CN108570459A - A kind of method of high-efficiency fermenting production recombinant bacteria laccase - Google Patents
A kind of method of high-efficiency fermenting production recombinant bacteria laccase Download PDFInfo
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Abstract
The invention discloses a kind of methods that high-efficiency fermenting produces recombinant bacteria laccase.This method is to be added that methanol, ethyl alcohol, Qula be logical, glycine, glycine betaine in recombination bacillus coli fermentation process, glycerine, any one or more tightening agent in sorbierite and reach.The present invention utilizes the coercion of tightening agent, delays the growth of thalline, reduces toxic action caused by the accumulation of acetic acid, the expression quantity of recombinant bacteria laccase is improved significantly.Use method provided by the invention, the fermenting and producing that recombinant bacteria laccase is carried out using recombination bacillus coli is above the highest recombinant bacteria laccase yield (recombinant protein yield) reported at present at total enzyme activity (total recombinant protein yield), intracellular enzyme activity (intracellular recombinant protein yield), three aspect of enzymatic activities (extracellular recombinant protein yield).
Description
Technical field
The invention belongs to biotechnologies, and in particular to arrive a kind of raising recombination bacillus coli fermenting production recombinant bacteria
The method of laccase yield.
Background technology
Laccase (laccase, Benzenediol：Oxygen oxidoreducing enzyme, EC1.10.3.2) it is a kind of polyphenol oxidase of cupric,
With the ceruloplasmin of ascorbic acid oxidase (ascorbieacidoxidase), mammal in plant
(ceruloplasmin) homologous, belong to a member in blue copper oxidase (bluecopperoxidase) family, it, which can be utilized, divides
Sub- oxygen aoxidizes the compound of a plurality of types of phenolic compounds (such as ferulic acid) and non-phenols as electron acceptor, and catalysis is anti-
The unique by-product answered is water, is known as most green, environmentally friendly biocatalyst.Laccase environmental contaminants improvement, food
Product processing industry, slurrying and paper industry, textile industry, soil the fields such as biological prosthetic and nanometer biotechnology have it is extensive
Application, such as remove pollutant, dye decolored, organic synthesis containing phenol, waste water decoloring, the clarification of beverage is controlled with color and luster,
The production of edible mushroom, medicinal fungus, the functional improvement of food component and vegetable food product protection etc..According to the difference in source, paint
Enzyme can be divided into fungal laccase and bacterial laccase.
Fungal laccase generally has higher enzyme activity, fermentation yield 50000-100000U/L, but fungal laccase pH suitable
Answer range relatively narrow, pH is more than 7 condition fast deactivation；Fungal laccase thermal stability is poor simultaneously, and optimum temperature is generally at 30 DEG C
50 DEG C or more of high temperature can also lose activity rapidly between to 45 DEG C.Therefore, although fungal laccase fermentation yield is higher,
The reason of its pH adaptability and thermal stability, limits the range of its application.Bacterial laccase has good thermal stability, enzyme
The features such as pH value in reaction range is wide, good to halogen stability.Meanwhile compared with fungi fermentation laccase, the Bacteria Culture period is short,
Required nutritional ingredient is simple, has the fermentation advantage that low energy consumption.However bacterial laccase fermentation yield is low at present, is extremely difficult to work
The requirement of industry fermentation level.
Due to being difficult the bacterium for obtaining ideal high yield laccase, recombinant technique is utilized, laccase gene is cloned and is transformed into
The bacterial hosts bacterium such as Escherichia coli or hay bacillus builds recombination engineering bacteria, by controlling fermentation condition, improves bacterium
The fermentation level of laccase is the strategy for solving the problems, such as bacterial laccase low output.Currently, it is thin to be improved Escherichia coli fermentation
The research of bacterium laccase, such as laccase gene codon optimization, the suitable promoter of selection improve mRNA stability, utilize fusion egg
In vain with molecular chaperones, by deleting the methods of engineering bacteria by-product route of synthesis, regulation and control fermentation condition realization High Density Cultivation,
Although improving the fermentation yield of recombinant bacteria laccase to a certain extent, compared with fungal laccase fermentation level, there are still
Prodigious gap, it is 4000-6000U/ that reports at present, which produces the maximum output of recombinant bacteria laccase using Escherichia coli fermentation,
L。
Invention content
The purpose of the present invention is for the above-mentioned of the prior art, provide a kind of utilization recombination bacillus coli high-efficiency fermenting life
The method for producing recombinant bacteria laccase.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of method of efficient production recombinant bacteria laccase, in the recombination bacillus coli fermentation process of production bacterial laccase
Add that methanol, ethyl alcohol, Qula be logical, any one or more side of body in glycine, glycine betaine, tween, sapn, glycerine, sorbierite
Compel agent and induced expression is carried out to laccase.
Preferably 1-10% is added in the 0-2h of the recombination bacillus coli fermentation of production bacterial laccase in method of the present invention
(v/v) methanol, Qula be logical or methanol and the logical combination of Qula.
Method of the present invention, the addition time preferred 0-0.5h of methanol, further preferred 0h, the preferred 4- of additive amount
8%, further preferred 6%.
Method of the present invention, the addition time preferred 0-0.5h that Qula is led to, further preferred 0h, additive amount are preferred
2-5%, further preferred additive amount are 4%.
The addition time that method of the present invention, methanol and Qula lead to co-induction agent is 0-0.5h, and additive amount is first
Alcohol 4-8%, Qula lead to 2-5%；And the two summation is no more than 10%.
Method of the present invention, methanol and Qula lead to the addition time preferred 0h of co-induction agent, the preferred first of additive amount
Alcohol 6%, Qula logical preferably 4%.
The method of the raising bacterial laccase expression quantity, further includes following steps：
(1) Recombinant organism of structure production recombinant bacteria laccase；
(2) the small bacterium of picking after cultivating the Recombinant organism for producing recombinant bacteria laccase 10-11 hours at 37 DEG C
It falls, accesses the 50ml LB liquid mediums containing kanamycins, 37 DEG C of overnight incubations of 180rpm obtain seed liquor；Wherein, described
The final concentration of 50ug/ml of kanamycins；
(3) seed liquor is taken to be transferred to the TB culture mediums containing kanamycins；
(4) the addition tightening agent, 30 DEG C, 180rpm shakes OD600=1.0；
(5) IPTG and GuSO4 is added to be induced, 16 DEG C, 180rpm, 20h；The final concentration of 100ug/ of wherein IPTG
Ml and GuSO4Final concentration of 0.25mM；
(6) 16 DEG C of standing 22h.
(7) centrifugation obtains thalline and supernatant, and supernatant is ectoenzyme, and thalline is with added with Cu2+The Tris-HCl of final concentration 1mM is slow
Fliud flushing is resuspended, ultrasonication, and 75 DEG C of heating 10min, 4 DEG C, 10000 × g centrifuges 30min and collects supernatant, and it is dead to obtain recombination
Die the crude enzyme liquid of paddy bacillus laccase.
As the preferred of the present invention, structure produces the death used when the Recombinant organism of recombinant bacteria laccase
Rice sprout spore laccase gene is to carry out the gene order after codon optimization for Escherichia coli, as shown in SEQ ID NO.1.
Advantageous effect：
Laccase is in the improvement of environmental contaminants, food processing industry, slurrying and paper industry, textile industry, the life of soil
The fields such as object reparation and nanometer biotechnology have a wide range of applications.Fungal laccase fermentation yield is higher, but pH stability and temperature
It is poor to spend stability, therefore, receives limitation in the actual use process.There is bacterial laccase thermal stability height, pH to adapt to model
It encloses the features such as wide, but bacterial laccase fermentation yield is low, fails to realize large-scale fermenting and producing.
The present invention utilizes that (tightening agent is by the way that one or more tightening agents are added in recombination bacillus coli fermentation process
Methanol, ethyl alcohol, Qula be logical, glycine, glycine betaine, glycerine, any one or more in sorbierite), it is effectively improved weight
The fermentation yield of group bacterial laccase.The present invention utilizes the coercion of tightening agent, delays the growth of thalline, reduces the heap of acetic acid
Toxic action caused by product, hence it is evident that improve the expression quantity of bacterial laccase.When using methanol as tightening agent, 500mL shake flask fermentations
Yield is increased to 11215U/L by original 1984U/L；Under 50L fermentation tank scales, total output reaches 114000U/L, hence it is evident that is higher than
The yield level (4000-6000U/L) of previously reported recombinant bacteria laccase.Meanwhile there is not recombinant bacteria laccase big in the past
The report of enterobacteria extracellular expression, using this patent provide tightening agent induced expression method, realize bacterial laccase
The effect of extracellular expression.When using methanol as tightening agent, extracellular recombinant bacteria laccase yield never detects in 500mL shaking flasks
Go out, is increased to 2578U/L；Under 50L fermentation tank scales, extracellular recombinant bacteria yield reaches 25000U/L.
When carrying out induced expression using the logical tightening agent of Qula or the logical collaboration stress of methanol/Qula, in total enzyme activity (gross weight
Histone yield), intracellular enzyme activity (intracellular recombinant protein yield), the aspect of enzymatic activities (extracellular recombinant protein yield) three have
Different improvement effects.
The present invention is directed to the problem of current recombinant bacteria laccase fermentation low output, provides one kind in recombination bacillus coli
Fermentation process, the method for adding tightening agent inducement efficient fermenting and producing recombinant bacteria laccase.Under 50L fermentation conditions, recombination is thin
Bacterium laccase fermentation level can reach 140000U/L or so, be 20 times or more of the highest bacterial laccase yield reported；Meanwhile
Recombinant bacteria laccase is realized in the extracellular expression effect of Escherichia coli, extracellular yield is up to 111000U/L or so.The present invention
The fermenting and producing period of the Escherichia coli recombinant bacteria laccase of offer is about 20h or so, and the fermentation period of fungal laccase is
96-168h.Therefore, the fermenting and producing mode of bacterial laccase provided by the invention, it is more energy saving compared with the fermenting and producing of fungal laccase
Advantage.
Description of the drawings：
Fig. 1 recombinates fmb-103 laccase gene expression vector establishment processes
Fig. 2 is not added with producing enzyme (intracellular) curve of tightening agent Escherichia coli fermentation production bacterial laccase
Fig. 3 Methanol Stress induces the producing enzyme curve of Escherichia coli fermentation production bacterial laccase
Fig. 4 Qulas lead to the producing enzyme curve of stress-inducing Escherichia coli fermentation production bacterial laccase
The producing enzyme curve of the logical collaboration stress-inducing Escherichia coli fermentation production bacterial laccase of Fig. 5 methanol/Qula
Specific implementation mode
The invention will be further elaborated by the following examples.
Embodiment 1：Produce the structure of the Recombinant organism of recombinant bacteria laccase
Gene sequence after dead rice sprout spore laccase gene codon (carrying out codon optimization for Escherichia coli) optimization
Row, as shown in SEQ ID NO.1；Amino acid sequence is as shown in SEQ ID NO.2.Entrust genome company's chemical synthesis gene.
According to the laccase gene sequence of optimization, two primers are designed, sense primer identifies sequence plus SacI, and downstream is drawn
Object is plus XhoI identifications sequence (underscore part is limitation enzyme recognition sequence):
Sense primer F-1：5′-CGCGAGCTCATGACACTTGAAAAATTTGTGGATGC-3′(SEQ ID NO.3),
Downstream primer R-1:5′-CCGCTCGAGTTATTTATGGGGATCAGTTATATC-3′(SEQ ID NO.4),
Each ingredient is added according to following PCR system, expands laccase gene:
PCR programs are：94℃3min；30×(94℃40s；53℃50s；72℃90s)；72℃10min.
Work PCR product Purification Kit PCR product is given birth to Shanghai, adds SacI, XhoI double digestion, is inactivated, ethyl alcohol is heavy
It forms sediment, ddH2O weights are molten, with suitable carrier pET-28a connections with identical limitation enzymic digestion, convert bacillus coli DH 5 alpha.From turn
Change the several bacterium colonies of random picking on tablet, access LB liquid medium, shaken cultivation extracts plasmid, electrophoresis, with electrophoresis in a small amount
The plasmid of lag is that template carries out PCR verifications, and Shanghai life work sequencing is sent to after determining successful connection.
Embodiment 2：It is not added under the conditions of tightening agent, expression and yield of the recombinant bacteria laccase in Escherichia coli
(1) fmb-L103 expression plasmids pET-28a-fmb-L103 will be contained and converts Bacillus coli expression host strain
BL21 (DE3) pLysS, picking petite after being cultivated 10-11 hours at 37 DEG C, access contain kanamycins (final concentration 50ug/
Ml 50ml LB liquid mediums), 37 DEG C of overnight incubations of 180rpm.
(2) according to 1:50 volume ratio takes seed liquor to be transferred to the 400mlTB containing kanamycins (final concentration 50ug/ml)
Culture medium, 30 DEG C, 180rpm.
(3) work as OD600=1.0, IPTG (final concentration 100ug/ml) and GuSO is added4(final concentration 0.25mM) is lured
It leads, 16 DEG C, 180rpm, 20h.
(4) 16 DEG C of standing 22h.
(5) centrifugation obtains thalline and supernatant, and supernatant is ectoenzyme, and thalline is with added with Cu2+The Tris-HCl of (final concentration 1mM)
Buffer solution is resuspended, ultrasonication (power 40W, ultrasonic 1s, be spaced 2s, 30min), 75 DEG C of heating 10min, 4 DEG C, and 10000
× g centrifuges 30min and collects supernatant, obtains dead paddy bacillus (Bacillus vallismortis) laccase of recombination recombination
(fmb-rL103) crude enzyme liquid is isolated and purified recombination laccase using the method for example 3, is surveyed using the method for example 3
Surely laccase activity is recombinated, intracellular total enzyme activity reaches 1984U/L, and recombination laccase protein total output is 251mg/L.It is extracellular not detect
To laccase activity.Producing enzyme curve is as shown in Fig. 2.
Embodiment 3：Recombinant bacteria laccase isolate and purify and enzyme activity determination
Using preheating, NTA (nickel column, GE Products) affinity chromatographys and ion exchange triplicity method to example 2
The fmb-rL103 crude enzyme liquids of middle acquisition are isolated and purified.
(1) heating just purifying.
Fmb-rL103 crude enzyme liquids first 75 DEG C of the heating water baths 10min, 8000rpm, 4 DEG C obtained in example 2, centrifugation
30min collects supernatant and carries out just purifying.
(2) NTA (nickel column, GE Products) affinity chromatography is further purified.
First purification of samples is further purified using NTA (nickel column, GE Products) affinity chromatography.
1. just adding 5mM imidazoles (final concentration) in purification of samples, enhance adsorption column.
2. before loading, chromatographic column is balanced with 20mM imidazoles.Sample crosses column material three times, reaches fmb-rL103 and affinity column
The well-bound purpose of material.
3. after end of the sample, eluting (respectively with 10 bed volumes) with 100mM imidazoles, collecting eluent.
(3) the last purifying of ion-exchange
By by the fmb-rL103 of ni-sepharose purification, it is concentrated by ultrafiltration, last purifying is carried out using ion-exchange, is used in combination
AKTA purify100 monitorings.
1. 5ml samples is taken to dialyse into Buffer A (20mM Tris-HCl, PH7.0), dialysis twice, is filtered with 0.22um
Membrane filtration
2. balancing GE Q-FF 1ml prepacked columns with Buffer A
3. 2ml samples are squeezed into loading ring (sample A280=3.025mg/ml), loading flow velocity 0.2ml/min
4. with Buffer A) wash miscellaneous, 1ml/min, 30ml
5. preparing 15%NaCl with Buffer A and Buffer B (20mM Tris+1M NaCl, PH7.0) carries out gradient
Elution, 1ml/min collect eluent, 2ml/ pipes, and survey enzyme activity.
Laccase activity measures slightly modified using 2,2 '-azine-two (3- ethyl-benzothiazole -6- sulfonic acid) (ABTS) methods：
Reaction system total volume 3mL, including 5.0 citrate-phosphate salt buffers of 2.45mL 0.2mol/L pH, 0.5mL 6mM
The ABTS and 50 μ l suitably diluted enzyme extracts of 5.0 citrate-phosphate salt buffers of pH, 45 DEG C of preceding 3min for measuring reaction
The incrementss of interior reaction solution light absorption value OD at 420nm do blank control to inactivate enzyme solution.1 μm of ol of interior generation per minute is anti-
The enzyme amount needed for object is answered to be defined as an enzyme activity unit.Laccase activity calculation formula：Laccase activity (U)=V is total × Δ OD/
(V enzymes × ε × Δ t × 10-6) × total enzyme enzyme solution extension rate；Wherein, ε=3.6 × 104mol/cm；Δt：3min； ΔOD：
The changing value of 3min internal absorbances OD；V is total：In enzyme reaction, the total volume of reaction solution；V enzymes：In enzyme reaction, the volume of enzyme solution.
Experiment is repeated 3 times, and is averaged.
Using above-mentioned isolation and purification method and enzyme activity determination method, the ratio enzyme of the recombination laccase fmb-rL103 of preparation is purified
Living is 7.9U/mg albumen, and the calculation formula for obtaining recombinating laccase protein yield (A) in 1 liter of zymotic fluid is：
A=X/7.9
Wherein, the unit of A is mg/L；X indicates the total enzyme activity of 1 liter of zymotic fluid, unit U/L.
Embodiment 4：It adds tightening agent methanol and improves recombinant bacteria laccase fermentation yield
(1) fmb-L103 expression plasmids pET-28a-fmb-L103 will be contained and converts Bacillus coli expression host strain
BL21 (DE3) pLysS, picking petite after being cultivated 10-11 hours at 37 DEG C, access contain kanamycins (final concentration 50ug/
Ml 50ml LB liquid mediums), 37 DEG C of overnight incubations of 180rpm.
(2) according to 1:50 volume ratio takes seed liquor to be transferred to the 400mlTB containing kanamycins (final concentration 50ug/ml)
Culture medium.
(3) methanol is added, 30 DEG C, 180rpm, about 12h shake OD600=1.0.
(4) IPTG (final concentration 100ug/ml) is added and GuSO4 (final concentration 0.25mM) is induced, 16 DEG C,
180rpm,20h。
(5) 16 DEG C of standing 22h.
(6) centrifugation obtains thalline and supernatant, and supernatant is ectoenzyme, and thalline is with added with Cu2+The Tris-HCl of (final concentration 1mM)
Buffer solution is resuspended, ultrasonication (power 40W, ultrasonic 1s, be spaced 2s, 30min), 75 DEG C of heating 10min, 4 DEG C, and 10000
× g centrifuges 30min and collects supernatant, obtains dead paddy bacillus (Bacillus vallismortis) laccase of recombination recombination
(fmb-rL103) crude enzyme liquid carries out isolating and purifying for crude enzyme liquid by embodiment 3, is then surveyed according to the method for example 3
Surely laccase activity is recombinated.
(7) for addition methanol as tightening agent, bacterial laccase producing enzyme curve reaches highest producing enzyme as shown in figure 3, fermentation 20h
Amount, total enzyme activity reach 114000U/L, and recombination laccase protein total output reaches 14430mg/L.Wherein intracellular enzyme activity is 89000U/
L, it is 11270mg/L that intracellular, which recombinates laccase protein yield,；Enzymatic activities are 25000U/L, and recombination laccase protein yield is
3160mg/L.Producing enzyme curve is as shown in Figure 3.
Embodiment 5：Add the logical raising recombinant bacteria laccase fermentation yield of tightening agent Qula
(1) fmb-L103 expression plasmids pET-28a-fmb-L103 will be contained and converts Bacillus coli expression host strain
BL21 (DE3) pLysS, picking petite after being cultivated 10-11 hours at 37 DEG C, access contain kanamycins (final concentration 50ug/
Ml 50ml LB liquid mediums), 37 DEG C of overnight incubations of 180rpm.
(2) according to 1:50 volume ratio takes seed liquor to be transferred to the 400mlTB containing kanamycins (final concentration 50ug/ml)
Culture medium.
(3) addition Qula is logical, and 30 DEG C, 180rpm, about 12h shake OD600=1.0.
(4) IPTG (final concentration 100ug/ml) is added and GuSO4 (final concentration 0.25mM) is induced, 16 DEG C,
180rpm,20h。
(5) 16 DEG C of standing 22h.
(6) centrifugation obtains thalline and supernatant, and supernatant is ectoenzyme, Tris-HCl of the thalline added with Cu2+ (final concentration 1mM)
Buffer solution is resuspended, ultrasonication (power 40W, ultrasonic 1s, be spaced 2s, 30min), 75 DEG C of heating 10min, 4 DEG C, and 10000
× g centrifuges 30min and collects supernatant, obtains the crude enzyme liquid of recombinant bacteria laccase (fmb-rL103), is carried out slightly by embodiment 3
Enzyme solution isolates and purifies, and then measures recombination laccase activity according to the method for example 3.
(7) addition Qula is logical is used as tightening agent, and bacterial laccase producing enzyme curve reaches most high yield as shown in figure 4, fermentation 20h
Enzyme amount, total enzyme activity reach 132000U/L, and recombination laccase protein total output reaches 16710mg/L.Wherein intracellular enzyme activity is
21000U/L, recombination laccase protein yield are 2660mg/L, enzymatic activities 111000U/L, and recombination laccase protein yield is
14050mg/L.Producing enzyme curve is as shown in Figure 4.
Embodiment 6：The logical two kinds of tightening agent co-inductions of methanol/Qula improve recombinant bacteria laccase fermentation yield
(1) fmb-L103 expression plasmids pET-28a-fmb-L103 will be contained and converts Bacillus coli expression host strain
BL21 (DE3) pLysS, picking petite after being cultivated 10-11 hours at 37 DEG C, access contain kanamycins (final concentration 50ug/
Ml 50ml LB liquid mediums), 37 DEG C of overnight incubations of 180rpm.
(2) according to 1:50 volume ratio takes seed liquor to be transferred to the 400mlTB containing kanamycins (final concentration 50ug/ml)
Culture medium.
(3) it adds methanol and Qula is logical, 30 DEG C, 180rpm, about 12h shake OD600=1.0.
(4) IPTG (final concentration 100ug/ml) and GuSO is added4(final concentration 0.25mM) is induced, 16 DEG C,
180rpm,20h。
(5) 16 DEG C of standing 22h.
(6) centrifugation obtains thalline and supernatant, and supernatant is ectoenzyme, Tris-HCl of the thalline added with Cu2+ (final concentration 1mM)
Buffer solution is resuspended, ultrasonication (power 40W, ultrasonic 1s, be spaced 2s, 30min), 75 DEG C of heating 10min, 4 DEG C, and 10000
× g centrifuges 30min and collects supernatant, obtains dead paddy bacillus (Bacillus vallismortis) laccase of recombination recombination
(fmb-rL103) crude enzyme liquid carries out isolating and purifying for crude enzyme liquid by embodiment 3, is then surveyed according to the method for example 3
Surely laccase activity is recombinated.
(7) addition methanol/Qula is logical meets tightening agent, and bacterial laccase producing enzyme curve reaches as shown in figure 5, fermentation 20h
Highest yield of enzyme, total enzyme activity reach 142000U/L, and recombination laccase protein total output reaches 17970mg/L, wherein intracellular enzyme activity
For 38000U/L, recombination laccase protein yield is 4810mg/L, enzymatic activities 104000U/L, and recombination laccase protein yield is
13160mg/L.Producing enzyme curve is as shown in Figure 5.
As shown in table 1, using the method provided in the present invention, the fermentation yield of recombinant bacteria laccase after tightening agent is added
With not plus tightening agent in total enzyme activity (total protein yield), intracellular enzyme activity (intracellular protein yield) and enzymatic activities (extracellular egg
White yield) aspect improve a lot.
Using method provided by the invention, the fermenting and producing of recombinant bacteria laccase is carried out using recombination bacillus coli, total
Enzyme activity (total recombinant protein yield), intracellular enzyme activity (intracellular recombinant protein yield), enzymatic activities (extracellular recombinant protein yield) three
Aspect is above the highest recombinant bacteria enzyme activity yield (recombinant protein yield) reported at present.
Table 1
Sequence table
<110>Agricultural University Of Nanjing
The bio tech ltd Dong Sheng of Taixing City
<120>A kind of method of high-efficiency fermenting production recombinant bacteria laccase
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1542
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
atgacacttg aaaaatttgt ggatgctctc ccaatcccag atacactaaa gccagtgcag 60
caatcaaaag aaaaaacata ctacgaagtc accatggagg aatgcactca tcagcttcac 120
cgcgatctcc ctccgacccg cctgtggggc tacaacggct tatttccggg ccctaccatt 180
gaggttaaaa gaaatgaaaa tgtatatgtg aaatggatga ataatcttcc ttccacgcat 240
ttccttccgg ttgatcacac cattcatcac agtgacagcc agcatgaaga acccgaagta 300
aagactgttg ttcatttaca tggcggcgtc acgccagatg acagcgacgg gtatccggag 360
gcttggtttt ccaaagactt tgaacaaaca ggaccttatt tcaaaagaga ggtttatcat 420
tatcctaatc agcagcgcgg ggctatattg tggtatcacg atcacgccat ggcgctcacc 480
aggctaaatg tctatgccgg acttgtcggc gcttatatca ttcatgatcc aaaggaaaaa 540
cgcttaaagc tgccttcagg cgaatacgat gtgccgcttc ttatcacaga ccgcacgatc 600
aatgaggacg gttctttgtt ttatccgagc ggaccggaaa atccttctcc gtcactgcct 660
aatccttcaa tcgttccggc tttttgcgga gaaaccatac tcgtcaacgg gaaggtatgg 720
ccatacttgg aagtcgagcc gaggaaatac cgattccgcg tcatcaacgc ctccaatacc 780
agaacctata atctgtcact cgataatggc ggagagttta ttcaggtcgg ttcagatgga 840
gggctcctgc cgcgatctgt taaattgaat tctttcagtc ttgcgcctgc tgaacgttac 900
gatatcatca ttgacttcac agcgtatgaa ggagaatcga tcattttggc aaacagcgcg 960
ggctgcggcg gtgacgtcaa tcctgaaaca gatgcgaata tcatgcaatt caaagtcaca 1020
aaaccattag cgcaacaaga cgaaagcaga aagccgaagt acctcgcctc atacccttcc 1080
gtacagcatg aaagaataca aaacatcaga acactaaaac tggcaggaac ccaagacaaa 1140
tacggcagac ccgtccttct gcttaataac aaacgctggc acgatcctgt cacagaagca 1200
ccaaaagccg gcacaactga aatatggtcc attatcaacc cgacacgcgg aacacatccg 1260
attcacctgc atctggtctc cttccgtgta ttagaccggc gtccgtttga tatcgcccgt 1320
tatcaagaaa gcggggaatt gtcctatacg ggtccggcta tcccgccgcc gccaagtgaa 1380
aagggatgga aagacacaat tcaagcgcat gcgggtgaag tcctgagaat cgcggcgaca 1440
ttcgggccgt acagcggacg atacgtatgg cactgccata ttcttgaaca tgaggactac 1500
gacatgatga gaccgatgga tataactgat ccccataaat aa 1542
<210> 3
<211> 513
<212> PRT
<213>Dead paddy bacillus fmb-103 (Bacillus vallismortisf mb-103)
<400> 3
Met Thr Leu Glu Lys Phe Val Asp Ala Leu Pro Ile Pro Asp Thr Leu
1 5 10 15
Lys Pro Val Gln Gln Ser Lys Glu Lys Thr Tyr Tyr Glu Val Thr Met
20 25 30
Glu Glu Cys Thr His Gln Leu His Arg Asp Leu Pro Pro Thr Arg Leu
35 40 45
Trp Gly Tyr Asn Gly Leu Phe Pro Gly Pro Thr Ile Glu Val Lys Arg
50 55 60
Asn Glu Asn Val Tyr Val Lys Trp Met Asn Asn Leu Pro Ser Thr His
65 70 75 80
Phe Leu Pro Val Asp His Thr Ile His His Ser Asp Ser Gln His Glu
85 90 95
Glu Pro Glu Val Lys Thr Val Val His Leu His Gly Gly Val Thr Pro
100 105 110
Asp Asp Ser Asp Gly Tyr Pro Glu Ala Trp Phe Ser Lys Asp Phe Glu
115 120 125
Gln Thr Gly Pro Tyr Phe Lys Arg Glu Val Tyr His Tyr Pro Asn Gln
130 135 140
Gln Arg Gly Ala Ile Leu Trp Tyr His Asp His Ala Met Ala Leu Thr
145 150 155 160
Arg Leu Asn Val Tyr Ala Gly Leu Val Gly Ala Tyr Ile Ile His Asp
165 170 175
Pro Lys Glu Lys Arg Leu Lys Leu Pro Ser Gly Glu Tyr Asp Val Pro
180 185 190
Leu Leu Ile Thr Asp Arg Thr Ile Asn Glu Asp Gly Ser Leu Phe Tyr
195 200 205
Pro Ser Gly Pro Glu Asn Pro Ser Pro Ser Leu Pro Asn Pro Ser Ile
210 215 220
Val Pro Ala Phe Cys Gly Glu Thr Ile Leu Val Asn Gly Lys Val Trp
225 230 235 240
Pro Tyr Leu Glu Val Glu Pro Arg Lys Tyr Arg Phe Arg Val Ile Asn
245 250 255
Ala Ser Asn Thr Arg Thr Tyr Asn Leu Ser Leu Asp Asn Gly Gly Glu
260 265 270
Phe Ile Gln Val Gly Ser Asp Gly Gly Leu Leu Pro Arg Ser Val Lys
275 280 285
Leu Asn Ser Phe Ser Leu Ala Pro Ala Glu Arg Tyr Asp Ile Ile Ile
290 295 300
Asp Phe Thr Ala Tyr Glu Gly Glu Ser Ile Ile Leu Ala Asn Ser Ala
305 310 315 320
Gly Cys Gly Gly Asp Val Asn Pro Glu Thr Asp Ala Asn Ile Met Gln
325 330 335
Phe Lys Val Thr Lys Pro Leu Ala Gln Gln Asp Glu Ser Arg Lys Pro
340 345 350
Lys Tyr Leu Ala Ser Tyr Pro Ser Val Gln His Glu Arg Ile Gln Asn
355 360 365
Ile Arg Thr Leu Lys Leu Ala Gly Thr Gln Asp Lys Tyr Gly Arg Pro
370 375 380
Val Leu Leu Leu Asn Asn Lys Arg Trp His Asp Pro Val Thr Glu Ala
385 390 395 400
Pro Lys Ala Gly Thr Thr Glu Ile Trp Ser Ile Ile Asn Pro Thr Arg
405 410 415
Gly Thr His Pro Ile His Leu His Leu Val Ser Phe Arg Val Leu Asp
420 425 430
Arg Arg Pro Phe Asp Ile Ala Arg Tyr Gln Glu Ser Gly Glu Leu Ser
435 440 445
Tyr Thr Gly Pro Ala Ile Pro Pro Pro Pro Ser Glu Lys Gly Trp Lys
450 455 460
Asp Thr Ile Gln Ala His Ala Gly Glu Val Leu Arg Ile Ala Ala Thr
465 470 475 480
Phe Gly Pro Tyr Ser Gly Arg Tyr Val Trp His Cys His Ile Leu Glu
485 490 495
His Glu Asp Tyr Asp Met Met Arg Pro Met Asp Ile Thr Asp Pro His
500 505 510
Lys
<210> 4
<211> 35
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
cgcgagctca tgacacttga aaaatttgtg gatgc 35
<210> 5
<211> 33
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ccgctcgagt tatttatggg gatcagttat atc 33
Claims (9)
1. a kind of method of efficient production recombinant bacteria laccase, it is characterised in that：In the recombination bacillus coli hair of production bacterial laccase
Add that methanol, ethyl alcohol, Qula be logical, any one in glycine, glycine betaine, tween, sapn, glycerine, sorbierite during ferment
Or various abiotic stress agent carries out induced expression to laccase.
2. according to the method described in claim 1, it is characterized in that the 0-2h of the recombination bacillus coli fermentation in production bacterial laccase
The methanol of 1-10% (v/v), Qula logical or methanol and the logical combination of Qula is added.
3. according to the method described in claim 2, it is characterized in that the addition time of methanol is 0-0.5h, additive amount 4-8%.
4. according to the method described in claim 3, it is characterized in that the addition time of methanol be 0h, additive amount 6%.
5. according to the method described in claim 2, it is characterized in that the addition time that Qula is led to is 0-0.5h, additive amount 2-
5%.
6. according to the method described in claim 4, it is characterized in that the addition time that Qula is led to is 0h, additive amount 4%.
7. according to the method described in claim 2, it is characterized in that the addition time that methanol and Qula lead to co-induction agent is 0-
0.5h, additive amount are methanol 4-8%, and Qula leads to 2-5%；And the two summation is no more than 10%.
8. according to the method described in claim 7, it is characterized in that the addition time that methanol and Qula lead to co-induction agent is 0h,
Additive amount is methanol 6%, Qula logical 4%.
9. according to any one of claim 1-8 raising bacterial laccase expression quantity method, it is characterised in that comprising with
Lower step：
(1) Recombinant organism of structure production recombinant bacteria laccase；
(2) picking petite after cultivating the Recombinant organism for producing recombinant bacteria laccase 10-11 hours at 37 DEG C, connects
Enter the 50ml LB liquid mediums containing kanamycins, 37 DEG C of overnight incubations of 180rpm obtain seed liquor；Wherein, the card that
The final concentration of 50ug/ml of mycin；
(3) seed liquor is taken to be transferred to the TB culture mediums containing kanamycins；
(4) tightening agent described in any one of addition claim 1-8,30 DEG C, 180rpm shakes OD600=1.0；
(5) IPTG and GuSO4 is added to be induced, 16 DEG C, 180rpm, 20h；The final concentration of 100ug/ml of wherein IPTG and
GuSO4Final concentration of 0.25mM；
(6) 16 DEG C of standing 22h.
(7) it is ectoenzyme that centrifugation, which obtains thalline and supernatant, supernatant, and thalline is with added with Cu2+The Tris-HCl buffer solutions of final concentration 1mM
It is resuspended, ultrasonication, 75 DEG C of heating 10min, 4 DEG C, 10000 × g centrifuges 30min and collects supernatant, obtains recombinant bacteria paint
The crude enzyme liquid of enzyme.
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