CN104593312B - The engineering strain of one plant of intracellular expression bacillus subtilis laccase and the abductive approach for realizing the extracellular release restructuring laccase of the bacterial strain - Google Patents
The engineering strain of one plant of intracellular expression bacillus subtilis laccase and the abductive approach for realizing the extracellular release restructuring laccase of the bacterial strain Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0061—Laccase (1.10.3.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03002—Laccase (1.10.3.2)
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Abstract
The engineering strain and the efficient abductive approach of extracellular laccase of one plant of intracellular expression bacillus subtilis laccase are realized, is related to the engineering strain of one plant of intracellular expression bacillus subtilis laccase and realizes that the bacterial strain efficiently discharges the abductive approach in restructuring laccase to culture medium.The preparation method of the engineering strain：First, the clone of the extraction of subtilis genomic dna and bacterial laccase gene；2nd, the structure of recombinant strains.Realize the abductive approach of the restructuring efficiently extracellular laccase of laccase：First, engineered strain is accessed into LB medium cultures to OD600For 0.8；2nd, 0.1mM IPTG and 0.1mM CuSO are added4, 25 DEG C, 120rpm cultivates 4h, then the static gas wave refrigerator at 30 DEG C.Restructuring intracellular laccase effectively can be discharged into culture medium by abductive approach provided by the present invention, laccase is expressed compared to traditional E. coli secretion, its Extracellular laccase yield is significantly improved, simple to operate, has good reference value for industrial-scale fermentation Extracellular laccase.
Description
Technical field
The present invention relates to the engineering strain of one plant of intracellular expression bacillus subtilis laccase and realize that the bacterial strain is extracellular
The abductive approach of release restructuring laccase.
Background technology
Laccase (EC 1.10.3.2) is a kind of cupric polyphenol oxidase, is subgroup maximum in blue multicopper oxidase.Enzyme profit
Use O2Substrate is catalyzed as electron acceptor, and course of reaction reclaimed water is unique accessory substance, is typical " green catalyst ".Paint
The substrate specificity of enzyme is quite varied, and so far, the application of laccase has covered environmental contaminants improvement, wood fibre material
Expect improvement, biosynthesis, paper industry, dye wastewater degradation, typical local food improvement etc. multiple fields.
Laccase is widely present in fungi, insect, plant, prokaryotes.The current research about fungal laccase is more deep
Enter, although but fungal laccase with higher redox potential, because the producing enzyme time is grown, stability is poor, and is unsatisfactory for
In the commercial Application condition of harshness.Main reason is that most of processing environment is at high temperature, high ionic strength and strong basicity
Extremity, so harsh condition are unfavorable for the existence of fungal laccase, or even completely inhibit the performance of its effect.Compared to it
Under, bacterial laccase growth for when it is short, heat endurance is high, the strong adaptability to environment, therefore, more conducively industrial extensive to push away
It is wide to use.CotA albumen is the exine albumen of bacillus, has laccase activity, and have than common fungus laccase
More excellent application speciality, such as high temperature resistant, the extreme condition such as strong basicity and enzyme inhibitor should more suitable for large-scale industry
With.Because CotA albumen is entrenched in bacillus outer wall, be unfavorable for albumen isolates and purifies and does large scale fermentation and application.
The heterogenous expression for realizing CotA by recombinant DNA technology can effectively avoid drawbacks described above to obtain substantial amounts of enzyme preparation, realize
A large amount of productions of CotA albumen.
Escherichia coli are the classical systems for being applied to protein hetero expression earliest.Its operating procedure is simple, and expression quantity is high,
Sustainable large scale fermentation, because Escherichia coli will not carry out complicated posttranslational modification (such as glycosylation, acyl group to heterologous protein
Change), the natural structure of destination protein script can be maintained, therefore be especially suitable for the heterogenous expression of protokaryon albumen.At present with large intestine bar
Bacterium is that the heterogenous expression albumen that host realizes is positioned at the thalline inside (at intracellular or periplasmic space) of host, it is necessary to carry out mostly
Clasmatosis and permeability dehydration effect obtain destination protein, processing step it is cumbersome and during easily loss target protein；And
Protein component complexity is various in Bacillus coli cells, is discharged into simultaneously in buffer solution with destination protein after broken born of the same parents, is unfavorable for target
The purifying of albumen.And the correct processing that the reductive condition of Escherichia coli intracellular is unfavorable for albumen is folded (especially rich in two
The destination protein of sulfide linkage), further, since Bacillus coli expression speed is quick, and target protein can not in time through cell membrane or
Correctly assemble and largely accumulate in intracellular, form fine and close, without protein active inclusion body and precipitate, it is necessary to carry out a series of denaturation
Renaturation process can just regain active destination protein.By contrast, the extracellular background proteins of Escherichia coli are few, more sharp
In the separation and purification of albumen.Therefore realize that the extracellular expression of destination protein can effectively simplify weight using E. coli secretion approach
The downstream treatment process of histone, realize industrialization production and the application of albumen.
The CotA albumen using Escherichia coli as host expresses is all located inside thalline (intracellular or periplasmic space) so far,
Need to carry out clasmatosis before protein purification procedures or permeate dehydration, the albumen of complex disposal process and intracellular can be with CotA albumen
Discharge simultaneously, increase purifying difficulty.Compared with intracellular protein, it is few that Escherichia coli itself are discharged into extracellular background proteins, because
CotA albumen is discharged into culture medium by this by Escherichia coli itself secretory pathway can effectively simplify CotA protein purification procedures,
And the stability and bioactivity of recombinant protein can be improved, maintain the N- ends authenticity of native protein.Have both at home and abroad each
Albuminoid realizes the extracellular expression in Escherichia coli.But remain as sky using the research of Escherichia coli extracellular expression CotA albumen
In vain.Currently with secreting, expressing original paper realize recombinant protein Escherichia coli secreting, expressing research in, protein expression yield compared with
It is low, do not utilize follow-up large-scale protein to obtain.Realizing the large-scale production of recombinant C otA laccases has important Research Significance
And application value.
With the development of dye industry, the kind and quantity of dyestuff are continuously increased.According to investigations, in dyeing process, dyestuff
Loss amount be about 10-20%.These industrial wastewater discharges among aquatic ecosystem, not only have impact on environment it is attractive in appearance,
Change the oxygen dissolved of water, very big pollution is more seriously caused to environment.Ecological toxicology association (ETAD) is to 400 kinds
Dyestuff finds that 90% dyestuff all has very high toxicity during being investigated.Most dyestuff due to it is complicated,
Chemical stability is high, degradability is low, and traditional physics, chemical down method cost are high, degradation efficiency is low, to intractable dyestuff
Degraded it is nearly unavailable, and cause solid waste, long-term accumulation can also cause secondary pollution to environment.By contrast, it is sharp
With enzyme preparation degradation of dye waste water not only rapidly and efficiently, it is easily operated, and nonhazardous acts on.Therefore, realize that recombinant C otA is painted
The large-scale production of enzyme has important Research Significance and application value.
The content of the invention
The present invention provides the engineering strain of one plant of intracellular expression bacillus subtilis laccase and realizes that the bacterial strain is extracellular
Express the abductive approach of laccase.
The construction method of the engineering strain of intracellular expression bacillus subtilis laccase of the present invention, enters according to the following steps
OK：
First, the clone of the extraction of subtilis genomic dna and bacterial laccase gene：
Extract bacillus subtilis LS02 genomic DNA, using genomic DNA as template, using primer LS02-F and
LS02-R enters performing PCR amplification, is connected to after amplified production is purified on pEASY-Blunt cloning vectors, obtains recombinant vector
PEASY/CotA, using pEASY/CotA as template, expanded using point mutation primer LS02-Mut-F and LS02-Mut-R, PCR complete
Plasmid, using full formula gold DMT enzymic digestion PCR primers, digestion product is then imported into Escherichia coli DMT competent cells, passed through
Blue hickie screening positive clone transformant, sequence verification, obtain carrier pEASY/CotA-Mut；
2nd, the structure of recombinant strains：
The carrier pEASY/CotA-Mut and expression vector pET-20b (+) that step 2 is obtained are respectively through NdeI and BamHI
Double digestion, the endonuclease bamhi CotA-Mut containing cohesive end is connected with pET-20b (+) by T4DNA ligases, structure weight
Group expression vector pET/CotA-His6, is transferred to Escherichia coli Top10 competent cells, is obtained by Amp antibiotic-screenings positive
Transformant and sequence verification, will be sequenced successful recombinant expression carrier and are transferred to e. coli bl21 (DE3), by antibiosis containing Amp
The LB solid mediums screening positive expression bacterial strain of element, is named as PSD.
Primer LS02-F and LS02-R sequence are as follows in step 1：
LS02-F：5’-GGAATTCCATATG(underlined sequence corresponds to NdeI enzymes to ACACTTGAAAAATTTGTGGATGC-3 '
Enzyme site)；
LS02-R：5’-CGCGGATCC(underlined sequence corresponds to BamHI digestions to GGTTTATGGGGATCAGTTATATCC-3 '
Site).
Primer LS02-Mut-F and LS02-Mut-R sequence are as follows in step 2：
LS02-Mut-F：5’-ATTGACTTCACAGCATAGAAGGAGAATCGATC-3 ' (is mutation position at mark
Point)；
LS02-Mut-R：5’-TATGCTGTGAAGTCAATGATGATATCATAAC-3 ' (being mutational site at mark).
The abductive approach of above-mentioned recombinant strains extracellular expression laccase is realized, is carried out according to the following steps：
First, recombinant strains PSD glycerol stocks are accessed in the LB culture mediums of the Amp containing 50mg/L, in 37 DEG C, 180rpm
Under the conditions of be incubated overnight 12~14h, obtain bacterium solution；
The 2nd, the bacterium solution that step 1 obtains is seeded to the fresh LB of the Amp containing 50mg/L according to 1% inoculum concentration,
30 DEG C, under the conditions of 180rpm culture to OD600For 0.8,0.1mM IPTG and 0.1mM CuSO are added4, it is placed under the conditions of 25 DEG C
120rpm cultivates 4h, then the static gas wave refrigerator 20h at 30 DEG C, i.e. induction obtains laccase.
Beneficial effects of the present invention：
The recombinant expression carrier pET/CotA-His6 that the present invention is built eliminates the pelB letters that pET20-b (+) carrier carries
Number peptide, and do not introduce the secretion element of other guiding recombinant C otA protein secretions expression, the expression bacterial strain obtained in theory
It is only used for cell inner expression of the laccase in Escherichia coli.But abductive approach provided by the present invention can realize the genetic engineering
The efficient extracellular expression recombinant C otA laccases of bacterial strain.The abductive approach is by adding appropriate copper sulphate into culture medium and micro-
Fiber differentiation bacterial strain significantly improves the ability of engineering strain extracellular expression restructuring laccase under aerobic condition.Method is simple,
Significant effect.The present invention has good reference value for improving the extracellular restructuring laccase yield of recombination bacillus coli.
The present invention provides application of the said gene engineering bacteria in synthetic dyestuffs decolouring.
Brief description of the drawings
Fig. 1 is the step 2 digestion products electrophoretogram of embodiment one；Fig. 2 builds for the step 2 of embodiment one
Recombinant expression carrier pET/CotA-His6 gel electrophoresis figure；Fig. 3 is recombinant expression carrier pET/CotA-His6 double enzymes
Cut proof diagram；Fig. 4 is recombinant expression carrier pET/CotA-His6 collection of illustrative plates；Fig. 5 is the distribution map of not isogeneous induction moment laccase；Fig. 6
For the SDS-PAGE of albumen in not isogeneous induction moment coli somatic；Fig. 7 is not isogeneous induction moment Escherichia coli bag
Contain the SDS-PAGE of body protein；Fig. 8 is not isogeneous induction moment Escherichia coli intracellular soluble protein SDS-PAGE electrophoresis
Figure；Fig. 9 is the SDS-PAGE of not isogeneous induction moment Escherichia coli inducing culture supernatant；When Figure 10 is not isogeneous induction
Carve colibacillus periplasm space protein SDS-PAGE electrophoretogram；Figure 11 is influence of the inducing temperature to the extracellular laccase amount of bacterial strain；
Figure 12 is influence of the copper ion concentration to the extracellular laccase amount of bacterial strain；Figure 13 is shadow of the IPTG concentration to the extracellular laccase amount of bacterial strain
Ring；Figure 14 is influence of the starting induction bacterium OD values to the extracellular laccase amount of bacterial strain；Figure 15 is the capacity of culture triangular flask to bacterium
The influence of the extracellular laccase amount of strain；Figure 16 is culture crude enzyme liquid (the phosphoric acid hydrogen two that buffer solution is pH=7.0 in neutral conditions
Sodium-citrate buffer solution) to the decolorization of 4 kinds of reactive dye；Figure 17 is that (buffer solution is culture crude enzyme liquid in the basic conditions
PH=9.0 Tris-HCl buffer solutions) to the decolorization of 4 kinds of reactive dye.
Embodiment
Technical solution of the present invention is not limited to act embodiment set forth below, in addition between each embodiment
Any combination.
Embodiment one：The structure of the engineering strain of present embodiment intracellular expression bacillus subtilis laccase
Method, carry out according to the following steps：
First, the clone of the extraction of subtilis genomic dna and bacterial laccase gene：
Extract bacillus subtilis LS02 genomic DNA, using genomic DNA as template, using primer LS02-F and
LS02-R enters performing PCR amplification, restriction enzyme site NdeI and BamhI is introduced at laccase gene sequence both ends, after amplified production is purified
It is connected on pEASY-Blunt cloning vectors, obtains recombinant vector pEASY/CotA, using pEASY/CotA as template, using point
Mutant primer LS02-Mut-F and LS02-Mut-R, PCR expand full plasmid, and the 927th bit base T on CotA DNA sequence dnas is dashed forward
Become base C, on the basis of the amino acid sequence of CotA albumen is not changed, remove the NdeI enzymes identification on CotA DNA sequence dnas
Sequence, using full formula gold DMT enzymic digestion PCR primers, digestion product is then imported into Escherichia coli DMT competent cells, passed through
Blue hickie screening positive clone transformant, sequence verification, obtain carrier pEASY/CotA-Mut；
2nd, the structure of recombinant strains：
The carrier pEASY/CotA-Mut and expression vector pET-20b (+) that step 1 is obtained are respectively through NdeI and BamHI
Double digestion, the endonuclease bamhi CotA-Mut containing cohesive end is connected with pET-20b (+) by T4DNA ligases, structure weight
Group expression vector pET/CotA-His6, is transferred to Escherichia coli Top10 competent cells, is obtained by Amp antibiotic-screenings positive
Transformant and sequence verification, will be sequenced successful recombinant expression carrier and are transferred to e. coli bl21 (DE3), by antibiosis containing Amp
The LB solid mediums screening positive expression bacterial strain of element, is named as PSD.
Primer LS02-F and LS02-R sequence are as follows in step 1：
LS02-F：5’-GGAATTCCATATG(underlined sequence corresponds to NdeI enzymes to ACACTTGAAAAATTTGTGGATGC-3 '
Enzyme site)；
LS02-R：5’-CGCGGATCC(underlined sequence corresponds to BamHI digestions to GGTTTATGGGGATCAGTTATATCC-3 '
Site).
Primer LS02-Mut-F and LS02-Mut-R sequence are as follows in step 2：
LS02-Mut-F：5’-ATTGACTTCACAGCATAGAAGGAGAATCGATC-3 ' (is mutation position at mark
Point)；
LS02-Mut-R：5’-TATGCTGTGAAGTCAATGATGATATCATAAC-3 ' (being mutational site at mark).
Bacillus subtilis LS02 is announced in patent (ZL201010599793.5) in step 1, and it is micro- to be preserved in China
Biological inoculum preservation administration committee common micro-organisms center, deposit number are CGMCC No.4261.Described CotA laccase bases
Because of Accession NO in sequence such as Genebank：Shown in GU972587.
Extracting genome DNA uses bacterial genomes extracts kit in step 1.
Escherichia coli Top10 competent cells and e. coli bl21 (DE3) are both from Tiangeng biochemical technology (Beijing)
Co., Ltd, point mutation are bought from Quan Shi King Companies with Escherichia coli DMT competent cells；Expression plasmid pET-20b (+) is
Merk Products.
Bacterial genomes extracts kit, glue reclaim kit and plasmid extraction kit are Omega Products；
TransStart fastpfu archaeal dna polymerases, pEASY-Blunt cloning vectors, DMT digestive ferments, DMT competent cells, albumen
Molecular weight standard is Quan Shi King Companies product；250bp DNA ladder Marker, NdeI quickcut restriction enzymes
Enzyme and BamHI quickcut restriction enzymes are all from precious biotech firm.
Present embodiment step 2 digestion products electrophoretogram such as Fig. 1, swimming lane M：250bp DNA Ladder Marker；1：
PET-20b (+) endonuclease bamhi 2 after purified：Digestion CotA-Mut DNA fragmentations after purified.
The recombinant expression carrier pET/CotA-His6 of present embodiment step 2 structure gel electrophoresis figure such as Fig. 2, wherein
Swimming lane M：250bp DNA Ladder Marker；1：Recombinant expression plasmid pET/CotA-His6.
Double digestion checking, wherein such as Fig. 3, swimming lane M are carried out to recombinant expression carrier pET/CotA-His6：250bp DNA
Ladder Marker；1：DNA products of the recombinant expression plasmid pET/CotA-His6 after NdeI and BamHI double digestions.
Recombinant expression carrier pET/CotA-His6 collection of illustrative plates is as shown in Figure 4.
Embodiment two：Present embodiment realizes the induction of bacterial strain extracellular expression laccase described in embodiment one
Method, carry out according to the following steps：
First, recombinant strains PSD glycerol stocks are accessed in the LB culture mediums of the Amp containing 50mg/L, in 37 DEG C, 180rpm
Under the conditions of be incubated overnight 12~14h, obtain bacterium solution；
The 2nd, the bacterium solution that step 1 obtains is seeded to the fresh LB of the Amp containing 50mg/L according to 1% inoculum concentration,
30 DEG C, under the conditions of 180rpm culture to OD600For 0.8,0.1mM IPTG and 0.1mM CuSO are added4, it is placed under the conditions of 25 DEG C
120rpm cultivates 4h, then the static gas wave refrigerator at 30 DEG C；From IPTG derivant timing is added, detected respectively every sampling in 4 hours
Culture medium supernatant, intracellular, periplasmic space laccase activity, while prepare protein sample and analyzed for SDS-PAGE.
Laccase activity assay method：Using spectrophotometer detection laccase and the light absorption value after substrate reactions, laccase is calculated
Vigor.The substrate is syringaldazine (SGZ), and reaction temperature is 30 DEG C, and buffer solution is that 0.2M disodium hydrogen phosphates-citric acid delays
Fliud flushing (pH 7.0).The enzyme amount that enzyme activity is defined as needed for 1 μM of substrate of oxidation per minute is an enzyme activity unit.
Fig. 5, which shows that recombinant bacterial strain PSD is intracellular, pericentral siphon is empty, to be seen and dynamic that culture medium supernatant laccase changes with induction time
Distribution situation.- ■-expression intracellulars in Fig. 5 ,-▲-culture medium supernatant is represented,Represent periplasmic space.Induction starting 8
Hour, laccase activity is detected in somatic cells, intracellular laccase activity reached peak value (162U/L) at 16 hours, then with luring
Leading the time incrementally gradually decreases down 3U/L；Periplasmic space laccase activity equally starts to detect at 8 hours in induction, and at 20 hours
Reach peak (40U/L), laccase activity starts slowly to decline after maintaining 8 hours.Nutrient solution supernatant is adding IPTG 12 hours
After detect laccase activity, supernatant laccase activity is persistently raised, and peak (403U/L) was reached at 36 hours and keeps stable afterwards
It is constant.
The SDS-PAGE of albumen is as shown in fig. 6, M in Fig. 6 in not isogeneous induction moment coli somatic：Protein
Molecular weight standard；1-9：Respectively Fiber differentiation 0, the mycoprotein of 4,8,12,16,20,24,28,32 hours.During not isogeneous induction
The SDS-PAGE of inclusion bodies of colibacillus albumen is carved as shown in fig. 7, M in Fig. 7：Protein molecular weight standard；1-9：Point
Wei not Fiber differentiation 0, the inclusion body protein of 4,8,12,16,20,24,28,32 hours.Not isogeneous induction moment Escherichia coli intracellular
Soluble protein SDS-PAGE is as shown in figure 8, M in Fig. 8：Protein molecular weight standard；1-9：Respectively Fiber differentiation
0th, the intracellular soluble protein of 4,8,12,16,20,24,28,32 hours.On not isogeneous induction moment Escherichia coli inducing culture
The SDS-PAGE of clear liquid is as shown in figure 9, M in Fig. 9：Protein molecular weight standard；1-9：Respectively Fiber differentiation 8,12,
16th, the Supernatant protein of 20,24,28,32,36 hours.Not isogeneous induction moment colibacillus periplasm space protein SDS-PAGE electricity
Swimming figure is as shown in Figure 10, M in Figure 10：Protein molecular weight standard；1-9：Respectively Fiber differentiation 0,4,8,12,16,20,24,
28th, the periplasmic space albumen of 32 hours.The strong of CotA protein bands corresponding to diverse location is relatively not difficult to find out by electrophoretogram
It is weak corresponding with laccase activity in Fig. 5 consistent.
Experiment 1：Influence of the inducing temperature to the extracellular laccase amount of bacterial strain
30 μ L glycerols bacterium are taken to access in 5mL LB culture mediums (Amp containing 50mg/L), 37 DEG C, 180rpm is incubated overnight 14h,
Next day is transferred to the fresh LB culture mediums (Amp containing 50mg/L) of 5mL, 30 DEG C, 180rpm to OD by 1% switching amount600=0.8, add
Enter 0.1mM IPTG, 0.1mM CuSO4, 120rpm cultivates 4h under the conditions of being respectively placed in 20 DEG C, 25 DEG C, 30 DEG C, is subsequently placed at 20
DEG C, 25 DEG C, 30 DEG C, static gas wave refrigerator 20h at 37 DEG C, determine culture medium supernatant laccase activity.As shown in figure 11, static temperature and training
Foster temperature plays material impact to the extracellular release for recombinating laccase, and the optimal inducing temperature finally established is that shake temperature is 25
DEG C, static gas wave refrigerator temperature is 30 DEG C.
Experiment 2：Influence of the copper ion concentration to the extracellular laccase amount of bacterial strain
30 μ L glycerols bacterium are taken to access in 5mL LB culture mediums (Amp containing 50mg/L), 37 DEG C, 180rpm is incubated overnight 14h,
Next day is transferred to the fresh LB culture mediums (Amp containing 50mg/L) of 5mL by 1% switching amount, and 30 DEG C of 180rpm are cultivated to OD600=0.8,
Add 0.1mM IPTG, be separately added into 0mM, 0.1mM, 0.5mM, 0.9mM, 1.3mM, 1.7mM, 2.1mM, 2.5mM, 2.9mM,
3.3mM CuSO4, 25 DEG C of 120rpm cultivate 4h, static gas wave refrigerator 20h at 30 DEG C, determine medium supernatant laccase activity.Pass through
Figure 12 can have found, with Cu2+The raising of concentration, extracellular laccase activity are obviously improved, and peak reaches 801U/L, and in culture medium
Do not add Cu2+When, culture medium laccase activity is detected as 0.Work as Cu2+Concentration persistently rises the rapid decline of Extracellular laccase activity, may
It is relevant with the copper ion resistance of Escherichia coli in itself.
Experiment 3：Influence of the IPTG concentration to the extracellular laccase amount of bacterial strain
30 μ L glycerols bacterium are taken to access in 5mL LB culture mediums (Amp containing 50mg/L), 37 DEG C, 180rpm is incubated overnight 14h,
Next day is transferred to the fresh LB culture mediums (Amp containing 50mg/L) of 5mL by 1% switching amount, and 30 DEG C of 180rpm are cultivated to OD600=
0.8, add 0.1mM CuSO4, it is separately added into 0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM IPTG, 25 DEG C of 120rpm
4h is cultivated, static gas wave refrigerator 20h at 30 DEG C, determines medium supernatant laccase activity.Derivant IPTG situation is being added without, still
Laccase activity so is detected in culture medium supernatant, because pET20-b (+) does not contain the lac of strict control background expression in itself
I gene, therefore understand some inevitable background expression.As shown in figure 13, with the raising of IPTG concentration, laccase activity
Slowly improve, when IPTG concentration is 0.1mM, laccase activity highest, reaches 285U/L, persistently raised with IPTG concentration, it is extracellular
Laccase activity declines therewith.
Experiment 4：Originate influence of the induction bacterium OD values to the extracellular laccase amount of bacterial strain
30 μ L glycerols bacterium are taken to access in 5mL LB culture mediums (Amp containing 50mg/L), 37 DEG C, 180rpm is incubated overnight 14h,
Next day is transferred to the fresh LB culture mediums (Amp containing 50mg/L) of 5mL by 1% switching amount, and 30 DEG C of 180rpm cultivate to OD respectively600=
0.4th, 0.6,0.8,1.0,1.2,1.4, add 0.1mM IPTG, 0.1mM CuSO4, it is 25 DEG C, 120rpm cultures 4h, quiet at 30 DEG C
20h is only cultivated, determines medium supernatant laccase activity.As shown in figure 14, when originating OD values less than or equal to 0.8, now bacterial strain
In growth logarithmic phase, growth conditions are good, metabolism it is vigorous, therefore Extracellular laccase activity with induction starter bacteria OD values increase and
Significantly improve, when OD values are between 0.8 to 1.2, strain growth speed slows down, and is now partial to enter stationary phase, extracellular paint
Enzymatic activity equally increases and risen with bacterium OD concentration, but ascensional range substantially slows down.When OD values are equal to 1.4, now bacterial strain
Growth metabolism speed substantially slows down, although bacteria concentration is very high when now adding derivant, because bacterial strain metabolic capability fails,
Therefore Extracellular laccase output capacity substantially weakens.
Experiment 5：Influence of the triangular flask size to the extracellular laccase amount of bacterial strain
50mL, 150mL, 250mL, 500mL triangular flask are chosen, is respectively charged into 1/10th LB Liquid Cultures of bottle product
Base (Amp containing 50mg/L).Seed liquor is transferred to the fresh LB (Amp containing 50mg/L) of same volume by 1% inoculum concentration, 30
DEG C, 180rpm is cultivated to OD600=0.8, add 0.1mM IPTG, 0.1mM CuSO4, 25 DEG C are placed in, is cultivated under the conditions of 120rpm
4h, then the static gas wave refrigerator 20h at 30 DEG C, determines culture medium supernatant laccase activity.Although as a result show 4 kinds of culture triangular flask institutes
The bacterium solution OD values obtained are consistent, but are increased with the volume of shaking flask, and Extracellular laccase activity also significantly improves (Figure 15).Utilize 500mL tri-
The Extracellular laccase activity that bottle culture laccase in angle obtains is 2 times of the Extracellular laccase activity that the culture of 50mL triangular flasks obtains.
Experiment 6：Recombinate the decolouring to 4 kinds of reactive dye in neutral and alkaline conditions of laccase crude enzyme liquid
Experiment reaction system is 6mL, sequentially added in system disodium hydrogen phosphate-citrate buffer solution (pH 7.0) or
Tris-HCl buffer solutions (pH 9.0), laccase (working solution concentration is 20U/L), amboceptor (final concentration of 0.1mM), dyestuff (are shown in Table
1).All measurements are averaged in triplicate, and dye decolored rate calculation formula is (A0-A)/A0× 100%, wherein A0To be first
Beginning dyestuff light absorption value, the light absorption value that A is determined by timing sampling.
The dye type of table 1, final concentration and maximum absorption wavelength
As a result show, under conditions of pH 7.0, restructuring laccase shows the ability of very strong decolouring dyestuff.In amboceptor second
Under the participation of acyl syringone, crude enzyme liquid to Congo red, crystal violet, active light blue, isatin four kinds of different structures synthetic dyestuffs
Percent of decolourization be respectively 80.12%, 87.70%, 81.89% and 98.97% (Figure 16, whereinRepresent Congo red ,- -table
Show crystal violet ,-▲-active light blue is represented ,-●-represent isatin).And under conditions of pH 9.0, restructuring laccase is still protected
Very high decoloring ability is held, the percent of decolourization to Congo red, crystal violet, active light blue, isatin is respectively 66.26%, 81.07%,
92.82% and 99.14% (Figure 17, wherein-■-expression is Congo red ,- -expression crystal violet ,-▲-represents that activity is bright
Indigo plant,Represent isatin).Laccase is recombinated to be higher than in neutrality the decoloring ability of active light blue and isatin under conditions of strong basicity
Under the conditions of to the decoloring abilities of both dyestuffs.Illustrate that efficient decolouring can be played in neutral and alkaline conditions by recombinating laccase
Ability, being decolourized in industrial dye waste water, application is upper to have very high application value.
Claims (1)
1. a kind of abductive approach for effectively realizing a large amount of extracellular expression laccases of Recombinant organism strain, it is characterised in that should
Method is realized on the basis of the Recombinant organism strain of one plant of intracellular expression bacillus subtilis laccase, bacterial strain
Preparation method is as follows：
First, the clone of the extraction of subtilis genomic dna and bacterial laccase gene：
Extract bacillus subtilis LS02 genomic DNA, using genomic DNA as template, using primer LS02-F 5 '-
GGAATTCCATATGACACTTGAAAAATTTGTGGATGC-3 ' and LS02-R 5 '-
CGCGGATCCGGTTTATGGGGATCAGTTATATCC-3 ' enters performing PCR amplification, and pEASY- is connected to after amplified production is purified
On Blunt cloning vectors, recombinant vector pEASY/CotA is obtained, using pEASY/CotA as template, using point mutation primer LS02-
Mut-F 5 '-ATTGACTTCACAGCATACGAAGGAGAATCGATC-3 ' and LS02-Mut-R 5 '-
GTATGCTGTGAAGTCAATGATGATATCATAAC-3 ', PCR expand full plasmid, using full formula gold DMT enzymic digestion PCR primers,
Digestion product is then imported into Escherichia coli DMT competent cells, by blue hickie screening positive clone transformant, sequencing is tested
Card, obtain carrier pEASY/CotA-Mut；By carrier pEASY/CotA-Mut and carrier pET-20b (+) respectively through NdeI and
BamHI double digestions, the endonuclease bamhi CotA-Mut containing cohesive end is connected with pET-20b (+) by T4DNA ligases,
Recombinant expression carrier pET/CotA-His6 is built, Escherichia coli Top10 competent cells is transferred to, is obtained by Amp antibiotic-screenings
Positive transformant and sequence verification, successful recombinant expression carrier will be sequenced and be transferred to e. coli bl21 (DE3), by containing
The LB solid mediums screening positive expression bacterial strain of Amp antibiotic, is named as PSD；
2nd, realize that the inductive condition of the extracellular release restructuring laccase of PSD bacterial strains is as follows
Recombinant strains PSD glycerol stocks are accessed in the LB culture mediums of the Amp containing 50mg/L, the mistake under the conditions of 37 DEG C, 180rpm
Night cultivates 12~14h, obtains bacterium solution；The bacterium solution being incubated overnight is seeded to the fresh LB of the Amp containing 50mg/L according to 1% inoculum concentration
Culture medium, cultivated under the conditions of 30 DEG C, 180rpm to OD600For 0.8,0.1mM IPTG and 0.1mM CuSO are added4, it is placed in 25
120rpm cultivates 4h under the conditions of DEG C, then the static gas wave refrigerator 20h at 30 DEG C, you can restructuring laccase is detected in culture medium supernatant
Activity.
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| CN107868790A (en) * | 2016-09-27 | 2018-04-03 | 常熟市中科兴华生物科技有限公司 | One kind tolerance organic solvent laccase derivative and preparation method thereof |
| CN106434707B (en) * | 2016-11-07 | 2019-08-06 | 吉林大学 | A kind of bacterial laccase gene, bacterial laccase and its application from hay bacillus ZNXH1 |
| CN106434706B (en) * | 2016-11-07 | 2019-08-06 | 吉林大学 | A kind of bacterial laccase gene, bacterial laccase and its application from hay bacillus ZXN4 |
| CN108570459B (en) * | 2018-04-10 | 2022-10-04 | 南京农业大学 | Method for producing recombinant bacterial laccase by high-efficiency fermentation |
| CN109161538B (en) * | 2018-09-29 | 2021-10-15 | 云南师范大学 | Lipase mutant with improved heat stability and application thereof |
| CN109439635B (en) * | 2018-10-23 | 2020-11-06 | 江南大学 | CotA laccase with improved catalytic efficiency and application thereof |
| CN116064429B (en) * | 2022-08-04 | 2023-11-03 | 四川省畜牧科学研究院 | Method for efficiently expressing laccase by recombinant escherichia coli |
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