CN109468288A - A kind of new blue multicopper oxidase of efficient degradation histamine - Google Patents
A kind of new blue multicopper oxidase of efficient degradation histamine Download PDFInfo
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- CN109468288A CN109468288A CN201811433209.1A CN201811433209A CN109468288A CN 109468288 A CN109468288 A CN 109468288A CN 201811433209 A CN201811433209 A CN 201811433209A CN 109468288 A CN109468288 A CN 109468288A
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- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
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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
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 calculatedm、Vmax。
It is transversal away from=- 1/Km, indulge intercept=1/Vmax
As shown in Figure 10, the K of the enzyme is calculated according to Lineweaver-Burk double-reciprocal plot methodm、VmaxRespectively
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
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ctacatgtgc atccccatct t 21
Claims (10)
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.
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CN110106153A (en) * | 2019-05-24 | 2019-08-09 | 江南大学 | A kind of blue multicopper oxidase mutant that salt tolerance improves |
CN112322608A (en) * | 2020-11-09 | 2021-02-05 | 江南大学 | Method for improving stability of copper-rich oxidase and capability of degrading biogenic amine |
CN112921010A (en) * | 2021-03-18 | 2021-06-08 | 江南大学 | Multi-copper oxidase recombinase suitable for fermented food |
CN114081120A (en) * | 2021-11-17 | 2022-02-25 | 大连工业大学 | Preparation of lactobacillus copper-rich oxidase and application of lactobacillus copper-rich oxidase in degradation of biogenic amine |
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CN110106153A (en) * | 2019-05-24 | 2019-08-09 | 江南大学 | A kind of blue multicopper oxidase mutant that salt tolerance improves |
CN112322608A (en) * | 2020-11-09 | 2021-02-05 | 江南大学 | Method for improving stability of copper-rich oxidase and capability of degrading biogenic amine |
CN112921010A (en) * | 2021-03-18 | 2021-06-08 | 江南大学 | Multi-copper oxidase recombinase suitable for fermented food |
CN114081120A (en) * | 2021-11-17 | 2022-02-25 | 大连工业大学 | Preparation of lactobacillus copper-rich oxidase and application of lactobacillus copper-rich oxidase in degradation of biogenic amine |
CN114081120B (en) * | 2021-11-17 | 2023-11-07 | 大连工业大学 | Preparation of lactobacillus multi-copper oxidase and application of lactobacillus multi-copper oxidase in degradation of biogenic amine |
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