CN101659960A - Biological preparation method of chitin deacetylase - Google Patents
Biological preparation method of chitin deacetylase Download PDFInfo
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- CN101659960A CN101659960A CN200910054879A CN200910054879A CN101659960A CN 101659960 A CN101659960 A CN 101659960A CN 200910054879 A CN200910054879 A CN 200910054879A CN 200910054879 A CN200910054879 A CN 200910054879A CN 101659960 A CN101659960 A CN 101659960A
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Abstract
The invention discloses a biological preparation method of chitin deacetylase derived from racemomucor, which comprises the following steps: (a) preparing a recombinant expression vector of the chitindeacetylase containing a gene sequence such as SEQ ID NO.1; (b) preparing a transformant containing the recombinant expression vector in the step (a); and (c) expressing and purifying the chitin deacetylase. In the invention, a gene specificity degenerate primer is designed, a reverse transcription and polymerase chain reaction and a rapid amplification technology of cDNA segment ends are adopted, and escherichia coli strains which can generate the chitin deacetylase are obtained by combining the DNA recombination method and utilizing the known pronucleus express vector. The invention provides a reliable source for biologically preparing the chitin deacetylase and has the advantages of short production period, low extraction cost, higher product enzyme yield, higher output, and the like.
Description
Technical field
The present invention relates to the enzyme engineering field of biotechnology, relate more specifically to utilize engineered means to obtain to produce the method that the chitin deacetylase engineering bacteria prepares acetyl enzyme.
Background technology
Chitin is a kind of polymer substance, is only second to Mierocrystalline cellulose at natural content, nearly 2,000 ten thousand tons of global shrimp and crab shells annual production.Chitin is water insoluble, diluted acid, diluted alkaline and other organic solvents, has limited its range of application to a great extent.What application was maximum at present is the acetyl derivative that takes off---the chitosan of chitin.
But; the process that the chitin deacetylate becomes chitosan must possess the condition of concentrated base (more than at least 40%) and comparatively high temps (more than 90 ℃); this has not only proposed very high requirement to production unit, and has run into the difficult problem of " how the concentrated base in the production process is handled ".The chitin output of China has surpassed 4000 tons of high pointes according to statistics, just arises at the historic moment at the research of chitin deacetylase to existing implacable contradiction between the great demand of chitin and the severe contamination in the chitin production in market.
Chitin deacetylase (chitin deacetylase; EC3.5.1.41) be the enzyme that a kind of catalysis chitin molecule removes ethanoyl, formation chitosan; this kind of enzyme is applied to the preparation of chitosan; not only can solve the problem of environmental pollution in the present chitosan production effectively; and can produce that present chemical method can not be produced, as to be applicable to special industry (medicine, environmental protection etc.) demand chitosan product, as the ethanoyl degree evenly, the chitosan product of molecular weight ranges narrowly distributing and Chitosan poly oligosaccharide etc. with specific ethanoyl position.But because this enzyme ubiquity not in organic sphere, so its source problem that comes is perplexing its application always.Around this problem, main progress is embodied in following aspect:
The chitin deacetylase of 1 originated from fungus
Yoshio in 1974 have at first found the existence of chitin deacetylase at first in the Lu Shi of zygomycetes Mucor, Kafezopulos has adopted ammonium sulfate precipitation, three gel chromatography bonded methods to separate and obtained electrophoretically pure chitin deacetylase after 20 years.After, detected the active existence of chitin deacetylase the microorganism such as investigators are mould from colter successively again, Aspergillus nidulans, bread mould, scopulariopsis brevicaulis, Cunninghamella bertholletiae and cereuisiae fermentum.
The chitin deacetylase of 2 bacterial origins
The yellow intelligent jasmine of Chinese scholar etc. has been found chitin deacetylase base enzymic activity in subtilis, and its enzymatic property is studied, and finds similar to the zymologic property of originated from fungus.
The chitin deacetylase in 3 pathogenic agent source
Nineteen ninety Japanology person in the Anthracnose Pathogen bacterium early discovery chitin deacetylase, and obtain the higher enzyme of purity with biochemical isolation technique separation, the enzyme of its zymologic property and originated from fungus has than big-difference.2006, the investigator again in pathogenic bacteria invasion and attack Ah clothing Palestine and China confirmed the existence of this enzyme.
Though chitin deacetylase is present in some organisms, its content all is very little.Rely on traditional biochemical technology, separate the main process that obtains chitin deacetylase and be: from mycelium or the nutrient solution of specific microorganism, adopt the isolating way of high speed centrifugation to obtain to contain the crude extract of chitin deacetylase, recombinant is used some kinds of column chromatography separation methods, to obtain the higher enzyme product of purity.This process length consuming time, yield are low, only can satisfy breadboard needs.So,, provide a valid approach for producing chitin deacetylase in a large number, fast with the engineering bacteria of engineered this enzyme of method construction expression.
Make up the enzyme engineering bacterial strain and at first need the special genes fragment.The gene fragment of different biogenetic derivations often to gene can express, expression of gene form and expression of gene condition play crucial effects.Yet, in case obtain good enzyme engineering bacterial strain, and then adopt the method for fermentation to obtain biological enzyme, so not only can solve the problem of biological material source, can also simplify the biological enzyme purge process that wastes time and energy greatly.
In order further to enlarge the range of application of chitin and chitosan and to protect its intellecture property, this area presses for the source problem that comes that solves chitin deacetylase, is its prerequisite and prepare biological enzyme with engineered ways and means.At present, China does not still have any patent about the chitin deacetylase preparation method.
Summary of the invention
For the length consuming time, the yield that overcome traditional biochemical technology, separate to obtain chitin deacetylase are low, only can satisfy the shortcoming of breadboard needs, the purpose of this invention is to provide a kind of method that can prepare chitin deacetylase fast, in a large number.
The technical problem that will solve required for the present invention can be achieved through the following technical solutions:
A kind of biological preparation method of chitin deacetylase, it comprises the following steps:
(a) preparation contains the recombinant expression vector of gene order as chitin deacetylase as described in the SEQ ID NO.1;
(b) preparation contains in steps the transformant of (a) described recombinant expression vector;
(c) expression and purifying chitin deacetylase.
In one embodiment, described step (a) is as follows:
1. choosing the Mucor racemosus mycelium that is in logarithmic phase is material, therefrom extracted total RNA;
2. use universal primer T17AP to carry out reverse transcription reaction, with the degenerated primer of gene specific (5 '-GATGATGGMCCYAACTGYTC-3 ', M=A/C, Y=C/T) and 3 ' end of universal primer AP amplification gene; According to the 3 ' final word that obtains, and the primer of design gene specific (5 '-TCGGCTACTGTTTTAGTCAGG-3 '), synthetic 5 ' end, first chain cDNA and the tailing; And then utilization gene-specific primer (5 '-ACCGTCTTGAATACCACGCAT-3 ') and AP primer amplification obtain 5 ' terminal cDNA; Splice 5 ' and 3 ' terminal cDNA sequence, thereby obtain the full-length cDNA of Mucor racemosus chitin deacetylase;
3. adopt the primer of PCR to be:
Forward 5 '-GCGGATCCCTTACTTCTGGCACAATTTTAC-3 ',
Reverse 5 '-CCGCTCGAGTTAAGACAGTAAAGAACCAAG-3 ' is a template with the full-length cDNA, carries out the PCR reaction, collects the dna fragmentation that contains described gene order such as SEQ ID NO.1;
4. with step 3. the dna fragmentation and the expression vector of gained carry out the double digestion effect, connect with dna ligase then, obtain the recombinant expression vector of chitin deacetylase.
In one embodiment, used restriction endonuclease is Bam I and Xho I.
In one embodiment, described expression vector is pET28a (the plasmid size is 5369bp), with the fragment between the sequence 158-198bp of restriction enzyme BamI and the excision of XhoI double digestion.
In one embodiment, the recombinant expressed zone of described expression vector comprises 6 Histidine sequence labels.
In one embodiment, described transformant is an e. coli bl21.
Beneficial effect of the present invention:
The contriver is through extensive and deep research and test, be found to: than other several mould (head molds, aspergillus and mould), all can detect in nutrient solution of Mucor racemosus and the mycelium higher, for the deacetylase activity of chitin crystal substrate, so wherein must contain encoding gene.The inventor is according to the general character of some other deacetylase gene of having found, designed the degenerated primer of gene specific, in conjunction with reverse transcription-polymerase chain reaction and rapid amplifying cDNA fragment end technology, from the total RNA of Mucor racemosus, caught the full-length cDNA of this gene.
The present invention has obtained (totally 2 of chitin deacetylase genes first from Mucor racemosus, be respectively Cda1 and Cda2), and preferably one is Cda2, and then obtained the recombinant expression vector of this gene, at the prokaryotic hosts e. coli expression, obtained chitin deacetylase.
The present invention designs the gene specific degenerated primer, adopt reverse transcription-polymerase chain reaction and rapid amplifying cDNA fragment end technology, in conjunction with the method for recombinant DNA, utilize existing prokaryotic expression carrier, obtained producing the coli strain of chitin deacetylase.For preparing chitin deacetylase, biology provides reliable source.
At home first at the prokaryotic cell prokaryocyte e. coli expression chitin deacetylase gene in Mucor racemosus source.Compare with traditional method of in organism, extracting, purifying obtains enzyme, have with short production cycle, extraction cost is low, product enzyme yield and output is than advantages such as height.
The inventor has further improved the expression efficiency of chitin deacetylase gene in intestinal bacteria through condition optimizing.
The present invention is directed to the inclusion body protein matter of sex change, realized the liquid chromatography on-column refolding.And contrast with the recombinant protein of another chitin deacetylase gene (Cda1) of Mucor racemosus.
Description of drawings
Further specify the present invention below in conjunction with the drawings and specific embodiments.
Fig. 1 represents the chitin deacetylase core sequence is inserted the recombinant plasmid that forms behind the expression vector pET28a.(Kan represents the kalamycin resistance mark to the pET28a-Cda2 recombinant plasmid; Ori represents replication orgin; LacI represents the aporepressor encoding sequence; Cda2 represents the chitin deacetylase encoding sequence; F1origin represents transcription initiation site).
Embodiment
In order to make technique means of the present invention, creation characteristic, to reach purpose and effect is easy to understand,, further set forth the present invention below in conjunction with concrete diagram.Should be understood that these case study on implementation only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to ordinary method, unspecified technology belongs to routine techniques.
Gene that the present invention adopts and cDNA clone and gene recombination method are not particularly limited, and can adopt the molecule clone technology of biology field routine.The main method of the present invention comprises: the bacterial screening that sets out, cDNA clone, DNA reorganization, prokaryotic expression and protein purification scheme.
Experiment 1
(1) selects suitable chitin deacetylase source bacterial strain
Can adopt conventional method to cultivate mould, choosing the Mucor racemosus mycelium that is in logarithmic phase is material, therefrom extracted total RNA; (Mucor racemosus) is material with Mucor racemosus, adopt the potato glucose liquid nutrient medium, 28 ℃, 200rpm shaking table concussion cultivation, collecting the mycelium time is 20h, separate and collection mycelium and nutrient solution part, mycelium for collection obtains adopts Trizol method (the total RNA extraction agent of unique UNIQ-10 post box) extracted total RNA.As substrate, measure the enzyme activity of chitin deacetylase, with the chitin crystal of part deacetylation to select the higher starting strain of this enzymic activity (or unit of activity number).The inventor all can detect the higher deacetylase activity for chitin crystal substrate through discovering in nutrient solution of Mucor racemosus and the mycelium, therefore, be suitable as starting strain.
The comparison of chitin deacetylase in nutrient solution of table 1 Mucor racemosus and the mycelium
| Nutrient solution chitin deacetylase total activity * ((the dried mycelium of U/g) | Mycelium chitin deacetylase total activity * * ((the dried mycelium of U/g) | |
| Mucor racemosus | ?97.2±4.2 | ??13.0±1.2 |
| Head mold | ?33.9±2.1 | ??12.7±0.8 |
| Aspergillus | ?14.9±2.0 | ??6.60±0.18 |
| Mould | ?27.3±1.3 | ??2.81±1.08 |
Annotate: the * data are from logarithmic growth latter stage, and * * data are from latter stage stationary phase.
Experiment 2
(2) chitin deacetylase full length cDNA clone
Design serial gene specific degenerated primer, obtain the full-length cDNA-Cda2 of chitin deacetylase gene with the method for reverse transcription-polymerase chain reaction and rapid amplifying cDNA end;
According to rapid amplifying cDNA end method, use universal primer T17AP to carry out reverse transcription reaction, with the degenerated primer of gene specific (5 '-GATGATGGMCCYAACTGYTC-3 ', M=A/C, Y=C/T) and 3 ' end of universal primer AP amplification gene; According to the 3 ' final word that obtains, and the primer of design gene specific (5 '-TCGGCTACTGTTTTAGTCAGG-3 '), synthesize 5, hold first chain cDNA and the tailing; And then utilization gene-specific primer (5 '-ACCGTCTTGAATACCACGCAT-3 ') and AP primer amplification obtain 5 ' terminal cDNA; Splice 5 ' and 3 ' terminal cDNA sequence, thereby obtain the full length cDNA sequence-Cda2 of Mucor racemosus chitin deacetylase, this cDNA total length is 1355bp, and described nucleotides sequence is identical with SEQ ID NO.2.
Correctly the clone obtains the key that the chitin deacetylase full-length cDNA is a construction of expression vector.The inventor is through research, the degenerated primers (being positioned at the gene middle part) that has synthesized gene specific, adopt molecule clone technology commonly used in the molecular biology, " angling " is to 2 chitin deacetylase full-length cDNAs, for necessary base has been established in follow-up work from the Mucor racemosus RNA storehouse of logarithmic phase.
One of full-length cDNA of Mucor racemosus source chitin deacetylase (being Cda2) and speculating acid sequence thereof.Mucor racemosus chitin deacetylase full-length cDNA-Cda2 and speculating acid sequence thereof, 67-1257bp is recombinant expressed used core sequence in the accompanying drawing, described nucleotides sequence is identical with SEQ ID NO.1.
Experiment 3
(3) DNA reorganization
Can adopt conventional DNA recombinant technology.But,,, use for the connection of DNA reorganization so need to use round pcr that required core sequence is separated owing to include 5 ' end and 3 ' end non-coding sequence and possible signal peptide sequence in the full length cDNA sequence.
With the method for PCR, intercepting genetic expression core sequence fragment from cDNA, and in two ends importing specific restriction enzyme action site and protection base; Use the fragment of BamI and XhoI restriction enzyme gained to carry out double digestion.
The primer of used PCR:
Forward primer CDA2-F 5 '-GCGGATCCCTTACTTCTGGCACAATTTTAC-3 '
Reverse primer CDA2-R 5 '-CCGCTCGAGTTAAGACAGTAAAGAACCAAG-3 '
Pcr amplification system (25 μ L): total DNA 2 μ L
10×buffer????????2.5μL
Forward primer CDA2-F 1 μ L
Reverse primer CDA2-R 1 μ L
dNTP??????????????1μL
Taq enzyme 0.5 μ L
ddH
2O?????????????17μL
??????????????????????????????
Cumulative volume 25 μ L
PCR response procedures: 94 ℃ of pre-sex change 4min; 94 ℃ of sex change 30s, 53 ℃ of renaturation 45s, 72 ℃ are extended 1.5min, 35 circulations; 72 ℃ postpone 10min.
The genetic expression core sequence that cuts from full-length cDNA is 1191bp, sees SEQ ID NO.1.
As shown in Figure 1, the recombinant plasmid that forms behind the expression vector pET28a is inserted the chitin deacetylase core sequence in expression.(Kan represents the kalamycin resistance mark to the pET28a-Cda2 recombinant plasmid; Ori represents replication orgin; LacI represents the aporepressor encoding sequence; Cda2 represents the chitin deacetylase encoding sequence; Fl origin represents transcription initiation site).The expression vector of selecting for use is pET28a (the plasmid size is 5369bp), with the fragment between the sequence 158-198bp of restriction enzyme BamI and the excision of XhoI double digestion.
Fragment with above-mentioned gained connects with dna ligase, obtains the recombinant expression plasmid of chitin deacetylase; The recombinant plasmid of gained is total up to 6526bp, and recombinant expressed zone comprises 6 Histidine sequence labels.
Experiment 4
(4) prokaryotic expression
Can adopt conventional e. coli bl21 system and pET carrier series.The establishment of optimum expression condition.
Recombinant expression plasmid is transformed into the defective e. coli host bacteria; Described defective type host bacterium is an e. coli bl21.
Made up and finished the pET28a plasmid that inserts the Cda2 gene, be transformed into e. coli bl21.As cultivating and the primary condition of abduction delivering, successively the temperature (16 ℃, 20 ℃, 25 ℃, 30 ℃, 37 ℃ and 42 ℃) when inducing, inductor IPTG concentration (0.05,0.1,0.5 and 1mM) and induction time (1,2,3 and 4h) have carried out optimization research with the LB substratum that contains kantlex.
The result as can be known, when culture temperature is that 37 ℃, IPTG concentration are 0.1mM, when inducing the back to express 1h, scan demonstration according to SDS-PAGE result, the chitin deacetylase of expression has accounted for more than 30% of total bacterial protein.
Experiment 5
(5) protein purification
For partly soluble chitin deacetylase, can adopt conventional affinity chromatography to separate.For inclusion body protein matter, can adopt conventional protein denaturation-renaturation technological line to separate.In another preference, the inventor has carried out a large amount of experiments to different protein denaturations-renaturation scheme, explores to have drawn and utilizes liquid phase chromatography the sex change solubilising protein to be carried out the protein purification scheme of on-column refolding.
The inclusion body purifying recombinant proteins:
Made up and finished the pET28a plasmid that inserts the Cda2 gene, be transformed into e. coli bl21.Culture temperature is that 37 ℃, IPTG concentration are 0.1mM, collect somatic cells after inducing back expression time 2h.With somatic cells fragmentation, washing with separate, treat sample after the dissolving, (the gradient volume uses 30ml to carry out refolding with 6-0M urea linear gradient on the Ni-Sepharose of AKTA fast protein liquid chromatography post, flow velocity is 0.5ml/min), (the gradient volume is 10ml with the target protein of 10-500mM imidazoles linear gradient elution refolding, flow velocity is 1ml/min), obtain electrophoretically pure chitin deacetylase.
The result shows that the protein vigor that recombinant C da2 gene obtains is higher than the protein that recombinant C da1 gene obtains, and in resulting two chitin deacetylase gene fragments, Cda2 is more suitable for the structure in recombinant vectors in Mucor racemosus in confirmation.
At home first at the prokaryotic cell prokaryocyte e. coli expression chitin deacetylase gene in Mucor racemosus source.Compare with traditional method of in organism, extracting, purifying obtains enzyme, have that growth cycle is short, extraction cost is low, product enzyme yield and output is than advantages such as height.
The inventor has further improved the expression efficiency of chitin deacetylase gene in intestinal bacteria through condition optimizing.
The present invention is directed to the inclusion body protein matter of sex change, realized the liquid chromatography on-column refolding.And contrast with the recombinant protein of another chitin deacetylase gene (Cda1) of Mucor racemosus.
Present method and routine biochemistry preparation method's comparison the results are shown in Table 2.
Table 2 present method and routine biochemistry preparation method's comparison
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that describes in the foregoing description and the specification sheets just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Attached: dna nucleotide sequence involved in the present invention is as follows
SEQUENCE?LISTING
＜110〉Shanghai Ocean University
＜120〉biological preparation method of chitin deacetylase
＜130〉specification sheets sequence table
<160>2
<170>PatentIn?version?3.3
<210>1
<211>1191
<212>DNA
<213>Mucor?racemosus
<400>1
cttactttct?ggcacaattt?tactacccaa?attgatccca?aaaacgtgtc?tgtaccttcg???60
attactcaaa?ccacaagcat?caatccctct?gaagaatgtg?tttattatag?ccctgatacc??120
tcattagttg?atatcgtctc?tgctgaatgg?cctactagct?ggcaaaaagc?tactaccaac??180
ggaatgaatg?aaacagctga?attcattgcc?atgtataact?ctattgattg?gaccaaggct??240
cccaatattc?cagtgcgtac?tctgcttgct?gacggttctt?taaacatgac?aggctattct??300
tcctcttctg?atcctgattg?ctggtggtct?gctagtggtt?gtactaagcc?caaaatttct??360
gacgcctata?cagatctggt?tcagtgtgca?gaacctgaga?cttggggctt?gacttacgat??420
gatggaccca?actgctctca?taacgctttt?tatgattacc?ttgaagagca?aaaattaaag??480
gctaccatgt?tttacattgg?ttctaatgtt?atcgattggc?cttatggtgc?tatgcgtggt??540
attcaagacg?gtcatcacat?tgcttgccac?acctggtctc?accctatgat?aactacattc??600
acaaaccaag?aagtgttggc?tgaattctat?ttctctttaa?aagctatcaa?actggccact????660
ggtttaacac?ctcgttattg?gcgccctcct?tatggtgata?ttgatgaccg?tgttcgttgg????720
attgctgctc?aactgggttt?gactgctgtt?atgtggaact?tggatacaga?cgattgggct????780
gctggcctga?ctaaaacagt?agccgatgtc?caaactgctt?atgatcaatt?cattcaaatg????840
ggcaagaacg?gtaccttcgc?tggtagtggt?aacattgtct?tggcgcatga?gatcaataat????900
gaaaccatga?gcctggccat?tcaaaatttg?cctagtattc?aagctgctta?caagcaagtc????960
attaatgtcg?caacttgttt?taatatatct?caaccttatt?tcgaagatta?tacatggttg???1020
gatgtattga?atggaacagc?tgcttcttct?actgccgtta?gtggagttcc?atctgctcag???1080
tctgcagcca?agtcaacctc?taccgacgcc?caatcttccg?atgctagcag?caatgctatt???1140
agttctatct?ttgctttgtt?tgcagtagct?cttggttctt?tactgtctta?a????????????1191
<210>2
<211>1355
<212>DNA
<213>Mucor?racemosus
<400>2
atgcaaatca?agacactggc?agcattgatt?gctatagctc?aaatagcaaa?tgaagtcgct?????60
gctgatctta?ctttctggca?caattttact?acccaaattg?atcccaaaaa?cgtgtctgta????120
ccttcgatta?ctcaaaccac?aagcatcaat?ccctctgaag?aatgtgttta?ttatagccct????180
gatacctcat?tagttgatat?cgtctctgct?gaatggccta?ctagctggca?aaaagctact????240
accaacggaa?tgaatgaaac?agctgaattc?attgccatgt?ataactctat?tgattggacc????300
aaggctccca?atattccagt?gcgtactctg?cttgctgacg?gttctttaaa?catgacaggc????360
tattcttcct?cttctgatcc?tgattgctgg?tggtctgcta?gtggttgtac?taagcccaaa????420
atttctgacg?cctatacaga?tctggttcag?tgtgcagaac?ctgagacttg?gggcttgact????480
tacgatgatg?gacccaactg?ctctcataac?gctttttatg?attaccttga?agagcaaaaa????540
ttaaaggcta?ccatgtttta?cattggttct?aatgttatcg?attggcctta?tggtgctatg????600
cgtggtattc?aagacggtca?tcacattgct?tgccacacct?ggtctcaccc?tatgataact????660
acattcacaa?accaagaagt?gttggctgaa?ttctatttct?ctttaaaagc?tatcaaactg?????720
gccactggtt?taacacctcg?ttattggcgc?cctccttatg?gtgatattga?tgaccgtgtt?????780
cgttggattg?ctgctcaact?gggtttgact?gctgttatgt?ggaacttgga?tacagacgat?????840
tgggctgctg?gcctgactaa?aacagtagcc?gatgtccaaa?ctgcttatga?tcaattcatt?????900
caaatgggca?agaacggtac?cttcgctggt?agtggtaaca?ttgtcttggc?gcatgagatc?????960
aataatgaaa?ccatgagcct?ggccattcaa?aatttgccta?gtattcaagc?tgcttacaag????1020
caagtcatta?atgtcgcaac?ttgttttaat?atatctcaac?cttatttcga?agattataca????1080
tggttggatg?tattgaatgg?aacagctgct?tcttctactg?ccgttagtgg?agttccatct????1140
gctcagtctg?cagccaagtc?aacctctacc?gacgcccaat?cttccgatgc?tagcagcaat????1200
gctattagtt?ctatctttgc?tttgtttgca?gtagctcttg?gttctttact?gtcttaatta????1260
agtctcatct?tttgatgatc?tccatttcat?aacacgtaaa?tcaataaagc?tcttattttt????1320
tatcctcgtt?tagacgtttc?aaaaaaaaaa?aaaaa???????????????????????????????1355
Claims (6)
1, a kind of biological preparation method of chitin deacetylase comprises the following steps:
(a) preparation contains the recombinant expression vector of gene order as chitin deacetylase as described in the SEQ ID NO.1;
(b) preparation contains in steps the transformant of (a) described recombinant expression vector;
(c) expression and purifying chitin deacetylase.
2, biological preparation method according to claim 1 is characterized in that described step (a) is as follows:
1. choosing the Mucor racemosus mycelium that is in logarithmic phase is material, therefrom extracted total RNA;
2. use universal primer T17AP to carry out reverse transcription reaction, with the degenerated primer 5 '-GATGATGGMCCYAACTGYTC-3 ' of gene specific (M=A/C, Y=C/T) and 3 ' end of universal primer AP amplification gene; According to the 3 ' final word that obtains, the primer 5 '-TCGGCTACTGTTTTAGTCAGG-3 ' of design gene specific, synthetic 5 ' end, first chain cDNA and the tailing; And then use gene-specific primer 5 '-ACCGTCTTGAATACCACGCAT-3 ' and AP primer amplification to obtain 5 ' terminal cDNA; Splice 5 ' and 3 ' terminal cDNA sequence, thereby obtain the full-length cDNA of Mucor racemosus chitin deacetylase;
3. adopt the primer of PCR to be:
Forward 5 '-GCGGATCCCTTACTTCTGGCACAATTTTAC-3 ',
Reverse 5 '-CCGCTCGAGTTAAGACAGTAAAGAACCAAG-3 ' is a template with the 2. described full-length cDNA of step, carries out the PCR reaction, collects the dna fragmentation that contains described gene order such as SEQ ID NO.1;
4. with step 3. the dna fragmentation and the expression vector of gained carry out the double digestion effect, connect with dna ligase then, obtain the recombinant expression vector of chitin deacetylase.
3, biological preparation method according to claim 2 is characterized in that described restriction endonuclease is BamI and Xho I.
4, biological preparation method according to claim 2 is characterized in that described recombinant expression vector is pET28a.
5, biological preparation method according to claim 1 is characterized in that the expression zone of described recombinant expression vector comprises 6 Histidine sequence labels.
6, biological preparation method according to claim 1 is characterized in that described transformant is an e. coli bl21.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642763A (en) * | 2013-11-16 | 2014-03-19 | 江南大学 | Cloning and analysis of P450 enzyme gene for 7alpha, 15alpha-dihydroxylation of DHEA |
| CN104109636A (en) * | 2014-06-30 | 2014-10-22 | 浙江树人大学 | Aspergillus versicolor SD-3 and its application in preparation of chitin deacetylase |
| CN109609485A (en) * | 2019-01-02 | 2019-04-12 | 吉林中粮生化有限公司 | A kind of chitin deacetylase and its application |
-
2009
- 2009-07-16 CN CN200910054879A patent/CN101659960A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642763A (en) * | 2013-11-16 | 2014-03-19 | 江南大学 | Cloning and analysis of P450 enzyme gene for 7alpha, 15alpha-dihydroxylation of DHEA |
| CN103642763B (en) * | 2013-11-16 | 2015-09-30 | 江南大学 | A kind of 7 α, the clone of 15 α dihydroxylation DHEA P450 enzyme genes and analysis |
| CN104109636A (en) * | 2014-06-30 | 2014-10-22 | 浙江树人大学 | Aspergillus versicolor SD-3 and its application in preparation of chitin deacetylase |
| CN109609485A (en) * | 2019-01-02 | 2019-04-12 | 吉林中粮生化有限公司 | A kind of chitin deacetylase and its application |
| CN109609485B (en) * | 2019-01-02 | 2022-06-28 | 吉林中粮生化有限公司 | Chitin deacetylase and application thereof |
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