CN102559624B - Thermostable mutant of pyrroloquiniline quinine-dependent glucose dehydrogenase and high-throughput screening method thereof - Google Patents

Abstract

The invention relates to a molecular structure of a directed evolution soluble pyrroloquiniline quinine-dependent glucose dehydrogenase (s-GDH) and a method for screening the thermostable mutant by adopting the high-throughput technology. The method is characterized by comprising the following steps: a high-capacity mutation library is constructed through an error-prone polymerase chain reaction (PCR) of the s-GDH, the filter paper soaked with a colored substrate is used to perform in-situ screening on a colony plate, and the colony direct conversion method is combined to obtain the mutant ofthe s-GDH, of which thermostability is increased. Compared with the wild-type s-GDH, the thermostability of the obtained s-GDH mutant is obviously increased. The obtained soluble pyrroloquiniline quinine-dependent glucose dehydrogenase with high thermostability lays the foundation for the further expansion of the application range of glucose dehydrogenase.

Description

A kind of thermostability mutant and high-throughput screening method thereof of pyrroloquinoline quinone dependent form Hexose phosphate dehydrogenase

Invention field

The present invention relates to mutant that a kind of new solubility pyrroloquinoline quinone dependent form Hexose phosphate dehydrogenase thermostability improves and the high-throughput screening method of screening thermostability mutant, belong to the gene engineering technology field of enzyme.

Background technology

Glucose concentration determination is very important in the clinical diagnosis of diabetes and management.At present, China has maturity-onset diabetes patient 9,700 ten thousand people approximately, also has 1.48 hundred million pre-diabetes patients, surmounted western developed country, become the maximum country of global diabetics, diabetes will be one of serious public health problems of following China, and blood sugar monitoring more and more comes into one's own.Blood sugar test detects in the market in autodiagnosis and has occupied nearly 16% the market share, data shows, present global diabetic subject has reached 3.66 hundred million people, and whole world blood sugar test market capacity is 5,000,000,000 dollars, and the diabetic subject of the developed countries such as 90% America and Europe carries out self-monitoring by blood glucose meter.Solubility pyrroloquinoline quinone dependent form Hexose phosphate dehydrogenase (EC 1.1.5.2, be called for short s-GDH), be a kind of can be the oxydo-reductase of gluconic acid with glucose oxidase, so the type enzyme can be used for detecting blood sugar.

Pyrroloquinoline quinone (PQQ) is the third the novel coenzyme behind flavin nucleotide (FMN, FAD) and nicotinamide nucleotide (NAD, NADP).In succession found again afterwards coenzyme topaquinone (TPQ), lysine tymsylqllinone (LTQ), the tryptophantryptophylquinone (TTQ) of some and PQQ similar, these four kinds of coenzyme also are called the quinoid coenzyme, are referred to as quinoprotein enzyme or quinone enzyme take PQQ, TPQ, LTQ, TTQ as the oxydo-reductase of coenzyme.Pyrroloquinoline quinone dependent form Hexose phosphate dehydrogenase is the main quinoprotein take PQQ as coenzyme of a class, because its unique character receives much concern.Quinoprotein extensively is present in the outer pericentral siphon of born of the same parents of gram-negative bacteria, be partially soluble in the outer pericentral siphon of born of the same parents, part links to each other with the outside surface of cytolemma, formed one with cytolemma on the outer pericentral siphon redox system of born of the same parents of respiratory chain phase coupling, the electron acceptor(EA) of transmission electronics to the cytolemma.The feature of the outer pericentral siphon redox system of this born of the same parents is to have shortened the path that electronics transmits, and has reduced substrate oxidation and product and has discharged required energy.

Pyrroloquinoline quinone dependent form Hexose phosphate dehydrogenase is different because of the position of its existence, and being divided into again film mating type is that m-GDH and water-soluble are s-GDH.M-GDH is present in the multiple gram-negative bacteria, such as the bacterium of acinetobacter, Rhodopseudomonas and acetic acid Pseudomonas, is positioned at the cytolemma outside surface of these bacteriums, but the direct oxidation D-Glucose generates lipid or gluconic acid.This enzyme is the quinoprotein that single subunit forms, and only the peptide chain by a 87kD consists of, and has 5 to wear membrane structure, for highly hydrophobic.Its Substratspezifitaet is higher, can the oxidation hexose except glucose, the monose such as pentose, but can the oxidation disaccharides.S-GDH is the solubility quinoprotein that derives from Acinetobacter calcoaceticus, is found in the periplasmic space of Acinetobacter calcoaceticus, only is found in acinetobacter at present.S-GDH is proved to be to be different from the Hexose phosphate dehydrogenase that the second PQQ of m-GDH relies on, and the two homology is very low.This enzyme is homodimer, and the relative molecular mass of each subunit is that 50,000, N end is a signal peptide that is comprised of 24 amino-acid residues, and is rear cut in the pericentral siphon outside being secreted into born of the same parents.This enzyme does not participate in the respiratory chain in the Acinetobacter calcoaceticus, and physiologic function is unknown.This enzyme can the various monose of catalysis and disaccharides be oxidized to corresponding ketone, ketone further is hydrolyzed into glyconic acid again, and s-GDH can offer electronics PMS (phenazine methosulfate), DCIP (2,6 dichlorophenol indophenol), WB (WursterShi is blue) short chain pantothenic acid such as ubiquinone Q1 and ubiquinone Q2 and some artificial electron acceptor(EA) such as N-methyl dimethoxy base phenylpyrazolone methyl sulfate and conductive polymers etc.

S-GDH compares with glucose oxidase with other Hexose phosphate dehydrogenase, has high reactive behavior, is quick on the draw; And coenzyme and protein binding closely need not react in the extra coenzyme (such as NAD) that adds; In addition, oxygen is not disturbed and artificial electron acceptor(EA) specificity widely by oxygen partial pressure can as the acceptor of electronics in the reaction process.These advantages make s-GDH in blood sugar test and microbody aspect important role and the application such as glucose sensor long-pending, fast response.

Although use s-GDH that the advantage of above-mentioned discussion is arranged, its thermostability is relatively poor, is easy to inactivation if in use run into the environment of comparatively high temps.Recently, shortcoming for poor heat stability, Sode etc. have carried out a series of rite-directed mutagenesis at Ser231 with PCR method, select the serine residue on 231 on polypeptide chain side-chain radical surface to replace respectively halfcystine, methionine(Met), leucine, aspartic acid, glutamine, Histidine and Methionin, all make Enzymic stability obtain raising to a certain degree, wherein the most obvious in the prerequisite stability inferior increase that keeps high vigor with Ser231Lys, compare with wild-type, 55 ℃ of transformation period have been improved 8 times, but still keep its constitutive enzyme vigor.According to the principle that improves stability by increasing by two bonding forces between the subunit, the additive method of employing has: 1. adopt glutaraldehyde that C, the N of two subunits end is carried out chemically crosslinked, 55 ℃ of transformation period reach 63min; 2. utilize engineered method, added one section in the centre of two subunits and linked peptide Glu-Leu-Gly-Thr-Arg-Gly-Ser-Ser-Arg-Val-Asp-Leu-Gln, transformation period 16min; 3. the surface in contact at two subunits suddenlys change, and Asn340Phe/Tyr418Ile and Thr416Val/Thr417Val increase the hydrophobic interaction power between subunit, transformation period 16min.Select the serine residue on 415 on sPQQGDH polypeptide chain side-chain radical surface to replace halfcystine, sPQQGDH after the sudden change compares with wild-type, under the prerequisite that keeps its original catalysis activity, 55 ℃ of thermostabilitys have increased by 30 times, 70 ℃ hatch 10min after, Ser415Cys is still keeping 90% vigor.

Orthogenesis belongs to irrational design, become a kind of strategy of external transformation enzyme molecule, need not understand in advance the factors such as the structure of protein, conservative site, catalyst mechanism, but create artificially special evolution conditions, in in-vitro simulated darwinian evolution process, by setting up random mutation library or recombination storehouse, pass through again suitable screening, obtain to have the protein that character obviously improves.The directed enzyme evolution technology has remedied the deficiency of site-directed mutagenesis technique to a certain extent, has very large using value.

The advantageous property of soluble glucose dehydrogenase makes its application at aspects such as blood sugar test and glucose sensors that great potential be arranged, but relatively poor thermostability has then limited its further application, therefore the thermostability that improves it by animal nutrition becomes an important directions in the s-GDH research, the solubility pyrroloquinoline quinone dependent form Hexose phosphate dehydrogenase that obtains high thermal stability has great importance, not only can widen the industrial applicability of enzyme, but and the relation of studying enzyme structure and stability.

Summary of the invention

The object of the invention is to the problem for soluble glucose dehydrogenase poor heat stability on the market, the task of invention is to provide by orthogenesis technology and high-throughout screening method to compare novel s-GDH mutant or the varient that thermostability is significantly improved with wild-type enzyme.

Technical scheme of the present invention: the s-GDH mutant that a kind of thermostability improves, it is characterized in that wild-type s-GDH is positioned at 111,263,270,331, one or more being substituted arranged in the amino acid in 347,416, and wherein these positions are corresponding to the known amino acid position of s-GDH wild-type sequence (SEQ ID NO:2) by Acinetobacter calcoaceticus Acinetobactercalcoaceticus L.M.D.79.41.The high-throughput screening method of s-GDH mutant, make up the heavy body sudden change library of s-GDH by fallibility PCR, the filter paper that utilization is soaked with coloured substrate carries out In situ Screening at the bacterium colony flat board, and obtains s-GDH clone in conjunction with the direct conversion method of bacterium colony, obtains the mutant that the s-GDH thermostability improves.

S-GDH compares with wild-type, and the thermostability of described s-GDH mutant has obtained to significantly improve.The solubility pyrroloquinoline quinone dependent form Hexose phosphate dehydrogenase that obtains high thermal stability is laid a good foundation for the range of application that further enlarges Hexose phosphate dehydrogenase.

The encode polynucleotide sequence of s-GDH mutain of the present invention and the host cell that contains the expression vector of this polynucleotide sequence and contain described expression vector also is the solution of the present invention.

The invention still further relates to the purposes of mutant of the present invention in the glucose detection method, especially by test strip or the glucose detection method of carrying out with biosensor.

Description of drawings

Fig. 1: the expression vector structure iron that contains wild-type or s-GDH mutant

Fig. 2: the agarose gel electrophoresis figure of fallibility PCR

Fig. 3: orthogenesis screening synoptic diagram

Fig. 4: wild-type and s-GDH mutant are 55 ℃ thermal stability analysis

Embodiment

Two kinds of diverse quinoprotein enzyme types with glucose dehydrogenase activity (film mating type and solvable type) are grouped under the EC 1.1.5.2 together, but this enzyme of two types and uncorrelated.

For the present invention, it is relevant only having solvable type Hexose phosphate dehydrogenase s-GDH, and its improved variants hereinafter is discussed.

This area knows that the wild-type dna sequence dna of s-GDH can be separated from the bacterial strain of acinetobacter (Acinetobacter).Prepreerence is to separate from Acinetobacter calcoaceticus bacterial strain LMD79.41.The dna sequence dna of this wild-type s-GDH and peptide sequence provide in SEQ ID NO:1 and SEQ ID NO:2 respectively.

Recombinant vectors of the present invention is expression vector, can use suitable carrier according to the purposes such as needs of Expression and purification, be suitable for colibacillary expression vector or be suitable for respectively the hyphomycetic expression vectors such as other prokaryotic cell prokaryocytes, yeast, mould such as withered grass bacterium such as pET series, pQE is serial, pMAL is serial, pGEX is serial etc.

Transformant of the present invention for example when preparation s-GDH, can use the thread funguss such as other prokaryotic cell prokaryocytes, yeast, mould such as intestinal bacteria, withered grass bacterium etc.Above-mentioned transformant can be prepared the recombinant vectors transfered cell by adopting the known methods such as electroporation, Calcium Chloride Method.In the concrete example as recombinant vectors and transformant, can enumerate the recombinant vectors pET28-gdh that provides among the following embodiment and the conversion e. coli bl21 (DE3) that obtains of carrier thus.

Along with the continuous foundation that creates the gene library method and perfect, set up a mutation library and become very conventional, become very crucial and set up a suitable high-throughput screening method, set up a general high-throughput screening method and be still at present a very large challenge.In the past few years, many screening methods that using value is arranged have appearred.The selection of catalytic activity is screened by phenotype or in solid phase and micropore often.The use of selective pressure often causes in the mutant occurring not affecting the sudden change of catalytic activity.

Therefore the mutant that the object of the invention screening thermostability improves selects thermal treatment as selective pressure.

S-GDH is intracellular enzyme, if will then need to carry out take cell as unit enzyme reaction alive by the direct screening of flat board, preliminary experiment finds that the BL21 of abduction delivering (DE3)/pET28-gdh also shows the activity of enzyme reaction identical with s-GDH, and the reaction substrate the color of the solution is taken off.In addition, express because the pET28a carrier has a small amount of leakage, when forming dull and stereotyped cultivation of screening of activated holoenzyme with the s-GDH that can make expression, the bacterium colony on the flat board gives expression to certain enzymic activity.More than 2 determined to change by substrate colors and carry out In situ Screening.

Because the most of dead of thalline in the heat treatment process, normal inoculation can't be grown, and in order to obtain positive muton, can adopt panel photocopy, and original position PCR or bacterium colony such as directly transform at the method, and the present invention adopts the direct conversion method of bacterium colony.

For the present invention, adopt the direct conversion method of bacterium colony to obtain the clone of positive muton.Thermal treatment is so that the part cellular lysate, and contained plasmid discharges, and with thermal treatment inactivation colony transformation host cell, makes the plasmid of positive muton obtain transforming by directly, thereby obtains its clone's.

After wild-type or s-GDH mutant are expressed in host cell, for separation and purification from culture obtains target protein, can make up known lock out operation and separate.Such as enumerating with ultrasonication, enzymic digestion, saltouing or solvent precipitation, dialysis, centrifugal, ultrafiltration, gel-filtration, SDS-PAGE, ion exchange chromatography, affinity chromatography etc.

The concrete scheme of this experiment is to utilize the His label of wild-type or s-GDH mutant C end amalgamation and expression to carry out affinity purification.

Because the ability of the synthetic PQQ of intestinal bacteria, can only obtain the recombinase protein of sPQQGDH, therefore measuring enzyme needs to add corresponding coenzyme prothetic group to be formed with the holoenzyme of functionally active before living.Enzyme liquid to pure enzyme or thick enzyme adds certain density PQQ and Ca ²⁺, coenzyme and zymoprotein are combined completely namely form activated holoenzyme solution, available enzyme assaying reaction alive.

As mentioned above, s-GDH can utilize multiple electron acceptor(EA), such as PMS (phenazine methosulfate), DCIP (2,6 dichlorophenol indophenol), WB (WursterShi is blue) short chain pantothenic acid such as ubiquinone Q1 and ubiquinone Q2 and some artificial electron acceptor(EA) such as N-methyl dimethoxy base phenylpyrazolone methyl sulfate and conductive polymers.By these electron acceptor(EA)s the activity of s-GDH oxidizing glucose is carried out indirect measurement.

The present invention adopts PMS and DCIP that the s-GDH activity is measured.

The enzyme activity determination principle of s-GDH oxidizing glucose is as follows:

D-Glucose+ox-PMS→D-glucono-δ-lactone+re-PMS

re-PMS+ox-DCIP→ox-PMS+re-DCIP

The DCIP solution of oxidation state is blue, under 600nm light absorption value is arranged, and the DCIP solution of reduction-state is colourless, and is therefore when reaction is carried out, blue for slowly taking off corresponding OD ₆₀₀Value can reduce gradually, passes through OD ₆₀₀The variation of value is carried out measure and calculation to enzyme work.

An enzyme unit alive (U) is under 25 ℃ of conditions, can make the enzyme amount of the glucose oxidase (or 1 μ mol DCIP reduction) of 1 μ mol in 1min.The calculation formula of enzyme unit alive is as follows in the unit volume enzyme solution:

Enzyme activity(U/ml)＝((ΔOD _test-ΔOD _blank)×df×Vt)/(t _min×k×Vs)

t _Min: reaction times length (2min)

Vt: total reaction volume (2ml)

Vs: the enzyme liquid of adding amasss (0.01ml)

The specific absorbance (1/ μ M) of k:DCIP under 600nm

Df: enzyme liquid extension rate

This area knows that stability can relate to different aspect, and difference is storing temp or storage time for example.The Short-range Temperature stress model is usually used in estimating stability.Stability of the present invention is estimated with this Short-range Temperature stress model, therefore is called thermostability.Thermostability is by the unstressed of test sample and have the s-GDH enzymic activity of stress to measure.By unstressed sample activity is set as 100%, the remaining activity after stress is processed can calculate by percentage.For concrete wild-type or s-GDH mutant, select 65 ℃ of transformation period of processing 30 minutes or measuring under 55 ℃.Use these conditions, process 30 minutes remaining its original activity of lower wild-type s-GDH below 15% for 65 ℃, and the s-GDH mutant remaining initial enzyme is lived after implementing this short-term stress model is much higher than 15%.

The sudden change s-GDH thermostability that the present invention obtains at last significantly promotes.Because the thermostability of the lifting of novel s-GDH is so might make the glucose assays in the various Application Areass obtain remarkable technical progress.Modified form s-GDH mutant of the present invention can huge advantage be used for the detection of biological sample glucose, especially by test strip or the detection undertaken by biosensor.

One of main application of improvement s-GDH varient of the present invention is for test strip, with monitoring diabetic subject's glucose level.As mentioned above, s-GDH is the huge advantage that surmounts glucose oxidase to the insensitivity of oxygen.Have now the novel s-GDH varient of the thermostability of raising, can better store and use.

The present invention also comprises the method for using glucose in s-GDH mutant of the present invention detection, mensuration or the measure sample.Especially preferred is to be characterised in that for detection of improving one's methods of glucose in the sample use sensor or test strip are carried out described detection, mensuration or the measurement of glucose.

For detection of or measure sample in the device of the glucose scope of the invention of also serving a ball, described device comprises s-GDH mutant according to the invention and needed other reagent of described measurement.

The s-GDH mutant of the present invention that thermostability improves can also be used for biosensor highly beneficially, with in the on-line monitoring sample or the glucose in the reactor.For this purpose, described s-GDH mutant for example can be used for coated having the insensitive glass electrode of oxygen of osmium complex, to measure more exactly glucose concn.

Further specify the present invention by following steps,

Step 1 fallibility PCR

The present invention adopts GeneMorph II fallibility PCR test kit to suddenly change, and by specification is described, and mutation rate is relevant with amount and PCR cycle number that the template that adopts adds, and the template amount is more, and cycle number is fewer, and mutation rate is lower, otherwise then mutation rate is higher.Take the recombinant vectors pET28-gdh (such as Fig. 1) that contains wild-type s-GDH gene (SEQ ID NO:2) as template, (5 '-GGAAGCTTATACATAGCCTTATAGG-3`) (=SEQ ID NO:6) carries out fallibility PCR as primer take F (5`-GCCCATGGAAAACATTTATTGGCT-3`) (=SEQ ID NO:5) and R, adopt 50 μ L reaction systems as follows

60ng/μL pET28-gdh 5μL；

250ng/ μ L primers F 0.5 μ L;

250ng/ μ L primer R 0.5 μ L;

10 * Mutazyme II damping fluid, 5 μ L;

40mM dNTP 1μL；

Mutazyme II archaeal dna polymerase 1 μ L;

ddH ₂O 37μL。

The PCR condition is: 95 ℃ of denaturation 3min;-95 ℃ of sex change 40s of 32 circulations, 56 ℃ of annealing 40s, 72 ℃ are extended 1.5min; 72 ℃ are extended 10min.The PCR product detects by 0.8% agarose gel electrophoresis, result such as Fig. 2.

Step 2 makes up heavy body sudden change library

Cut the glue recovery and carry out respectively the double digestion reaction of NcoI/HindIII with carrier pET28 utilizing DNA glue to reclaim test kit (AxyPrep DNA Gel Extraction Kit) behind the fallibility PCR product electrophoresis.Enzyme is cut the fragment of rear recovery and is carried out concentration determination by 260nm place absorbance.Be that 10: 1 ratio is mixed with purpose fragment and carrier segments according to molar ratio, utilize the T4 ligase enzyme under 12 ℃ of conditions, to connect 10h.To connect product utilization PCR recovery test kit (AxyPrep PCR cleanup Kit) and carry out the desalination recovery, at last with 10 μ L deionized water dissolvings, exert an influence with the ion pair electricity conversion that prevents damping fluid in the connection procedure.Then the product electricity be will connect and efficient competent cell E.coli BL21 (DE3), electric shock condition: 200 Ω, 25 μ F, 2.5kV transformed.

To restore cell and coat uniformly on the screening flat board, and cultivate 12～16h, treat that bacterium colony grows for 37 ℃.Screening is dull and stereotyped: 0.5% yeast powder (Yeast Extract), 1% Tryptones (Tryptone), 1% sodium-chlor, 10mM calcium chloride, 1.5% agar powder, mixing is cooled to about 55 ℃ at 121 ℃ of sterilization 20min, add the PQQ that final concentration 30 μ g/ml kantlex and 1mM sterilize after filtration, be down flat plate.

Step 3 high flux screening thermostability mutant

Utilize flat board that high-throughout screening is carried out in s-GDH thermostability sudden change library, concrete grammar is as follows:

The filter paper that 1) will newly break a seal carries out cutting by dull and stereotyped big or small diameter in the clean platform of aseptic behaviour for subsequent use;

2) liquid at the bottom of the preparation enzyme reaction, 10mM MOPS (pH 7.0), 20mM glucose, 0.6mM PMS, 0.3mM DCIP mixes;

3) library that will suddenly change placed in 60 ℃ the baking oven thermal treatment 30 minutes, then was cooled to room temperature;

4) then the filter paper complete wetting reaction solution that will cut out carefully is laid on it on flat board in sudden change library and picks up behind the 5s.Enzyme reaction can occur in the part that contacts with thalline on the filter paper, and the white spot of appearance will occur fading; If because thermal treatment causes inactivation of enzyme, then bacterium colony can be dyed to mazarine, and colour-change can not occur in its corresponding filter paper part, and therefore according to this colour-change, the bacterium colony that filter paper is faded then might be the mutational site with high thermal stability;

5) owing to the most of dead of thalline in the heat treatment process, normal inoculation can't be grown, therefore the possible muton that will choose directly joins in the competence of BL21 (DE3), utilizes the heat shock method to transform, (namely adopting direct conversion method to obtain s-GDH clone);

6) bacterium colony that grows behind the twice transformation is carried out the multiple sieve of thermostability checking:

7) will sieve again the mutant strain that the stability that obtains is improved and cultivate, and extract plasmid, by giving birth to worker's order-checking, sequencing result is as follows,

Specifically can be referring to the gene order SEQ ID NO:3 of s-GDH mutant and the aminoacid sequence SEQ ID NO:4 of s-GDH mutant.

Step 4 wild-type or s-GDH mutant efficiently expressing in intestinal bacteria

BL21 (DE3) recombinant bacterium of picking wild-type or s-GDH mutant is in the 5mlLB substratum from the fresh LB flat board, 37 ℃, the 200rpm incubated overnight is as seed liquor, be inoculated in the 30mlLB substratum (30 μ g/mlkanamycin) with 1: 100 ratio, 37 ℃, cultivate under the 200rpm condition.When bacterium liquid is cultured to OD ₆₀₀Reach at 0.8 o'clock, induce with 0.05mM IPTG, then under 25 ℃ of conditions, continue to be cultured to initial stage stationary phase.

Nutrient solution after inducing is abandoned supernatant with the centrifugal 5min of 5,000rpm.50mMTris-Cl (pH 8.0) solution with precooling cleans 2-3 time.Use again the resuspended bacterial sediment of 50mMTris-Cl (pH 8.0) (adding before use 1mM PMSF) of 20ml precooling.Then use the ultrasonic disruption cell, working conditions is power 200W, working hour 5s, and intermittent time 7s carries out 30 times altogether, and cell should remain in the ice bath in this process, in order to avoid the thermally denature of albumen.

With broken liquid sample 13, the centrifugal 10min of 000rpm separates solvable and soluble component.Soluble supernatant is crude enzyme liquid.

The purifying of step 5 wild-type or s-GDH mutant

Owing to the His label is arranged on the pET28 carrier, so wild-type or s-GDH mutant that abduction delivering obtains be the C end with the fusion rotein of His label, can carry out each chromatography purification of parent by Ni-NTA.

The nutrient solution of fermentation ends with the centrifugal 5min of 5,000rpm, is abandoned supernatant and obtained thalline, in the concentrated level pad that is suspended in precooling of 2 times of volumes (the 20mM imidazoles, pH 8.0 for 20mM Tris-Cl, 0.5M NaCl).Then use the ultrasonic disruption cell, working conditions is power 200W, working hour 5s, and intermittent time 7s carries out 30 times altogether, and cell should remain in the ice bath in this process, in order to avoid the thermally denature of albumen.With all broken liquid samples 13, the centrifugal 10min of 000rpm is to separate solvable and soluble component, and supernatant is loading sample to be purified.

First wash pillar with 3-5 times of column volume level pad, flow rate control is at 1ml/min, to A ₂₈₀Till being stabilized in below 0.01.The sample of processing is squeezed into pipeline by pump, the about 0.5ml/min of coutroi velocity; Continue to wash pillar with 3-5 times of column volume level pad, flow rate control is at 1ml/min, to A again ₂₈₀Till being stabilized in below 0.01.

Wash pillar with elutriant I (the 60mM imidazoles, pH 8.0 for 20mM Tris-Cl, 0.5M NaCl), to remove nonspecific foreign protein, the about 0.5ml/min of coutroi velocity continues to be eluted to A ₂₈₀Reach minimum value and remain unchanged; Then use elutriant II (the 200mM imidazoles, pH 8.0 for 20mM Tris-Cl, 0.5M NaCl) flushing pillar, the target protein of wash-out absorption, flow rate control is at 1ml/min.

The elution fraction that will contain target protein utilize the Millipore ultra-filtration centrifuge tube (30,000MWCO) carried out concentrated and purifying again to protein solution behind wild-type or the s-GDH mutant purifying.At first the elute soln of pending wild-type or s-GDH mutant is concentrated, carried out holoenzyme and hatch, then transfer among Sample reservoir (noticing that the rifle head does not touch filter membrane) and the Filtrate vial that packs into; Then utilize ultra-filtration centrifuge tube to wherein unnecessary PQQ, the high imidazoles of high salt removes in Ca2+ and the elution buffer.Then 4 ℃ of lower centrifugal 10min of 5,000rpm add the displacement damping fluid to 5ml again 5, the centrifugal 10min of 000rpm, and triplicate, to the salt ion Ex-all, but last proper extension centrifugation time makes protein solution concentrated, saves backup in 4 ℃.

The enzyme activity assay of step 6 wild-type or s-GDH mutant

Enzyme liquid adding final concentration to pure enzyme or thick enzyme is 600nM PQQ and 1mM Ca ²⁺, and under 25 ℃, hatch 30min, coenzyme and zymoprotein are combined completely namely form activated holoenzyme solution, available enzyme assaying reaction alive.Because PQQ is combined closely with zymoprotein, just be difficult for losing in case form holoenzyme, therefore just do not need to add again coenzyme in follow-up preservation or the reaction.

Reaction totally is 2ml, comprises 10mM MOPS (pH 7.0), 20mM glucose, and 0.6mM PMS, 0.06mMDCIP and 10 μ l are the enzyme liquid of dilution suitably.The reaction reagent mother liquor joined in the 1cm cuvette successively mix, under 25 ℃, react, and timing begins, read the OD that records 1min and 3min by UV-9100 type spectrophotometer ₆₀₀, and with per minute OD ₆₀₀The speed of response of variation about 0.1 is advisable, and the speed of reaction can be by carrying out suitable dilution to enzyme liquid.Easily decompose because the aqueous solution of DCIP itself is met photo-labile, long-time placement also can be faded gradually, therefore replaces 10 μ L enzyme liquid with 10 μ L deionized waters, also records the OD of 1min and 3min ₆₀₀Value is as negative control.

An enzyme unit alive (U) is under 25 ℃ of conditions, can make the enzyme amount of the glucose oxidase (or 1 μ mol DCIP reduction) of 1 μ mol in 1min.The calculation formula of enzyme unit alive is as follows in the unit volume enzyme solution:

Enzyme activity(U/ml)＝((ΔOD_test-ΔOD_blank)×df×Vt)/(t _min×k×Vs)

t _Min: reaction times length (2min)

Vt: total reaction volume (2ml)

Vs: the enzyme liquid of adding amasss (0.01ml)

The specific absorbance (1/ μ M) of k:DCIP under 600nm

Df: enzyme liquid extension rate.

The enzyme activity assay of wild-type or s-GDH mutant, with the fermented liquid ultrasonic disruption cell that cultivation obtains, working conditions is power 200W, working hour 5s, intermittent time 7s carries out 30 times altogether, cell should remain in the ice bath in this process, in order to avoid the thermally denature of albumen.

With broken liquid sample 13, the centrifugal 10min of 000rpm separates solvable and soluble component.Soluble supernatant is crude enzyme liquid.

In thick enzyme, add 600nM PQQ and 1mM Ca ²⁺, and under 25 ℃, hatch 30min, and then in 65 ℃ of water-baths, hatch half an hour, measure residual activity, the result is as follows,

By form as seen, the residual activity of s-GDH mutant is compared and will greatly be improved with the residual activity of wild-type s-GDH.

The thermal stability analysis of step 7 wild-type or s-GDH mutant

The thermostability of s-GDH is to estimate by its transformation period under 55 ℃.The holoenzyme solution of 1ml 3 μ g/ml wild-types or s-GDH mutant is hatched in 55 ℃ of water-baths, and every the 10min sampling, then ice bath 2min at room temperature recovers half an hour, measures its enzyme and lives, and compares with initial enzyme work before the water-bath and is residual activity.When enzyme work is reduced to half that initial enzyme is lived, when namely residual activity was 50%, the used time was the transformation period of s-GDH under 55 ℃.Transformation period, the result was as follows,

By form as seen, the transformation period of wild-type s-GDH is compared with the s-GDH mutant and wants much shorter.

＜110〉converge peaceful bio tech ltd in Deqing, Zhejiang

＜120〉a kind of thermostability mutant and high-throughput screening method thereof of pyrroloquinoline quinone dependent form Hexose phosphate dehydrogenase

<160>6

<210>1

<211>1437

<212>DNA

＜213〉Acinetobacter calcoaceticus (Acinetobactercalcoaceticus L.M.D. 79.41)

<400>1

ATGAATAAAC ATTTATTGGC TAAAATTGCT TTATTAAGCG CTGTTCAGCT 50 AGTTACACTC TCAGCATTTG CTGATGTTCC TCTAACTCCA TCTCAATTTG 100 CTAAAGCGAA ATCAGAGAAC TTTGACAAGA AAGTTATTCT ATCTAATCTA 150 AATAAGCCGC ATGCTTTGTT ATGGGGACCA GATAATCAAA TTTGGTTAAC 200 TGAGCGAGCA ACAGGTAAGA TTCTAAGAGT TAATCCAGAG TCGGGTAGTG 250 TAAAAACAGT TTTTCAGGTA CCAGAGATTG TCAATGATGC TGATGGGCAG 300 AATGGTTTAT TAGGTTTTGC CTTCCATCCT GATTTTAAAA ATAATCCTTA 350 TATCTATATT TCAGGTACAT TTAAAAATCC GAAATCTACA GATAAAGAAT 400 TACCGAACCA AACGATTATT CGTCGTTATA CCTATAATAA ATCAACAGAT 450 ACGCTCGAGA AGCCAGTCGA TTTATTAGCA GGATTACCTT CATCAAAAGA 500 CCATCAGTCA GGTCGTCTTG TCATTGGGCC AGATCAAAAG ATTTATTATA 550 CGATTGGTGA CCAAGGGCGT AACCAGCTTG CTTATTTGTT CTTGCCAAAT 600 CAAGCACAAC ATACGCCAAC TCAACAAGAA CTGAATGGTA AAGACTATCA 650 CACCTATATG GGTAAAGTAC TACGCTTAAA TCTTGATGGA AGTATTCCAA 700 AGGATAATCC AAGTTTTAAC GGGGTGGTTA GCCATATTTA TACACTTGGA 750 CATCGTAATC CGCAGGGCTT AGCATTCACT CCAAATGGTA AATTATTGCA 800 GTCTGAACAA GGCCCAAACT CTGACGATGA AATTAACCTC ATTGTCAAAG 850 GTGGCAATTA TGGTTGGCCG AATGTAGCAG GTTATAAAGA TGATAGTGGC 900 TATGCTTATG CAAATTATTC AGCAGCAGCC AATAAGTCAA TTAAGGATTT 950 AGCTCAAAAT GGAGTAAAAG TAGCCGCAGG GGTCCCTGTG ACGAAAGAAT 1000 CTGAATGGAC TGGTAAAAAC TTTGTCCCAC CATTAAAAAC TTTATATACC 1050 GTTCAAGATA CCTACAACTA TAACGATCCA ACTTGTGGAG AGATGACCTA 1100 CATTTGCTGG CCAACAGTTG CACCGTCATC TGCCTATGTC TATAAGGGCG 1150 GTAAAAAAGC AATTACTGGT TGGGAAAATA CATTATTGGT TCCATCTTTA 1200 AAACGTGGTG TCATTTTCCG TATTAAGTTA GATCCAACTT ATAGCACTAC 1250 TTATGATGAC GCTGTACCGA TGTTTAAGAG CAACAACCGT TATCGTGATG 1300 TGATTGCAAG TCCAGATGGG AATGTCTTAT ATGTATTAAC TGATACTGCC 1350 GGAAATGTCC AAAAAGATGA TGGCTCAGTA ACAAATACAT TAGAAAACCC 1400 AGGATCTCTC ATTAAGTTCA CCTATAAGGC TAAGTAA 1437

<210>2

<211>478

<212>PRT

＜213〉Acinetobacter calcoaceticus (Acinetobactercalcoaceticus L.M.D. 79.41)

<400>2

Met Asn Lys His Leu Leu Ala Lys Ile Ala Leu Leu Ser Ala Val Gln Leu Val Thr Leu

5 10 15 20

Ser Ala Phe Ala Asp Val Pro Leu Thr Pro Ser Gln Phe Ala Lys Ala Lys Ser Glu Asn

25 30 35 40

Phe Asp Lys Lys Val Ile Leu Ser Asn Leu Asn Lys Pro His Ala Leu Leu Trp Gly Pro

45 50 55 60

Asp Asn Gln Ile Trp Leu Thr Glu Arg Ala Thr Gly Lys Ile Leu Arg Val Asn Pro Glu

65 70 75 80

Ser Gly Ser Val Lys Thr Val Phe Gln Val Pro Glu Ile Val Asn Asp Ala Asp Gly Gln

85 90 95 100

Asn Gly Leu Leu Gly Phe Ala Phe His Pro Asp Phe Lys Asn Asn Pro Tyr Ile Tyr Ile

105 110 115 120

Ser Gly Thr Phe Lys Asn Pro Lys Ser Thr Asp Lys Glu Leu Pro Asn Gln Thr Ile Ile

125 130 135 140

Arg Arg Tyr Thr Tyr Asn Lys Ser Thr Asp Thr Leu Glu Lys Pro Val Asp Leu Leu Ala

145 150 155 160

Gly Leu Pro Ser Ser Lys Asp His Gln Ser Gly Arg Leu Val Ile Gly Pro Asp Gln Lys

165 170 175 180

Ile Tyr Tyr Thr Ile Gly Asp Gln Gly Arg Asn Gln Leu Ala Tyr Leu Phe Leu Pro Asn

185 190 195 200

Gln Ala Gln His Thr Pro Thr Gln Gln Glu Leu Asn Gly Lys Asp Tyr His Thr Tyr Met

205 210 215 220

Gly Lys Val Leu Arg Leu Asn Leu Asp Gly Ser Ile Pro Lys Asp Asn Pro Ser Phe Asn

225 230 235 240

Gly Val Val Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro Gln Gly Leu Ala Phe Thr

245 250 255 260

Pro Asn Gly Lys Leu Leu Gln Ser Glu Gln Gly Pro Asn Ser Asp Asp Glu Ile Asn Leu

265 270 275 280

Ile Val Lys Gly Gly Asn Tyr Gly Trp Pro Asn Val Ala Gly Tyr Lys Asp Asp Ser Gly

285 290 295 300

Tyr Ala Tyr Ala Asn Tyr Ser Ala Ala Ala Asn Lys Ser Ile Lys Asp Leu Ala Gln Asn

305 310 315 320

Gly Val Lys Val Ala Ala Gly Val Pro Val Thr Lys Glu Ser Glu Trp Thr Gly Lys Asn

325 330 335 340

Phe Val Pro Pro Leu Lys Thr Leu Tyr Thr Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro

345 350 355 360

Thr Cys Gly Glu Met Thr Tyr Ile Cys Trp Pro Thr Val Ala Pro Ser Ser Ala Tyr Val

365 370 375 380

Tyr Lys Gly Gly Lys Lys Ala Ile Thr Gly Trp Glu Asn Thr Leu Leu Val Pro Ser Leu

385 390 395 400

Lys Arg Gly Val Ile Phe Arg Ile Lys Leu Asp Pro Thr Tyr Ser Thr Thr Tyr Asp Asp

405 410 415 420

Ala Val Pro Met Phe Lys Ser Asn Asn Arg Tyr Arg Asp Val Ile Ala Ser Pro Asp Gly

425 430 435 440

Asn Val Leu Tyr Val Leu Thr Asp Thr Ala Gly Asn Val Gln Lys Asp Asp Gly Ser Val

445 450 455 460

Thr Asn Thr Leu Glu Asn Pro Gly Ser Leu Ile Lys Phe Thr Tyr Lys Ala Lys

465 470 475

<210>3

<211>1437

<212>DNA

＜213〉Acinetobacter calcoaceticus (Acinetobactercalcoaceticus L.M.D. 79.41)

<400>3

ATGAATAAAC ATTTATTGGC TAAAATTGCT TTATTAAGCG CTGTTCAGCT 50 AGTTACACTC TCAGCATTTG CTGATGTTCC TCTAACTCCA TCTCAATTTG 100 CTAAAGCGAA ATCAGAGAAC TTTGACAAGA AAGTTATTCT ATCTAATCTA 150 AATAAGCCGC ATGCTTTGTT ATGGGGACCA GATAATCAAA TTTGGTTAAC 200 TGAGCGAGCA ACAGGTAAGA TTCTAAGAGT TAATCCAGAG TCGGGTAGTG 250 TAAAAACAGT TTTTCAGGTA CCAGAGATTG TCAATGATGC TGATGGGCAG 300 AATGGTTTAT TAGGTTTTGC CTGCCATCCT GATTTTAAAA ATAATCCTTA 350 TATCTATATT TCAGGTACAT TTAAAAATCC GAAATCTACA GATAAAGAAT 400 TACCGAACCA AACGATTATT CGTCGTTATA CCTATAATAA ATCAACAGAT 450 ACGCTCGAGA AGCCAGTCGA TTTATTAGCA GGATTACCTT CATCAAAAGA 500 CCATCAGTCA GGTCGTCTTG TCATTGGGCC AGATCAAAAG ATTTATTATA 550 CGATTGGTGA CCAAGGGCGT AACCAGCTTG CTTATTTGTT CTTGCCAAAT 600 CAAGCACAAC ATACGCCAAC TCAACAAGAA CTGAATGGTA AAGACTATCA 650 CACCTATATG GGTAAAGTAC TACGCTTAAA TCTTGATGGA AGTATTCCAA 700 AGGATAATCC AAGTTTTAAC GGGGTGGTTA GCCATATTTA TACACTTGGA 750 CATCGTAATC CGCAGGGCTT AGCATTCACT CCAAATTGTA AATTATTGCA 800 GTCTGAACAC GGCCCAAACT CTGACGATGA AATTAACCTC ATTGTCAAAG 850 GTGGCAATTA TGGTTGGCCG AATGTAGCAG GTTATAAAGA TGATAGTGGC 900 TATGCTTATG CAAATTATTC AGCAGCAGCC AATAAGTCAA TTAAGGATTT 950 AGCTCAAAAT GGAGTAAAAG TAGCCGCAGG GGTCCCTGTG ATGAAAGAAT 1000 CTGAATGGAC TGGTAAAAAC TTTGTCCCAC CATTAAAACC TTTATATACC 1050 GTTCAAGATA CCTACAACTA TAACGATCCA ACTTGTGGAG AGATGACCTA 1100 CATTTGCTGG CCAACAGTTG CACCGTCATC TGCCTATGTC TATAAGGGCG 1150 GTAAAAAAGC AATTACTGGT TGGGAAAATA CATTATTGGT TCCATCTTTA 1200 AAACGTGGTG TCATTTTCCG TATTAAGTTA GATCCAACTT ATAGCATTAC 1250 TTATGATGAC GCTGTACCGA TGTTTAAGAG CAACAACCGT TATCGTGATG 1300 TGATTGCAAG TCCAGATGGG AATGTCTTAT ATGTATTAAC TGATACTGCC 1350 GGAAATGTCC AAAAAGATGA TGGCTCAGTA ACAAATACAT TAGAAAACCC 1400 AGGATCTCTC ATTAAGTTCA CCTATAAGGC TAAGTAA 1437

<210>4

<211>478

<212>PRT

＜213〉Acinetobacter calcoaceticus (Acinetobactercalcoaceticus L.M.D. 79.41)

<400>4

Met Asn Lys His Leu Leu Ala Lys Ile Ala Leu Leu Ser Ala Val Gln Leu Val Thr Leu

5 10 15 20

Ser Ala Phe Ala Asp Val Pro Leu Thr Pro Ser Gln Phe Ala Lys Ala Lys Ser Glu Asn

25 30 35 40

Phe Asp Lys Lys Val Ile Leu Ser Asn Leu Asn Lys Pro His Ala Leu Leu Trp Gly Pro

45 50 55 60

Asp Asn Gln Ile Trp Leu Thr Glu Arg Ala Thr Gly Lys Ile Leu Arg Val Asn Pro Glu

65 70 75 80

Ser Gly Ser Val Lys Thr Val Phe Gln Val Pro Glu Ile Val Asn Asp Ala Asp Gly Gln

85 90 95 100

Asn Gly Leu Leu Gly Phe Ala Phe His Pro Glu Phe Lys Asn Asn Pro Tyr Ile Tyr Ile

105 110 115 120

Ser Gly Thr Phe Lys Asn Pro Lys Ser Thr Asp Lys Glu Leu Pro Asn Gln Thr Ile Ile

125 130 135 140

Arg Arg Tyr Thr Tyr Asn Lys Ser Thr Asp Thr Leu Glu Lys Pro Val Asp Leu Leu Ala

145 150 155 160

Gly Leu Pro Ser Ser Lys Asp His Gln Ser Gly Arg Leu Val Ile Gly Pro Asp Gln Lys

165 170 175 180

Ile Tyr Tyr Thr Ile Gly Asp Gln Gly Arg Asn Gln Leu Ala Tyr Leu Phe Leu Pro Asn

185 190 195 200

Gln Ala Gln His Thr Pro Thr Gln Gln Glu Leu Asn Gly Lys Asp Tyr His Thr Tyr Met

205 210 215 220

Gly Lys Val Leu Arg Leu Asn Leu Asp Gly Ser Ile Pro Lys Asp Asn Pro Ser Phe Asn

225 230 235 240

Gly Val Val Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro Gln Gly Leu Ala Phe Thr

245 250 255 260

Pro Asn Cys Lys Leu Leu Gln Ser Glu His Gly Pro Asn Ser Asp Asp Glu Ile Asn Leu

265 270 275 280

Ile Val Lys Gly Gly Asn Tyr Gly Trp Pro Asn Val Ala Gly Tyr Lys Asp Asp Ser Gly

285 290 295 300

Tyr Ala Tyr Ala Asn Tyr Ser Ala Ala Ala Asn Lys Ser Ile Lys Asp Leu Ala Gln Asn

305 310 315 320

Gly Val Lys Val Ala Ala Gly Val Pro Val Met Lys Glu Ser Glu Trp Thr Gly Lys Asn

325 330 335 340

Phe Val Pro Pro Leu Lys Pro Leu Tyr Thr Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro

345 350 355 360

Thr Cys Gly Glu Met Thr Tyr Ile Cys Trp Pro Thr Val Ala Pro Ser Ser Ala Tyr Val

365 370 375 380

Tyr Lys Gly Gly Lys Lys Ala Ile Thr Gly Trp Glu Asn Thr Leu Leu Val Pro Ser Leu

385 390 395 400

Lys Arg Gly Val Ile Phe Arg Ile Lys Leu Asp Pro Thr Tyr Ser Ile Thr Tyr Asp Asp

405 410 415 420

Ala Val Pro Met Phe Lys Ser Asn Asn Arg Tyr Arg Asp Val Ile Ala Ser Pro Asp Gly

425 430 435 440

Asn Val Leu Tyr Val Leu Thr Asp Thr Ala Gly Asn Val Gln Lys Asp Asp Gly Ser Val

445 450 455 460

Thr Asn Thr Leu Glu Asn Pro Gly Ser Leu Ile Lys Phe Thr Tyr Lys Ala Lys

<210>5

<211>24

<212>DNA

＜213〉artificial sequence

<400>5

GCCCATGGAAAACATTTATTGGCT

<210>6

<211>25

<212>DNA

＜213〉artificial sequence

<400>6

GGAAGCTTATACATAGCCTTATAGG

Claims (5)

1. the s-GDH mutant that improves of a thermostability, it is characterized in that wild-type s-GDH is positioned at 111,263,270,331, have one in the amino acid in 347,416 and be substituted, wherein these positions are corresponding to the known amino acid position of s-GDH wild-type sequence by Acinetobacter calcoaceticus Acinetobactercalcoaceticus L.M.D.79.41; The aspartic acid that is positioned at 111 among the wild-type s-GDH is replaced by L-glutamic acid, the glycine that is positioned at 263 is replaced by halfcystine, the glutamine that is positioned at 270 is replaced by Histidine, the Threonine that is positioned at 331 is replaced by methionine(Met), the Serine that is positioned at 347 is replaced by proline(Pro), and the Threonine that is positioned at 416 is replaced by Isoleucine.

2. the polynucleotide of a separation, the s-GDH mutant of described polynucleotide encoding claim 1.

3. recombinant vectors is characterized in that being loaded with the polynucleotide of separation claimed in claim 2.

4. a transformant is characterized in that being transformed and being made by recombinant vectors claimed in claim 3.

One kind for detection of or measure sample in the device of glucose, described device comprises the s-GDH mutant in the claim 1.

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