CN105602968B - A kind of esterase and its application - Google Patents
A kind of esterase and its application Download PDFInfo
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- CN105602968B CN105602968B CN201610086094.8A CN201610086094A CN105602968B CN 105602968 B CN105602968 B CN 105602968B CN 201610086094 A CN201610086094 A CN 201610086094A CN 105602968 B CN105602968 B CN 105602968B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01073—Feruloyl esterase (3.1.1.73)
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Abstract
The present invention relates to a kind of esterase gene of acquisition and its clonal expression and applications, belong to bioengineering field.Disclose its substrate specificities.The esterase has extensive effect, has important industrial application value.
Description
Technical field
A kind of esterase of clonal expression of the present invention, and disclose its nucleotide sequence and amino acid sequence and zymologic property and
Using belonging to industrial microorganism field.
Background technique
Feruloyl esterase (Feruloyl esterases, FAEs) is also known as Ferulic acid esterase, it is a kind of carboxy-lesterase, is
The ester bond in Ferulic acid methylester, oligosaccharide esterase and polysaccharide ferulic acid ester can be hydrolyzed, the enzyme of free ferulic acid is discharged.It can cut off
Crosslinking in cell wall between polysaccharide-polysaccharide, polysaccharide-lignin is conducive to the degradation of polysaccharide and lignin in cell wall substance
Release, therefore have broad application prospects in food, feed and paper industry.In the food industry, it is degraded using esterase
Ferulic acid ester bond in plant cell wall, the available free ferulic acid for having medical value and healthcare function.And vegetalitas is former
Material becomes loose by the processing cell wall of esterase, and the raw material as feed industry is easier by poultry digestibility and utilization, as
The raw material of pulp and paper industry can reduce the use of pulping process chemicals, reduce pollution, and be conducive to subsequent handling
Progress.
1987, feruloyl esterase was found for the first time in Streptomyces olivochromogenes.Study table
Bright fungi, bacterium, yeast can secrete esterase, and presently found microbes producing cellulase has aspergillus niger (Aaspergillus
Niger), streptomycete (Streptomyces avermitilis), clostridium (Clostridium thermocellum), lactic acid bar
Bacterium (Lactobacilli sp.), pseudomonad (Pseudomonas fluorescens) etc., but most esterases are from true
It is separated in bacterium.Research finds that Yue Shi lactobacillus (Lactobacillus johnsonii) has stronger Ah acid's esterase active,
And therefrom clonal expression goes out gene (the Biochemical Properties of Two of 2 feruloyl esterases
Cinnamoyl Esterases Purified from a Lactobacillus johnsonii Strain Isolated
From Stool Samples of Diabetes-Resistant Rats, Applied and environmental
Microbiology, 2009,5 (15), 5018-5024).By the characteristic research to Yue Shi lactobacillus, which may be there are also it
Its esterase.This patent discloses a kind of novel Yue Shi lactobacillus esterases.
Summary of the invention
The present invention is cloned from Yue Shi lactobacillus has obtained a novel esterase gene, different using colibacillus engineering
Source expression, discloses its relevant enzymatic property.And the application of ferulic acid extraction is carried out with it.
Technical scheme is as follows:
1, bacterial strain
The source bacterial strain of esterase gene of the present invention are as follows: Lactobacillus johnsonii ATCC11506 is (from the U.S.
ATCC strain library purchase).
2, the clone of esterase gene
Extract Lactobacillus johnsonii ATCC11506 phage gene group total DNA.Specific primer is designed,
Using PCR method, esterase gene overall length encoder block sequence is amplified.And construction recombination plasmid.
3, esterase expression and purification
Recombinant plasmid is imported in BL21 Escherichia coli, inducing expression.Crude enzyme liquid is obtained after bacterial cell disruption, it is cold after repurity
Freeze drying for standby.
4, esterase zymologic property
Influence of the pH to esterase enzyme activity of the present invention is studied by substrate of Ferulic acid methylester.
Influence of the temperature to esterase enzyme activity of the present invention is studied by substrate of Ferulic acid methylester.
Fe is determined by substrate of Ferulic acid methylester2+、Ag+、Fe3+、K+、Ca2+、Cu2+、Mg2+、Mn2+、Zn2+、Ba2+Ion
Influence to enzyme activity.
The substrate specificities of esterase are analyzed: substrate used has ethyl hexanoate, certain herbaceous plants with big flowers acetoacetic ester, ethyl butyrate, ethyl caprilate, second
Acetoacetic ester, ethylene certain herbaceous plants with big flowers acid esters, Vinyl crotonate, vinyl laurate, vinyl pivalate, vinyl benzoate, first
Base vinyl acrylate, vinyl propionate, vinyl propionate, vinyl acetate, Glyceryl tributyrate, gamma-valerolactone, acetic acid fourth
Ester, three-O- acetyl group-D glucals, glyceryl triacetate, α-D (+) five acetyl glucose, geranyl acetate, acetic acid positive third
Ester, methylvinyl acetate, methyl lactate, dihydrojasmonate, methyl caprylate, methyl caproate, 2 bromopropionic acid methyl esters, bromine second
Sour methyl esters, methyl (R)-(-)-mandelic acid, bromo-acid methyl esters, cinnamic acid Bian ester, acetic acid -2- naphthalene ester, propionic acid naphthalene ester, acetic acid benzene
Ester, butyric acid naphthalene ester, 4- nitrobenzophenone caprylate, acetic acid -1- naphthalene ester, 2,6- dihydroxy-methyl 4 methylbenzoate, naphthols third
Acid esters, methyl-mandelic acid ester, ferulic acid ethyl ester, Ferulic acid methylester, chlorogenic acid, Rosmarinic acid, NSC 619661, caffeic acid phenethyl
Ester, p-Coumaric Acid methyl esters.
Enzyme activity determination method are as follows: 4ml reaction system, include 3ml phosphate buffer, 1ml substrate solution (4mg/ml substrate),
The enzyme of 5ug freeze-drying, water-bath temperature control react 4h, 100 DEG C of boiling water bath heating termination in 3 minutes reactions.It is examined with high performance liquid chromatography
Survey substrate reduction amount, high performance liquid chromatography detection condition are as follows: 10% acetonitrile of 0-15min, 90% water (0.1% formic acid)；15-
100% acetonitrile of 20min, 0 water；20-30min10% acetonitrile, 90% water (0.1% formic acid), detector are 3300 type of Alltech
Evaporative light scattering detector.The influence of temperature, pH, ion, substrate is all made of the system.
5. the ferulic acid release rate analysis using desizing wheat bran as raw material, after adding the esterase.
Desizing wheat bran prepares (Xylan-hydrolysing enzymes from according to the method for document
Streptomyces spp.Enzyme and Microbial Technology, 1988,10 (7): 403-409.).In wheat bran
The total amount of ferulic acid calculates (Preparation of fer μ lic acid from agric with the ferulic acid that alkalinity extraction obtains
μltural wastes:Its improved extraction and purification.Journal of Agricμ
Ltural and Food Chemistry, 2008,56 (17): 7644-7648.).Ferulic acid release rate is the enzymatic hydrolysis of ferulic acid
Burst size accounts for the percentage for the ferulic acid total amount that alkali carries take
Specific embodiment
Embodiment 1
The present embodiment is that the clone of esterase gene of the present invention and colibacillus engineering construct.
1, the extraction of Lactobacillus johnsonii ATCC11506DNA
Lactobacillus johnsonii ATCC11506 bacterial strain is cultivated 12 hours in LB culture medium, 12000r/
Min is centrifuged 10 minutes and obtains thallus, extracts using bacterial genomes DNA extraction agent box (TaKaRa company) according to its operation
Lactobacillus johnsonii ATCC11506 phage gene group total DNA, it is spare to put refrigerator.
2, prepared by E. coli competent
(1) inoculation E.coli DH5 α and BL21 (DE3) is respectively in the 250mL shaking flask containing 20mL LB culture medium, and 37
DEG C, 200rpm/min overnight incubation.
(2) it is inoculated in 50mL LB culture medium by 1% inoculum concentration, 37 DEG C of cultures to OD600 about 0.6 (about 2~3h).
(3) bacterium solution is transferred in the centrifuge tube of 50mL pre-cooling, places 30min, 8000rpm/min, 4 DEG C of centrifugations on ice
5min。
(4) supernatant is abandoned, the 0.1mol/L CaCl2 solution of 5mL pre-cooling is added, so that thallus is suspended, places 20min on ice,
8000rpm/min, 4 DEG C of centrifugation 5min.It is repeated 2 times.
(5) supernatant is abandoned, the 0.1mol/LCaCl2 solution of 1.5mL pre-cooling is added, containing 15% glycerol, gently suspended bacteria
Body, then 100 μ L are dispensed into 1.5mL centrifuge tube, and -70 DEG C of Storage in refrigerator are spare.
3, the clone of esterase gene
(1) design of primers
The whole genome sequence and known Yue Shi Bacillus acidi lactici esterase of correlation Yue Shi lactobacillus in ncbi database are analyzed,
Design primer sequence are as follows:
Primer 1:5'GCCGGAGCTCATGCTATTTAAGACAAGTGATAA 3'
Primer 2: 5'GCCGTCTAGATTACTTTAAAATATCTTTTAACTTT 3'
(2) PCR amplification
With two primers synthesized above, the genomic DNA with Lactobacillus johnsonii ATCC11506 is
Template carries out PCR amplification.
Amplification system in this step are as follows:
Amplification program are as follows:
94 DEG C, 10min
94 DEG C, 30sec；55 DEG C, 30sec；72 DEG C of 1min react 30 circulations
72 DEG C, 10min
PCR product obtains the gene order of the esterase after sending Hua Da gene sequencing, as shown in SEQ ID NO:1.According to this
The amino acid sequence that gene order obtains is as shown in SEQ ID NO:2.
(3) double digestion and connection
II plasmid of pCold- and PCR product are subjected to double digestion, digestion system are as follows: 4 μ of 10 × cut buffer3 μ l, DNA
L, enzyme Sac I and Xba I each 0.5 μ l, 2 μ l of sterile water totally 30 μ l.1h is cut in 37 DEG C of water-baths.DNA fragmentation is cloned into pCold-
On II Vector, and it is transformed into E.coli DH5 α competent cell.Linked system: 10 × DNA ligase buffer 2.5
μ l, 8 μ l of DNA fragmentation, 2 μ l, T4DNA ligase of carrier DNA 1 μ l, 11.5 μ l of sterile water totally 25 μ l.It is connected under 16 DEG C of water-baths
12h-16h。
(4) it converts
Step: 1 is added 100 μ l DH5 α competent bacterias in linked system, light to mix, ice bath 30min.2 are put into preheating
42 DEG C of water-baths in, place 90s carry out heat shock processing.3 ice bath 2min immediately.4 are added the not antibiotic LB culture of 1ml
Liquid, 37 DEG C of culture 1h make bacterium recover.5 are uniformly coated on 6 cultures on antibiotic LB culture medium grows fine for 24 hours.Choose list
Bacterium colony carries out bacterium colony PCR, and recombinant plasmid is extracted in nucleic acid electrophoresis verifying.Recombinant plasmid is imported in BL21 E. coli competent,
It saves backup.
Embodiment 2
The present embodiment is the inducing expression of esterase of the present invention and isolates and purifies.
1, add 500 μ l recombinant bacterium (BL21 Escherichia coli) liquid to 50ml shaking flask.37 DEG C of culture 2.5h are stood at 15 DEG C
0.5h.Again plus 20 μ l IPTG, cold-induction culture is for 24 hours at 15 DEG C.Fermentation obtains fermentation liquid and is centrifuged to obtain thallus, disodium hydrogen phosphate-
Sodium dihydrogen phosphate buffer (20mmol/L, pH7.0) redissolves thallus, and Ultrasonic Cell Disruptor is broken, and supernatant is collected by centrifugation and obtains slightly
Enzyme solution.
2, the crude enzyme liquid for obtaining step 1 carries out ni-sepharose purification using the operation of 150 protein purification system of AKTA avant,
It all puts the tetra- root canal road A1, A2, B1, B2 into water, system flow20ml/min flow velocity is set, is exhausted.Later A1
It putting into and combines in liquid, B1 is put into eluent, and it is primary being exhausted, then system flow 1ml/min, flow are set
Path (column position 3), delta pressure 0.3, pre-pressure 0.5, Gradient 0, inset
A1 balance fills pillar ten minutes later.Then loading crude enzyme liquid elutes destination protein with the high concentration imidazole buffer B1 of 500mM,
The albumen wash-out being adsorbed on ion column is got off, collection has the eluent of enzyme activity to be concentrated with super filter tube (30-kDa), obtains
To the enzyme of purifying.Enzyme after purification is freeze-dried spare.
Embodiment 3
The present embodiment is the optimum temperature and temperature stability of esterase of the present invention.As shown in table 1, with Ferulic acid methylester
For substrate, substrate is lauched the enzyme that bath 4h measures esterase in 20-70 DEG C of different temperature condition from the phosphate buffer that pH is 6.0
It is living, as a result, it has been found that the esterase enzyme activity highest measured under the conditions of 30 DEG C.Esterase of the present invention is placed in 40,50,60 and 70
It is handled 3 hours at DEG C, enzyme activity is not lost substantially under the conditions of 40 and 50 DEG C of discovery, remaining after 3 hours when temperature reaches 60 DEG C
Enzyme activity is 45% when starting, after temperature is higher than 70 DEG C, enzyme complete deactivation after 5 hours.
Influence of 1 temperature of table to esterase enzyme activity
Embodiment 4
The present embodiment is the optimum pH and pH stability of esterase of the present invention.As shown in table 2, it is with Ferulic acid methylester
Substrate, by substrate in pH3-9, the enzyme activity of 40 DEG C of water-bath 4h measurement esterases, as a result, it has been found that the esterase enzyme measured under the conditions of 6 pH
Highest living.Influence of the pH to esterase stability of the present invention is studied by substrate of Ferulic acid methylester, esterase is respectively placed in pH
The remaining enzyme activity of measurement in 96 hours is handled in the buffer that value is 3-9 and finds that pH value enzyme activity in 6-7 is more stable, and pH value is lower than 5
Or residue enzyme activity deficiency originates when higher than 8 50%.
Table 2 is influence of the pH to esterase enzyme activity
Embodiment 5
The present embodiment is influence of the different metal ions to esterase enzyme activity.The enzyme purified in embodiment is placed in concentration
Its remaining enzyme activity discovery Fe is measured in 4ml solution for the different metal ions of 10mM after ten minutes2+, Ag+And Fe3+Ionization
After ten minutes, enzyme activity loss is larger, and other ions influence little.The results are shown in Table 3.
Influence of 3 metal ion of table to enzymatic activity
Embodiment 6
The present embodiment is the response characteristic of esterase and different substrates, is listed in Table 4 below.As can be seen from Table 4, which has
Extensive substrate shows very strong vigor for aromatic series esters, belongs to feruloyl esterase.
Activity of 4 esterase of table to different substrates
Embodiment 7
The present embodiment is the application that esterase hydrolyzed desizing wheat bran produces ferulic acid.200g wheat bran is added and purifies and freeze dry
Esterase 40mg after dry, adds water 1000ml, pH naturally, 40 DEG C keep the temperature 12 hours, measure ferulic acid release rate be 90.3%.
Claims (1)
1. a kind of method for preparing ferulic acid, which is characterized in that the method is SEQ ID NO:2 using amino acid sequence
Esterase is hydrolyzed substrate and ferulic acid is prepared；The described method includes: taking 200g desizing wheat bran, it is added and purifies and freeze dry
Esterase 40mg after dry, adds water 1000ml, pH naturally, 40 DEG C keep the temperature 12 hours, measure ferulic acid release rate be 90.3%.
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| CN106434598B (en) * | 2016-09-07 | 2019-12-06 | 江南大学 | Esterase and application thereof |
| CN106167794B (en) * | 2016-09-07 | 2019-11-26 | 江南大学 | A kind of esterase and its application |
| CN106399275B (en) * | 2016-09-07 | 2019-11-26 | 江南大学 | A kind of esterase and its application |
| CN106434599B (en) * | 2016-09-07 | 2019-11-26 | 江南大学 | A kind of esterase and its application |
| CN106167795B (en) * | 2016-09-07 | 2019-12-24 | 江南大学 | Esterase and application thereof |
| CN106399274B (en) * | 2016-09-07 | 2019-12-10 | 江南大学 | Esterase and application thereof |
| CN115992059B (en) * | 2022-07-06 | 2024-05-28 | 南京财经大学 | Lactobacillus johnsonii for producing feruloyl esterase and application thereof in relieving ulcerative colitis |
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| CN102796673A (en) * | 2012-08-24 | 2012-11-28 | 黄河三角洲京博化工研究院有限公司 | Feruloyl esterase production strain and method for producing feruloyl esterase by using same |
| CN104673767A (en) * | 2015-03-31 | 2015-06-03 | 河南工业大学 | Method for producing feruloyl esterase |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102796673A (en) * | 2012-08-24 | 2012-11-28 | 黄河三角洲京博化工研究院有限公司 | Feruloyl esterase production strain and method for producing feruloyl esterase by using same |
| CN104673767A (en) * | 2015-03-31 | 2015-06-03 | 河南工业大学 | Method for producing feruloyl esterase |
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