CN108570459B - Method for producing recombinant bacterial laccase by high-efficiency fermentation - Google Patents
Method for producing recombinant bacterial laccase by high-efficiency fermentation Download PDFInfo
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Abstract
The invention discloses a method for producing recombinant bacterial laccase by high-efficiency fermentation. The method is achieved by adding any one or more stress agents of methanol, ethanol, triton, glycine, betaine, glycerol and sorbitol in the fermentation process of the recombinant escherichia coli. The invention utilizes the stress action of the stress agent to delay the growth of thalli and reduce the toxic action caused by the accumulation of acetic acid, and the expression quantity of the recombinant bacterial laccase is obviously improved. By using the method provided by the invention, the fermentation production of the recombinant bacterial laccase is carried out by using the recombinant Escherichia coli, and the total enzyme activity (total recombinant protein yield), the intracellular enzyme activity (intracellular recombinant protein yield) and the extracellular enzyme activity (extracellular recombinant protein yield) are all higher than the highest recombinant bacterial laccase yield (recombinant protein yield) reported at present.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for improving the yield of recombinant bacterial laccase produced by fermentation of recombinant escherichia coli.
Background
Laccase (laccase, benzenediol: oxygen oxidoreductase, ec 1.10.3.2) is a copper-containing polyphenol oxidase, is homologous to ascorbic acid oxidase (ascorbyl oxidase) in plants and ceruloplasmin (ceruloplasmin) in mammals, belongs to a member of the family of blue copper oxidases (bluecopperoxidases), can oxidize various types of phenolic compounds (such as ferulic acid) and non-phenolic compounds by using molecular oxygen as an electron acceptor, and the only byproduct of the catalytic reaction is water, which is known as the greenest and environmentally-friendly biocatalyst. Laccase has wide application in the fields of treatment of environmental pollutants, food processing industry, pulping and paper making industry, textile industry, soil bioremediation, nano biotechnology and the like, such as removal of phenol-containing pollutants, dye decolorization, organic matter synthesis, wastewater decolorization, clarification and color control of beverages, production of edible fungi and medicinal fungi, improvement of food component functionality, plant food protection and the like. Laccase can be classified into fungal laccase and bacterial laccase, depending on the source.
The fungal laccase generally has higher enzyme activity, the fermentation yield is 50000-100000U/L, but the pH adaptation range of the fungal laccase is narrower, and the condition that the pH is more than 7 is quickly inactivated; meanwhile, the fungal laccase has poor heat stability, and the activity of the fungal laccase can be rapidly lost at the most suitable temperature of more than 50 ℃ between 30 ℃ and 45 ℃. Thus, fungal laccases, although high in fermentation yield, have limited the range of applications due to their pH adaptation and thermostability. The bacterial laccase has the characteristics of good thermal stability, wide pH value range of enzyme reaction, good stability to halogen and the like. Meanwhile, compared with the fungal fermentation laccase, the bacterial culture period is short, the required nutrient components are simple, and the fermentation energy consumption is low. However, the fermentation yield of the prior bacterial laccase is low, and the requirement of industrial fermentation level is difficult to achieve.
Because bacteria with ideal high laccase yield are difficult to obtain, the laccase gene is cloned and transformed into bacterial host bacteria such as escherichia coli or bacillus subtilis and the like by utilizing a recombination technology to construct recombinant genetic engineering bacteria, and the fermentation level of the bacterial laccase is improved by controlling fermentation conditions, so that the method is a strategy for solving the problem of low bacterial laccase yield. At present, researches for improving escherichia coli fermentation bacterial laccase, such as laccase gene codon optimization, selection of a proper starter, mRNA stability improvement, utilization of fusion protein and molecular chaperone, deletion of an engineering bacteria byproduct synthesis path, realization of high-density culture by regulating and controlling fermentation conditions and the like, have been carried out, although the fermentation yield of the recombinant bacterial laccase is improved to a certain extent, a great difference still exists compared with the fermentation level of fungal laccase, and the highest yield of the currently reported recombinant bacterial laccase produced by escherichia coli fermentation is 4000-6000U/L.
Disclosure of Invention
The invention aims to provide a method for producing recombinant bacterial laccase by utilizing recombinant Escherichia coli through high-efficiency fermentation in the prior art.
The purpose of the invention can be realized by the following technical scheme:
a method for efficiently producing recombinant bacterial laccase is characterized in that any one or more stress agents of methanol, ethanol, triton, glycine, betaine, tween, span, glycerol and sorbitol are added in the fermentation process of recombinant Escherichia coli for producing the bacterial laccase to perform induced expression on the laccase.
The method of the invention preferably adds 1-10% (v/v) of methanol, triton or a combination of methanol and triton in 0-2h of the recombinant escherichia coli fermentation for producing the bacterial laccase.
In the method of the present invention, the addition time of methanol is preferably 0 to 0.5 hour, more preferably 0 hour, and the addition amount is preferably 4 to 8%, more preferably 6%.
In the method of the present invention, the addition time of triton is preferably 0 to 0.5 hour, more preferably 0 hour, and the addition amount is preferably 2 to 5%, more preferably 4%.
According to the method, the adding time of the methanol and the triton composite inducer is 0-0.5h, the adding amount of the methanol is 4-8%, and the adding amount of the triton is 2-5%; and the sum of the two is not more than 10%.
According to the method, the adding time of the methanol and triton composite inducer is preferably 0h, the adding amount of the methanol and the triton composite inducer is preferably 6 percent, and the adding amount of the triton is preferably 4 percent.
The method for improving the expression level of the bacterial laccase further comprises the following steps:
(1) Constructing escherichia coli genetic engineering bacteria for producing recombinant bacterial laccase;
(2) Culturing the Escherichia coli genetic engineering bacteria producing recombinant bacterial laccase at 37 ℃ for 10-11 hours, selecting microcolonies, inoculating 50ml of LB liquid culture medium containing kanamycin, and culturing overnight at 180rpm and 37 ℃ to obtain seed liquid; wherein, the final concentration of the kanamycin is 50ug/ml;
(3) Taking a seed solution and transferring the seed solution to a TB culture medium containing kanamycin;
(4) Adding the stress agent, 30 ℃,180rpm, shaking to OD600=1.0;
(5) Adding IPTG and GuSO4 for induction at 16 ℃ and 180rpm for 20h; wherein the final concentration of IPTG is 100ug/ml and GuSO 4 Final concentration 0.25mM;
(6) Standing at 16 ℃ for 22h.
(7) Centrifuging to obtain thallus and supernatant, wherein the supernatant is extracellular enzyme, and the thallus is added with Cu 2+ And (3) carrying out heavy suspension on Tris-HCl buffer solution with the final concentration of 1mM, carrying out ultrasonic crushing, heating at 75 ℃ for 10min, centrifuging at 4 ℃ and 10000 Xg for 30min, and collecting supernatant to obtain crude enzyme solution of the recombinant dead bacillus vallismortis laccase.
As the optimization of the invention, the gene sequence of the dead botrytis cinerea laccase used for constructing the escherichia coli genetic engineering bacteria for producing the recombinant bacterial laccase is obtained by performing codon optimization on escherichia coli, and is shown as SEQ ID NO. 1.
Has the advantages that:
laccase has wide application in the fields of environmental pollutant treatment, food processing industry, pulping and paper making industry, textile industry, soil bioremediation, nano biotechnology and the like. Fungal laccase fermentation yields are high, but the pH stability and temperature stability are poor, and therefore, practical use is limited. The bacterial laccase has the characteristics of high thermal stability, wide pH application range and the like, but the bacterial laccase has low fermentation yield, and large-scale fermentation production cannot be realized.
The invention effectively improves the fermentation yield of the recombinant bacterial laccase by adding one or more stress agents (the stress agent is any one or more of methanol, ethanol, triton, glycine, betaine, glycerol and sorbitol) in the fermentation process of the recombinant Escherichia coli. The invention utilizes the stress action of the stress agent to delay the growth of the bacteria, reduces the toxic action caused by the accumulation of acetic acid and obviously improves the expression quantity of the bacterial laccase. When methanol is used as a stress agent, the fermentation yield of a 500mL shake flask is increased to 11215U/L from the original 1984U/L; under the condition of 50L fermentation tank scale, the total yield reaches 114000U/L, which is obviously higher than the yield level (4000-6000U/L) of the recombinant bacterial laccase reported in the past. Meanwhile, no report of extracellular expression of the recombinant bacterial laccase in escherichia coli exists in the past, and the effect of the extracellular expression of the bacterial laccase is realized by adopting the method for inducing expression by the stress agent provided by the patent. When methanol is used as a stress agent, the yield of extracellular recombinant bacterial laccase in a 500mL shake flask is never detected and is increased to 2578U/L; the yield of extracellular recombinant bacteria reaches 25000U/L under the scale of a 50L fermentation tank.
When the expression is induced by using a triton stress agent or methanol/triton synergistic stress, the improvement effect on the total enzyme activity (total recombinant protein yield), the intracellular enzyme activity (intracellular recombinant protein yield) and the extracellular enzyme activity (extracellular recombinant protein yield) is different.
The invention provides a method for inducing high-efficiency fermentation to produce recombinant bacterial laccase by adding a stress agent in the fermentation process of recombinant escherichia coli aiming at the problem of low fermentation yield of the conventional recombinant bacterial laccase. Under the condition of a 50L fermentation tank, the fermentation level of the recombinant bacterial laccase can reach about 140000U/L, which is more than 20 times of the reported highest bacterial laccase yield; meanwhile, the extracellular expression effect of the recombinant bacterial laccase in escherichia coli is realized, and the extracellular yield can reach about 111000U/L. The fermentation production period of the escherichia coli recombinant bacterial laccase provided by the invention is about 20 hours, and the fermentation period of the fungal laccase is 96-168 hours. Therefore, the fermentation production mode of the bacterial laccase provided by the invention has the advantage of energy conservation compared with the fermentation production of the fungal laccase.
Description of the drawings:
FIG. 1 construction process of recombinant fmb-103 laccase gene expression vector
FIG. 2 enzyme production (intracellular) curves for bacterial laccase production by E.coli fermentation without addition of stress agents
FIG. 3 enzyme production curve for producing bacterial laccase by methanol stress induced fermentation of Escherichia coli
FIG. 4 enzyme production curve for producing bacterial laccase by fermentation of Aspergillus under stress induction
FIG. 5 shows enzyme production curves for producing bacterial laccase by methanol/triton synergistic stress induction of Escherichia coli fermentation
Detailed Description
The invention is further illustrated by the following examples.
Example 1: construction of Escherichia coli genetic engineering bacteria for producing recombinant bacterial laccase
The gene sequence of the death rice-bud spore laccase gene codon (codon optimization aiming at escherichia coli) after optimization is shown as SEQ ID NO. 1; the amino acid sequence is shown as SEQ ID NO. 2. The gene company was requested to chemically synthesize the gene.
Two primers were designed based on the optimized laccase gene sequence, the upstream primer plus the SacI recognition sequence, the downstream primer plus the XhoI recognition sequence (the underlined part is the restriction enzyme recognition sequence):
an upstream primer F-1:5' -CGCGAGCTCATGACACTTGAAAAATTTGTGGATGC-3′(SEQ ID NO.3),
The downstream primer R-1CTCGAGTTATTTATGGGGATCAGTTATATC-3′(SEQ ID NO.4),
Adding the components according to the following PCR system to amplify laccase gene:
the PCR procedure was: 3min at 94 ℃;30 × (94 ℃ 40s, 53 ℃ 50s, 72 ℃ 90 s); 10min at 72 ℃.
Purifying PCR product with Shanghai PCR product purification kit, performing double digestion with SacI and XhoI, inactivating, precipitating with ethanol, and adding ddH 2 O redissolving, with the appropriate amount of vector pET-2 digested with the same restriction enzymes8a, and transforming Escherichia coli DH5 alpha. Randomly selecting several colonies from a transformation plate, inoculating an LB liquid culture medium, carrying out shaking culture, extracting plasmids in small quantity, carrying out electrophoresis, carrying out PCR verification by taking the plasmids with the electrophoresis lag as a template, and sending the plasmids to Shanghai for sequencing after the connection is successful.
Example 2: expression and yield of recombinant bacterial laccase in escherichia coli without adding stress agent
(1) Escherichia coli expression host strain BL21 (DE 3) pLysS was transformed with plasmid pET-28a-fmb-L103 containing fmb-L103 expression, cultured at 37 ℃ for 10-11 hours, then microcolonies were picked, inoculated into 50ml LB liquid medium containing kanamycin (50 ug/ml final concentration), and cultured overnight at 180rpm 37 ℃.
(2) The seed solution was transferred to 400ml of TB medium containing kanamycin (50 ug/ml final concentration) at 30 ℃ and 180rpm according to a volume ratio of 1.
(3) When OD is measured 600 =1.0, add IPTG (final concentration 100 ug/ml) and GuSO 4 (final concentration: 0.25 mM) was used for induction at 16 ℃ and 180rpm,20h.
(4) Standing at 16 ℃ for 22h.
(5) Centrifuging to obtain thallus and supernatant, wherein the supernatant is extracellular enzyme, and the thallus is added with Cu 2+ Carrying out resuspension on Tris-HCl buffer solution (with the final concentration of 1 mM), carrying out ultrasonication (with the power of 40W, ultrasonication of 1s, interval of 2s and 30min), heating at 75 ℃ for 10min, heating at 4 ℃, centrifuging at 10000 Xg for 30min, and collecting supernatant to obtain crude enzyme solution of recombinant killed Bacillus vallismortis laccase (fmb-rL 103), separating and purifying the recombinant laccase by adopting the method of example 3, measuring the activity of the recombinant laccase by adopting the method of example 3, wherein the total intracellular enzyme activity reaches 1984U/L, and the total yield of the recombinant laccase protein is 251mg/L. No laccase activity was detected extracellularly. The enzyme production curve is shown in FIG. 2.
Example 3: separation and purification of recombinant bacterial laccase and enzyme activity determination
The fmb-rL103 crude enzyme solution obtained in example 2 was separated and purified by a combination of preheating, NTA (Nickel column, product of GE) affinity chromatography, and ion exchange.
(1) Heating for primary purification.
The crude fmb-rL103 enzyme solution obtained in example 2 was first heated in a water bath at 75 ℃ for 10min,8000rpm, at 4 ℃, centrifuged for 30min, and the supernatant was collected for primary purification.
(2) Further purification by affinity chromatography using NTA (Nickel column, product of GE).
The primary purified sample was further purified by NTA (nickel column, GE) affinity chromatography.
(1) 5mM imidazole (final concentration) was added to the initial purified sample and the adsorption column was reinforced.
(2) Before loading, the column was equilibrated with 20mM imidazole. The sample passes through the column material for three times, and the aim of fully combining fmb-rL103 with the affinity column material is fulfilled.
(3) After the end of the loading, the column was eluted with 100mM imidazole (10 bed volumes each) and the eluates were collected.
(3) Final purification by ion exchange
fmb-rL103 purified by nickel column was concentrated by ultrafiltration, and the final purification was performed by ion exchange and monitored by AKTA purify 100.
(1) 5ml of the sample was dialyzed into Buffer A (20 mM Tris-HCl, pH 7.0), dialyzed twice, and filtered through a 0.22um filter
(2) Balancing GE Q-FF 1ml prepacked column with Buffer A
(3) 2ml of sample was driven into the loading ring (sample A280=3.025 mg/ml) at a loading flow rate of 0.2ml/min
(4) Washing with Buffer A), 1ml/min,30ml
(5) 15% NaCl was prepared with Buffer A and Buffer B (20mM Tris +1M NaCl, PH7.0) and gradient eluted at 1ml/min, and the eluates were collected at 2 ml/tube and assayed for enzyme activity.
Laccase activity was determined using the 2,2' -azino-bis (3-ethylbenzothiazole-6-sulfonic Acid) (ABTS) method with minor modifications: the total volume of the reaction system was 3mL, including 2.45mL of 0.2mol/L of pH 5.0 citrate-phosphate buffer, 0.5mL of 6mM ABTS and 50. Mu.l of crude enzyme extract diluted with pH 5.0 citrate-phosphate buffer, and the increase of OD at 420nm in the first 3min of the reaction was measured at 45 ℃ to use the inactivated enzyme solution as a blank. The amount of enzyme required to produce 1. Mu. Mol of reactant per minute was defined as one unit of enzyme activity. The laccase enzyme activity calculation formula is as follows: laccase activity (U) = Vtotal multiplied by delta OD/(Venzyme multiplied by epsilon multiplied by delta t multiplied by 10-6) multiplied by total enzyme dilution times; wherein, epsilon =3.6 × 104mol/cm; Δ t:3min; Δ OD: change value of absorbance OD within 3min; v total: the total volume of the reaction solution in the enzyme reaction; v, enzyme V: volume of enzyme solution in the enzyme reaction. The experiment was repeated 3 times and the average was taken.
By adopting the separation and purification method and the enzyme activity determination method, the specific enzyme activity of the purified recombinant laccase fmb-rL103 is 7.9U/mg protein, and the calculation formula of the yield (A) of the recombinant laccase protein in 1 liter of fermentation liquid is as follows:
A＝X/7.9
wherein, the unit of A is mg/L; x represents the total enzyme activity of 1 liter of fermentation broth and has the unit of U/L.
Example 4: method for improving fermentation yield of recombinant bacterial laccase by adding stress agent methanol
(1) Escherichia coli expression host strain BL21 (DE 3) pLysS was transformed with plasmid pET-28a-fmb-L103 containing fmb-L103 expression, cultured at 37 ℃ for 10 to 11 hours, then microcolonies were picked up, inoculated into 50ml of LB liquid medium containing kanamycin (final concentration 50 ug/ml), and cultured at 180rpm at 37 ℃ overnight.
(2) The seed solution was transferred to 400ml of TB medium containing kanamycin (50 ug/ml final concentration) at a volume ratio of 1.
(3) Methanol was added, 30 ℃,180rpm, shaking for about 12h to OD600=1.0.
(4) Induction was carried out by adding IPTG (final concentration 100 ug/ml) and GuSO4 (final concentration 0.25 mM) at 16 ℃ and 180rpm for 20h.
(5) Standing at 16 ℃ for 22h.
(6) Centrifuging to obtain thallus and supernatant, wherein the supernatant is extracellular enzyme, and the thallus is treated with Cu 2+ Resuspending in Tris-HCl buffer (final concentration of 1 mM), ultrasonication (power 40W, ultrasonication 1s, interval 2s, 30min), heating at 75 ℃ for 10min, heating at 4 ℃, centrifuging at 10000 Xg for 30min, collecting supernatant, obtaining crude enzyme solution of recombinant killed Bacillus vallismortis laccase (fmb-rL 103), separating and purifying the crude enzyme solution by the implementation example 3, and then determining the activity of the recombinant laccase according to the method of the example 3.
(7) Methanol is added as a stress agent, the enzyme production curve of the bacterial laccase is shown in figure 3, the fermentation is carried out for 20 hours, the maximum enzyme yield is achieved, the total enzyme activity reaches 114000U/L, and the total yield of the recombinant laccase protein reaches 14430mg/L. Wherein the intracellular enzyme activity is 89000U/L, and the yield of the intracellular recombinant laccase protein is 11270mg/L; the extracellular enzyme activity is 25000U/L, and the yield of the recombinant laccase protein is 3160mg/L. The enzyme production curve is shown in FIG. 3.
Example 5: stress agent triton is added to improve the fermentation yield of recombinant bacterial laccase
(1) An E.coli expression host strain BL21 (DE 3) pLysS was transformed with the fmb-L103-containing expression plasmid pET-28a-fmb-L103, and after culturing at 37 ℃ for 10 to 11 hours, microcolonies were picked up and inoculated into 50ml of LB liquid medium containing kanamycin (final concentration of 50 ug/ml), and cultured overnight at 180rpm 37 ℃.
(2) The seed solution was transferred to 400ml of TB medium containing kanamycin (50 ug/ml final concentration) at a volume ratio of 1.
(3) Triton was added at 30 ℃,180rpm, shaken for about 12h to OD600=1.0.
(4) Induction was carried out by adding IPTG (final concentration 100 ug/ml) and GuSO4 (final concentration 0.25 mM) at 16 ℃ and 180rpm for 20h.
(5) Standing at 16 ℃ for 22h.
(6) Centrifugation is carried out to obtain thalli and supernatant, the supernatant is extracellular enzyme, the thalli is resuspended by Tris-HCl buffer solution added with Cu < 2+ > (the final concentration is 1 mM), ultrasonication (the power is 40W, the ultrasound is carried out for 1s, the interval is 2s and 30min), heating is carried out at 75 ℃ for 10min, centrifugation is carried out at 10000 Xg for 30min, the supernatant is collected, crude enzyme liquid of the recombinant bacterial laccase (fmb-rL 103) is obtained, separation and purification of the crude enzyme liquid are carried out by implementing example 3, and then the activity of the recombinant laccase is determined according to the method of example 3.
(7) The triton is added as a stress agent, a bacterial laccase enzyme production curve is shown in figure 4, the fermentation is carried out for 20 hours, the highest enzyme yield is achieved, the total enzyme activity reaches 132000U/L, and the total yield of the recombinant laccase protein reaches 16710mg/L. Wherein the intracellular enzyme activity is 21000U/L, the yield of the recombinant laccase protein is 2660mg/L, the extracellular enzyme activity is 111000U/L, and the yield of the recombinant laccase protein is 14050mg/L. The enzyme production curve is shown in FIG. 4.
Example 6: methanol/triton two stress agents are cooperatively induced to improve fermentation yield of recombinant bacterial laccase
(1) Escherichia coli expression host strain BL21 (DE 3) pLysS was transformed with plasmid pET-28a-fmb-L103 containing fmb-L103 expression, cultured at 37 ℃ for 10 to 11 hours, then microcolonies were picked up, inoculated into 50ml of LB liquid medium containing kanamycin (final concentration 50 ug/ml), and cultured at 180rpm at 37 ℃ overnight.
(2) The seed solution was transferred to 400ml of TB medium containing kanamycin (50 ug/ml final concentration) at a volume ratio of 1.
(3) Methanol and triton were added, 30 ℃,180rpm, about 12h shaking to OD600=1.0.
(4) IPTG (final concentration 100 ug/ml) and GuSO were added 4 (final concentration: 0.25 mM) was used for induction at 16 ℃ and 180rpm for 20h.
(5) Standing at 16 ℃ for 22h.
(6) Centrifuging to obtain thalli and supernatant, wherein the supernatant is extracellular enzyme, resuspending the thalli by using Tris-HCl buffer solution added with Cu < 2+ > (the final concentration is 1 mM), carrying out ultrasonic disruption (the power is 40W, the ultrasonic is 1s, the interval is 2s and 30min), heating at 75 ℃ for 1 min, carrying out centrifugation at 10000 Xg for 30min, collecting the supernatant, obtaining crude enzyme liquid of recombinant killed Bacillus vallisportis laccase (fmb-rL 103), carrying out separation and purification of the crude enzyme liquid by implementing example 3, and then determining the activity of the recombinant laccase according to the method of example 3.
(7) The addition of methanol/triton meets the stress agent, the enzyme production curve of the bacterial laccase is shown in figure 5, the fermentation is carried out for 20 hours, the highest enzyme yield is achieved, the total enzyme activity reaches 142000U/L, the total yield of the recombinant laccase protein reaches 17970mg/L, the intracellular enzyme activity is 38000U/L, the yield of the recombinant laccase protein is 4810mg/L, the extracellular enzyme activity is 104000U/L, and the yield of the recombinant laccase protein is 13160mg/L. The enzyme production curve is shown in FIG. 5.
As shown in Table 1, the fermentation yield of the recombinant bacterial laccase enzyme after the addition of the stress agent is greatly improved compared with the fermentation yield of the recombinant bacterial laccase enzyme without the addition of the stress agent in terms of total enzyme activity (total protein yield), intracellular enzyme activity (intracellular protein yield) and extracellular enzyme activity (extracellular protein yield).
By using the method provided by the invention, the fermentation production of the recombinant bacterial laccase is carried out by using the recombinant Escherichia coli, and the total enzyme activity (total recombinant protein yield), the intracellular enzyme activity (intracellular recombinant protein yield) and the extracellular enzyme activity (extracellular recombinant protein yield) are all higher than the highest enzyme activity yield (recombinant protein yield) of the recombinant bacteria reported at present.
TABLE 1
Sequence listing
<110> Nanjing university of agriculture
TAIXING DONGSHENG BIO-TECH Co.,Ltd.
<120> method for producing recombinant bacterial laccase by high-efficiency fermentation
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gggctcctgc cgcgatctgt taaattgaat tctttcagtc ttgcgcctgc tgaacgttac 900
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<212> PRT
<213> Bacillus vallisportisf mb-103 (Bacillus vallisportisf 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 (8)
1. A process for producing a recombinant bacterial laccase enzyme, characterized in that 0-2h of fermentation of a recombinant Escherichia coli producing the bacterial laccase enzyme is supplemented with 1-10% v/v of methanol or triton or a combination of methanol and triton.
2. The process according to claim 1, characterized in that the addition time of methanol is 0-0.5h and the amount is 4-8% v/v.
3. The process according to claim 2, characterized in that the methanol is added for 0h at 6% v/v.
4. The method according to claim 1, characterized in that the addition time of triton is 0-0.5h and the addition amount is 2-5% v/v.
5. The method according to claim 4, wherein the addition time of triton is 0h and the addition amount is 4% v/v.
6. The method according to claim 1, wherein the addition time of the methanol and triton composite inducer is 0-0.5h, the addition amount is 4-8% v/v of methanol, and 2-5% v/v of triton; and the sum of the two does not exceed 10% v/v.
7. The method according to claim 6, wherein the addition time of the composite inducer of methanol and triton is 0h, the addition amount is 6% v/v of methanol and 4% v/v of triton.
8. The method according to any one of claims 1 to 7, characterized by comprising the steps of:
(1) Constructing escherichia coli genetic engineering bacteria for producing recombinant bacterial laccase;
(2) Culturing Escherichia coli genetic engineering bacteria producing recombinant bacterial laccase at 37 ℃ for 10-11 hours, then selecting microcolonies, inoculating 50ml LB liquid culture medium containing kanamycin, and culturing overnight at 180rpm and 37 ℃ to obtain seed liquid; wherein the final concentration of the kanamycin is 50ug/ml;
(3) Taking a seed solution and transferring the seed solution to a TB culture medium containing kanamycin;
(4) Adding methanol or triton or a combination of methanol and triton as claimed in any one of claims 1-7, 30 ℃,180rpm, shaking to OD600=1.0;
(5) Adding IPTG and GuSO 4 Inducing at 16 deg.C, 180rpm,20h; wherein the final concentration of IPTG is 100ug/ml and GuSO 4 Final concentration 0.25mM;
(6) Standing for 22h at 16 ℃;
(7) Centrifuging to obtain thallus and supernatant, wherein the supernatant is extracellular enzyme, and the thallus is added with Cu 2+ And (3) carrying out heavy suspension on Tris-HCl buffer solution with the final concentration of 1mM, carrying out ultrasonic crushing, heating at 75 ℃ for 10min, carrying out centrifugation at 10000 Xg for 30min at 4 ℃, and collecting supernatant to obtain crude enzyme solution of the recombinant bacterial laccase.
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