CN102559624A

CN102559624A - Thermostable mutant of pyrroloquiniline quinine-dependent glucose dehydrogenase and high-throughput screening method thereof

Info

Publication number: CN102559624A
Application number: CN2012100549838A
Authority: CN
Inventors: 徐志南; 于怡; 黄磊; 朱建忠; 杨叶东
Original assignee: ZHEJIANG DEQING HUINING BIOTECHNOLOGY CO Ltd
Current assignee: ZHEJIANG DEQING HUINING BIOTECHNOLOGY CO Ltd
Priority date: 2012-03-05
Filing date: 2012-03-05
Publication date: 2012-07-11
Anticipated expiration: 2032-03-05
Also published as: CN102559624B

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 of the 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 two mutants and high-throughput screening method thereof of PQQ dependent form Hexose phosphate dehydrogenase

Invention field

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

Background technology

Glucose concentration determination is very important in the clinical diagnosis of mellitus 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, mellitus 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 at OBD 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 developed countries such as 90% America and Europe carries out self-monitoring through blood glucose meter.Solubility PQQ 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 glucono-with glucose oxidase, so the type enzyme can be used for detecting blood sugar.

PQQ (PQQ) is the third the novel coenzyme behind flavin nucleotide (FMN, FAD) and nicotinamide nucleotide (NAD, NADP).Found coenzyme topaquinone (TPQ), lysine tymsylqllinone (LTQ), the tryptophantryptophylquinone (TTQ) of some and PQQ similar afterwards again in succession; These four kinds of coenzyme also are called the quinoid coenzyme, are that the oxydo-reductase of coenzyme is referred to as quinoprotein enzyme or quinone enzyme with PQQ, TPQ, LTQ, TTQ.PQQ dependent form Hexose phosphate dehydrogenase be one type main be the quinoprotein of coenzyme with PQQ, because of 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 respiratory chain outer pericentral siphon redox system of link coupled born of the same parents mutually, the electron acceptor(EA) of transmission electronics to the cytolemma.The characteristic of the outer pericentral siphon redox system of this born of the same parents is the path of having shortened electron transport, has reduced substrate oxidation and product and has discharged required energy.

PQQ dependent form Hexose phosphate dehydrogenase is different because of the position of its existence, and being divided into film mating type again is that m-GDH and water-soluble type are s-GDH.M-GDH is present in the multiple gram-negative bacteria, like the bacterium of acinetobacter, Rhodopseudomonas and acetic acid Pseudomonas, is positioned at the cytolemma outside surface of these bacteriums, but direct oxidation D-glucose generates lipid or glucono-.This enzyme is the quinoprotein that single subunit is formed, and only the peptide chain by a 87kD constitutes, and has 5 to wear membrane structure, and is hydrophobic for highly.Its Substratspezifitaet is higher, can the oxidation hexose except glucose, 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, and discovery is only arranged in acinetobacter at present.S-GDH is proved to be to be different from the Hexose phosphate dehydrogenase that second kind of PQQ of m-GDH relies on, and the two homology is very low.This enzyme is a homodimer, and the relative molecular mass of each subunit is 50,000, and the N end is a signal peptide of being made up of 24 amino-acid residues, and the back is excised in the pericentral siphon outside being secreted into born of the same parents.This enzyme is not participated 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 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. with electronics.

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

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 to 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 polypeptied chain side-chain radical surface to replace halfcystine, methionine(Met), leucine, aspartic acid, Stimulina, Histidine and Methionin respectively, all make the stability of enzyme obtain raising to a certain degree; Wherein increase the most obvious with Ser231Lys stability under the prerequisite that keeps high vigor; Compare with wild-type, 55 ℃ of transformation period have been improved 8 times, but still keep its constitutive enzyme vigor.According to improving stable principle through increasing by two bonding forces between the subunit, the additive method of employing has: 1. adopt LUTARALDEHYDE that C, the N end of two subunits are 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 polypeptied 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 factors such as proteinic structure, conservative site, catalyst mechanism in advance, but create special evolution conditions artificially; In in-vitro simulated darwinian evolution process; Through setting up random mutation library or recombination storehouse, pass through suitable screening again, obtain to have the protein that character obviously improves.Enzyme orthogenesis technology has remedied the deficiency of site-directed mutagenesis technique to a certain extent, has very big 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 arranged; But relatively poor thermostability has then limited its further application; Therefore become an important directions in the s-GDH research through its thermostability of biotechnology means raising; The solubility PQQ dependent form Hexose phosphate dehydrogenase that obtains high thermal stability has great importance; Not only can widen the industrial applicability of enzyme, and can study enzymatic structure and stability relationship.

Summary of the invention

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

Technical scheme of the present invention: the s-GDH two mutants 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 two mutants; Make up the heavy body sudden change library of s-GDH through fallibility PCR; The filter paper that utilization is soaked with coloured substrate carries out the original position screening on the bacterium colony flat board, and combines the direct conversion method of bacterium colony to obtain s-GDH clone, obtains the two mutants that the s-GDH thermostability improves.

S-GDH compares with wild-type, and the thermostability of described s-GDH two mutants has obtained to significantly improve.The solubility PQQ 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.

Polynucleotide sequence and the expression vector that contains this polynucleotide sequence and the host cell that contains said expression vector of s-GDH mutain of the present invention of encoding also is scheme of the present invention.

The invention still further relates to the purposes of two mutants of the present invention in the glucose detection method, especially through the 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 two mutants

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

Fig. 3: orthogenesis screening synoptic diagram

Fig. 4: wild-type and s-GDH two mutants 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 having only solvable type Hexose phosphate dehydrogenase s-GDH, and hereinafter is discussed its improvement variant.

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 from Acinetobacter calcoaceticus bacterial strain LMD79.41, to separate.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 an expression vector; Can be according to expressing and the purposes such as needs of purifying are used appropriate carriers, for example pET series, pQE is serial, pMAL is serial, pGEX series etc. is suitable for colibacillary expression vector or be suitable for hyphomycetic expression vectors such as other prokaryotic cell prokaryocytes, yeast, mould such as withered grass bacterium respectively.

Transformant of the present invention for example when preparation s-GDH, can use thread funguss such as other prokaryotic cell prokaryocytes, yeast, mould such as intestinal bacteria, withered grass bacterium etc.Above-mentioned transformant can prepare the recombinant vectors transfered cell through adopting known method 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 transformed into escherichia coli BL21 (DE3) that obtains of carrier thus.

Along with the continuous foundation of creating the gene library method and perfect; Set up a sudden change storehouse 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 a very big challenge at present.In the past few years, many screening methods that using value is arranged have appearred.The selection of catalytic activity is screened through phenotype or in solid phase and micropore often.The use of selective pressure often causes in the two mutants occurring not influencing the sudden change of catalytic activity.

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

S-GDH is an intracellular enzyme; If will then need be that unit carries out enzyme reaction alive with the cell through 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, can make the color fade of reaction substrate solution.In addition, express because the pET28a carrier has a spot of leakage, when being formed with dull and stereotyped cultivation of screening of active 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 and carry out the original position screening through substrate colors.

Because the thalline major part is dead in the heat treatment process, normal inoculation can't be grown, and in order to obtain positive muton, can adopt dull and stereotyped the photomechanical printing, and original position PCR or bacterium colony such as directly transform at 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 makes 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 through directly, thereby obtains its clone's.

After wild-type or s-GDH two mutants are expressed in host cell,, can make up known lock out operation and separate for separation and purification from culture obtains target protein.For example can enumerate with ultrasonication, enzymic digestion, saltout 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 two mutants C end amalgamation and expression to carry out affinity purification.

Because intestinal bacteria are synthesized the ability of PQQ, can only obtain the recombinase protein of sPQQGDH, therefore measure the enzyme corresponding coenzyme prothetic group of interpolation that needs before alive to be formed with the holoenzyme of functionally active.Enzyme liquid to pure enzyme or thick enzyme adds certain density PQQ and Ca ²⁺, make coenzyme and zymoprotein combine promptly to be formed with active holoenzyme solution completely, available enzyme assaying reaction alive.

As stated; S-GDH can utilize multiple electron acceptor(EA); Like 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.Through 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 following:

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 going back ortho states is colourless, and is therefore when reaction is carried out, blue for slowly taking off, accordingly OD ₆₀₀Value can reduce gradually, through OD ₆₀₀The variation of value to enzyme work measure calculating.

An enzyme unit alive (U) is under 25 ℃ of conditions, in 1min, can make the enzyme amount of the glucose oxidase (or 1 μ mol DCIP reduction) of 1 μ mol.The calculation formula of enzyme unit alive is following 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.Short-term thermal stress model is usually used in estimating stability.Therefore stability of the present invention be called thermostability with this short-term thermal stress model evaluation.Thermostability is through the unstressed of test sample and have the s-GDH enzymic activity of stress to measure.Through unstressed sample activity is set at 100%, the remaining activity after stress is handled can calculate by percentage.For concrete wild-type or s-GDH two mutants, select 65 ℃ of processing 30 minutes or measure the transformation period under 55 ℃.Use these conditions, handle 30 minutes remaining its original activity of following wild-type s-GDH below 15% for 65 ℃, and the s-GDH two mutants 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 two mutants of the present invention can huge advantage be used for the detection of biological sample glucose, especially through test strip or the detection carried out through biosensor.

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

The present invention also comprises the method for using glucose in s-GDH two mutants of the present invention detection, mensuration or the measure sample.Especially preferredly be, be used for improving one's methods of test sample glucose and be characterised in that, use transmitter or test strip to carry out said detection, mensuration or the measurement of glucose.

Be used for detecting or the device of the measure sample glucose scope of the invention of also serving a ball, said device comprises s-GDH two mutants according to the invention and needed other reagent of said measurement.

The s-GDH two mutants 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 drum.For this purpose, said s-GDH two mutants for example can be used for encapsulating the insensitive glass electrode of the oxygen with osmium complex, to measure glucose concn more exactly.

Further specify the present invention through following steps,

Step 1 fallibility PCR

The present invention adopts GeneMorph II fallibility PCR test kit to suddenly change, and by specification is said, and mutation rate is relevant with amount and PCR cycle number that the template that is adopted adds, and the template amount is many more, and cycle number is few more, and mutation rate is low more, otherwise then mutation rate is high more.Recombinant vectors pET28-gdh (like Fig. 1) to contain wild-type s-GDH gene (SEQ ID NO:2) is a template; (5 '-GGAAGCTTATACATAGCCTTATAGG-3`) (=SEQ ID NO:6) is that primer carries out fallibility PCR with F (5`-GCCCATGGAAAACATTTATTGGCT-3`) (=SEQ ID NO:5) and R; Adopt 50 μ L reaction systems following

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 preparatory sex change 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 through 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 the double digestion reaction of NcoI/HindIII respectively utilizing DNA glue to reclaim test kit (AxyPrep DNA Gel Extraction Kit) behind the fallibility PCR product electrophoresis with carrier pET28.Enzyme is cut the fragment that reclaims the back and is carried out concentration determination through 260nm place absorbance.Is 10: 1 mixed with purpose fragment and carrier segments according to the amount of substance ratio, utilizes 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,, exert an influence with the pair ion electricity conversion that prevents damping fluid in the connection procedure at last with 10 μ L deionized water dissolvings.The product electricity be will connect then 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 final concentration 30 μ g/ml kantlex and 1mM PQQ, fall dull and stereotyped through filtration sterilization.

Step 3 high flux screening thermostability two mutants

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

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 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, was cooled to room temperature then;

4) filter paper that will cut out soaks into reaction solution fully, it carefully is laid on the flat board in sudden change library picks up behind the 5s then.Enzyme reaction can take place 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 enzyme inactivation, then bacterium colony can be dyed to mazarine, and colour-change can not take place 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) because thalline major part death 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, (promptly 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 the stability that obtains again has the mutant strain of raising to cultivate, and extracts plasmid, and by giving birth to worker's order-checking, sequencing result is following,

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

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

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

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

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 two mutants

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

With 5, the centrifugal 5min of 000rpm abandons supernatant and obtains thalline with the nutrient solution of fermentation ends, concentrates in the level pad that is suspended in precooling (the 20mM imidazoles, pH 8.0 for 20mM Tris-Cl, 0.5M NaCl) with 2 times of volumes.Use the ultrasonic disruption cell then, working conditions is power 200W, working hour 5s, and quiescent interval 7s carries out 30 times altogether, and cell should remain in the ice bath in this process, in order to avoid proteic thermally denature.With all broken liquid samples 13, the centrifugal 10min of 000rpm is to separate solvable and soluble component, and supernatant is last all article to be purified.

Earlier 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 handling is squeezed into pipeline by pump, the about 0.5ml/min of control flow 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 control flow velocity continues to be eluted to A ₂₈₀Reach minimum value and remain unchanged; Use elutriant II (the 200mM imidazoles, pH 8.0 for 20mM Tris-Cl, 0.5M NaCl) flushing pillar then, 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) protein solution behind wild-type or the s-GDH two mutants purifying has been carried out concentrating and purifying once more.At first the elute soln of pending wild-type or s-GDH two mutants is concentrated, carried out holoenzyme and hatch, transfer to then among the Sample reservoir (noticing that the rifle head does not touch filter membrane) and the Filtrate vial that packs into; Utilize ultra-filtration centrifuge tube to wherein unnecessary PQQ then, the high imidazoles of high salt removes in Ca2+ and the elution buffer.4 ℃ down 5, the centrifugal 10min of 000rpm adds the displacement damping fluid then to 5ml again 5, the centrifugal 10min of 000rpm, and triplicate, to the salt ion Ex-all, but last proper extension centrifugation time concentrates protein solution, and is subsequent use in 4 ℃ of preservations.

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

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

Reacting total system 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, through the OD of 1min and 3min under the UV-9100 type spectrophotometer reading and recording ₆₀₀, and with PM OD ₆₀₀The speed of response of variation about 0.1 is advisable, and the speed of reaction can be through carrying out suitable dilution to enzyme liquid.Be prone to 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 notes the OD of 1min and 3min ₆₀₀Value is as negative control.

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 two mutants is used the ultrasonic disruption cell with the fermented liquid that cultivation obtains, and working conditions is power 200W; Working hour 5s, quiescent interval 7s carries out 30 times altogether; Cell should remain in the ice bath in this process, in order to avoid proteic thermally denature.

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

Visible by form, the residual activity of s-GDH two mutants is compared and will be improved greatly with the residual activity of wild-type s-GDH.

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

The thermostability of s-GDH is to estimate through its transformation period under 55 ℃.The holoenzyme solution of 1ml 3 μ g/ml wild-types or s-GDH two mutants is hatched in 55 ℃ of water-baths, and every ice bath 2min at room temperature recovers half a hour then at a distance from the 10min sampling, 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 lives, promptly residual activity is 50% o'clock, and the used time is the transformation period of s-GDH under 55 ℃.Transformation period, the result was following,

Visible by form, the transformation period of wild-type s-GDH is compared with the s-GDH two mutants and wants much shorter.

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< 120>a kind of thermostability two mutants and high-throughput screening method thereof of PQQ dependent form Hexose phosphate dehydrogenase

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< 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

1. the s-GDH two mutants that improves of a thermostability; 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 by Acinetobacter calcoaceticus Acinetobactercalcoaceticus L.M.D.79.41.

2. s-GDH two mutants as claimed in claim 1; The aspartic acid that it is characterized in that being positioned among the wild-type s-GDH 111 is replaced by L-glutamic acid; The glycocoll that is positioned at 263 is replaced by halfcystine, and the Stimulina that is positioned at 270 is replaced by Histidine, and 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.

3. isolating polynucleotide, the s-GDH two mutants of said polynucleotide encoding claim 2.

4. a recombinant vectors is characterized in that being loaded with the described isolating polynucleotide of claim 3.

5. a transformant is characterized in that being transformed and being processed by the described recombinant vectors of claim 4.

6. an application rights requires the method for glucose in 3 the detection of s-GDH two mutants, mensuration or the measure sample, and said method comprises makes said sample contact with said two mutants.

7. according to the method for claim 6, its characteristic also is to use transmitter or test strip device to carry out said detection, mensuration or the measurement of glucose.

8. one kind is used for detecting or the device of the glucose of measure sample, and said device comprises the s-GDH two mutants in the claim 2.

9. the method for a high flux screening s-GDH two mutants as claimed in claim 1 is characterized in that utilizing the filter paper that is soaked with coloured substrate on the bacterium colony flat board, to carry out the original position screening.

10. the method for high flux screening s-GDH two mutants according to claim 9, the positive muton that screening obtains obtains its clone's through the direct conversion method of bacterium colony.