ultrasonic time 12min, work Make 6s, interval 6s, power 400W, bacterium solution remains ice bath in ultrasonic procedure.4 DEG C are pressed after broken, 12000rpm centrifugations 10min collects supernatant precipitation respectively.The SDS-PAGE protein for carrying out that resolving gel concentration is 8% is precipitated to supernatant respectively Electrophoresis, expression of the detection DBAT mutant enzymes in expression system E.coliBL21.

2, protein purification

After SDS-PAGE is detected, by previous action again inducible protein.Cell pyrolysis liquid is substituted for (50mM NaH2PO4,300mM sodium chloride, 10mM imidazoles, 1mM PMSF, pH8.0).Supernatant is collected by centrifugation in smudge cells.

Chromatographic column (1cm × 10cm) is first installed, column material dosage is 5~10mg/l mL, with cell pyrolysis liquid balance columns Son.The upper clear enzyme solution loading hanging column that centrifugation is obtained rinses nickel column to remove non-specific binding with the buffer solution A of 5 times of column volumes Foreign protein, connect appropriate albumen efflux and mark, spectrophotometric determination albumen concentration, be carried out at the same time SDS-PAGE inspection Survey the elution profile of foreign protein.Town column is thoroughly eluted with 5~10 times of column volumes of buffer B after having eluted, after having eluted The 20% ethyl alcohol flushing of 5~10 times of column volumes of town column is placed on 4 DEG C of preservations.

Protein eluate in collecting pipe by the verification of SDS-PAGE protein electrophoresis is incorporated in super filter tube (10KDa) Carry out 4 DEG C of ultrafiltration concentrations.9mL concentrating buffer solution (20mM acid sodium, 200mM chlorinations are added when eluent is concentrated into about 1mL or so Sodium and 10% glycerine) continue to concentrate, it is repeated twice.Enzyme solution is finally concentrated into 1mL or so and glycerine is added to final concentration of 20%, It is sub-packed in strain preservative tube, is placed in -80 DEG C of preservations.

(2) the enzyme activity analysis of mutant enzyme

1, determination of protein concentration

The measurement of protein content is carried out using Bradford methods.Cardinal principle is when Coomassie brilliant blue protein is in acid item When being combined under part, the maximum light absorption value wavelength of dyeing liquor is transferred to 595nm by 465nm, while its color switchs to blue by brown. By the light absorption value of determination sample and the light absorption value of reference standard albumen, albumen concentration is extrapolated.

(1) preparation of Coomassie Brillant Blue solution

It weighs 50mg Coomassie brilliant blues G250 and is completely dissolved in 50mL ethyl alcohol (95%) to being completely dissolved, 100mLH is added₃P0₄ (85%), distilled water is added to 1L, 4 DEG C are stirred overnight, and brown bottle is kept in dark place spare in 4 DEG C after filter paper filtering.

(2) bovine serum albumin(BSA) (BSA) standard curve

Standard curve is drawn using BSA as standard protein.Prepare 10mg/mL BSA solution, respectively draw 0,0.1,0.5, 0.75,1.0,1.25,1.5,2mL and water is added to complement to 10mL, obtain a concentration of 0,0.1,0.5,0.75,1.0,1.25,1.5, The BSA solution of 2mg/mL is separately added into 2.5mL Coomassie Brillant Blue solutions after respectively drawing 500 μ L, stands at room temperature after mixing 5min, measures absorbance value at 595nm, and reading keeps stablizing in 3~5min.Obtained BSA standard curves.

Linear fit is carried out by experimental data, obtains curvilinear equation：Y=0.1514X-0.0158, R2=0.9946.It measures When testing protein liquid concentration need to be within the scope of 0.1~2mg/mL.

2, enzyme activity determination (be subject to wild type)

(1) recombinase catalyst system and catalyzing is arranged

DBAT enzyme activity units define：Under the conditions of 30 DEG C, 1 μM of 10-DBA of conversion per minute generates the institute of Bakating III The enzyme amount needed is defined as 1U.

According to the variable in different purpose change systems, general system is set as：

Mg²⁺400 μM of 1mM, 10-DAB, 400 μM of acetyl coenzyme A, after purification 10 μ L, Buffer of recombinase be settled to 400 μ L。

(2) Bakating III production quantity is measured

After sample-adding, mixing is gently shaken.1h is reacted at 30 DEG C.After reaction, 400 μ L acetonitriles are added immediately to terminate instead It answers.0.22 μm of filtering with microporous membrane sample carries out HPLC-UV detections.

3, HPLC-UC measures Bakating III (be subject to wild enzyme)

(1) HPLC conditions：Qualitative to Bakating III and 10-DAB progress, quantitative using HPLC-UV, major parameter is as follows： Column temperature：Room temperature；Detection wavelength：227nm；Mobile phase：MeCN：H₂O(50:50)；Flow velocity：1.0mL/min；Sample size：20μL.

(2) preparation of standard solution：Precise Bakating III and 10-DBA standard items are appropriate respectively, methanol dissolving, It is each configured to 1mg/mL mother liquors.

(3) preparation of sample solution：After the completion of catalysis reaction, isometric acetonitrile is added, shakes up, terminates reaction.It uses again 0.45 μm of filtering with microporous membrane, takes filtrate, you can.

(4) standard curve：Precision draws 2.5 μ L of Bakating III standard items mother liquor, 5 μ L, 10 μ L, 15 μ L, 20 μ L, uses chromatography Grade methanol constant volume is to 10mL.After filtering, above-mentioned chromatographic condition is pressed respectively and is measured, replication 3 times is averaged.With standard items The sample size (X) of Bakating III is abscissa, and integrating peak areas value (Y) is ordinate, draws standard curve, and to measured Data carry out linear regression analysis.

Similarly draw 10-DAB standard curves.

(5) investigation of precision：It takes Bakating III standard solution, precision to draw 20 μ L continuous sample introductions 5 times, presses respectively upper It states chromatographic condition to be detected, investigates the precision of method.

(6) sample recovery rate：Oneself knows the sample of content to precision weighing, appropriate that Bakating III reference substance is added, and prepares for examination Product solution is measured by above-mentioned chromatographic condition and assay method, the rate of recovery is calculated according to the following formula respectively：

As a result with analysis：

As shown in Figure 1, wild type DBAT has an impact its solubility expression and expression quantity after amino acid substitution occurs. After identical concentration IPTG inductions, all mutant still are able to normal expression.Wherein, D166A and H162AR363H (are abbreviated as HARH soluble-expression amount) is reduced.By contrast, D166H, R363H and D166HR363H (being abbreviated as DHRH) soluble-expression Amount all increases.The result shows that the sites R363, which are substituted for His or Ala, can increase the soluble-expression of DBAT, the sites D166 are replaced It reduces at Ala soluble-expression amounts, then increases when being substituted for His.

Respectively by these mutation inductions, expression and purifying (Fig. 2), zymologic property measurement is carried out.

The characterization analysis of embodiment 4DBAT enzymes

(1) measurement of kinetic parameter

Using 10-DAB and acetyl coenzyme A as substrate, in pH 7.4,0.5mol/L sodium phosphate buffers, in 30 DEG C of reactions Under the conditions of 1h, according to above-mentioned enzyme activity determination method, mutant enzyme product bar Ka Ting under 2~10mmol/L concentration of substrate is measured The production quantity of III, replication 3 times.Km, Vmax and kcat of enzyme are calculated using Lineweaver-Burk double-reciprocal plot methods.

(2) measurement of mutant enzyme optimal reactive temperature

Appropriate mutant enzyme after purification is taken to be added in sodium phosphate buffer (0.5mol/L, pH 7.4), and different Under the conditions of temperature (10 DEG C~60 DEG C, be spaced 10 DEG C), 6 reaction systems are respectively set, measure its optimal reactive temperature, 10-DAB Concentration with acetyl coenzyme A is 400 μm of ol/L 10-DAB, with reference to its enzyme activity of said determination, replication 3 times.With mutation Enzyme highest enzyme activity measured under condition of different temperatures is 100%, the relative value mapping that other values compare therewith.

(3) measurement of the optimal reaction pH of mutant enzyme

In order to measure the optimum pH of mutant enzyme, we in normal conditions, using the sucrose of 1.2M as 30 DEG C of water-baths of substrate After reacting 60min, the optimal pH of saccharose phosphorylation enzymatic hydrolysis reaction is groped.By mutant enzyme after purification different Enzymatic reaction is carried out under the conditions of pH (4.0~9.0) and measures its optimum pH, and used buffer solution is slow for 0.5mol/L acetate Fliud flushing, pH (4.0~5.0)；0.5mol/L sodium phosphate buffers, pH (6.0~8.0)；0.5mol/L Tris-HCl buffer solutions, PH (8.0~9.0).And with reference to its enzyme activity of said determination, replication 3 times.The highest one group of reaction buffer pH value of enzyme activity Enzyme activity is set as 100% for the optimum pH of the enzyme, and different pH value and enzyme relative activity relation curve are made as standard.

As a result with analysis：

(1) zymologic property of mutant enzyme measures

Using the mutant enzyme of above-mentioned purifying, respectively using acetyl coenzyme A and 10-DAB as substrate, zymologic property measurement is carried out, To study these design and rational sites whether can effect as expected.

(1)D166A

Influences of the mutant D166A to pH.Find out, enzyme optimum response pH is 7, this is consistent with wild type DBAT.But In acid range, vigor decline is more apparent, this and wild type have bigger difference again.Such as in pH=6, wild type The opposite enzyme activity of DBAT is about 70%, and D166A is less than 50%；In pH=8, the opposite enzyme activity and D166A of wild type DBAT Quite.From the point of view of enzyme is to the adaptation angle of environment pH, Asp belongs to acidic amino acid, and Ala belongs to aliphatic amino acid, Asp166 Site mutation is unfavorable for keeping enzymatic activity, reduces adaptability at Ala.It is presumed that nucleophillic attack occurs for His162, need to take by force The hydrogen of alcoholic extract hydroxyl group on 10-DAB is taken, and under acidic environment, this mechanism is very likely interfered, to make enzyme activity reduce.

In addition, we have investigated the optimum temperature of D166A.Show D166 site mutations at Ala, not to thermal adaptability There is apparent influence.

(2)D166H

Further to verify the microenvironment whether sites D166 provide for active residue H162 reaction, it is contemplated that, acid Acidic amino acid Asp, which is substituted for basic amino acid (such as His) equally, can influence adaptability of the enzyme to environment pH.We continue to determine The optimal pH of D166H.The optimal pH of D166H is 7, this is consistent with mutant D166A with wild type DBAT.The difference is that When pH=6, the opposite enzyme activity of D166H is also retained in 80% or more, this is apparently higher than wild type DBAT and D166A；And in pH=8 When, the enzyme activity of D166H but has decreased to 50% or so, this is less than wild type DBAT and D166A.It is therefore believed that The sites D166 not only maintain the higher order conformation of albumen, but also when nucleophillic attack occurs for His162, are provided for one Fixed microenvironment.

In addition, we test the optimal reactive temperature of D166H.As can be seen that its optimum temperature is 30 DEG C, this and it is wild Type DBAT is consistent with D166A.But at a lower temperature, moreover it is possible to higher enzyme activity is kept, we guess, after D166 is mutated into His, More conducively maintain its conformation.

(3)R363H

Influences of the pH to mutant R363H enzyme activity.Find out, the property and wild type that R363H is shown are entirely different, the enzyme Optimum response pH is 3, this and wild type DBAT fall far short.In acid range, the vigor of R363H is higher, and in alkaline ring In border, activity completely disappears, this and wild type are entirely different.

The pKa of His is 6.04, belongs to weak base acidic amino acid.The pKa of Arg is 12.48, belongs to basic amino acid.In alkalinity Under environment, after Arg363 site mutations are at His, it is unfavorable for the protonation of His, so that enzymatic activity substantially reduces.This also says It is bright other than environment pH has an impact enzymatic activity, the amino acid residue (D166 and R363) near enzyme active center is similarly His provides the microenvironment for its protonation, this also determines adaptability of the enzyme to environment pH to a certain extent.

In addition, the influence we have studied temperature to R363H enzymatic activitys.It is found that the optimum temperature of R363H is 30 DEG C, this It is consistent with wild type.But under higher temperature (such as 30~50 DEG C), R363H can also keep 70% or more enzymatic activity, and wild Raw type drops to 50% or less.This shows that R363H possesses higher thermal adaptability.

(4)H162AR363H

According to the acyl group principle of transfer that His is catalyzed, we construct double mutational site mutant H162AR363H, wherein Original active residue H162A is substituted, while introducing new quasi- active residue His in the position of R363.Zymologic property measures, Show that H162AR363H is active, i.e., the active residue newly introduced can normally play a role.We further study pH Influence to its enzyme activity.It is found that the optimal pH of H162AR363H is 6, and under acidic environment (when such as pH=4~6), still With higher vigor.It is compared with His162, Ala162 improves the microenvironment near active residue, increases in higher environment Adaptability under pH.

Influence of the temperature to H162AR363H.It is found that its optimum temperature is 30 DEG C, this and wild type DBAT and R363H are simultaneously There is no difference.

(5)D166HR363H

His residues protonate first, then capture the electronics of carbonyl carbon on acetyl coenzyme A.Using site-directed mutagenesis technique, 2 sites His are introduced in DBAT simultaneously, this and original His162 are together, it is most likely that promote enzymatic activity.

We are only to have studied pH to its active influence.It is found that the Optimal pH of mutant D166HR363H is 6, this and H162AR363H is consistent and R363H (pH=3) and wild type DBAT (pH=7) differences are larger.Speculate that this and His166 residues are same Sample improves reaction microenvironment, illustrates that His162, His166 and His363 form microenvironment each other, His under acidic environment more Easily protonation, to start nucleophillic attack.

Influence of the temperature to D166HR363H enzymatic activitys.It is found that its optimum temperature is 30 DEG C.This and above-mentioned all mutation Body is the same, shows the adaptability that the mutation in these sites cannot change enzyme molecule to temperature.

(6) the zymetology aerodynamic constant of rationality transformation enzyme

According to the acyl group transfer mechanism of DBAT, by utilizing analysis to non-natural acry radical donor, it is believed that acyl group supplies The activity of distance and enzyme of the ketonic oxygen apart from active residue on body presents linearly related.In wild type DBAT, H162, D166 Distance with R363 apart from acetyl coenzyme A is suitable, so this is then just constructed just at the starting point of enzyme design and rational The enzyme of 5 design and rationals such as H162AR363H, D166A, D166H, R363H and D166HR363H.

By great expression, after purification, we carry out the properties such as Michaelis constant, the catalytic efficiency of above-mentioned mutant It characterizes (table 2).The result shows that in H162AR363H, single catalytic site H363 can equally complete to be catalyzed, and show and WT phases When catalytic efficiency.After the active residue of enzyme is increased to two, i.e. simple point mutation D166H and R363H, the efficiency constant ratio of enzyme Wild type increases 11 times and 12 times, and catalytic efficiency is similar.After continuing to increase to three, the efficiency constant of enzyme improves 39 times, Show that the mutation for improving two vigor has addition or synergistic effect, it can further be promoted by, which simply adding up, urges Change activity.

The catalytic efficiency of 2 mutant of table characterizes

(2) non-natural substrates are expanded

(1)H162AR363H

Although acetyl coenzyme A can be used as the acry radical donor of H162AR363H, and its transformation efficiency and WT are suitable.But test Acry radical donor 1, acry radical donor 2, acry radical donor 3, acry radical donor 4, acry radical donor 5, acry radical donor 6 and acry radical donor 7 cannot It is utilized by mutant.Speculate that these compounds generate aldehydes and active residue is caused to be destroyed.

(2)D166H

Mutant D166H is extremely low to the transformation efficiency of acry radical donor 2, acry radical donor 3, acry radical donor 4 and acry radical donor 6, It can hardly be utilized.But there is higher catalytic efficiency (Fig. 3) to acry radical donor 1, acry radical donor 5 and acry radical donor 7.From figure It can be seen that, with being incremented by for additive amount, transformation efficiency also accordingly increases.

(3)R363H

Mutant R363H can using acry radical donor 1, acry radical donor 2, acry radical donor 3, acry radical donor 4, acry radical donor 5, Acry radical donor 6 and acry radical donor 7.Wherein, there is higher catalytic efficiency (Fig. 4) to acry radical donor 1 and acry radical donor 7.

We further analyze Preferences of the mutant R363H to different acyl donor.It is found that R363H supplies acyl group Body 1 and acry radical donor 5 have higher catalytic activity, followed by acry radical donor 7.This is similar to the DBAT of wild type, we Think that the side-chain structure of acry radical donor is more complicated, the efficiency for being converted to product is higher.

(4)D166HR363H

We equally test double-mutant D166HR363H to acry radical donor utilization power.The result shows that the mutant It more can efficiently utilize acry radical donor 1, acry radical donor 2, acry radical donor 3, acry radical donor 4, acry radical donor 5, acry radical donor 6 With acry radical donor 7.Wherein, when the additive amount of acry radical donor 6 is more than 1% and when 7 additive amount of acry radical donor is more than 2.5%, production Amount is gradually reduced (Fig. 5).Show that the mutant has different tolerances to different acry radical donors.

Similarly, we further analyze Preferences of the mutant D166HR363H to different acyl donor.It is found that D166HR363H has higher catalytic activity to acry radical donor 1, acry radical donor 5 and acry radical donor 7.This and wild type DBAT, R363H are similar.

(3) evolution line

Kcat/Km is the catalytic efficiency constant (specificity constant) of enzyme.For same substrate, Kcat/Km values are bigger, indicate that the activity of enzyme is bigger.Using acetyl coenzyme A as acry radical donor, mutant H162AR363H and WT phase When showing that the sites R363 can be as new catalytic site.And D166H and R363H improve about 11 times and 12 than WT respectively Times, this shows that the two sites can be designed as new active residue.D166 and R363 combine, and about 39 are improved than WT Times, this not only shows that design to enzyme and transformation are reasonable, but also has further confirmed that in DBAT catalysis, and His is hair The Key residues of dynamic nucleophillic attack increase the number of His in reasonable range, can increase the ketonic oxygen of acry radical donor by To the chance of the attack of the imidazole group of His, to increase the activity of enzyme.

In addition, our that these design enzyme carry out the test of non-natural acry radical donor.The result shows that H162AR363H pairs Majority cannot be catalyzed, thus it is speculated that this is because these enol esters produce aldehydes, the H363 residues newly introduced be destroyed, to lead Activation declines.And for D166H, R363H and D166HR363H, it improves to varying degrees to these non-naturals The utilization rate (Fig. 6) of substrate.Wherein, wild type is no to acry radical donor 7 active, and these three mutant energy more efficient It utilizes.The utilization rate of acry radical donor 1 has been respectively increased 74,100 and 143 times, thus it is speculated that at the active His of greater number, carbonyl Oxygen is easier by nucleophillic attack.

(4) conclusion

The catalysis activity that enzyme is quickly and effectively promoted by design and rational is always the research hotspot of protein engineering field. It can not only be provided for the structure-function relationship for the enzyme molecule that sharpens understanding can concrete case, while can also be carried for industrial production For outstanding new enzyme source.In this chapter, the method that we utilize molecular docking fast and accurately finds target in enzyme active center Site, i.e. D166 and R363.By site-directed mutagenesis technique, mutant is obtained, and property representation is carried out to mutant.Wherein, Using acetyl coenzyme A as acry radical donor, the catalytic efficiency and wild type of H162AR363H is suitable, and D166H, R363H and The catalytic efficiency of D166HR363H improves 11 times, 12 times and 39 times than wild type.For non-natural substrates, these mutant Different property is shown, majority can equally increase efficiency.The transformation demonstrated again for this for activated centre is one The method of rapid, the efficient evolution enzymatic activity of kind.

In addition, the acylation of DBAT catalysis has its Key residues His162 leading.Do not changing the molten of enzyme molecule Under the premise of agent channel, it can be superimposed the efficiency of enzyme by effective range, only simply increasing His numbers, because This, this example also becomes a great representative case.On the basis of understanding enzymatic structure-functional relationship, by enzymatic activity The transformation at center will provide effective guidance for the following protein engineering.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications, Equivalent substitute mode is should be, is included within the scope of the present invention.

Claims (10)

1. a kind of DBAT mutant enzymes R363H, it is characterised in that：The DBAT mutant enzymes R363H is that amino acid sequence is SEQ ID NO：363rd amino acids of 2 DBAT enzymes change into His by Arg；Its amino acid sequence is SEQ ID NO：14.

2. the gene of coding DBAT mutant enzymes R363H described in claim 1, it is characterised in that：Its nucleotides sequence is classified as SEQ ID NO：13.

3. a kind of recombinant plasmid in host cell inner expression DBAT mutant enzymes R363H described in claim 1.

4. recombinant plasmid according to claim 3, it is characterised in that：The recombinant plasmid carries claim 2 institute The gene stated.

5. a kind of engineered strain, it is characterised in that：The engineered strain carries the recombinant plasmid described in claim 4.

6. engineered strain according to claim 5, it is characterised in that：The engineered strain is TBL21-R363H, place Main bacterium is e. coli bl21.

7. applications of the DBAT mutant enzymes R363H described in claim 1 in catalysis 10-DAB synthesis Bakating IIIs.

8. application according to claim 7, it is characterised in that：Acyl group in the catalysis 10-DAB synthesis Bakating IIIs Donor is acetyl coenzyme A, and the catalytic efficiency ratio DBAT enzymes of the DBAT mutant enzymes R363H improve 12 times.

9. application according to claim 7, it is characterised in that：Acyl group in the catalysis 10-DAB synthesis Bakating IIIs Donor is vinyl acetate, and the utilization rate of the vinyl acetate improves 100 times.

10. application according to claim 7, it is characterised in that：Acyl in the catalysis 10-DAB synthesis Bakating IIIs Base donor is butyl acetate, isobutyl acetate, sec-butyl acetate, pentyl acetate, isoamyl acetate or methylvinyl acetate.