CN106497896A - A kind of restructuring carboxy-lesterase DMWf18 558 in deep-sea source and its encoding gene and application - Google Patents
A kind of restructuring carboxy-lesterase DMWf18 558 in deep-sea source and its encoding gene and application Download PDFInfo
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Abstract
The present invention relates to a kind of deep-sea source carboxy-lesterase DMWf18 558 and its encoding gene and application.558 encoding genes of carboxy-lesterase DMWf18 are obtained by the screening of grand genome, and its nucleotide sequence and amino acid sequence are respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.After by this carboxylesterase gene heterogenous expression, the catalysis activity highest when substrate is p-nitrophenol capronate (C6), enzyme activity is up to 367U/mg.558 catalyzing hydrolysis temperature ranges of carboxy-lesterase DMWf18 are 15～40 DEG C, preferably 15～35 DEG C；Catalyzing hydrolysis pH value is 3.0～10.0, preferably 6.5～9.0；Catalyzing hydrolysis NaCl concentration is in 0.5～3M, preferably 0.5 1M.The esterase can be widely applied to the fields such as grease hydrolysis, pharmaceutical synthesis metabolism, food processing, wastewater treatment, detergent industry.
Description
Technical field
The invention belongs to genetic engineering field, and in particular to a kind of halmeic deposit source carboxy-lesterase, its encoding gene
And its application.
Background technology
Lipid hydrolyzing enzyme includes carboxy-lesterase (EC3.1.1.1) and lipase (EC3.1.1.3), is widely present in microorganism
In, the hydrolysis and synthesis of ester type compound can be catalyzed.Short chain acyl glycerine of the carboxy-lesterase major catalytic less than 10 carbon, fat
Long acyl glycerine of the fat enzyme major catalytic more than 10 carbon.Lipid hydrolyzing enzyme has many good characteristics, such as catalytic reaction not
Need confactor, hand-type to select specificity high, have wider substrate spectrum, and keep high stability in organic solvent
Deng.Lipid hydrolyzing enzyme is important catalyst in industrial production, can be widely applied to chiral drug catalysis, leather silk spinning raw material and takes off
The aspects such as fat, wastewater treatment, detergent industry and food processing.
There are many microbe-derived lipase to be commercially produced at present, and each side in productive life
Face is applied.And carboxy-lesterase is used as a kind of important industrial enzymes, the type and quantity being applied in actual production
Also extremely limited.The excavation and accumulation of novel carboxylic acid esterase resource is to meet the industry such as food and chemical industry increasingly to increase carboxy-lesterase
The necessary condition of long demand.The technology that the present invention is built with grand genome and Carboxylesterase Activity is screened, obtains deep-sea source
Novel carboxylic acid esterase, and study its zymologic property.
Content of the invention
It is an object of the invention to provide a kind of new deep-sea source carboxy-lesterase, its encoding gene and preparation method thereof, should
Carboxy-lesterase can be used for esters degraded and living things catalysis and the conversion of other ester type compounds.
The present invention is screened using tributyrin as substrate from the grand genomic library of Pacific seamounts halmeic deposit
Carboxylesterase Activity, obtains a kind of new carboxy-lesterase DMWf18-558, the nucleotide sequence such as SEQ ID of its encoding gene
Shown in No.1, its amino acid sequence is as shown in SEQ ID No.2.
The carboxylesterase sequence is carried out in GenBank Homology search, similar property highest is also grand genome
The esterase in source, similitude are 82% (its number of registration in GenBank databases is AFB82690).Phylogenetic Analysis are tied
Fruit shows that carboxy-lesterase DMWf18-558 belongs to the iv family in hydrolysis of lipid enzyme family.Speculated according to amino acid sequence, carboxylic
The catalytic center of acid esters enzyme DMWf18-558 is by serine, glutamic acid and histidine (amino acid position is 144,238 and 268) group
Into wherein serine is located at glycine, aspartic acid, serine, alanine and glycine (amino acid position is 142 to 146)
In the conserved sequence of composition, therefore carboxy-lesterase DMWf18-558 belongs to iv family GDSAG subfamilies.Oxonium ion hole is located at 75
With 76 two glycine.In sum, DMWf18-558 should be a newcomer in lipid hydrolyzing enzyme iv family.
Under the premise of esterase DMWf18-558 protein actives are not affected, can be to SEQ ID NO:Shown in 2 away from catalysis in
The amino acid sequence of heart amino acid position (amino acid position of preferably 142-146,238 and 268) carry out various replacements, interpolation and/
Or lack the derived protein that one or several amino acid acquisitions have esterase DMWf18-558 activity.According to art technology
Common knowledge, the BA of protein is closely related with its functional domain.In general, only occur in function
The site mutation of domain may produce impact to the 2 and 3 dimensional organization of protein, so as to affect its BA.And
For occurring in the amino acid sites away from functional domain (preferably away from the amino acids position of 142-146,238 and 268), by
Protein function conformation is not involved in this region, thus indivedual point mutation of amino acid will not be produced to the BA of protein
Raw substantial effect such that it is able to the basic biological function for retaining crude protein.Described can retain crude protein substantially
Biological function refer to derived protein have more than 80% esterase DMWf18-558 BA, preferably have
The BA of more than 90% esterase DMWf18-558, the biology more preferably with more than 95% esterase DMWf18-558
Learn activity.
Preferred esterase DMWf18-558 mutant have at least with SEQ ID NO:Amino acid sequence 90% shown in 2 with
On homology, more preferably have at least more than 95% homology, most preferably have at least more than 99% homology.
In the same manner, present invention also offers the gene order of coding amino acid sequence as shown in SEQ ID NO.2, itself and SEQ
Nucleotide sequence shown in ID NO.1 is consistent；The present invention is also provided to removing 424- in the nucleotide sequence shown in SEQ ID NO.1
438th, other nucleotides outside the nucleotides of 712-714 and 802-804 positions are replaced, add and/or lack one or several cores
Thuja acid can retain substantially the mutant gene of esterase DMWf18-558 biological activity of albumen so as to obtain coding.Preferred esterase
DMWf18-558 mutant genes have at least with SEQ ID NO:The homology of the nucleotide sequence more than 90% shown in 1, more
It is preferred that having at least more than 95% homology, most preferably there is at least more than 99% homology.
Using gene clone technology, the esterase DMWf18-558 genes being cloned into can be connected on suitable carrier, and
Conversion is transfected into prokaryotes or eucaryote host expresses Prepare restructuring esterase DMWf18-558.Suitable prokaryotes place
Master includes that various bacteriums such as E.coli etc., suitable eucaryote host include yeast (such as methanol yeast) and mammalian cell
(such as Chinese hamster ovary cell) etc., it is preferred to use prokaryotic expression system E.coli.
The protokaryon or eukaryotic expression of the various commercially viable purchase that suitable carrier is well known to those skilled in the art is carried
Body, prokaryotic expression carrier such as pET serial carriers, pQE serial carriers；Yeast expression carrier pPICZ- α-A, pHIL-D2, pPIC9,
pHIL-S1(Invitrogen Corp.San Diego.California.USA)；Animal cell expression vectors pSVK3, pMSG
(Amersham Pharmacia Biotech Inc.USA) etc..One preferred example is that the present invention is screened carboxy-lesterase
The encoding gene of DMWf18-558 is connected on coli expression carrier pET28a (Novagen), and is transformed into Escherichia coli
In Rosetta (DE3), go out highly active Recombinant esterase through abduction delivering.
Present invention also offers carboxy-lesterase DMWf18-558 or the Host Strains of carboxy-lesterase DMWf18-558 can be expressed exist
Industrial application, for example, can be used for being catalyzed ester-type hydrolysis.By esterase activity determine show, carboxy-lesterase DMWf18-558 or
The above-mentioned Host Strains that can express carboxy-lesterase DMWf18-558 can be used for hydrolyzing short-chain fatty acid ester, such as C2-C8 short carbon chains fat
Fat acid esters.Preferred short-chain aliphatic ester is the p-nitrophenyl phenolic ester with C2-C8 short carbon chains, such as p-nitrophenol acetic acid
Ester, p-nitrophenol butyrate, p-nitrophenol capronate or p-nitrophenol caprylate etc., wherein substrate are p-nitrophenol
Catalysis activity highest during capronate (C6), enzyme activity reach 367U/mg.
Carboxy-lesterase DMWf18-558 catalyzing hydrolysis temperature range is 15～40 DEG C, preferably 15～35 DEG C；The hydrolysis
PH value be 3.0～10.0, preferably 6.5～9.0.1h is incubated below 20 DEG C, more than 70% remnant enzyme activity can be kept；?
1h is incubated in 30 DEG C, more than 50% remnant enzyme activity can be kept.The novel carboxylic acid esterase and its encoding gene that the present invention is provided exists
Prepared by medicine, food processing and flavor improvement, wastewater treatment, washing industry have important application potential.
Description of the drawings
Policapram gel electrophoresis analysis figures of the Fig. 1 for purification of carboxylic acids esterase DMWf18-558.
Substrate specificity figures of the Fig. 2 for carboxy-lesterase DMWf18-558.C2：P-nitrophenol acetic acid esters；C4：P-nitrophenyl
Oxyphenisatin acid esters, C6：P-nitrophenol capronate；C8：P-nitrophenol caprylate；C10：P-nitrophenol decylate；C12：Right
Nitrophenol dodecanoate；C14：P-nitrophenol myristinate；C16：P-nitrophenol Palmitate；When definition substrate is C6
Measured value is 100%.
Fig. 3 is carboxy-lesterase DMWf18-558 optimal reactive temperature figures.
Fig. 4 is schemed for carboxy-lesterase DMWf18-558 optimal reactions pH.
Fig. 5 is carboxy-lesterase DMWf18-558 optimal reaction NaCl concentration figures.
Fig. 6 be bivalent cation to carboxy-lesterase DMWf18-558 activity influence figures.
Fig. 7 is organic solvent and detergent to carboxy-lesterase DMWf18-558 activity influence figures.
Temperature stability figures of the Fig. 8 for carboxy-lesterase DMWf18-558.
Specific embodiment
The acquisition of 1 carboxy-lesterase DMWf18-558 encoding genes of embodiment
Halmeic deposit sample is gathered from Pacific seamounts edge by the visual multitube sampler in deep-sea.Grand genomic library structure
Build and adopt CopyControlTMHTP fosmid library production kit(Epicentre
Biotechnologies, the U.S.), host strain is E.coli EPI300 (Epicentre Biotechnologies, U.S.
State), carrier is pCC2FOS fosmid vector (Epicentre Biotechnologies, the U.S.).Through pulsed field gel electrophoresis
Detection, Insert Fragment size are 36～48kb.Take 10 μ l libraries bacterium solutions and be diluted to 100 μ l, coat carboxy-lesterase screening flat board,
30 DEG C are cultivated 2 days.Culture medium prescription is LB culture mediums (10g/L tryptones, 5g/L yeast extracts, 10g/L sodium chloride, pH
7.2)；After sterilizing, chloramphenicol is added under aseptic condition so as to final concentration of 12.5 μ g ml-1.
Picking monoclonal is simultaneously packed, and fosmid packings library is sequenced, ORFs is predicted and annotate after splicing
Gene, therefrom screens lipid hydrolyzing enzyme related gene.By Blastx (http://blast.ncbi.nlm.nih.gov/) ratio
Homology to known esterase gene sequence in sequence and database.Analysis is compared through database and obtains DMWf18-558 coding bases
Cause, size is 909bp, and base composition is：141A (15.51%), 146T (16.06%), 331C (36.41%) and 291G
(32.01%), its nucleotide sequence such as SEQ ID No:Shown in 1.Encoding proteins size be 302 amino acid residues, its amino
Acid sequence such as SEQ ID No:Shown in 2.The amino acid sequence is carried out in GenBank Homology search, similar property highest
Carboxy-lesterase also be grand genome, similitude is 82%, its number of registration in GenBank databases be AFB82690.
Phylogenetic Analysis result shows that carboxy-lesterase DMWf18-558 belongs to the iv man in hydrolysis of lipid enzyme family
Race.Speculate that the catalytic center of carboxy-lesterase DMWf18-558 is by serine, glutamic acid and histidine (ammonia according to amino acid sequence
Base acid position be 144,238 and 268) constitute, wherein serine be located at glycine, aspartic acid, serine, alanine and sweet ammonia
In the conservative region that acid (amino acid position is 142 to 146) is constituted, therefore carboxy-lesterase DMWf18-558 belongs to iv family
GDSAG subfamilies.And oxonium ion hole is located at 75 and 76 two glycine.
In sum, DMWf18-558 should be a newcomer in lipid hydrolyzing enzyme iv family.
The structure of the recombinant expression plasmid and recombinant bacterial strain of 2 carboxy-lesterase DMWf18-558 of embodiment
The carboxy-lesterase DMWf18-558 encoding genes that the present invention is obtained are cloned on expression vector, are built recombinant expressed
Bacterial strain.The open reading frame sequence of the esterase gene that the ORF analyses based on NCBIORF Finder are obtained, design amplification esterase are complete
The upstream primer 558F (5 '-TCGCGGATCCATGGCGAGTCCACAGCTCC-3 ', BamHI) of gene and downstream primer 558R
For packing fosmid Library PCR amplifications, (5 '-ATTTGCGGCCGCCTAGCGTGCGGCGGC-3 ', XhoI), confirms that gene is complete
Long sequence.Using the method construction expression plasmid of enzyme cutting clone, that is, BamHI and XhoI double digestion PCR primers are used, piece after purification
The plasmid pET28a connections of Duan Yujing BamHI and XhoI double digestions, using CaCl2During conversion method is converted to E.coliDH5 α, card
That chloramphenicol resistance screening positive clone.The plasmid that positive colony is extracted using plasmid extraction kit (Axygen, the U.S.), warp
BamHI and XhoI double digestions are identified, obtain the DNA fragmentation of 1000bp or so, be carboxy-lesterase DMWf18-558 through sequencing identification
Encoding gene.Recombinant expression plasmid is transformed in E.coliRosetta (DE3) expression bacterial strains, construction expression recombinant bacterial strain.
Embodiment 3 is using recombinant strains expression restructuring carboxy-lesterase DMWf18-558
The 3ml recombinant strains for building are transferred to 100ml contains 20 μ g/ml kanamycins and 34 μ g/ml chlorine are mould
In the LB fluid nutrient mediums of element, 37 DEG C of shaken cultivations to OD6000.6 is reached, adds the IPTG of final concentration of 0.5mM to be induced
Expression, proceeds to 20 DEG C with 150r/min shaken cultivations 8h.Low-temperature centrifugation collects thalline, is resuspended in (the 500mM chlorinations of NTA-10 solution
Sodium, 10mM imidazoles, 20mM Tris hydrochloric acid, pH 8.0) in, carry out ultrasonic disruption process on ice.On low-temperature centrifugation is collected
Clearly, using NTA-Ni2+Affinity column chromatography purifies expressing protein.Expressed recombinant protein contains 6 × His tag of N-terminal, affable
Inhale on post with layer is adsorbed onto, through the imidazole solution gradient elution of variable concentrations, collect eluent.Detect through SDS-PAGE, obtain
Arrive electrophoretically pure restructuring carboxy-lesterase DMWf18-558, molecular weight 36kDa or so (Fig. 1).Protein compression is determined with Lowry methods
Degree, obtains the expression of about 28.9mg/100ml zymotic fluids.
The Activity determination of the restructuring carboxy-lesterase DMWf18-558 of embodiment 4
The carboxy-lesterase DMWf18-558 activity that purifying is determined using p-nitrophenol caproic acid ester process.Concrete operations：1ml is anti-
System is answered to include that the own phenols acids of 1mM p-nitrophenyls, 100mM Tricine buffer solutions (pH 9.0) and 48ng pure enzyme proteins (are
The diluted purifying enzyme liquids of 10 μ l), using ultra-violet and visible spectrophotometer (Beckman DU800 types, the U.S.) in 25 DEG C of bars
METHOD FOR CONTINUOUS DETERMINATION light absorption value A under part4052min, uses the enzyme liquid of inactivation to be used for returning to zero as control.One enzyme activity unit is defined as
The enzyme amount produced from p-nitrophenol ester catalysis required for l μm of ol p-nitrophenol per minute.The esterase active for measuring is
367U/mg.
The restructuring carboxy-lesterase DMWf18-558 substrate specificity analyses of embodiment 5
The substrate specificity analysis of carboxy-lesterase DMWf18-558 adopts system：100mM Tricine buffer solution (pH
9.0), 1mM substrates, add 48ng pure enzyme proteins, METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 25 DEG C4052min.Determine the substrate that adopts for：
P-nitrophenol acetic acid esters (C2), p-nitrophenol butyrate (C4), p-nitrophenol capronate (C6), p-nitrophenol octanoic acid
Ester (C8), p-nitrophenol decylate (C10), p-nitrophenol dodecanoate (C12), p-nitrophenol myristinate (C14)
With p-nitrophenol Palmitate (C16).Show after measured, carboxy-lesterase DMWf18-558 is shorter to acyl group carbochain or longer
P-nitrophenyl phenolic ester (C2, C4, C6, C8, C10, C12 and C14) has catalysis activity, and wherein substrate is p-nitrophenol caproate
(C6) catalysis activity highest when, it is more difficult to hydrolyze p-nitrophenol Palmitate (C16) (Fig. 2).As a result show, carboxy-lesterase
DMWf18-558 has higher catalysis activity, and the hydrolysis vigor for short-chain lipid to the shorter lipid material of acyl group carbochain
It is better than long-chain lipid.
The restructuring carboxy-lesterase DMWf18-558 optimum reaction conditionses analyses of embodiment 6
Carboxy-lesterase DMWf18-558 optimal reactive temperatures are determined in the range of 15～60 DEG C.Concrete operations are：100mM
Tricine buffer solutions (pH 9.0), 1mM p-nitrophenol capronates add 48ng pure enzyme proteins, respectively 15,20,25,30,
35th, METHOD FOR CONTINUOUS DETERMINATION light absorption value A under the conditions of 40,45,50,55 and 60 DEG C4052min.Measurement result shows carboxy-lesterase DMWf18-
558 range of reaction temperature is 15～40 DEG C, 25 DEG C of optimal reactive temperature (Fig. 3).
Carboxy-lesterase DMWf18-558 optimal reactions pH is determined in the range of 3.0～10.0.Concrete operations are：In different pH
1mM p-nitrophenols capronate and 48ng pure enzyme proteins, METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 25 DEG C is added in buffer solution3482min.
Determining the buffer solution for using is：100mM citric acid-sodium citrate buffer solutions (pH 5.0～6.0), 100mM potassium dihydrogen phosphates-hydrogen
Sodium oxide molybdena buffer solution (pH 6.0～7.5), 100mM Tricine buffer solutions (pH 7.5～9.0) and 100mM 2- cyclohexylaminos
Ethyl sulfonic acid-sodium hydrate buffer solution (pH 9.0～10.0).Measurement result shows, carboxy-lesterase DMWf18-558 pH 3.0～
Active in the range of 10.0, optimal reaction pH is 9.0 (Fig. 4).
The NaCl concentration of carboxy-lesterase DMWf18-558 optimal reactions is determined in the range of 0.5～5M.Concrete operations are：
100mM Tricine buffer solutions (pH 9.0), 1mM p-nitrophenol capronates add 48ng pure enzyme proteins, respectively 0.5,1,
2nd, under the conditions of 3,4 and 5M NaCl, METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 25 DEG C4052min.Measurement result shows carboxy-lesterase
The NaCl reaction densities scope of DMWf18-558 is 0.5～3M, and optimal reaction concentration is 0.5M (Fig. 5).
The restructuring carboxy-lesterase DMWf18-558 zymetology stability analyses of embodiment 7
Bivalent cation to the measure concrete operations of carboxy-lesterase DMWf18-558 activity influences is：Divide in reaction system
Jia Ru not 10mM Co2+、Cu2+、Ca2+、Mg2+、Zn2+、Sr2+、Mn2+、Ni2+、Ba2+With ethylenediamine tetra-acetic acid (EDTA), enzyme is determined
Activity.Surveying enzyme activity system is：100mM Tricine buffer solutions (pH 9.0), 1mM p-nitrophenol capronates, the pure enzyme eggs of 48ng
In vain, METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 25 DEG C4052min.Measurement result shows that the metal ion for being added can suppress carboxy-lesterase
The activity of DMWf18-558, but its activity remains at more than 40%, in Mg2+In the presence of relative activity keep more than 80% (figure
6).
Organic solvent and detergent to the measure concrete operations of carboxy-lesterase DMWf18-558 activity influences are：In reactant
15% (v/v) organic solvent (isopropanol, acetonitrile, ethanol, methyl alcohol, acetone, dimethyl sulfoxide (DMSO) and dimethyl methyl is separately added in system
Acid amides) and 1% detergent (w/v or v/v) (SDS, polysorbas20, Tween 80 and Triton X-100), determine the activity of enzyme.Survey and live
System is：100mM Tricine buffer solutions (pH 9.0), 1mM p-nitrophenol capronates, 48ng pure enzyme proteins, at 25 DEG C
METHOD FOR CONTINUOUS DETERMINATION light absorption value A4052min.Measurement result shows that SDS and Tween 80 can suppress the work of carboxy-lesterase DMWf18-558
Property (relative activity<10%), and under 20 existence condition of acetonitrile dimethyl sulfoxide (DMSO) and Tween, DMWf18-558 remain to keep compared with
Strongly active (>50%；Fig. 7).
Carboxy-lesterase DMWf18-558 activity thermal stability determination concrete operations be：Enzyme is placed in 10,20,30,40,50
With 60 DEG C at be incubated 1h, determine enzyme activity.Live body system is：100mM Tricine buffer solutions (pH 9.0), 1mM is to nitro
Phenol capronate, 48ng pure enzyme proteins, METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 25 DEG C4052min.Measurement result shows, carboxy-lesterase
DMWf18-558 is incubated 1h below 20 DEG C, can keep more than 70% remnant enzyme activity；1h is incubated in 30 DEG C, 50% can be kept
Above remnant enzyme activity (Fig. 8).
Claims (10)
1. a kind of carboxy-lesterase, is the protein with following (1) or (2) feature：
(1), its amino acid sequence is consistent with sequence shown in Seq ID NO.2；
(2), the amino acid sequence away from the amino acid position of 142-146,238 and 268 shown in SEQ ID NO.2 is carried out various
Replace, add and/or lack one or several amino acid after obtain and can retain substantially the derivative of crude protein biological function
Protein.
2. carboxy-lesterase according to claim 1, it is characterised in that：The described derived protein with Carboxylesterase Activity
Matter has the homology at least with the amino acid sequence more than 90% shown in SEQ ID NO.2.
3. the gene of carboxy-lesterase described in claim 1 is encoded, and its nucleotide sequence is as shown in SEQ ID NO.1；Or be to SEQ
Other nucleotides in nucleotide sequence shown in ID NO.1 in addition to the nucleotides of 424-438,712-714 and 802-804 position are carried out
Replace, add and/or lack one or several nucleotides and can retain substantially the work of carboxy-lesterase protein biological so as to obtain coding
The mutant gene of property.
4. gene according to claim 3, it is characterised in that：Described carboxy-lesterase mutant gene have at least with
SEQ ID NO:The homology of the nucleotide sequence more than 90% shown in 1.
5. the carrier of gene described in any one of claim 3-4 is carried.
6. carrier according to claim 5, it is characterised in that：Described carrier is selected from pET serial carriers, and pQE series is carried
Body, Yeast expression carrier pPICZ- α-A, pHIL-D2, pPIC9, pHIL-S1, animal cell expression vectors pSVK3 or pMSG.
7. a kind of host, its carrier described in any one of claim 5-6 is inverted or transfected prokaryotic is biological or eucaryote
Host obtains.
8. host according to claim 7, which is bacterium, yeast or mammalian cell.
9. the Host Strains that can express carboxy-lesterase described in carboxy-lesterase described in claim 1 or claim 7 are in catalysis ester
Application in class hydrolysis.
10. application according to claim 9, it is characterised in that described esters are short-chain aliphatic ester.
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| CN105505898A (en) * | 2015-12-28 | 2016-04-20 | 国家海洋局第二海洋研究所 | Carboxylesterase sourcing from deep sea, and coding gene and application thereof |
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| CN113444705A (en) * | 2021-06-29 | 2021-09-28 | 武汉华美生物工程有限公司 | High-purity and high-activity Ces1C protein, and expression vector and application thereof |
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