CN103937763A - R112W type ketoamine oxidase, encoding gene thereof and application - Google Patents

Abstract

The invention discloses R112W type ketoamine oxidase, encoding gene thereof and application. The provided R112W type ketoamine oxidase is protein described by (a) or (b): (a) protein consisting of the 1st to the 438th amino acid residues from N terminal of a sequence 3 in a sequence table; and (b) protein consisting of amino acid residues shown as the sequence 3 in the sequence table. Compared with conventional ketoamine oxidase, the provided R112W type ketoamine oxidase is substantially improved in enzyme activity and has important application value.

Description

R112W type Ketoamine oxidase and encoding gene thereof and application

Technical field

The present invention relates to a kind of R112W type Ketoamine oxidase and encoding gene and application.

Background technology

Maillard reaction, is called again non-enzymatic catalysis glycosylation, is the common reaction of a class nature.Reducing sugar (modal is glucose) reacts under normal temperature or high temperature with aminocompound, generates Amadori product, and is further cross-linked and obtains advanced glycosylation end product and melanoid class material.This reaction plays an important role in the processing of food and storage process, and to the color and luster of food, local flavor and nutritive ingredient etc. has important impact.Research is in recent years found, also has in vivo Maillard reaction, the product of reaction and the mankind's aging, and the illnesss such as diabetic complication have close relationship, thereby are paid close attention to widely.

Ketoamine oxidase (fructosyl amine oxidases, is abbreviated as FAOXs) is that a class is present in the deglycosylating enzyme in microbe, can catalyzed degradation Amadori product, conventionally generate aminocompound, glucosone and hydrogen peroxide.At the beginning of this enzyme is found, be applied at first the detection of diabetes, the detection based on FAOXs at present has become " golden index " in diabetes diagnosis and detection.Meanwhile, this proteinoid enzyme is in food quality control, and the aspects such as detergent additives and treating diabetes have also shown huge application prospect.But known Ketoamine oxidase all can only act on small molecules substrate at present, as glycosylation amino acid or glycosylation dipeptides, this has also greatly limited the application of this enzyme.

The Ketoamine oxidase Amadoriase II that derives from Aspergillus fumigatus Aspergillusfumiga tus is separated first in 1997, can directly act on a kind of substrate glycosylation amantadine (fructosyl-adamantanamine) of larger molecular weight.The crystalline structure of this enzyme was in the news in 2008, crystalline structure shows, there are two flexible Loop regions (amino acid residue sequence from the residue region of N-terminal 58-66 position and the residue region of 110-119 position) on this enzyme surface, be positioned near substrate feeder connection, play an important role in Binding Capacity process.

Summary of the invention

The object of this invention is to provide a kind of R112W type Ketoamine oxidase and encoding gene and application.

R112W type Ketoamine oxidase provided by the invention is following (a) or (b):

(a) protein being formed from N-terminal the 1st to 438 amino acids residues by sequence in sequence table 3;

(b) protein being formed by the amino-acid residue shown in sequence in sequence table 3.

The gene of described R112W type Ketoamine oxidase of encoding also belongs to protection scope of the present invention.

Described gene is can following 1) or 2) or 3) or 4) or 5) DNA molecular:

1) coding region is if sequence in sequence table 4 is from the DNA molecular as shown in the 1st to 1314 Nucleotide of 5 ' end;

2) coding region is if sequence in sequence table 4 is from the DNA molecular as shown in the 1st to 1344 Nucleotide of 5 ' end;

3) DNA molecular of coding region as shown in sequence in sequence table 4;

4) under stringent condition with 1) or 2) or the 3 DNA sequence dnas hybridization that limit and the DNA molecular of code for said proteins;

5) with 1) or 2) or 3 DNA sequence dnas that limit there is the DNA molecular of 90% above homology and code for said proteins.

Above-mentioned stringent condition can be at 6 × SSC, in the solution of 0.5%SDS, under 65 ° of C, hybridizes, and then uses 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively to wash film once.

The recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contain described gene all belong to protection scope of the present invention.

The recombinant expression vector that available existing expression vector establishment contains described gene.Described expression vector also can comprise 3 ' end untranslated region of foreign gene, comprises the DNA fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.While using described gene constructed recombinant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter, they can be used alone or are combined with other promotor; In addition, while using gene constructed recombinant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, but must be identical with the reading frame of encoding sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation region or structure gene.

Described recombinant vectors specifically can be the recombinant plasmid that the multiple clone site of described gene insertion pEAS-1a plasmid is obtained.

Described recombinant bacterium specifically can be described recombinant vectors is imported to the recombinant bacterium that e. coli bl21 obtains.

The present invention also protects described protein in the application of preparing in Ketoamine oxidase.

The present invention also protects the application of described protein in degraded glycosylation substrate.Described glycosylation substrate specifically can be glycosylation poly-lysine or glycosylation amantadine.

The present invention also protects described recombinant bacterium in the application of producing in described protein.

When applying described recombinant bacterium and preparing described protein, can adopt with the following method: described recombinant bacterium is fermented, collect tunning, in tunning, contain described protein.

Described method specifically comprises the steps:

(1) described recombinant bacterium is cultured to OD _600nmbe that to add IPTG at 0.5 o'clock and make its concentration be 0.5mM, continue 30 DEG C, 250rpm and cultivate 6 hours, collect thalline;

(2) thalline step (1) being obtained carries out ultrasonication, collects supernatant liquor;

(3) supernatant liquor step (2) being obtained is splined in conjunction with Ni ²⁺hiTrapTM Chelating HP post, first use elutriant first (containing the 20mM sodium phosphate buffer of 500mM sodium-chlor and 30mM imidazoles, pH7.4) remove foreign protein, then use elutriant second (containing the 20mM sodium phosphate buffer of 500mM sodium-chlor and 400mM imidazoles, pH7.4) wash-out target protein, solution after the post excessively of collection elutriant second, is the solution that contains described protein.

The present invention also protects the application of described protein as Ketoamine oxidase.

Compared with existing Ketoamine oxidase, the enzymic activity of R112W type Ketoamine oxidase provided by the invention improves greatly, has great using value.

Brief description of the drawings

Fig. 1 is the structural representation of recombinant plasmid pEAS-1a-CT-AMAII.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.

PUC18-cI857/pR-SRRz-rrnB plasmid, is called for short pEAS-1a plasmid, is a kind of thermal induction autothermic cracking plasmid.Mention the reference of pUC18-cI857/pR-SRRz-rrnB plasmid: Xu, L., et al., Heat-inducibleautolytic vector for high-throughput screening.BioTechniques, 2006.41 (3): p.319-322..

E. coli bl21: purchased from Novagen.

Wild-type Ketoamine oxidase Amadoriase II is made up of from N-terminal the 1st to 438 amino acids residues the sequence 1 of sequence table.The coding region of wild-type Ketoamine oxidase Amadoriase II gene is if the sequence 2 of sequence table is from as shown in the 1st to 1314 Nucleotide of 5 ' end.

The structure of embodiment 1, recombinant plasmid

One, the structure of recombinant plasmid pEAS-1a-CT-AMAII

1, the sequence 2 of composition sequence table is from the double chain DNA molecule shown in the 1st to 1314 Nucleotide of 5 ' end.

2,, using double chain DNA molecule synthetic step 1 as template, with the primer pair of AMA-for and CT-AMA-rev composition, adopt the Deep of high-fidelity archaeal dna polymerase carries out pcr amplification, obtains pcr amplification product.

AMA-for:5'-AGAG gGATCCgATGGCGGTAACCAAGTCA-3'(wherein underscore part is BamHI restriction endonuclease recognition site);

CT-AMA-rev：

5 '-ATTC cTGCAGtTA (wherein underscore part is Pst I restriction endonuclease recognition site to TAACTTGGAAATATCTC-3 ', is His in square frame ₆the encoding sequence of label, runic represents connection peptides).

3, with the pcr amplification product of restriction enzyme BamH I and Pst I double digestion step 2, reclaim enzyme and cut product.

4,, with restriction enzyme BamHI and Pst I double digestion pEAS-1a plasmid, reclaim the carrier framework of about 5400bp.

5, the carrier framework of the enzyme of step 3 being cut to product and step 4 is connected, and obtains recombinant plasmid

The expression vector of pEAS-1a-CT-AMAII(wild-type Ketoamine oxidase AmadoriaseII gene).According to sequencing result, recombinant plasmid pEAS-1a-CT-AMAII is carried out to structrual description as follows: between the BamHI of pEAS-1a plasmid and Pst I restriction enzyme site, inserted the double chain DNA molecule shown in the sequence 2 of sequence table.Recombinant plasmid

The structural representation of pEAS-1a-CT-AMAII is shown in Fig. 1.

Two, recombinant plasmid pEAS-1a-CT-AMAII(R112W) structure

1, taking recombinant plasmid pEAS-1a-CT-AMAII as template, carry out pcr amplification with the primer pair of EP-for and R112W-rev composition, obtain pcr amplification product.

EP-for：5'-TTACGAATTCGAGCTCGGTACCCG-3'；

R112W-rev：5'-CTCACCGGGCCTGAC CCAGACTCCAAGGCGGTCCA-3'。

2, taking recombinant plasmid pEAS-1a-CT-AMAII as template, carry out pcr amplification with the primer pair of R112W-for and EP-rev composition, obtain pcr amplification product.

R112W-for：5'-GACCGCCTTGGAGTC TGGGTCAGGCCCGGTGAGG-3'；

EP-rev：5'-ACGGCCAGTGCCAAGCTTGCATGCCT-3'。

3, simultaneously using the pcr amplification product of the pcr amplification product of step 1 and step 2 as template, carry out pcr amplification with the primer pair of EP-for and EP-rev composition, obtain pcr amplification product.

EP-for and EP-rev correspond respectively to the upstream and downstream of double chain DNA molecule shown in sequence 2 in recombinant plasmid pEAS-1a-CT-AMAII.

4, with the pcr amplification product of restriction enzyme BamH I and Pst I double digestion step 3, reclaim enzyme and cut product.

5,, with restriction enzyme BamHI and Pst I double digestion pEAS-1a plasmid, reclaim the carrier framework of about 5400bp.

6, the carrier framework of the enzyme of step 4 being cut to product and step 5 is connected, and obtains recombinant plasmid pEAS-1a-CT-AMAI I(R112W).According to sequencing result, to recombinant plasmid pEAS-1a-CT-AMAI I(R112W) to carry out structrual description as follows: between the BamH of pEAS-1a plasmid I and Pst I restriction enzyme site, insert the double chain DNA molecule shown in the sequence 4 of sequence table.Compared with the sequence 2 of sequence table, the difference of the sequence 4 of sequence table is only double chain DNA molecule shown in the sequence of sequence table 2 to be suddenlyd change for " TGG " by " cgt " from 5 ' end 334-336 position Nucleotide.Protein shown in the sequence 3 of the DNA molecule encode sequence table shown in the sequence 4 of sequence table.Compared with the sequence 1 of sequence table, the difference of the sequence 3 of sequence table is only the sequence of sequence table 1 to be suddenlyd change for " W " by " R " from N-terminal the 112nd amino acids residue.Protein called after R112W type Ketoamine oxidase Amadoriase II by the sequence of sequence table 3 from N-terminal the 1st to 438 amino acids residue compositions.

The preparation of embodiment 2, wild-type Ketoamine oxidase Amadoriase II and R112W type Ketoamine oxidase Amadoriase II

One, the preparation of wild-type Ketoamine oxidase Amadoriase II

1, recombinant plasmid pEAS-1a-CT-AMAII is imported to e. coli bl21, obtain recombinant bacterium.

Single colony inoculation of the recombinant bacterium 2, step 1 being obtained is the LB liquid nutrient medium containing 50 μ g/ml Ampicillin Trihydrates in 15ml, 30 DEG C, 250rpm incubated overnight.

3, culture system step 2 being obtained is inoculated in the volume ratio of 1:40 the LB liquid nutrient medium that 400ml contains 50 μ g/ml Ampicillin Trihydrates; 30 DEG C, 250rpm are cultured to OD _600nmbe that to add IPTG at 0.5 o'clock and make its concentration be 0.5mM, continue 30 DEG C, 250rpm and cultivate; From adding IPTG to start timing, after 6 hours, collect thalline.

4, thalline step 3 being obtained carries out ultrasonication (ultrasonication power 200W, ultrasonic 4 seconds stop 3 seconds, totally 99 times), and the then centrifugal 25min of 15000rpm collects supernatant liquor.

5, the supernatant liquor obtaining by the membrane filtration step 4 of 0.22 μ m lower protein combination, collects filtrate.

6, get the filtrate that step 5 obtains, utilize protein purification equipment ( explorerTM station), loading is extremely in conjunction with Ni ²⁺hiTrapTM Chelating HP post (purchased from peace agate West Asia (Amersham Biosciences, volume 1mL), first use 15ml elutriant first (containing the 20mM sodium phosphate buffer of 500mM sodium-chlor and 30mM imidazoles, pH7.4) remove foreign protein, then use 3ml elutriant second (containing the 20mM sodium phosphate buffer of 500mM sodium-chlor and 400mM imidazoles, pH7.4) wash-out target protein, solution after the post excessively of collection elutriant second, is protein liquid first (fusion rotein that contains wild-type Ketoamine oxidase Amadoriase II and His label).

Two, the preparation of R112W type Ketoamine oxidase Amadoriase II

With recombinant plasmid pEAS-1a-CT-AMAI I(R112W) replacement recombinant plasmid pEAS-1a-CT-AMAI I, other same step 1, solution after the post excessively of collection elutriant second, is protein liquid second (fusion rotein that contains R112W type Ketoamine oxidase Amadoriase II and His label).

The enzyme of embodiment 3, wild-type Ketoamine oxidase Amadoriase II and R112W type Ketoamine oxidase Amadoriase II is lived

One, the preparation of glycosylation pattern substrate

1, the preparation of glycosylation poly-lysine

(purchased from Sigma-Aldrich, catalog number is P8954-500MG to take poly-lysine mixture according to mol ratio 5:6; Each polylysine molecule contains 3-13 Methionin) and glucose, add in 200mM sodium phosphate buffer (pH7.5), the final concentration of glucose is 0.75M, mixes rear 60 DEG C of water-baths 6 hours, obtains glycation product (containing glycosylation poly-lysine).

2, the preparation of glycosylation amantadine

Take amantadine (purchased from Sigma-Aldrich according to mol ratio 5:6, catalog number is A1260-5G) and glucose, be added to the water, the final concentration of glucose is 0.75M, impel amantadine to dissolve by dripping the sodium hydroxide solution of 1M, mix rear 121 DEG C of reactions 15 minutes, obtain glycosylation amantadine.

Two, the enzyme activity determination of wild-type Ketoamine oxidase Amadoriase II and R112W type Ketoamine oxidase Amadoriase II

1, preparation feedback liquid

Get the horseradish peroxidase of 2.7 purpurogallin units (purpurogallin unit), the 4-AA of 0.45mM, N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-m-Tolylamine (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine) of 0.5mM and 100 μ l glycosylation substrates (being 2 the system obtaining of the system that obtains of 1 of step 1 or step 1), be dissolved in the potassium phosphate buffer (pH8.0) of 3ml100mM.

2, prepare diluted protein solution

Be diluted to 50 times of volumes by the protein liquid first that the potassium phosphate buffer (pH8.0) of 100mM is prepared embodiment 2, obtain diluted protein solution first.

Be diluted to 50 times of volumes by the protein liquid second that the potassium phosphate buffer (pH8.0) of 100mM is prepared embodiment 2, obtain diluted protein solution second.

3, determination of protein concentration

Detect respectively the protein concentration in diluted protein solution first and diluted protein solution second, concrete grammar is as follows:

Detect the absorbance value at 280nm place, then calculate according to the following formula:

Protein concentration (mg/ml)=A ₂₈₀/ (ε × b);

A ₂₈₀-diluted protein solution is at the absorbance value at 280nm place; B-light path, cm; ε-optical extinction coefficient.

Optical extinction coefficient, can calculate according to the following formula:

$\mathrm{\ϵ}=\frac{{5700n}_{\mathrm{Trp}}+{1300n}_{\mathrm{Tyr}}}{\mathrm{Mw}}$

N _trpthe number of tryptophan residue in-albumen;

N _tyrthe number of tyrosine residues in-albumen

Mw-molecular weight of albumen.

The protein concentration of diluted protein solution first is 0.247mg/ml.The protein concentration of diluted protein solution second is 0.133mg/ml.

4, the enzyme activity determination of wild-type Ketoamine oxidase Amadoriase II and R112W type Ketoamine oxidase AmadoriaseII

(1) live for the enzyme of glycosylation amantadine

The reaction solution that 2 μ l diluted protein solution first (or diluted protein solution second) and 50 μ l steps 1 are obtained mixes, and detects under 37 DEG C of conditions by microplate reader, and mixed solution in 555nm place absorption photometric value over time.

Calculating enzyme mechanism alive by absorbancy is: Ketoamine oxidase Amadoriase II glycosylation substrate generates glucosone and the hydrogen peroxide of equimolar ratio, hydrogen peroxide reacts with 4-AA and N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-m-Tolylamine under the catalysis of horseradish peroxidase, and every bimolecular hydrogen peroxide participation reaction can generate a part the quinones dyestuff of special absorption at 555nm place.

Quinone dyestuff generating rate (μ molcm ^-3min ^-1)=A ₅₅₅/ (ε × b)

A ₅₅₅the time dependent curve of the absorbance of-reaction system at 555nm place, the slope of linear portion, min ^-1;

ε-optical extinction coefficient, for the quinone dyestuff generating in this reaction, optical extinction coefficient is 39.2cm ²/ μ mol; B-light path, cm.

A unit of enzyme activity is defined as under above-mentioned reaction conditions, and in 1min, catalysis generates the needed enzyme amount of 0.5 μ mol quinones substance (i.e. 1 μ mol hydrogen peroxide).

The enzyme of diluted protein solution first is lived as 0.124U/ml.The enzyme of diluted protein solution second is lived as 0.791U/ml.

(2) live for the enzyme of glycosylation poly-lysine

The reaction solution that 10 μ l diluted protein solution first (or diluted protein solution second) and 30 μ l step 1 are obtained mixes, and detects under 37 DEG C of conditions by microplate reader, and mixed solution in 555nm place absorption photometric value over time.

Calculate by absorbancy mechanism and the enzyme same steps of method of calculation alive (1) that enzyme is lived.

The enzyme of diluted protein solution first is lived as 1.48mU/ml.The enzyme of diluted protein solution second is lived as 2.07mU/ml.

5,, according to the result of step 3 and step 4, calculate the enzyme of wild-type Ketoamine oxidase Amadoriase II and R112W type Ketoamine oxidase Amadoriase II and live.

Wild-type Ketoamine oxidase AmadoriaseII lives as 0.5U/mg to the enzyme of substrate glycosylation amantadine.It is 11.9 ± 1.1 to the enzyme of substrate glycosylation amantadine ratio alive that R112W type Ketoamine oxidase Amadoriase II lives with wild-type Ketoamine oxidase Amadoriase II to the enzyme of substrate glycosylation amantadine.

Wild-type Ketoamine oxidase Amadoriase II lives as 6.0mU/mg to the enzyme of substrate glycosylation poly-lysine.It is 2.6 ± 0.1 to the enzyme of substrate glycosylation poly-lysine ratio alive that R112W type Ketoamine oxidase Amadoriase II lives with wild-type Ketoamine oxidase Amadoriase II to the enzyme of substrate glycosylation poly-lysine.

Claims (10)

1. a protein is following (a) or (b):

(a) protein being formed from N-terminal the 1st to 438 amino acids residues by sequence in sequence table 3;

(b) protein being formed by the amino-acid residue shown in sequence in sequence table 3.

2. the gene of protein described in coding claim 1.

3. gene as claimed in claim 2, is characterized in that: described gene is following 1) or 2) or 3) or 4) or 5) DNA molecular:

1) coding region is if sequence in sequence table 4 is from the DNA molecular as shown in the 1st to 1314 Nucleotide of 5 ' end;

2) coding region is if sequence in sequence table 4 is from the DNA molecular as shown in the 1st to 1344 Nucleotide of 5 ' end;

3) DNA molecular of coding region as shown in sequence in sequence table 4;

4) under stringent condition with 1) or 2) or the 3 DNA sequence dnas hybridization that limit and the DNA molecular of code for said proteins;

5) with 1) or 2) or 3 DNA sequence dnas that limit there is the DNA molecular of 90% above homology and code for said proteins.

4. contain recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium of gene described in claim 2 or 3.

5. recombinant vectors as claimed in claim 4, is characterized in that: described recombinant vectors is the recombinant plasmid that the multiple clone site of gene insertion pEAS-1a plasmid described in claim 2 or 3 is obtained.

6. recombinant bacterium as claimed in claim 4, is characterized in that: described recombinant bacterium is that recombinant plasmid claimed in claim 5 is imported to the recombinant bacterium that e. coli bl21 obtains.

Described in claim 1 protein in the application of preparing in Ketoamine oxidase.

8. the application of protein in degraded glycosylation substrate described in claim 1.

9. the application of recombinant bacterium in protein described in production claim 1 described in claim 6.

Described in claim 1 protein as the application of Ketoamine oxidase.