CN109468288A

CN109468288A - A kind of new blue multicopper oxidase of efficient degradation histamine

Info

Publication number: CN109468288A
Application number: CN201811433209.1A
Authority: CN
Inventors: 方芳; 周朝晖; 李铁桥; 徐洁; 卢丽玲; 陈坚
Original assignee: GUANGDONG PRB BIO-TECH Co Ltd; Jiangnan University
Current assignee: GUANGDONG PRB BIO-TECH Co Ltd; Jiangnan University
Priority date: 2018-11-28
Filing date: 2018-11-28
Publication date: 2019-03-15

Abstract

The invention discloses a kind of new blue multicopper oxidases of efficient degradation histamine, belong to technical field of molecular biology.Blue multicopper oxidase sequence provided by the invention is as shown in SEQ ID NO.1.The enzyme has the performance of stronger degradation biological amine, degradation spectrum is wide, and can degrade tryptamines, phenyl ethylamine, putrescine, cadaverine, histamine, tyrasamine, spermidine, especially for the good degrading effect of histamine, interior histamine degradation rate reaches 82% for 24 hours, has important commercial application potentiality.

Description

A kind of new blue multicopper oxidase of efficient degradation histamine

Technical field

The present invention relates to a kind of new blue multicopper oxidases of efficient degradation histamine, belong to technical field of molecular biology.

Background technique

Biogenic amine is a kind of biologically active Small-molecule basic compounds, is widely present in fermented food and containing wine In the fermented beverage of essence, mainly formed by corresponding amino acid by the decarboxylation of microorganism, or by aldehyde, letones in ammonia The effect of base acid transaminase is lower to be generated.After the exogenous biological amine of human body excess intake, headache, nausea, palpitaition, disordered breathing can be generated Equal allergic reactions, even threat to life when serious, and also a variety of biogenic amines are jointly in the presence of the toxic effect that can reinforce biogenic amine.Food Common biogenic amine mainly includes putrescine, cadaverine, spermine, spermidine, tyrasamine, phenyl ethylamine and histamine in product, wherein tyrasamine and group The content highest of amine, it is maximum to the potential hazard of human body.

Biogenic amine in control and reduction fermented product, there are mainly three types of methods at present: 1, controlling raw material；2, control fermentation The microorganism of generation amine in system；3, enzymic degradation.First two method has certain limitation, for example will affect certain high eggs The flavor of food and drinks one gets without pay product improves equipment and energy consumption cost.And the third method by into fermented food add biogenic amine degrading enzyme, Food nutrition material damage is not caused, does not generate new toxicant.Existing biogenic amine degrading enzyme includes amine oxidase and amine Dehydrogenase, both enzymes have three: 1, the biogenic amine type degraded is few, and the efficiency for histamine of degrading is lower；2, optimal pH Partial neutral is not suitable for being applied in acid fermentation food；3, activity is easy to receive the inhibition of some drugs or compound.

Blue multicopper oxidase (Multicopper oxidase, MCO) is a kind of family containing CuO-2 layer, part of more copper Biogenic amine can be catalyzed and generate corresponding aldehyde, ammonia and water by oxidizing ferment, achieve the purpose that degradation biological amine.There is presently no reported It is capable of the blue multicopper oxidase of efficient degradation biogenic amine, especially histamine in the environment of acid saliferous.Therefore, it finds a kind of efficient The novel blue multicopper oxidase of degradation histamine, it is particularly important for reducing the biogenic amine in acid fermentation food.

Summary of the invention

Of the invention provides a kind of novel more copper for deriving from lactobacillus fermenti (Lactobacillus fermentum) Oxidizing ferment can be used for degradation biological amine, especially histamine.

The first purpose of the invention is to provide a kind of blue multicopper oxidases, contain amino acid shown in SEQ ID NO.1 Sequence.

A second object of the present invention is to provide the genes for encoding the blue multicopper oxidase.

In one embodiment of the invention, the gene of the coding blue multicopper oxidase contains shown in SEQ ID NO.2 Nucleotide sequence.

Third object of the present invention is to provide a kind of genetic engineering bacteriums, express the blue multicopper oxidase.

In one embodiment of the invention, the genetic engineering bacterium is host with bacterium or fungal cell.

In one embodiment of the invention, the genetic engineering bacterium is host with e. coli bl21.

In one embodiment of the invention, the genetic engineering bacterium is using pET series plasmids as expression vector.

In one embodiment of the invention, the genetic engineering bacterium is using pET-28a as expression vector.

Fourth object of the present invention is to provide the construction method of the genetic engineering bacterium, is that will encode more copper oxidations The gene of enzyme is connect with carrier, is converted into Bacillus coli cells.

In one embodiment of the invention, the method is to connect sequence shown in SEQ ID NO.2 and pET-28a It connects, converts into e. coli bl21.

Fifth object of the present invention is to provide a kind of production method of blue multicopper oxidase, the method is by the gene Engineering bacteria is seeded in culture medium, in 16~20 DEG C cultivate 16~for 24 hours, collect somatic cells, smudge cells obtain enzyme solution.

In one embodiment of the invention, the genetic engineering bacterium is seeded in TB culture medium by the method, training It supports to OD₆₀₀When being 0.6~0.8, IPTG and copper ion induction is added.

In one embodiment of the invention, the genetic engineering bacterium is seeded in TB culture medium by the method, training It supports to OD₆₀₀When being 0.6~0.8, the copper ion induction of the IPTG and 0.5~1mM of final concentration of 0.5~1mM is added.

Sixth object of the present invention is to provide application of the blue multicopper oxidase in terms of degradation biological amine.

In one embodiment of the invention, the application includes the Content of Biogenic Amines reduced in fermented food.

In one embodiment of the invention, the application is specifically to reduce in soy sauce in terms of biogenic amine in field of food Application.

In one embodiment of the invention, the application is that the blue multicopper oxidase is added in soy sauce, degradation Biogenic amine in soy sauce.

In one embodiment of the invention, the biogenic amine includes tryptamines, phenyl ethylamine, putrescine, cadaverine, histamine, junket At least one of amine, spermine, spermidine.

The preparation containing the blue multicopper oxidase is also claimed in the present invention.

The utility model has the advantages that the optimal pH of the enzyme is 3.5, and optimum temperature is the present invention provides a kind of new blue multicopper oxidase 50 DEG C, copper ion can promote enzyme activity to improve, and enzyme activity is made to improve 3 times.The present invention is by the blue multicopper oxidase in lactobacillus fermenti source The heterogenous expression in Escherichia coli, the blue multicopper oxidase recombinase of acquisition have the performance of stronger degradation biological amine, Neng Gou Degradation tryptamines, phenyl ethylamine, putrescine, cadaverine, histamine, tyrasamine, spermidine, the biogenic amine in the system of total amine about 720mg/L in for 24 hours Degradation rate is up to 42%, wherein degradation histamine effect is best, interior histamine degradation rate reaches 82% for 24 hours, has important industrial application Potentiality.

Detailed description of the invention

Fig. 1 is the proof diagram of blue multicopper oxidase gene in L.fermentum；Wherein, M Marker；1 negative control； Blue multicopper oxidase gene in 2L.fermentum；

Fig. 2 is temperature on blue multicopper oxidase (being abbreviated as MCOF enzyme) active influence；

Fig. 3 is influence of the temperature to MCOF stability；

Fig. 4 is influence of the pH to MCOF enzymatic activity；

Fig. 5 is influence of the pH to MCOF stability；

Fig. 6 is influence of the different metal ions to MCOF enzyme activity；

Fig. 7 is influence of the various concentration copper ion to MCOF enzyme activity；

Fig. 8 is tolerance of the MCOF to various concentration (w/v, g/100mL) NaCl；

Fig. 9 is tolerance of the MCOF to various concentration (v/v, mL/100mL) ethyl alcohol；

Figure 10 is the Lineweaver-Burk double reciprocal plot of MCOF.

Embodiment 1

According to Lactobacillus fermentum strain 47-7genome in NCBI (GenBank accession number are as follows: NZ_CP017712.1 the gene order of the blue multicopper oxidase in), design primer, with the lactobacillus fermenti of this research department's preservation (Lactobacillus fermentum) genome is template, expands blue multicopper oxidase gene.Primer needed for amplification is as follows:

Upstream: ATGAATGAACCAGTTTTCGATTC

Downstream: CTACATGTGCATCCCCATCTT

PCR response procedures: 95 DEG C of initial denaturation 3min；30s is denaturalized at 95 DEG C, anneal 30s at 55 DEG C, extends at 72 DEG C 1min30s, 34 circulations；Extend 10min at 72 DEG C, is kept the temperature at 4 DEG C.

Agarose gel electrophoresis verifying is carried out by 1% after amplification, as shown in Figure 1, it is 1500bp that amplification, which obtains size, The genetic fragment of left and right, it is in the same size with target gene, show to produce PCR containing blue multicopper oxidase gene in L.fermentum Object purifying sequencing, sequencing result is as shown in SEQ ID NO.2.

Embodiment 2

(1) vector construction: the gene and pET-28a that embodiment 1 is obtained use restriction enzyme EcoRI, HindIII Double digestion reacts 45min in 37 DEG C of metal baths.Genetic fragment and plasmid after digestion is after recovery purifying, and in molar ratio 4 The ratio of~10:1 mixes, and is attached reaction by Solution I ligase, connects overnight in 16 DEG C of metal baths.

(2) building of recombinant bacterium: the connection that the preparation of 5 μ L steps (1) is added into escherichia coli jm109 competent cell produces Object stands 30min in ice after evenly mixing.It is taken out immediately after 42 DEG C of water-bath 90s and places 2-5min in ice.700 μ L are added LB culture is based on 37 DEG C, 200rmin^-1Shaken cultivation 1h.4000r·min^-1It is centrifuged 2min, most of supernatant is discarded, stays Thallus is resuspended in 100 μ L or so supernatant.Bacterium solution is spread evenly across plate containing kanamycin, is placed in 37 DEG C of incubators overnight Culture.Next day picking single colonie screens positive transformant by bacterium colony PCR.Digestion and sequence verification.Select sequencing correctly weight Group plasmid is converted into e. coli bl21 competent cell, is obtained recombinant bacterium, is named as pET-28a-MCOF/BL21.

(3) preparation of crude enzyme liquid: recombinant bacterium pET-28a-MCOF/BL21 is inoculated into LB culture medium, in 37 DEG C, It is incubated overnight under the conditions of 220r/min.Seed liquor is transferred in TB culture medium by 1% inoculum concentration, in 37 DEG C, 220r/min Under the conditions of culture to OD₆₀₀It is 0.6~0.8, the Cu of final concentration of 1mM is added²⁺And the IPTG of final concentration of 0.1mM is added, 20 DEG C shaken cultivation 20h.Gained fermentation liquid is centrifuged 15min in 4 DEG C, 8000r/min, bacterial sediment object is collected, uses 20mmol L^-1Thallus is resuspended by cell washing 2 times, then with phosphate buffer for the phosphate buffer solution of pH 7.0.

Using wild mushroom (Lactobacillus fermentum) as control, Lactobacillus fermentum is connect Kind into MRS culture medium, in 37 DEG C of stationary cultures 18~for 24 hours.Seed liquor is transferred in MRS culture medium by 1% inoculum concentration, It is inoculated into MRS culture medium for 24 hours in 37 DEG C of stationary cultures, in 37 DEG C of stationary cultures 18~for 24 hours, collects thallus.

Thallus suspension is placed on ice-water bath, using ultrasonic disruption thalline cell, ultrasonic power 35%, vibration 2s stop 4s, until solution is limpid.By broken liquid in 4 DEG C, 10000rmin^-1It is centrifuged 30min, collects thick enzyme needed for supernatant is The enzyme activity of liquid, crude enzyme liquid is 484.2U/L, improves 258 times or so compared with wild mushroom (original lactobacillus fermenti).

(4) preparation of crude enzyme liquid: embodiment 1 is obtained into crude enzyme liquid after ultrasonic disruption, it is spare to cross 0.45 μ L filter membrane.Albumen Purifying: clean nickel column with the deionized water of 5~10 times of column volumes under the flow velocity of 5mL/min；Add under the flow velocity of 5mL/min The Binding buffer for entering about 8~10 times of column volumes, balances nickel column；The loading under the flow velocity of 1mL/min；In 5mL/min Flow velocity under the Binding buffer of about 8~10 times of column volumes is added, balance nickel column；With not under the flow velocity of 5mL/min Phosphate buffer with gradient concentration containing imidazoles elutes nickel column, and eluent is collected with centrifuge tube, washes through SDA-PAGE detection determination De- condition, obtains pure protein.It is sequenced, obtains amino acid sequence shown in SEQ ID NO.1.

Embodiment 3

(1) influence of the temperature to enzymatic activity and stability

By the obtained blue multicopper oxidase of purifying and substrate respectively under the conditions of different temperature (25,35,40,45,50,55, 60,70,80 DEG C) reaction assay enzyme activity, with highest enzyme activity for 100%, the opposite enzyme activity calculated at each temperature determines enzyme most Suitable reaction temperature.

By enzyme solution after purification under the conditions of each temperature warm bath, take appropriate enzyme solution and substrate in optimal reactive temperature every 3h Lower reaction measures influence of the condition of different temperatures to enzyme activity stability.

As shown in Fig. 2, MCOF, when temperature is 30~60 DEG C, enzyme activity is up to 50% or more, when temperature is 50 DEG C, relatively Enzyme activity is up to 100%；As shown in figure 3, MCOF, when being lower than 55 DEG C, after keeping the temperature 3h, enzyme activity is still able to maintain 30% or more.

(2) influence of the pH to enzymatic activity and stability

Under the conditions of the optimum temperature of enzyme, enzyme activity of the enzyme under condition of different pH is measured using visible absorption measuring method, Determine optimal reaction pH.The condition of different pH is respectively: 1. 50mmol/L HAc-NaAc buffer, pH 2.5~5.0,2. 50mmol/L phosphate buffer pH 5.5~7.0,3. 50mmol/L Tris-HCl buffer, pH 7.5~8.5.

Blue multicopper oxidase after purification is stored in respectively under above-mentioned three kinds of pH parts, appropriate enzyme solution is taken every 3h, with substrate It is reacted under optimum temperature, the enzyme activity measured with 0h measures influence of the condition of different pH to enzyme activity stability for 100%.

As shown in Figures 4 and 5, the optimal pH of MCOF is 3.5, and 3h is placed at pH 3~4, and enzyme activity keeps 45% or more.

(3) influence of the metal ion to enzyme activity

Enzyme solution is mixed with the metal ion buffer of final concentration of 1mmol/L, is kept the temperature under temperature stable condition 10min measures enzyme activity.Enzyme activity is measured as 100% to be not added with metal ion, determines influence of the metal ion to enzyme activity.

As shown in fig. 6, compared with the enzyme for being not added with metal ion, Cu²⁺Enzyme activity can significantly be promoted, improve enzyme activity 2.8 times, Fe²⁺Inhibit the effect of enzyme activity most obvious, enzyme activity is made to be reduced to the 57% of the enzyme for not adding metal ion.As shown in fig. 7, It is added to the Cu of 0.1~1mM²⁺Afterwards, enzyme activity is with Cu²⁺The increase of concentration and increase, work as Cu²⁺When concentration is 0.1mM, opposite enzyme Living is 98%；Cu²⁺When concentration is 0.3mM, opposite enzyme activity is 127%；Work as Cu²⁺When concentration is 0.5mM, opposite enzyme activity is 171%；Work as Cu²⁺When concentration is 0.7mM, opposite enzyme activity is 208%；Work as Cu²⁺When concentration is 1mM, opposite enzyme activity is 296%.? It is added to the Cu of 1~8mM²⁺Afterwards, enzyme activity is with Cu²⁺The increase of concentration and reduce, work as Cu²⁺When concentration is 2mM, opposite enzyme activity is 106%；Work as Cu²⁺When concentration is 4mM, opposite enzyme activity is 44%, the Cu more than 4mM²⁺It can inhibit enzyme activity.

(4) influence of the NaCl to enzyme activity stability

Enzyme solution is kept the temperature 1h (control group warm bath under similarity condition) in the NaCl buffer of different quality containing, is measured Enzyme activity under corresponding conditions measures enzyme activity with control (being not added with the enzyme solution of NaCl) as 100%, calculates each NaCl mass fraction item Opposite enzyme activity, determines influence of the NaCl to enzyme activity stability under part.As shown in figure 8, the NaCl of different quality containing is to the steady of enzyme It is qualitative to have an impact, wherein 18% NaCl is minimum to the stability influence of enzyme, placed in the buffer containing 18% NaCl 1h, enzyme activity are still able to maintain 40%.

(5) influence of the ethyl alcohol to blue multicopper oxidase enzyme activity

Enzyme solution is kept the temperature 1h (control group warm bath under similarity condition) in the ethyl alcohol buffer of different volumes score, is measured Enzyme activity under corresponding conditions measures enzyme activity with control as 100%, calculates the opposite enzyme activity under the conditions of each ethyl alcohol, determine ethyl alcohol pair The influence of enzyme activity stability.

As shown in figure 9, concentration of alcohol is higher, the influence to enzyme is bigger, (the ethyl alcohol in Japanese soy sauce under 2.5% ethyl alcohol Concentration is 2.5% or so) 1h is placed, enzyme activity is still able to maintain 70% or more.

(6) enzyme kinetics constant

Under the conditions of most suitable pH, the ABTS (0.05~0.5mM) of various concentration is added, and under optimal reactive temperature, Enzyme activity is measured at 420nm respectively, then according to calculation formula, K is calculated_m、V_max。

It is transversal away from=- 1/K_m, indulge intercept=1/V_max

As shown in Figure 10, the K of the enzyme is calculated according to Lineweaver-Burk double-reciprocal plot method_m、V_maxRespectively 1.30mmol/L, 7.67 × 10^-2mmol/(L·min)。

Embodiment 5

Blue multicopper oxidase enzyme solution is added to each biogenic amine monoamine system, 37 DEG C of reaction 48h, biogenic amine list with dense 100U/L eventually It is 50mg/L that monoamine is dense eventually in amine system；Control group replaces the crude enzyme liquid in experimental group with phosphate buffer.In biogenic amine list In amine system, degradation rate of the MCOW to biogenic amine:

30% > phenyl ethylamine > cadaverine > tryptamines > 10% of MCOF: histamine > 50% > junket ammonia > putrescine > spermidine >

Wherein, best to histamine degradation effect, degradation rate is up to 52%.

Degradation of 1 MCOF of table to the biogenic amine in biogenic amine monoamine system

Embodiment 6

Blue multicopper oxidase enzyme solution is added separately to 37 DEG C of biogenic amine mixed system reactions for 24 hours with dense 500U/L eventually, biogenic amine The ratio of biogenic amine is close to the biogenic amine in soy sauce in mixed system, wherein tryptamines 20mg/L, phenyl ethylamine 150mg/L, putrescine 50mg/L, cadaverine 50mg/L, spermidine 50mg/L, histamine 2 00mg/L, junket ammonia 200mg/L.Control group is taken with phosphate buffer For the crude enzyme liquid in experimental group.Biogenic amine degradation results are as shown in table 2, and wherein histamine degradation effect is best, interior for 24 hours to organize amine degradation Rate reaches 82%.

2 MCOF of table is in biogenic amine mixed system to the degradation of biogenic amine

Embodiment 7

Blue multicopper oxidase enzyme solution is added in soy sauce with dense 500U/L eventually, 37 DEG C are reacted for 24 hours, control group PBS buffer solution Replace the crude enzyme liquid in experimental group, biogenic amine degradation results are as shown in table 3 below, and the amount for organizing amine degradation is most.

Degradation of 3 MCOF of table in soy sauce

Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.

SEQUENCE LISTING

<110>Guangdong ZhuJiangqiao Biotechnology Co., Ltd

Southern Yangtze University

<120>a kind of new blue multicopper oxidase of efficient degradation histamine

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 518

<212> PRT

<213>artificial sequence

<400> 1

Met Asn Glu Pro Val Phe Asp Ser Tyr Phe Tyr Asp Glu Gln Ala Phe

1 5 10 15

Asn Leu His Asp Ala Ala Tyr Lys Pro Leu Lys Ala Ala Gln Ala Pro

20 25 30

Ala Thr Pro Leu Asn Ile Pro Pro Leu Leu Thr Pro Asp Lys Val Asp

35 40 45

Gly Asp Asp Val Tyr Tyr Thr Val Thr Ala Gln Ala Gly Ala Thr Gln

50 55 60

Leu Leu Pro Gly Glu Lys Thr Lys Thr Trp Gly Phe Asn Ala Ser Met

65 70 75 80

Leu Gly Gln Thr Val Val Phe Glu Arg Gly Lys Thr Tyr His Met His

85 90 95

Leu Val Asn His Leu Pro Glu Val Thr Thr Phe His Trp His Gly Leu

100 105 110

Glu Ile Pro Gly Pro Ile Glu Asp Gly Gly Cys His Ala Pro Val Tyr

115 120 125

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

130 135 140

Thr Ala Trp Leu His Ala His Pro Cys Pro Glu Thr Ala Tyr Gln Val

145 150 155 160

Trp Gln Gly Leu Ala Thr Met Ala Ile Ile His Asp Gln Glu Glu Ala

165 170 175

Ser Leu Pro Leu Pro His Thr Tyr Gly Val Asp Asp Leu Pro Leu Ile

180 185 190

Leu Gln Asp Arg Asn Phe His Ala Gly Asn Gln Phe Asp Tyr Arg Ala

195 200 205

Asp Tyr Asp Pro Asp Gly Val Gln Gly Asp Thr Ala Val Ile Asn Ala

210 215 220

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

225 230 235 240

Ile Leu Asn Gly Ser Asn Arg Arg Glu Tyr Arg Leu His Phe Ser Asp

245 250 255

Asp Leu Thr Phe Thr Gln Ile Gly Ser Asp Leu Ser Phe Leu Pro His

260 265 270

Pro Val Lys Leu Lys Lys Leu Met Thr Thr Cys Ala Glu Arg Gln Glu

275 280 285

Ile Val Val Asp Phe Ala Gly Tyr Gln Pro Gly Asp Thr Val Thr Leu

290 295 300

Tyr Ser Asp Asp Ser Pro Leu Val Glu Phe Arg Ile His Glu Phe Thr

305 310 315 320

Pro Ala Gly Glu Glu Leu Pro Thr Thr Leu Thr Lys Ile Asp Tyr Pro

325 330 335

Thr Pro Asp Pro Ser Leu Pro Val Lys Gln Val Val Met Ser Gly Met

340 345 350

Asp Glu Thr Val Met Ile Asp Gly Lys Lys Phe Gln Met Asp Arg Ile

355 360 365

Asp Tyr Thr Met Pro Met Gly Lys Cys Gln Leu Trp Asp Ile Thr Asn

370 375 380

Thr Asn Asp Met Asn Gly Gly Met Ile His Pro Phe His Met His Gly

385 390 395 400

Cys Ala Phe Glu Ile Val Ser Arg Asn Gly Gln Glu Pro Tyr Pro Phe

405 410 415

Glu His Gly Leu Asn Asp Thr Val Ala Val Asn Pro Gly Glu His Val

420 425 430

Ile Ile Lys Val Tyr Phe Gln Val Pro Gly Val Phe Met Tyr His Cys

435 440 445

His Ile Ile Glu His Glu Asp Gly Gly Met Met Ala Gln Leu Lys Val

450 455 460

Val Asp Pro Ala Ala Pro Asp Arg Glu Tyr His Leu Leu Asn His Met

465 470 475 480

Thr Leu Met Glu Ala Phe Ala Lys Glu Arg Gly Val Thr Met Asp Glu

485 490 495

Leu Trp Leu Gly Gly Met Asp Ser Tyr Lys Lys Met Gly Met His Met

500 505 510

His His His His His His

515

<210> 2

<211> 1557

<212> DNA

<213>artificial sequence

<400> 2

atgaatgaac cagttttcga ttcctacttt tacgatgagc aagccttcaa ccttcacgat 60

gcggcctaca agccactgaa ggcggctcaa gcacccgcga cgcccctcaa cattccgccc 120

ctgttaaccc cagacaaggt ggacggcgac gacgtgtact acaccgttac cgcccaggcg 180

ggggcaaccc agctattgcc gggggaaaag accaagacct ggggctttaa cgcctcaatg 240

ctggggcaga cggtggtctt tgagcgcggc aagacttacc acatgcacct ggtcaaccac 300

ctaccggaag tgacgacctt ccactggcac ggactagaga ttcccgggcc aattgaagac 360

ggcggctgcc acgccccggt ttacccgggc gaggcccgcg acgtgacctt taagatcatt 420

cagccggccg ccaccgcctg gctccacgcc cacccctgcc cggaaacggc ctaccaggtt 480

tggcagggac tggcgacgat ggcgatcatt cacgaccaag aagaagctag tttgcccctg 540

ccgcacactt acggagttga tgacctgccg ttgatcttgc aggaccgcaa cttccacgcg 600

ggcaaccagt ttgattaccg ggccgattac gaccctgatg gcgtccaggg tgacacggcg 660

gtgattaacg ccaccgtcaa tccttacttt gacgtcacca cccagcgcct gcggttgcga 720

attttaaacg gctctaaccg ccgcgagtac cggctccact tctcggacga cttaaccttc 780

acccaaattg gctcggacct gagctttttg ccccacccgg tcaagttaaa gaagctgatg 840

actacctgtg ccgagcgcca ggagatcgtg gtcgactttg ccggctacca gccgggcgac 900

acggttaccc tgtactccga cgacagtccg ctggtcgagt tccggatcca cgagtttacg 960

ccggcaggag aggagttgcc caccaccctg accaagattg attacccgac cccagacccg 1020

agcctacccg tcaagcaggt ggtgatgtct gggatggacg agacggtgat gattgacggc 1080

aagaagttcc agatggaccg gatcgattac acgatgccga tgggcaagtg ccagctttgg 1140

gacattacca acaccaacga catgaatggc gggatgattc accccttcca catgcacggc 1200

tgcgcctttg agatcgtctc ccgtaacggc caggaaccct acccgtttga acacggactt 1260

aacgacaccg tcgccgttaa cccgggcgaa cacgtcatca tcaaggtcta cttccaagta 1320

ccgggggtct ttatgtacca ttgtcacatt atcgaacacg aagatggcgg gatgatggcc 1380

caactaaagg ttgtcgaccc ggccgccccg gaccgggagt accacttgct caaccatatg 1440

accctgatgg aggcctttgc caaggagcgg ggcgtcacga tggatgaact ctggcttggt 1500

gggatggact cctacaagaa gatggggatg cacatgcatc atcaccacca ccactag 1557

<210> 3

<211> 23

<212> DNA

<213>artificial sequence

<400> 3

atgaatgaac cagttttcga ttc 23

<210> 4

<211> 21

<212> DNA

<213>artificial sequence

<400> 4

ctacatgtgc atccccatct t 21

Claims

1. a kind of blue multicopper oxidase, which is characterized in that contain amino acid sequence shown in SEQ ID NO.1.

2. encoding the gene of blue multicopper oxidase described in claim 1.

3. expressing the cell of blue multicopper oxidase described in claim 1.

4. a kind of genetic engineering bacterium, which is characterized in that with e. coli bl21 be host, express more copper described in claim 1 Oxidizing ferment.

5. genetic engineering bacterium according to claim 4, which is characterized in that using pET series plasmids as expression vector.

6. a kind of method of genetic engineering bacterium described in building claim 4, which is characterized in that the gene of blue multicopper oxidase will be encoded It connect, is converted into Bacillus coli cells with carrier.

7. a kind of production method of blue multicopper oxidase, which is characterized in that genetic engineering bacterium described in claim 4 or 5 to be inoculated with Into culture medium, in 16~20 DEG C cultivate 16~for 24 hours, collect somatic cells, smudge cells obtain enzyme solution.

8. application of the blue multicopper oxidase described in claim 1 in terms of degradation biological amine.

9. blue multicopper oxidase described in claim 1 reduces the application in soy sauce in terms of biogenic amine in field of food.

10. the preparation containing blue multicopper oxidase described in claim 1.