CN104212785A - High-density fermentation method of engineering bacteria containing nitrilase - Google Patents

High-density fermentation method of engineering bacteria containing nitrilase Download PDF

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CN104212785A
CN104212785A CN201410394475.3A CN201410394475A CN104212785A CN 104212785 A CN104212785 A CN 104212785A CN 201410394475 A CN201410394475 A CN 201410394475A CN 104212785 A CN104212785 A CN 104212785A
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fermentation
engineering bacteria
sequence
nitrilase
high density
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薛亚平
郑裕国
柳志强
刘兆巍
王应鹏
徐喆
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Zhejiang University of Technology ZJUT
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Zhejiang University of Technology ZJUT
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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
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/05—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in nitriles (3.5.5)
    • C12Y305/05001—Nitrilase (3.5.5.1)

Abstract

The invention discloses a high-density fermentation method of engineering bacteria containing nitrilase. The method adopts a novel recombinant Escherichia coli fermentation and supplementary material culture medium formula, and uses a method of DO-STAT coupling step-by-step induction and gradual increase of the speed stage by stage. The method not only meets the need for mixing power in the culture process with biomass increase, effectively controls the growth rate of thalli, and prevents disadvantages of produced acid accumulation caused by fast growth of thalli, feedback inhibition and culture medium waste. In addition, the induced mode of batch-by-batch lactose supplement addition avoids the high viscosity of the fermentation broth caused by high concentration of lactose, resulting in resistance of oxygen transfer and mass transfer, but also improves the induction strength. The high-density fermentation technology of the invention increase the biomass of recombinant Escherichia coli from 10.35g DCW / L to 70g DCW / L, volume enzyme activity from 18205U / L to 103530U / L, and the fermentation level by 4.7 times.

Description

A kind of fermentation process in high density containing nitrilase gene engineering bacteria
(1) technical field
The present invention relates to and a kind ofly produce the high-density culture of the recombination bacillus coli of nitrilase and the high-efficiency expression method of goal gene.
(2) background technology
Nitrilase can realize the organic carboxyl acid of an one-step hydrolysis synthesis correspondence of organonitrile compound.Realize cyan-hydrolysis reaction conditions by nitrilase gentle, reaction efficiency is high, and has higher regioselectivity and stereoselectivity.In recent years, utilizing nitrilase to carry out cyan-hydrolysis synthesis organic carboxyl acid has become the focus of research, lot of documents, patent reports the application of nitrilase in organic carboxyl acid synthesis.The more important thing is, many discoveries two nitrile to the nitrilase of regioselective catalytic vigor, for atorvastatin side chain crucial chiral intermediate (R)-4-cyano-3-hydroxy butyric acid (Organic Process Research & Development 2006, 10, 661-665), lyrica chiral intermediate (3S)-3-cyano group-5-methylhexanoic acid (Journal of Molecular Catalysis B:Enzymatic41, 2006:75 – 80), gabapentin intermediate 1-cyanocyclohexanoic guanidine-acetic acid (US2005009157A1), the foundation of the critical medication intermediate enzymatic clarification techniques such as L-methyldopa has huge directive function.
Amygdalic acid (mandelic acid, MA), also known as making mandelic acid, is chiral molecules, two kinds of enantiomers.The pure R-MA of photochemistry (R-MA) is a kind of important fine-chemical intermediate and chiral precursor.Be widely used in the synthesis of multiple optics drugs, as the microbiotic such as cynnematin, semisynthetic penicillin, antitumor drug, slimming medicine, agricultural chemicals and other drug.In addition, R-MA or important chiral resolving agent and chiral catalyst, be called as " omnipotent " resolving agent, wherein particularly outstanding to the fractionation of alcamines medicine.Along with the continuous discovery of the novelty teabag of R-MA, its market requirement is also growing with each passing day.
The production technology of R-MA mainly contains Optical Instruments Industry method and asymmetric chemistry synthesis method.Wherein the catalyzer that adopts of asymmetric chemistry synthesis method is expensive, production cost is high, and the e.e.% of products therefrom (65%-85%) on the low side, the synthesis of chiral drug cannot be used for.Therefore, at present, industrial main based on chemical resolution method.Namely first synthesize racemic mandelic acid, then adopt diastereomer salt-pepper noise Split Method to obtain having optically active amygdalic acid.But the theoretical yield of the method is only 50%, and reagent used has certain toxicity, and this causes the waste of resource and the pollution of environment to a certain extent.By comparison, biological catalysis chiral synthesize R-MA has the advantages such as catalysis is efficient, highly selective, mild condition.Therefore extensive concern is subject to.Particularly using nitrilase as biological catalyst, the method for asymmetric hydrolysis racemize mandelonitrile, has a extensive future, and has become the enzyme technology that various countries fall over each other to develop.
Gabapentin (Gabapentin) is a kind of new antiepileptic medicine, and its chemistry 1-aminomethyl-cyclohexyl acetic acid by name, molecular formula is C 19h 17nO 2.Gabapentin is the analog of the important neurotransmitter-γ-aminobutyric acid (GABA) of the one in mammalian body, plays pharmacological action mainly through the metabolism changing GABA, especially obvious to the result for the treatment of of severe epilepsy.Tolerance is good at high doses, and be not combined with plasma proteins, toxicity is lower, and the transformation period is longer, and side effect is little.Therefore, since listing, the market share increases gradually, has leapt to world's best-selling drugs Top 100 at present.
Gabapentin molecular structure is simple, and the same carbon atom of a six-ring connects two substituting groups.Therefore, mainly with six-ring compound be starting raw material synthesis gabapentin.At present, many investigators with pimelinketone, hexahydrobenzaldehyde, methylenecyclohexane, cyanobenzene etc. for development of raw materials has gone out tens of synthesis paths.What these synthesis paths used is all simple chemical process.They respectively have quality: some reactions steps is tediously long; Some severe reaction conditions.These synthesis paths of having reported for work, majority is not also suitable for suitability for industrialized production.Than other synthetic methods, the people (EP0414262A2) such as Jennings, Rex Allen reported the method from 1-cyanocyclohexanoic base acetonitrile synthesis gabapentin in 1991, be a good production ways.
Reagent and condition: (1) Py/TiCl 4(2) NaCN/EtOH/H 2o (3) 1. (g, 10psi)/EtOH/PhCH 32. H2O/1mol/L/NaOH/PhCH 3(4) 50%NaOH/MeOH/CH 2cl 2, 37%HCl (5) 10%Rh-C/H 2(5) 10%Rh-C/H 2(50psi)/MeOH/i-PrOH (6) Raney Ni (50%in H 2o)/MeOH/50%NaOH/H 2(180psi), i-PrOH/THF.
The cheaper starting materials of this synthesis path is easy to get, and reactions steps is less, and its committed step is that 1-cyanocyclohexanoic base acetonitrile regioselective hydrolyis generates 1-cyanocyclohexanoic guanidine-acetic acid.Current employing chemical hydrolysis, namely 90% the vitriol oil, stoichiometric number hour at 200 DEG C.This chemical method also exists poor selectivity, yield is low, severe reaction conditions, many defects such as economy is low, environmental pollution is serious.These defects greatly limit the industrialization suitability in this path.And the catalytic condition of the high catalytic selectivity of nitrilase, high catalysis activity, gentleness provides direction for addressing this problem.
The industrial applications of nitrilase method catalysis, its key is efficient enzyme production technology.And high-density culture provides direction for realizing this goal.High-density culture refers to apply the fermentation density that certain culture technique and device improve thalline, make cell density comparatively Nostoc commune Vanch improve significantly, the final specific production rate improving specific product.Compared with conventional cultivation technique, high-density culture technology has many advantages.As improve space-time yield; Reduce reactor volume; Shorten culture cycle etc.Current high-density culture technology has been used successfully to the production in enormous quantities of a series of high value low yield albumen such as Interferon, rabbit, G CFS, interleukin-, tethelin, rhIGF-1.
The factor affecting high-density culture is a lot, as: dissolved oxygen, temperature, pH, CO 2, medium component, metabolic by-prods, fermentation broth viscosity, heat transfer, mass transfer, froth breaking etc.And for the high-density culture of recombination bacillus coli, the problem of its most critical is the high expression of restraining effect caused by metabolic by-prods Acetic Acid Accumulation and suitable inductive technology induction goal gene.The present invention is just for the Exploration & stu dy that these problems are launched.
(3) summary of the invention
The object of the invention is to provide a kind of method of the recombination bacillus coli high-density culture containing nitrilase gene, and namely DO-STAT feed supplement coupling divides one-step inducing, and progressively puies forward high-revolving high density fermentation technology, makes nitrilase at E. coli.
The technical solution used in the present invention is:
The invention provides a kind of fermentation process in high density containing nitrilase gene engineering bacteria, described method is: the seed liquor that the engineering bacteria containing nitrilase gene obtains through enlarged culturing is seeded to fermentor tank with the inoculum size of volumetric concentration 2-4% (preferably 3%), in 37 DEG C, 500rpm, blowing air is cultured to fermented liquid dissolved oxygen 15% ± 5% than 1-2vvm (preferred 1.3vvm) condition bottom fermentation, start to adopt DO-STAT mode to add supplemented medium, maintain fermented liquid oxygen dissolving value 15% ± 5%, adjustment mixing speed is 600-700rpm (preferred 700rpm), fermentation is to OD 600when reaching 20-40 (preferably 30), adjustment temperature, to 25-35 DEG C (preferably 30 DEG C), adds final concentration 10-25g/L lactose (preferred 20g/L) and carries out first time induction, fermentation is to OD 600after reaching 70-110 (preferably 90), adjustment rotating speed is to 720-780rpm (preferred 750rpm), fermentation is to OD 600reach 110-130 (preferably 120), again add 10-15g/L (preferred 14g/L) lactose and carry out chain induction, fermentation is to OD 600after reaching 115-140 (preferably 125), adjustment rotating speed, to 780-850rpm (preferred 800rpm), continues fermentation to OD 600after reaching 150-190 (preferably 170), adjustment rotating speed is to 900-1300rpm (preferred 1000rpm), maintaining fermentation rotating speed is under 900-1300rpm (preferred 1000rpm), temperature 25-35 DEG C of (preferably 30 DEG C) condition, continues fermentation to OD 600value and when no longer rising in 3h than enzyme work or start to decline, stops fermentation, puts tank, and obtain fermented liquid, maintaining fermented liquid pH value in fermenting process is 6.0-7.0 (preferably 6.5).
Add the fermention medium containing final concentration 50g/L kantlex in described fermentor tank, described fermention medium consists of: peptone 10-20g/L, yeast powder 5-15g/L, NaCl 5-15g/L, glycerine 5-20g/L, (NH 4) 2sO 43-10g/L, KH 2pO 40.5-3g/L, K 2hPO 43H 2o 1.0-5g/L, MgSO 47H 2o 0.1-1.0g/L, solvent is water, and pH value is 6.0-7.0; Described fermention medium composition is preferably: peptone 15g/L, yeast powder 12g/L, NaCl 10g/L, glycerine 12g/L, (NH 4) 2sO 45g/L, KH 2pO 41.36g/L, K 2hPO 43H 2o 2.28g/L, MgSO 47H 2o 0.375g/L, solvent is water, and pH value is 6.5;
Described supplemented medium consists of: peptone 50-120g/L, and yeast extracts 30-80g/L, glycerine 300-900g/L, citric acid 1-8g/L, ammonium sulfate 1-10g/L, K 2hPO 41-8g/L, NaCl 1-10g/L, MgSO 41-10g/L, solvent is water, and pH value is 6.0-7.0.Described supplemented medium composition is preferably: peptone 75g/L, yeast extracts 50g/L, glycerine 500g/L, citric acid 3g/L, ammonium sulfate 5g/L, K 2hPO 43g/L, NaCl 5g/L, MgSO 45g/L, solvent is water, and pH value is 6.5.
Further, described seed liquor is prepared as follows: the engineering bacteria containing nitrilase gene is seeded to LB solid medium, cultivates 1-2 days at 37 DEG C, obtains inclined-plane thalline; Picking inclined-plane thalline is seeded in LB liquid nutrient medium, 37 DEG C, cultivate 8 ~ 12h under 150rpm, obtains seed liquor.
Further, the described engineering bacteria containing nitrilase gene is for template with Acidovorax facilis (Acidovorax facilis) ZJB09122 genomic dna, with sequence 1 and sequence 2 for primer, the expression plasmid containing restructuring nitrilase is obtained by PCR method, and then with the expression plasmid containing restructuring nitrilase for template, with sequence 3 and sequence 4 for primer, build the engineering bacteria containing restructuring nitrilase gene by PCR method;
Sequence 1:5 '-AAT gGATCCaTGGTTTCGTATAACAGCAAG-3 ';
Sequence 2:5 '-AGG gTCGACcTACTTTGCTGGGACCGG-3 ';
Sequence 3:5 '-GAGCAC gTTcAGCCGCTGTCCAAAT-3 ';
Sequence 4:5 '-CGGCTG aACgTGCTCCCAGCAGTTC-3 '.
In addition, engineering bacteria containing nitrilase gene of the present invention can also with Bacillus foecalis alkaligenes (Alcaligenes faecalis) ZJUTB10 genomic dna for template, with sequence 5 and sequence 6 for primer, the expression plasmid containing restructuring nitrilase is obtained by PCR method, and then with the expression plasmid containing restructuring nitrilase for template, with sequence 7 and sequence 8 for primer, build the engineering bacteria containing restructuring nitrilase gene by PCR method;
Sequence 5:5 '-AAT gGATCCaTGCAGACAAGAAAAATCGTC-3 ';
Sequence 6:5 '-AGG gTCGACtCAGGACGGTTCTTGCAC-3 ';
Sequence 7:5 '-CATGGAAGAGATC nNNtTCGCTAAGGCTATC-3 ';
Sequence 8:5 '-GATAGCCTTAGCGAA nNNgATCTCTTCCATG-3 '.
Further, in described fermenting process, stream adds volumetric concentration 8% ammoniacal liquor and volumetric concentration 10% phosphate aqueous solution to maintain fermented liquid pH value is 6 ~ 7.
Beneficial effect of the present invention is: the fermentation process in high density that the invention provides a kind of nitrilase engineering bacteria, the method adopts brand-new recombination bacillus coli fermentation and feed-batch culture based formulas, one-step inducing is divided by adopting DO-STAT coupling, and progressively put forward high-revolving method stage by stage, both met in culturing process along with biomass increases the needs to power of agitator, effectively control again the growth velocity of thalline, prevent the too fast product acid accumulation caused of thalli growth, feedback inhibition, the drawbacks such as substratum waste.In addition, by adopting batch induction mode adding lactose, both avoided the too high caused fermentation broth viscosity of lactose concn to increase, cause and pass oxygen, mass transfer is obstructed problem, turn improve induction intensity, utilize high density fermentation technology of the present invention, the biomass of recombination bacillus coli is increased to 70g DCW/L by 10.35g DCW/L, the work of volume enzyme is increased to 103530U/L from 18205U/L, makes fermentation level improve 4.7 times.
(4) accompanying drawing explanation
Course of fermentation curve when Fig. 1 is permanent dissolved oxygen 5%.
Course of fermentation curve when Fig. 2 is permanent dissolved oxygen 15%.
Fig. 3 is permanent dissolved oxygen 15% and the course of fermentation curve stirred stage by stage.
Fig. 4 is that initial induced concentration optimizes comparison diagram.
Fig. 5 is induction final concentration optimization comparison diagram.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
LB liquid nutrient medium forms: peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, and solvent is water, pH nature.
LB solid medium forms: peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, agar 1.5-2.0%, and solvent is water, pH nature.
Embodiment 1: build the engineering bacteria containing nitrilase gene
The present invention adopts Rapid nucleic acid extraction apparatus and microbial genome to extract test kit (MP company of the U.S.) and extracts Acidovorax facilis (Acidovorax facilis) ZJB09122 genomic dna.This culture presevation is in China typical culture collection center, and deposit number is CCTCC No.M 209044, discloses in the patent CN101629192B of previously application.
According to Acidovorax facilis nitrilase gene and the homology analysis of report on NCBI, devise a pair cloning primer in order to the AcN gene that increases:
Upstream primer (primer 1):
AcN(F)5’-AAT GGATCCATGGTTTCGTATAACAGCAAG-3’
Downstream primer (primer 2):
AcN(R)5’-AGG GTCGACCTACTTTGCTGGGACCGG-3’
For the ease of the structure of prokaryotic expression carrier, in 5 ' end primer and 3 ' end primer, introduce Nco I, Xho I restriction enzyme sites (underscore part) respectively.
Be that template carries out polymerase chain reaction (PCR) with Acidovorax facilis (Acidovorax facilis) ZJB09122 genomic dna.PCR system is: template (~ 10ng/ μ L) 1 μ L, upstream primer (25 μMs) 1 μ L, downstream primer (25 μMs) 1 μ L, dNTPs (2.5mM) 1 μ L, 10 × Buffer for pfu 5 μ L, pfu archaeal dna polymerase (5U/ μ L) 1 μ L, ddH 2o 40 μ L.PCR reaction process is: (1) 95 DEG C of denaturation 5min, (2) 94 DEG C of sex change 45s, (3) 55 DEG C of annealing 1.5min, (4) 72 DEG C extend 3min, 35 circulations are repeated in step (2)-(4), (5) 72 DEG C are continued to extend 10min, are cooled to 4 DEG C.Glue reclaims PCR primer.
PCR primer after recovery and pET28b (+) carry out double digestion with Nco I and Xho I respectively, after about enzyme cuts 6h, reclaim digestion products, utilize T4 ligase enzyme to connect 16h at 16 DEG C, obtain recombinant expression plasmid pET28b (+)-AcN.Expression vector pET28b (+)-AcN is converted into E.coli BL21 (DE3) recipient bacterium, coat on the LB agar plate containing kantlex (final concentration 50mg/L), after 37 DEG C of overnight incubation, flat board grows bacterium colony (yellow-white circular colonies).Random picking mono-clonal, extract plasmid after cultivating and check order, result shows to obtain original positive colony E.coli BL21 (DE3)/pET28b (+)-NIT.
According to Quikchange directed mutagenesis method, devise pair of primers (underscore part is mutational site):
Primer 3-upstream primer:
F168V(F)5’-GAGCAC GTTCAGCCGCTGTCCAAAT-3’
Primer 4-downstream primer:
F168V(R)5’-CGGCTG AACGTGCTCCCAGCAGTTC-3’
With pET28b-AcN plasmid for template, (PCR reaction parameter is: 94 DEG C of denaturation 4min to carry out pcr amplification; 98 DEG C of sex change 10s, 55 DEG C of annealing 15s, 72 DEG C extend 6min, repeat 30 circulations; 72 DEG C are continued to extend 10min).The PCR primer DpnI enzyme reclaimed after amplification is cut 3 hours, E.coli BL21 (DE3) recipient bacterium is converted into after digestion products purifying, coat on the LB agar plate containing kantlex (final concentration 50mg/L), after 37 DEG C of overnight incubation, random picking colony extracts plasmid and checks order, show to obtain positive colony E.coli BL21 (DE3)/pET28b (+)-AcNIT, namely obtain the engineering bacteria containing nitrilase.
Embodiment 2: build bacterial strain E.coli BL21 (DE3)/pET28b (+)-AfNIT
The present invention adopts Rapid nucleic acid extraction apparatus and microbial genome to extract test kit (MP company of the U.S.) and extracts Bacillus foecalis alkaligenes (Alcaligenes faecalis) ZJUTB10 genomic dna.This culture presevation is in China typical culture collection center, and deposit number is CCTCC No.M 208168, preservation date on October 22nd, 2008, discloses in the patent CN101392276B of previously application.
According to nitrilase gene and the homology analysis of report on NCBI, devise a pair cloning primer in order to the AfN gene that increases:
Upstream primer (primer 5):
AfN(F)5’-AAT GGATCCATGCAGACAAGAAAAATCGTC-3’
Downstream primer (primer 6):
AfN(R)5’-AGG GTCGACTCAGGACGGTTCTTGCAC-3’
For the ease of the structure of prokaryotic expression carrier, in 5 ' end primer and 3 ' end primer, introduce Nco I, Xho I restriction enzyme sites (underscore part) respectively.
Be that template carries out polymerase chain reaction (PCR) with Bacillus foecalis alkaligenes (Alcaligenes faecalis) ZJUTB10 genomic dna.PCR system is: template 20ng, upstream primer 0.5 μM, downstream primer 0.5 μM, dNTPs50 μM, 10 × Buffer for pfu, pfu archaeal dna polymerase 2U.PCR reaction process is: (1) 94 DEG C of denaturation 5min, (2) 94 DEG C of sex change 45s, (3) 55 DEG C of annealing 45s, (4) 72 DEG C extend 2min, 35 circulations are repeated in step (2)-(4), (5) 72 DEG C are continued to extend 10min, are cooled to 4 DEG C.Glue reclaims PCR primer.
PCR primer after recovery and pET28b (+) carry out double digestion with Nco I and Xho I respectively, after about enzyme cuts 6h, reclaim digestion products, utilize T4 ligase enzyme to connect 16h at 16 DEG C, obtain recombinant expression plasmid pET28b (+)-AfN.Expression vector pET28b (+)-AfN is converted into E.coli BL21 (DE3) recipient bacterium, coat on the LB agar plate containing kantlex (final concentration 50mg/L), after 37 DEG C of overnight incubation, flat board grows bacterium colony (yellow-white circular colonies).Random picking mono-clonal, extracts plasmid and checks order, show to obtain original positive colony E.coli BL21 (DE3)/pET28b (+)-NIT1 after cultivating.
According to Quikchange fixed point saturation mutation method, devise pair of primers (underscore part is mutational site): upstream primer (primer 7):
Q196(F)5’-CATGGAAGAGATC NNNTTCGCTAAGGCTATC-3’
Downstream primer (primer 8):
Q196(R)5’-GATAGCCTTAGCGAA NNNGATCTCTTCCATG-3’
With pET28b (+)-AfN plasmid for template, (PCR reaction parameter is: 94 DEG C of denaturation 4min in performing PCR amplification; 98 DEG C of sex change 10s, 55 DEG C of annealing 15s, 72 DEG C extend 8min, repeat 30 circulations; 72 DEG C are continued to extend 10min).The PCR primer DpnI enzyme reclaimed after amplification is cut 3 hours, E.coli BL21 (DE3) recipient bacterium is converted into after digestion products purifying, coat on the LB agar plate containing kantlex (final concentration 50mg/L), after 37 DEG C of incubated overnight, random picking colony carries out in access LB substratum, in 37 DEG C, incubated overnight under 150rpm, obtain seed liquor, after with in the inoculum size of volumetric concentration 2% access LB substratum, in 37 DEG C, under 150rpm after 4h, add lactose (final concentration 5g/L) induction, in 28 DEG C, after 150rpm bottom fermentation cultivates 8h, collect fermented liquid, in 4 DEG C, centrifugal 5min under 9000rpm, obtain thalline.Be that substrate carries out catalyzed reaction with mandelonitrile, compare its than enzyme alive and catalytic selectivity, mutant strain E.coli BL21 (DE3)/pET28b (+)-AfNIT that the strain vigor that finally screens is higher.Through order-checking, Bacillus foecalis alkaligenes (Alcaligenes faecalis) ZJUTB10 nitrilase expressed by this mutant strain, its 196 is Serine S (nucleotide sequence becomes TCA from CAG) (before sudden change, the gene pool call number of gene order is HQ407378) from glutamine Q-spoiling, obtains the engineering bacteria containing nitrilase.
Comparative example 1: the application of batch fermentation culture technique in fermentation culture nitrilase gene engineering bacteria
1, experimentation:
(1) seed liquor preparation: E.coliBL21 (DE3)/pET28b (+)-AcNIT slant strains containing nitrilase gene built in Example 1, inoculate a ring in being equipped with in the 500mL shaking flask of 100mL containing the LB liquid nutrient medium of final concentration 5g/L kantlex, in 37 DEG C, incubated overnight under 150rpm, obtains seed liquor.
(2) fermentor tank batch culture: seed liquor step (1) obtained is with the inoculum size of 3% (v/v), be inoculated in and 3L be housed containing in the 5L fermentor tank of final concentration 50g/L kantlex fermention medium, in 37 DEG C, 500rpm, ventilation ratio 1.3vvm condition bottom fermentation is cultivated, and the phosphate aqueous solution of ammoniacal liquor and volumetric concentration 10% that fermenting process stream adds volumetric concentration 8% regulates pH to maintain 6.5.OD is surveyed every 2h sampling 600and the content of glycerine and acetic acid.Fermention medium consists of: peptone 15g/L, yeast powder 12g/L, NaCl 10g/L, glycerine 12g/L, (NH 4) 2sO 45g/L, KH 2pO 41.36g/L), K 2hPO 43H 2o 2.28g/L, MgSO 47H 2o 0.375g/L, solvent is water, and pH value is 6.5.
(3) lactose-induced: batch fermentation 4h, temperature regulating to 30 DEG C, adds lactose (final concentration 12.5g/L) induction., OD alive every 2h sampling and measuring enzyme 600, acetic acid content and glycerol content.
(4) stop tank and collect thalline: after induction, continue fermentation to OD 600value and than enzyme work in 3h not rising or when starting to decline, stop fermentation, put tank, obtain fermented liquid.In 4 DEG C, under 9000rpm, centrifugal 8min, abandons supernatant liquor, collects wet thallus.
2, experimental result:
Experimental result shows, and adopt the technology of batch fermentation, biomass reaches 10.35g DCW/L, and living than enzyme reaches 1759U/g DCW, and volume enzyme is lived and reached 18205U/L.This fermentation level efficiency is lower, therefore, it may be necessary high-density culture technology and improves fermentation level further.
Comparative example 2: the application of NEW TYPE OF COMPOSITE supplemented medium in nitrilase gene engineering bacteria high density fermentation
1, experimentation:
(1) seed liquor preparation: E.coliBL21 (DE3)/pET28b (+)-AcNIT slant strains containing nitrilase gene built in Example 1, inoculate a ring in being equipped with in the 500mL shaking flask of 100mL containing the LB liquid nutrient medium of final concentration 5g/L kantlex, in 37 DEG C, incubated overnight under 150rpm, obtains seed liquor.
(2) fermentor tank batch culture: seed liquor step (1) obtained is with the inoculum size of 3% (v/v), be inoculated in and 3L be housed containing in the 5L fermentor tank of final concentration 50g/L kantlex fermention medium, in 37 DEG C, 500rpm, ventilation ratio 1.3vvm condition bottom fermentation is cultivated, and the phosphate aqueous solution of ammoniacal liquor and volumetric concentration 10% that fermenting process stream adds volumetric concentration 8% regulates pH to maintain 6.5.OD is surveyed every 2h sampling 600and the content of glycerine and acetic acid.Fermention medium consists of: peptone 15g/L, yeast powder 12g/L, NaCl 10g/L, glycerine 12g/L, (NH 4) 2sO 45g/L, KH 2pO 41.36g/L, K 2hPO 43H 2o 2.28g/L, MgSO 47H 2o 0.375g/L, solvent is water, and pH value is 6.5.
(3) after batch fermentation 7-8h, when oxygen dissolving value rises to rapidly 15% ± 5%, supplemented medium is added with the speed constant speed of 20mL/h, supplemented medium consists of: peptone 75g/L, and yeast extracts 50g/L, glycerine 500g/L, citric acid 3g/L, ammonium sulfate 5g/L, K 2hPO 43g/L, NaCl 5g/L, MgSO 45g/L, solvent is water, and pH value is 6.5.Improve rotating speed to 700rpm, in fermenting process, stream adds 8% (v/v) ammoniacal liquor and 10% (v/v) phosphate aqueous solution and adjusts pH to maintain 6.5.OD is surveyed every 2h sampling 600and the content of glycerine and acetic acid.To add volumetric concentration 50% aqueous glycerin solution for contrast under similarity condition.
(4) lactose-induced: to treat OD 600when value reaches 30, temperature regulating maintains 30 DEG C, adds lactose (final concentration 34g/L) induction., OD alive every 2h sampling and measuring enzyme 600, acetic acid content and glycerol content.
(5) stop tank and collect thalline: after induction, continue fermentation to OD 600value and than enzyme work in 3h not rising or when starting to decline, stop fermentation, put tank, obtain fermented liquid.In 4 DEG C, under 9000rpm, centrifugal 8min, abandons supernatant liquor, collects wet thallus.
2, experimental result:
Experimental result shows, and the biomass adopting NEW TYPE OF COMPOSITE supplemented medium is 37.43g DCW/L, and add merely the method for volumetric concentration 50% aqueous glycerin solution than traditional employing, its biomass improves 58.80%.Volume enzyme is lived and is reached 39226U/L, adds the method for simple 50% aqueous glycerin solution than traditional employing, and volume enzyme is lived and improve 41.52%.
Comparative example 3: oxygen dissolving value fluctuation area is the application of DO-STAT fed-batch fermentation technology in nitrilase gene engineering bacteria high density fermentation under 5% ± 2.5%
1, experimentation:
(1) seed liquor preparation: with comparative example 2.
(2) fermentor tank batch culture: with comparative example 2.
(3) DO-STAT feed-batch culture: after batch fermentation 7-8h, when oxygen dissolving value rises to rapidly suddenly 5% ± 2.5%, starts to adopt DO-STAT mode to add supplemented medium, controls oxygen dissolving value 5% ± 2.5%.Improve rotating speed to 700rpm.In fermenting process, stream adds 8% (v/v) ammoniacal liquor and 10% (v/v) phosphate aqueous solution and adjusts pH to maintain 6.5.OD is surveyed every 2h sampling 600and the content of glycerine and acetic acid.Described supplemented medium consists of: peptone 75g/L, and yeast extracts 50g/L, glycerine 500g/L, citric acid 3g/L, ammonium sulfate 5g/L, K 2hPO 43g/L, NaCl 5g/L, MgSO 45g/L, solvent is water, and pH value is 6.5.
(4) lactose-induced: to treat OD 600when value reaches 30, temperature regulating maintains 30 DEG C, adds lactose (final concentration 34g/L) induction., OD alive every 2h sampling and measuring enzyme 600, acetic acid content and glycerol content.
(5) stop tank and collect thalline: after chain induction, continue fermentation to OD 600value and than enzyme work in 3h not rising or when starting to decline, stop fermentation, put tank, obtain fermented liquid.
In 4 DEG C, under 9000rpm, centrifugal 8min, abandons supernatant liquor, collects wet thallus.
2, experimental result:
Adopt DO-STAT fed-batch fermentation, oxygen dissolving value fluctuation area is set in 5% ± 2.5%.Course of fermentation curve is shown in Fig. 1, glycerol concentration fluctuation change, and acetic acid content accumulates continuously and healthily, be up to 20g/L, far beyond an inhibiting minimum concentration (5g/L), biomass is rapidly increased to 38.32g DCW/L, but i.e. basic stagnation after 24h.This may be due to oxygen dissolving value maintain lower level time, Metabolism of E. coli activity is very active, and specific growth rate is higher (is up to 0.6h -1).But cause acetic acid while thalline grows fast to accumulate in a large number, had a strong impact on colibacillary growth, shortened the time of thalline exponential growth.
Comparative example 4: oxygen dissolving value fluctuation area is the application of DO-STAT fed-batch fermentation technology in nitrilase gene engineering bacteria high density fermentation under 15% ± 5%
1, experimentation:
(1) seed liquor preparation: with comparative example 2.
(2) fermentor tank batch culture: with comparative example 2.
(3) SO-STAT feed-batch culture: after batch fermentation 7-8h, when oxygen dissolving value rises to rapidly suddenly 15% ± 5%, starts to adopt DO-STAT mode to add supplemented medium and controls oxygen dissolving value 15% ± 5%.Improve rotating speed to 700rpm.Described supplemented medium consists of: peptone 75g/L, and yeast extracts 50g/L, glycerine 500g/L, citric acid 3g/L, ammonium sulfate 5g/L, K 2hPO 43g/L, NaCl 5g/L, MgSO 45g/L, solvent is water, and pH value is 6.5.In fermenting process, stream adds 8% (v/v) ammoniacal liquor and 10% (v/v) phosphoric acid and adjusts pH to maintain 6.5.OD is surveyed every 2h sampling 600and the content of glycerine and acetic acid.
(4) lactose-induced: with comparative example 3.
(5) stop tank and collect thalline: with comparative example 3.
2 experimental results:
Adopt DO-STAT fed-batch fermentation, oxygen dissolving value fluctuation area is set in 15% ± 5%.Course of fermentation curve is shown in Fig. 2.Compared with testing with comparative example 3, this technique thalli growth in early stage is comparatively slow, and specific growth rate is lower, and (when fermenting to 15h, comparative example 3 biomass is 26.54g.Comparative example 4 biomass is 20.31g DCW/L), but the Acetic Acid Accumulation of feed phase greatly reduces (maximum concentration is 5g/L).The minimizing of Acetic Acid Accumulation amount makes extend 10h the exponential phase of growth of thalline, and final biomass reaches 54.31g DCW/L, and volume enzyme is lived and reached 63468U/L.In comparative example 3, control dissolved oxygen fluctuation area is 5% ± 2.5%, and biomass improves 39.12%, and volume enzyme is lived and improve 49.34%.
Comparative example 5:DO-STAT feed supplement, and progressively propose the application of high-revolving high density fermentation technology in nitrilase gene engineering bacteria high density fermentation
1 experimentation:
(1) seed liquor preparation: with comparative example 2.
(2) fermentor tank batch culture: with comparative example 2.
(3) SO-STAT feed-batch culture: after batch fermentation 7-8h, when oxygen dissolving value rises to rapidly suddenly 15% ± 5%, start to adopt DO-STAT mode to add supplemented medium and maintain fermented liquid oxygen dissolving value 15% ± 5%, adjustment mixing speed is 700rpm.Supplemented medium consists of: peptone 75g/L, and yeast extracts 50g/L, glycerine 500g/L, citric acid 3g/L, ammonium sulfate 5g/L, K 2hPO 43g/L, NaCl 5g/L, MgSO 45g/L, solvent is water, and pH value is 6.5.Treat that fermentation is to OD 600reach 90, rear adjustment rotating speed is to 750rpm.Treat that fermentation is to OD 600reach 125, rear adjustment rotating speed is to 800rpm; Continue fermentation to OD 600reach 170, rear adjustment rotating speed is to 1000rpm.Maintaining fermentation rotating speed is under 1000rpm, temperature 30 DEG C of conditions, continues fermentation.In fermenting process, stream adds 8% (v/v) ammoniacal liquor and 10% (v/v) phosphate aqueous solution and adjusts pH to maintain 6.5.OD is surveyed every 2h sampling 600and the content of glycerine and acetic acid.
(4) lactose-induced: with comparative example 3.
(5) stop tank and collect thalline: with comparative example 3.
2 experimental results:
For regulating the specific growth rate of thalline further in threshold range, controlling the growing amount of acetic acid, under the prerequisite controlling permanent dissolved oxygen 15% ± 5%, taking the method that sublevel segmentation stirs.The method slow speed of revolution in early stage, low power of agitator, prevents the too fast growth of thalline and produces acid, and along with the increase of biomass, the power of agitator that thalline continued growth needs constantly increases, and progressively improves rotating speed, increases power of agitator.The results are shown in Figure 3, the growing amount utilizing this technology to reach acetic acid controls below inhibition concentration (lower than 2g/L), thus the exponential phase of growth of thalline is extended 4h again, and biomass reaches 69.35g DCW/L, and volume enzyme is lived and reached 79380U/L.DO-STAT fed-batch fermentation technology simple in comparative example 4, biomass improves 27.69%, and volume enzyme is lived and improve 25.07%.
Embodiment 3:DO-STAT feed supplement coupling divides one-step inducing, and progressively proposes the application () of high-revolving high density fermentation technology in nitrilase gene engineering bacteria high density fermentation
1 experimentation:
(1) seed liquor preparation: E.coli BL21 (DE3)/pET28b (+)-AcNIT slant strains containing nitrilase gene built in Example 1, inoculate a ring in being equipped with in the 500mL shaking flask of 100mL containing the LB liquid nutrient medium of final concentration 5g/L kantlex, in 37 DEG C, incubated overnight under 150rpm, obtains seed liquor.
(2) fermentor tank batch culture: seed liquor step (1) obtained is with the inoculum size of 3% (v/v), be inoculated in and 3L be housed containing in the 5L fermentor tank of final concentration 50g/L kantlex fermention medium, in 37 DEG C, 500rpm, ventilation ratio 1.3vvm condition bottom fermentation is cultivated, and the phosphate aqueous solution of ammoniacal liquor and volumetric concentration 10% that fermenting process stream adds volumetric concentration 8% regulates pH to maintain 6.5.OD is surveyed every 2h sampling 600and the content of glycerine and acetic acid.Fermention medium consists of: peptone 15g/L, yeast powder 12g/L, NaCl 10g/L, glycerine 12g/L, (NH 4) 2sO 45g/L, KH 2pO 41.36g/L, K 2hPO 43H 2o 2.28g/L, MgSO 47H 2o 0.375g/L, solvent is water, and pH value is 6.5.
(3) SO-STAT feed-batch culture: after batch fermentation 7-8h, when oxygen dissolving value rises to rapidly suddenly 15% ± 5%, start to adopt DO-STAT mode to add supplemented medium and maintain fermented liquid oxygen dissolving value 15% ± 5%, adjustment mixing speed is 700rpm.Supplemented medium consists of: peptone 75g/L, and yeast extracts 50g/L, glycerine 500g/L, citric acid 3g/L, ammonium sulfate 5g/L, K 2hPO 43g/L, NaCl 5g/L, MgSO 45g/L, solvent is water, and pH value is 6.5.Treat that fermentation is to OD 600reach 90, rear adjustment rotating speed is to 750rpm.Treat that fermentation is to OD 600reach 125, rear adjustment rotating speed is to 800rpm; Continue fermentation to OD 600reach 170, rear adjustment rotating speed is to 1000rpm.Maintaining fermentation rotating speed is under 1000rpm, temperature 30 DEG C of conditions, continues fermentation.In fermenting process, stream adds 8% (v/v) ammoniacal liquor and 10% (v/v) phosphate aqueous solution and adjusts pH to maintain 6.5.OD is surveyed every 2h sampling 600and the content of glycerine and acetic acid.
(4) lactose-induced: to adopt the mode in batches added, that is: OD 600when reaching 30, it is lactose-induced first to add 20g/L, adjusts the temperature to 30 DEG C.Treat the logarithmic growth later stage, OD 600when reaching 120, again add 14g/L lactose, chain induction., OD alive every 2h sampling and measuring enzyme 600, acetic acid content and glycerol content.
(5) stop tank and collect thalline: after induction, continue fermentation to OD 600value and than enzyme work in 3h not rising or when starting to decline, stop fermentation, put tank, obtain fermented liquid.In 4 DEG C, under 9000rpm, centrifugal 8min, abandons supernatant liquor, collects wet thallus.
2 experimental results:
Experimental result display adopts the mode of in batches inducing, and biomass reaches 70g DCW/L, and volume enzyme is lived and reached 103530U/L.Than the once induction in comparative example 5, volume enzyme is lived and is improve 30.42%.Meanwhile, this high density fermentation technology, compared with the traditional batch culture technology used in comparative example 1, biomass is increased to 70g DCW/L by 10.35g DCW/L.The work of volume enzyme is increased to 103530U/L from 18205U/L.Fermentation level is made to improve 4.7 times.
Embodiment 4:DO-STAT feed supplement coupling divides one-step inducing, and progressively proposes the application (two) of high-revolving high density fermentation technology in nitrilase gene engineering bacteria high density fermentation
1 experimentation:
(1) seed liquor preparation: with embodiment 3;
(2) fermentor tank batch culture: seed liquor step (1) obtained is with the inoculum size of 3% (v/v), be inoculated in and 3L be housed containing in the 5L fermentor tank of final concentration 50g/L kantlex fermention medium, in 37 DEG C, 500rpm, ventilation ratio 1.3vvm condition bottom fermentation is cultivated, and the phosphate aqueous solution of ammoniacal liquor and volumetric concentration 10% that fermenting process stream adds volumetric concentration 8% regulates pH to maintain 6.5.OD is surveyed every 2h sampling 600and the content of glycerine and acetic acid.Fermention medium consists of: peptone 10g/L, yeast powder 5g/L, NaCl 5g/L, glycerine 5g/L, (NH 4) 2sO 43g/L, KH 2pO 40.5g/L, K 2hPO 43H 2o 1.0g/L, MgSO 47H 2o 0.1g/L, solvent is water, and pH value is 6.0;
(3) SO-STAT feed-batch culture: with embodiment 3;
(4) lactose-induced: with embodiment 3;
(5) stop tank and collect thalline: with embodiment 3;
2 experimental results:
Experimental result display adopts this DO-STAT feed supplement coupling to divide one-step inducing, and progressively puies forward high-revolving high density fermentation technology, and biomass reaches 65.48g DCW/L, and volume enzyme is lived and reached 87209U/L.
Embodiment 5:DO-STAT feed supplement coupling divides one-step inducing, and progressively proposes the application (three) of high-revolving high density fermentation technology in nitrilase gene engineering bacteria high density fermentation
1 experimentation:
(1) seed liquor preparation: with embodiment 3;
(2) fermentor tank batch culture: seed liquor step (1) obtained is with the inoculum size of 3% (v/v), be inoculated in and 3L be housed containing in the 5L fermentor tank of final concentration 50g/L kantlex fermention medium, in 37 DEG C, 500rpm, ventilation ratio 1.3vvm condition bottom fermentation is cultivated, and the phosphate aqueous solution of ammoniacal liquor and volumetric concentration 10% that fermenting process stream adds volumetric concentration 8% regulates pH to maintain 6.5.OD is surveyed every 2h sampling 600and the content of glycerine and acetic acid.Fermention medium consists of: peptone 20g/L, yeast powder 15g/L, NaCl 15g/L, glycerine 20g/L, (NH 4) 2sO 410g/L, KH 2pO 43g/L, K 2hPO 43H 2o 5g/L, MgSO 47H 2o 1.0g/L, solvent is water, and pH value is 7.0;
(3) SO-STAT feed-batch culture: with embodiment 3;
(4) lactose-induced: with embodiment 3;
(5) stop tank and collect thalline: with embodiment 3;
2 experimental results:
Experimental result display adopts this DO-STAT feed supplement coupling to divide one-step inducing, and progressively puies forward high-revolving high density fermentation technology, and biomass reaches 67.21g DCW/L, and volume enzyme is lived and reached 89558U/L.
Embodiment 6:DO-STAT feed supplement coupling divides one-step inducing, and progressively proposes the application (four) of high-revolving high density fermentation technology in nitrilase gene engineering bacteria high density fermentation
1 experimentation:
(1) seed liquor preparation: with embodiment 3;
(2) fermentor tank batch culture: with embodiment 3;
(3) SO-STAT feed-batch culture: after batch fermentation 7-8h, when oxygen dissolving value rises to rapidly suddenly 15% ± 5%, start to adopt DO-STAT mode to add supplemented medium and maintain fermented liquid oxygen dissolving value 15% ± 5%, adjustment mixing speed is 700rpm.Supplemented medium consists of: peptone 50g/L, and yeast extracts 30g/L, glycerine 300g/L, citric acid 1g/L, ammonium sulfate 1g/L, K 2hPO 41g/L, NaCl 1g/L, MgSO 41g/L, solvent is water, and pH value is 6.0.Treat that fermentation is to OD 600reach 90, rear adjustment rotating speed is to 750rpm.Treat that fermentation is to OD 600reach 125, rear adjustment rotating speed is to 800rpm; Continue fermentation to OD 600reach 170, rear adjustment rotating speed is to 1000rpm.Maintaining fermentation rotating speed is under 1000rpm, temperature 30 DEG C of conditions, continues fermentation.In fermenting process, stream adds 8% (v/v) ammoniacal liquor and 10% (v/v) phosphate aqueous solution and adjusts pH to maintain 6.0.OD is surveyed every 2h sampling 600and the content of glycerine and acetic acid.
(4) lactose-induced: with embodiment 3;
(5) stop tank and collect thalline: with embodiment 3
2 experimental results:
Experimental result display adopts this NEW TYPE OF COMPOSITE supplemented medium and adopts DO-STAT feed supplement coupling to divide one-step inducing, and progressively puies forward high-revolving high density fermentation technology, and biomass reaches 60.98g DCW/L, and volume enzyme is lived and reached 67626U/L.
Embodiment 7:DO-STAT feed supplement coupling divides one-step inducing, and progressively proposes the application (five) of high-revolving high density fermentation technology in nitrilase gene engineering bacteria high density fermentation
1 experimentation:
(1) seed liquor preparation: with embodiment 3;
(2) fermentor tank batch culture: with embodiment 3;
(3) SO-STAT feed-batch culture: after batch fermentation 7-8h, when oxygen dissolving value rises to rapidly suddenly 15% ± 5%, start to adopt DO-STAT mode to add supplemented medium and maintain fermented liquid oxygen dissolving value 15% ± 5%, adjustment mixing speed is 700rpm.Supplemented medium consists of: peptone 120g/L, and yeast extracts 80g/L, glycerine 900g/L, citric acid 8g/L, ammonium sulfate 10g/L, K 2hPO 48g/L, NaCl 10g/L, MgSO 410g/L, solvent is water, and pH value is 7.0.Treat that fermentation is to OD 600reach 90, rear adjustment rotating speed is to 750rpm.Treat that fermentation is to OD 600reach 125, rear adjustment rotating speed is to 800rpm; Continue fermentation to OD 600reach 170, rear adjustment rotating speed is to 1000rpm.Maintaining fermentation rotating speed is under 1000rpm, temperature 30 DEG C of conditions, continues fermentation.In fermenting process, stream adds 8% (v/v) ammoniacal liquor and 10% (v/v) phosphate aqueous solution and adjusts pH to maintain 7.0.OD is surveyed every 2h sampling 600and the content of glycerine and acetic acid.
(4) lactose-induced: with embodiment 3;
(5) stop tank and collect thalline: with embodiment 3
2 experimental results:
Experimental result display adopts this NEW TYPE OF COMPOSITE supplemented medium and adopts DO-STAT feed supplement coupling to divide one-step inducing, and progressively puies forward high-revolving high density fermentation technology, and biomass reaches 66.45g DCW/L, and volume enzyme is lived and reached 70699U/L.
Embodiment 8:DO-STAT feed supplement coupling divides one-step inducing, and progressively proposes the application (six) of high-revolving high density fermentation technology in nitrilase gene engineering bacteria high density fermentation
1 experimentation:
(1) seed liquor preparation: with embodiment 3;
(2) fermentor tank batch culture: with embodiment 3;
(3) SO-STAT feed-batch culture: after batch fermentation 7-8h, when oxygen dissolving value rises to rapidly suddenly 15% ± 5%, start to adopt DO-STAT mode to add supplemented medium and maintain fermented liquid oxygen dissolving value 15% ± 5%, adjustment mixing speed is 600rpm.Supplemented medium consists of: peptone 75g/L, and yeast extracts 50g/L, glycerine 500g/L, citric acid 3g/L, ammonium sulfate 5g/L, K 2hPO 43g/L, NaCl 5g/L, MgSO 45g/L, solvent is water, and pH value is 6.5.Treat that fermentation is to OD 600reach 70, rear adjustment rotating speed is to 720rpm.Treat that fermentation is to OD 600reach 115, rear adjustment rotating speed is to 780rpm; Continue fermentation to OD 600reach 150, rear adjustment rotating speed is to 900rpm.Maintaining fermentation rotating speed is under 900rpm, temperature 30 DEG C of conditions, continues fermentation.In fermenting process, stream adds 8% (v/v) ammoniacal liquor and 10% (v/v) phosphate aqueous solution and adjusts pH to maintain 6.5.OD is surveyed every 2h sampling 600and the content of glycerine and acetic acid.
(4) lactose-induced: to adopt the mode in batches added, that is: OD 600when reaching 20, it is lactose-induced first to add 10g/L, adjusts the temperature to 25 DEG C.Treat the logarithmic growth later stage, OD 600when reaching 110, again add 10g/L lactose, chain induction., OD alive every 2h sampling and measuring enzyme 600, acetic acid content and glycerol content.
(5) stop tank and collect thalline: with embodiment 3.
2 experimental results:
Experimental result display adopts this NEW TYPE OF COMPOSITE supplemented medium and adopts DO-STAT feed supplement coupling to divide one-step inducing, and progressively puies forward high-revolving high density fermentation technology, and biomass reaches 67.50g DCW/L, and volume enzyme is lived and reached 76596U/L.
Embodiment 9:DO-STAT feed supplement coupling divides one-step inducing, and progressively proposes the application (seven) of high-revolving high density fermentation technology in nitrilase gene engineering bacteria high density fermentation
1 experimentation:
(1) seed liquor preparation: with embodiment 3;
(2) fermentor tank batch culture: with embodiment 3;
(3) SO-STAT feed-batch culture: after batch fermentation 7-8h, when oxygen dissolving value rises to rapidly suddenly 15% ± 5%, start to adopt DO-STAT mode to add supplemented medium and maintain fermented liquid oxygen dissolving value 15% ± 5%, adjustment mixing speed is 700rpm.Supplemented medium consists of: peptone 75g/L, and yeast extracts 50g/L, glycerine 500g/L, citric acid 3g/L, ammonium sulfate 5g/L, K 2hPO 43g/L, NaCl 5g/L, MgSO 45g/L, solvent is water, and pH value is 6.5.Treat that fermentation is to OD 600reach 110, rear adjustment rotating speed is to 780rpm.Treat that fermentation is to OD 600reach 140, rear adjustment rotating speed is to 850rpm; Continue fermentation to OD 600reach 190, rear adjustment rotating speed is to 1300rpm.Maintaining fermentation rotating speed is under 1300rpm, temperature 30 DEG C of conditions, continues fermentation.In fermenting process, stream adds 8% (v/v) ammoniacal liquor and 10% (v/v) phosphate aqueous solution and adjusts pH to maintain 6.5.OD is surveyed every 2h sampling 600and the content of glycerine and acetic acid.
(4) lactose-induced: to adopt the mode in batches added, that is: OD 600when reaching 40, it is lactose-induced first to add 25g/L, adjusts the temperature to 35 DEG C.Treat the logarithmic growth later stage, OD 600when reaching 130, again add 15g/L lactose, chain induction., OD alive every 2h sampling and measuring enzyme 600, acetic acid content and glycerol content.
(5) stop tank and collect thalline: with embodiment 3
2 experimental results:
Experimental result display adopts this NEW TYPE OF COMPOSITE supplemented medium and adopts DO-STAT feed supplement coupling to divide one-step inducing, and progressively puies forward high-revolving high density fermentation technology, and biomass reaches 42g DCW/L, and volume enzyme is lived and reached 52590U/L.
Embodiment 10: nitrilase is catalyzing and synthesizing gabapentin important intermediate---the application in 1-cyanocyclohexanoic guanidine-acetic acid
1 experimentation:
Get the wet thallus that in 50g embodiment 3, fermentation obtains, be suspended in 1L, 200mM, in the PBS damping fluid of pH 7.0, stir and be placed in 35 DEG C of thermostat water baths, arranging agitator speed is 200rpm, adds 148.2g substrate (1-cyanocyclohexanoic base acetonitrile) and starts reaction.
2 experimental results:
Experimental result shows, and reaction 8h, transformation efficiency reaches 92%, and selectivity reaches 99.9%.Further according to above-mentioned experimental technique, reaction system is amplified to 5L, reaction 9h, transformation efficiency reaches 90%, and selectivity reaches 99.9%.The prolongation reaction times can further improve transformation efficiency.As can be seen here, this nitrilase has high regioselective catalytic ability, is applicable to large-scale industrial production, solves the predicament in gabapentin building-up process, has great using value.
Embodiment 11:DO-STAT feed supplement coupling divides one-step inducing, and progressively proposes the application of high-revolving high density fermentation technology in the recombination bacillus coli of the nitrilase of high-density culture product highly-solid selectively hydrolysis mandelonitrile.
1 experimentation:
(1) seed liquor preparation: in Example 2 build containing nitrile hydrolyzable group because of E.coliBL21 (DE3)/pET28b (+)-AfNIT bacterium inclined-plane, inoculate a ring in 100mL being housed containing in the 500mL shaking flask of final concentration 5g/L kantlex LB liquid nutrient medium, in 37 DEG C, incubated overnight under 150rpm, obtains seed liquor.
(2) fermentor tank batch culture: by cultured seed liquor with the inoculum size of 3% (v/v), is inoculated in and is equipped with in the 5L fermentor tank of 3L containing the fermention medium of final concentration 50g/L kantlex.In 37 DEG C, 500rpm, ventilation ratio 1.3vvm condition bottom fermentation is cultivated.Stream adds the ammoniacal liquor of 8% and the phosphorus acid for adjusting pH of 10% maintains 7.0.OD is surveyed every 2h sampling 600and the content of glycerine and acetic acid.
(3) DO-STAT feed-batch culture: after batch fermentation 7-8h, when oxygen dissolving value rises to rapidly suddenly 15% ± 5%, start to adopt DO-STAT mode to add supplemented medium, control oxygen dissolving value 15% ± 5%, adjustment mixing speed is 700rpm, ferments to OD 600when reaching 90, adjustment rotating speed, to 750rpm, then ferments to OD 600when reaching 125, adjustment rotating speed, to 800rpm, continues fermentation to OD 600when reaching 170, adjustment rotating speed is to 1000rpm.In fermenting process, stream adds 8% (v/v) ammoniacal liquor and 10% (v/v) phosphoric acid and adjusts pH to maintain 7.0.OD is surveyed every 2h sampling 600and the content of glycerine and acetic acid.
(4) lactose-induced: maintaining fermentation revolution is under 1000rpm, temperature 37 DEG C, ventilation ratio 1.3vvm condition, ferments to fermented liquid OD 600when value reaches 30, temperature regulating maintains 30 DEG C, adds lactose (final concentration 20g/L) induction.Treat OD 600when value reaches 120, add lactose (final concentration 16g/L) chain induction., OD alive every 2h sampling and measuring enzyme 600, acetic acid content and glycerol content.
(5) stop tank and collect thalline: after chain induction, continue fermentation to OD 600value and than enzyme work in 3h not rising or when starting to decline, stop fermentation, put tank, obtain fermented liquid.In 4 DEG C, under 9000rpm, centrifugal 8min, abandons supernatant liquor, collects wet thallus.
2 experimental results:
Experimental result shows, and adopts DO-STAT feed supplement coupling to divide one-step inducing, and progressively puies forward high-revolving high density fermentation technology, make biomass from the 14.5g DCW/L of batch fermentation, be increased to 73.3g DCW/L.Than enzyme work from 180U/g DCW, be increased to 540U/g DCW.Show thus, this DO-STAT feed supplement coupling divides one-step inducing, and progressively puies forward the recombination bacillus coli high-density culture that high-revolving high density fermentation technology also can be applicable to produce other nitrilases.

Claims (10)

1. the fermentation process in high density containing nitrilase gene engineering bacteria, it is characterized in that described method is: the seed liquor that the engineering bacteria containing nitrilase gene obtains through enlarged culturing is seeded to fermentor tank with the inoculum size of volumetric concentration 2 ~ 4%, in 37 DEG C, 500rpm, blowing air be cultured to fermented liquid dissolved oxygen 15% ± 5% than 1-2vvm condition bottom fermentation, start to adopt DO-STAT mode to add supplemented medium, maintain fermented liquid oxygen dissolving value 15% ± 5%, adjustment mixing speed is 600 ~ 700rpm, ferments to OD 600when reaching 20 ~ 40, adjustment temperature to 25 ~ 35 DEG C, add final concentration 10 ~ 25g/L lactose and carry out first time induction; Fermentation is to OD 600after reaching 70 ~ 110, adjustment rotating speed to 720 ~ 780rpm; Fermentation is to OD 600reach 110-130, again add final concentration 10 ~ 15g/L lactose and carry out chain induction; Fermentation is to OD 600after reaching 115 ~ 140, adjustment rotating speed to 780 ~ 850rpm, continues fermentation to OD 600after reaching 150 ~ 190, adjustment rotating speed to 900 ~ 1300rpm; Maintaining fermentation rotating speed is under 900 ~ 1300rpm, temperature 25 ~ 35 DEG C of conditions, continues fermentation to OD 600value and when no longer rising in 3h than enzyme work or start to decline, stops fermentation, puts tank, and obtain fermented liquid, maintaining fermented liquid pH value in fermenting process is 6.0 ~ 7.0; Add the fermention medium containing final concentration 50g/L kantlex in described fermentor tank, described fermention medium consists of: peptone 10 ~ 20g/L, yeast powder 5 ~ 15g/L, NaCl5 ~ 15g/L, glycerine 5 ~ 20g/L, (NH 4) 2sO 43 ~ 10g/L, KH 2pO 40.5 ~ 3g/L, K 2hPO 43H 2o 1.0 ~ 5g/L, MgSO 47H 2o 0.1 ~ 1.0g/L, solvent is water, and pH value is 6.0-7.0; Described supplemented medium consists of: peptone 50-120g/L, and yeast extracts 30 ~ 80g/L, glycerine 300 ~ 900g/L, citric acid 1 ~ 8g/L, ammonium sulfate 1 ~ 10g/L, K 2hPO 41 ~ 8g/L, NaCl 1 ~ 10g/L, MgSO 41 ~ 10g/L, solvent is water, and pH value is 6.0 ~ 7.0.
2., as claimed in claim 1 containing the fermentation process in high density of nitrilase gene engineering bacteria, it is characterized in that described maintenance fermented liquid oxygen dissolving value is 15% ± 5%, adjustment mixing speed is 700rpm, ferments to OD 600reach after 90s, adjustment rotating speed is to 750rpm; Fermentation is to OD 600after reaching 125, adjustment rotating speed, to 800rpm, continues fermentation to OD 600after reaching 170, adjustment rotating speed is to 1000rpm.
3., as claimed in claim 1 containing the fermentation process in high density of nitrilase gene engineering bacteria, it is characterized in that described fermented liquid OD 600when value reaches 30, add final concentration 20g/L lactose and induce.
4., as claimed in claim 1 containing the fermentation process in high density of nitrilase gene engineering bacteria, it is characterized in that described inducing culture is to OD 600when value reaches 120, again add final concentration 14g/L lactose and carry out chain induction.
5., as claimed in claim 1 containing the fermentation process in high density of nitrilase gene engineering bacteria, it is characterized in that described fermention medium consists of: peptone 15g/L, yeast powder 12g/L, NaCl 10g/L, glycerine 12g/L, (NH 4) 2sO 45g/L, KH 2pO 41.36g/L, K 2hPO 43H 2o 2.28g/L, MgSO 47H 2o 0.375g/L, solvent is water, and pH value is 6.5.
6. as claimed in claim 1 containing the fermentation process in high density of nitrilase gene engineering bacteria, it is characterized in that described feed supplement used medium consists of: peptone 75g/L, yeast extracts 50g/L, glycerine 500g/L, citric acid 3g/L, ammonium sulfate 5g/L, K 2hPO 43g/L, NaCl 5g/L, MgSO 45g/L, solvent is water, and pH value is 6.5.
7. as claimed in claim 1 containing the fermentation process in high density of nitrilase gene engineering bacteria, it is characterized in that described seed liquor is prepared as follows: the engineering bacteria containing nitrilase gene is seeded to LB solid medium, cultivate 1 ~ 2 day at 37 DEG C, obtain inclined-plane thalline; Picking inclined-plane thalline is seeded to the LB liquid nutrient medium containing final concentration 50g/L, 37 DEG C, cultivate 12 ~ 24h under 150rpm, obtains seed liquor.
8. as claimed in claim 7 containing the fermentation process in high density of nitrilase gene engineering bacteria, it is characterized in that the described engineering bacteria containing nitrilase gene is for template with Acidovorax facilis (Acidovorax facilis) ZJB09122 genomic dna, with sequence 1 and sequence 2 for primer, the expression plasmid containing restructuring nitrilase is obtained by PCR method, and then with the expression plasmid containing restructuring nitrilase for template, with sequence 3 and sequence 4 for primer, build the engineering bacteria containing restructuring nitrilase by PCR method;
Sequence 1:5 '-AAT gGATCCaTGGTTTCGTATAACAGCAAG-3 ';
Sequence 2:5 '-AGG gTCGACcTACTTTGCTGGGACCGG-3 ';
Sequence 3:5 '-GAGCACGTTCAGCCGCTGTCCAAAT-3 ';
Sequence 4:5 '-CGGCTG aACgTGCTCCCAGCAGTTC-3 '.
9. as claimed in claim 7 containing the fermentation process in high density of nitrilase gene engineering bacteria, it is characterized in that the described engineering bacteria containing nitrilase gene is for template with Bacillus foecalis alkaligenes (Alcaligenes faecalis) ZJUTB10 genomic dna, with sequence 5 and sequence 6 for primer, the expression plasmid containing restructuring nitrilase is obtained by PCR method, and then with the expression plasmid containing restructuring nitrilase for template, with sequence 7 and sequence 8 for primer, build the engineering bacteria containing restructuring nitrilase by PCR method;
Sequence 5:5 '-AAT gGATCCaTGCAGACAAGAAAAATCGTC-3 ';
Sequence 6:5 '-AGG gTCGACtCAGGACGGTTCTTGCAC-3 ';
Sequence 7:5 '-CATGGAAGAGATC nNNtTCGCTAAGGCTATC-3 ';
Sequence 8:5 '-GATAGCCTTAGCGAA nNNgATCTCTTCCATG-3 '.
10., as claimed in claim 1 containing the fermentation process in high density of nitrilase gene engineering bacteria, it is characterized in that in described fermenting process adding volumetric concentration 8% ammoniacal liquor and volumetric concentration 10% phosphate aqueous solution by stream, to maintain fermented liquid pH value be 6.5.
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104762248A (en) * 2015-03-25 2015-07-08 江南大学 Genetically engineered bacteria for highly yielding isoamylase and fermentation process thereof
CN104962546A (en) * 2015-06-19 2015-10-07 浙江工业大学 Immobilization method for cells containing glucose isomerase
CN107267433A (en) * 2017-07-17 2017-10-20 江南大学 A kind of structure and its fermentation process in high density of nitrilase recombinant strains
CN109112122A (en) * 2018-09-29 2019-01-01 山东鲁抗医药股份有限公司 A kind of derivational expression method in tyrosine phenol lyase fermentation process
CN109294968A (en) * 2017-07-25 2019-02-01 格兰柏生化科技有限公司 A kind of mass production techniques of animal nucleic acid vaccine
CN110229774A (en) * 2019-07-02 2019-09-13 山东国力生物科技有限公司 A kind of arginine decarboxylase gene engineering bacteria and its high density fermentation culture method
CN111172140A (en) * 2020-01-21 2020-05-19 浙江工业大学 Nitrilase mutant and application thereof in preparation of anti-epileptic drug intermediate
CN111471668A (en) * 2020-02-28 2020-07-31 浙江工业大学 Nitrilase mutant and application thereof in preparation of 1-cyanocyclohexylacetic acid
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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
吴乔等: "腈水解酶重组菌发酵产酶条件的优化和应用", 《化学与生物工程》 *
金利群等: "重组大肠杆菌产腈水解酶的培养条件优化", 《食品与发酵工程》 *

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CN104762248B (en) * 2015-03-25 2018-02-23 江南大学 A kind of genetic engineering bacterium and its zymotechnique of high yield isoamylase
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CN113025601A (en) * 2019-12-25 2021-06-25 上海奥博生物医药技术有限公司 Nitrilase promoter optimized expression and application
CN111172140A (en) * 2020-01-21 2020-05-19 浙江工业大学 Nitrilase mutant and application thereof in preparation of anti-epileptic drug intermediate
CN111172140B (en) * 2020-01-21 2022-04-19 浙江工业大学 Nitrilase mutant and application thereof in preparation of anti-epileptic drug intermediate
CN111471668A (en) * 2020-02-28 2020-07-31 浙江工业大学 Nitrilase mutant and application thereof in preparation of 1-cyanocyclohexylacetic acid
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