CN107586766A - A kind of bacillus amyloliquefaciens chitosan enzyme and its preparation method and application - Google Patents
A kind of bacillus amyloliquefaciens chitosan enzyme and its preparation method and application Download PDFInfo
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- CN107586766A CN107586766A CN201711013149.3A CN201711013149A CN107586766A CN 107586766 A CN107586766 A CN 107586766A CN 201711013149 A CN201711013149 A CN 201711013149A CN 107586766 A CN107586766 A CN 107586766A
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- chitosan
- bacillus amyloliquefaciens
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Abstract
The invention discloses a kind of bacillus amyloliquefaciens chitosan enzyme and its preparation method and application.The present invention is according to Pichia pastoris codon-bias, the chitosan enzyme coding gene sequence in bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is obtained using full genome synthetic method, the nucleotide sequence after optimization is as shown in SEQ ID NO.2.Efficient secretory expression further is carried out to the chitosan enzyme encoding gene of optimization this described using pichia yeast expression system, obtains bacillus amyloliquefaciens chitosan enzyme, its amino acid sequence is as shown in SEQ ID NO.1.The chitosan enzyme that the present invention obtains has higher hydrolysing activity to chitosan substrate, crude enzyme liquid caused by shake flask fermentation is the hydrolysis ability with 1mL (protein content about 0.23mg) degraded 5g chitosans, and same amount of chitosan of degrading about needs alpha amylase 150mg, efficiency improves more than 600 times in theory；With good prospects for commercial application.
Description
Technical field
The invention belongs to chitosan enzyme technical field, and in particular to a kind of bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens) chitosan enzyme and its preparation method and application.
Background technology
Chitosan enzyme (Chitosanases, EC.3.2.1.132) is widely present in ancient bacterium, bacterium and eucaryote,
It is distributed in glycoside hydrolase (Glycoside Hydrolases, GH) family 3,5,7,8,46,75 and 80, wherein, family
46th, chitosan enzyme is only included in 75 and 80.Industrially, due to the specific chitosan enzyme of shortage economical and efficient, often use
The non-specific commodity enzyme hydrolyzing chitosan such as protease and cellulase is to prepare chitosan oligosaccharide.Due to being hydrolyzed with chitosan enzyme
The enzyme of activity proportion in these commercial enzymes is extremely low, and larger with enzyme amount, the production cost of chitosan oligosaccharide also accordingly increases.Cause
This, there is an urgent need to develop a series of chitosan enzyme of economical and efficients to meet the needs of industrial chitosan oligosaccharide produces.
The content of the invention
It is an object of the present invention to provide a kind of bacillus amyloliquefaciens chitosan enzyme and its preparation method and application；It is intended to
A kind of specific chitosan enzyme of economical and efficient is provided.
Present invention technical scheme used to achieve the above object is as follows：
The present invention by being optimized to the codon of bacillus amyloliquefaciens chitosan enzyme (GH46 families) encoding gene,
So that it realizes secreting, expressing in Pichia pastoris, so as to efficiently quickly obtain the high bacillus amyloliquefaciens of hydrolysing activity
Chitosan enzyme.
A kind of bacillus amyloliquefaciens chitosan enzyme provided by the invention, its amino acid sequence is as shown in SEQ ID NO.1：
SEQ ID NO.1：
LEKREAEAAGLNKDQKRRAEQLTSIFENGKTEIQYGYVEALDDGRGYTCGRAGFTTATGDALEVVEVYT
KAVPNNKLKKY
LPELRRLAKDESDDISNLKGFASAWRSLGNDKAFRAAQDKVNDSLYYQPAMERSENAGLKTALAKAVMY
DTVIQHGDGDD
PDSFYALIKRTNKKMGGSPKDGTDEKKWLNKFLDVRYDDLMNPSDEDTQDEWRESVARVDVFRDIVKEK
NYNLNGPIHVR
SSEYGNFTIQ。
Present invention also offers a kind of bacillus amyloliquefaciens chitosanase gene, above-mentioned bacillus amyloliquefaciens are encoded
Chitosan enzyme.Preferably, the nucleotide sequence of the bacillus amyloliquefaciens chitosanase gene is as shown in SEQ ID NO.2：
SEQ ID NO.2：
ctcgagaagagagaggctgaggctgctggtttgaacaaggaccaaaagagaagagctgagcaattgacttccatctt
cgagaacggtaagactgagatccaatacggttacgttgaggctttggacgacggtagaggttacacttgtggtagag
ctggtttcactactgctactggtgacgctttggaggttgttgaggtttacactaaggctgttccaaacaacaagttg
aagaagtacttgccagagttgagaagattggctaaggacgagtccgacgacatctccaacttgaagggtttcgcttc
cgcttggagatccttgggtaacgacaaggctttcagagctgctcaagacaaggttaacgactccttgtactaccaac
cagctatggagagatccgagaacgctggtttgaagactgctttggctaaggctgttatgtacgacactgttatccaa
cacggtgacggtgacgacccagactccttctacgctttgatcaagagaactaacaagaagatgggtggttccccaaa
ggacggtactgacgagaagaagtggttgaacaagttcttggacgttagatacgacgacttgatgaacccatccgacg
aggacactcaagacgagtggagagagtccgttgctagagttgacgttttcagagacatcgttaaggagaagaactac
aacttgaacggtccaatccacgttagatcctccgagtacggtaacttcactatccaataagcggccgcg。
Present invention also offers the recombinant expression carrier for including above-mentioned bacillus amyloliquefaciens chitosanase gene.
Preferably, the expression vector is pGBG1, and expression vector pGBG1 is obtained in the following way：
By carrying out codon optimization to the signal peptide sequence (referring to shown in SEQ ID NO.4) in expression vector pPIC9,
The new signal peptide sequence (referring to shown in SEQ ID NO.5) for being adapted to express in Pichia pastoris is obtained, and utilizes Nsi I/Xho
I double digestions substitute the signal peptide sequence shown in SEQ ID NO.5 signal peptide shown in original SEQ ID NO.4, are expressed
Carrier pGBG1.Expression vector pGBG1 can make destination protein secreting, expressing more efficient in Pichia pastoris.
Wherein, expression vector pPIC9 and Pichia pastoris GS115 is commercially produced product.
The preparation method of bacillus amyloliquefaciens chitosan enzyme of the present invention is：By above-mentioned recombinant expression carrier
(for example, chitosanase gene of the nucleotide sequence as shown in SEQ ID NO.2 is built into expression vector) imports Pichia pastoris
Cell, induce and obtain the chitosan enzyme of secreting, expressing.Preferably, the Pichia pastoris is GS115.
Further, the specific preparation process of bacillus amyloliquefaciens chitosan enzyme of the present invention includes：(1) according to complete
The Preference that red yeast codons use, password is carried out to the chitosanase gene original series from bacillus amyloliquefaciens
Son optimization；(2) gene after optimization is carried out fully synthetic and built into expression vector pGBG1；(3) to the expression vector of structure
After carrying out linearization for enzyme restriction, give up the fragment containing resistant gene, reclaim the fragment containing chitosanase gene and import complete red
In yeast cells GS115, induce and obtain the chitosan enzyme of secreting, expressing.
Chitosan substrate is hydrolyzed using the chitosan enzyme of induced expression thick enzyme supernatant and uses MALDI-TOF mass spectrums
Method is analyzed the degree of polymerization and composition of product.As a result show, bacillus amyloliquefaciens chitosan enzyme of the present invention
The degraded that chitosan can be individually used for prepares chitosan oligosaccharide；Or the bacillus amyloliquefaciens chitosan enzyme is with removing solution starch gemma
Other chitosan enzymes or chitinase outside bacillus chitosan enzyme are used in mixed way, Synergistic degradation chitosan or chitin.
Present invention also offers application of the above-mentioned bacillus amyloliquefaciens chitosan enzyme in chitosan or chitin degrading.
Preferably, bacillus amyloliquefaciens chitosan enzyme is individually used for degradation of chitosan and prepares chitosan oligosaccharide；Or the solution starch
Bacillus chitosan enzyme is used in mixed way with other chitinases or chitosan enzyme, Synergistic degradation chitosan or chitin.
Screen the enzyme system with chitosan hydrolyzate enzymatic activity present inventor's early stage in numerous food level commercial enzyme
Found during agent, alpha-amylase has preferable chitosan hydrolyzate active (the thick enzyme dry powder of 1g about can thoroughly hydrolyze 30g chitosans).I
Speculate, it may be possible to the production strain of alpha-amylase, such as bacillus amyloliquefaciens or bacillus subtilis, during the fermentation
Generate chitosan enzyme.Therefore, it is used for degrade chitosan or chitin to obtain safer efficient chitosan enzyme, we choose
One chitosan enzyme for belonging to the family of glycoside hydrolase 46 from bacillus amyloliquefaciens, according to the inclined of Pichia pastoris codon
Good property, its encoding gene is obtained by full genome synthetic method and secreting, expressing is carried out in Pichia pastoris.The chitosan of expression
Enzyme can substitute the large-scale production that existing commodity alpha-amylase is used for chitosan oligosaccharide or chitin oligo saccharide etc..
Compared with prior art, beneficial effects of the present invention are：
1. the chitosanase gene of the present invention derives from bacillus amyloliquefaciens, pichia pastoris phaff expression system is used
Secreting, expressing.Bacillus amyloliquefaciens have been used for the system of food enzyme preparation alpha-amylase, 1,4 beta-glucanase, protease etc.
Standby, the enzyme security in its source is secure, and pichia pastoris phaff (Pichia pastoris) expression system also by with
In the expression of the food-grade enzyme preparation such as lactase and phospholipase C, its own is also used for the production (GB2760- of zytase
2014).Therefore, using the chitosanase gene in bacillus amyloliquefaciens source in Pichia pastoris secreting, expressing, its product shell
Dextranase can turn into the production enzyme preparation of food-grade chitosan oligosaccharide or chitin oligo saccharide.
2. the present invention is obtained using full genome synthetic method according to Pichia pastoris codon-bias and understands starch gemma bar
Chitosan enzyme coding gene sequence in bacterium (Bacillus amyloliquefaciens), the nucleotide sequence such as SEQ after optimization
Shown in ID NO.2.Further the chitosan enzyme encoding gene of optimization this described is carried out using pichia yeast expression system efficient
Secreting, expressing, bacillus amyloliquefaciens chitosan enzyme is obtained, its amino acid sequence is as shown in SEQ ID NO.1.The present invention obtains
Chitosan enzyme have higher hydrolysing activity to chitosan substrate, through determination of activity and conversion, crude enzyme liquid caused by shake flask fermentation
There is the hydrolysis ability of 1mL crude enzyme liquids (protein content about 0.23mg) degraded 5g chitosans, and same amount of shell of degrading gathers
Sugar about needs alpha-amylase 150mg, and efficiency improves more than 600 times in theory.Therefore, compared with commodity alpha-amylase, the present invention
The chitosan enzyme efficiency of secreting, expressing is improved more than 600 times, and there is replacement existing goods enzyme to prepare the huge of chitosan oligosaccharide for scale
Big application potential, there is good prospects for commercial application.
Brief description of the drawings
Fig. 1 is the recombinant expression carrier bacsn-pGBG1 and its digestion products gel electrophoresis spectrum in the embodiment of the present invention；
Fig. 2 is the Pichia yeast engineering zymotic fluid of the chitosanase gene containing bacillus amyloliquefaciens in the embodiment of the present invention
The SDS-PAGE collection of illustrative plates of supernatant, wherein, arrow meaning is target product；
Fig. 3 is the high-efficient liquid phase chromatogram of chitosan oligosaccharide COS-94-BACSN in the embodiment of the present invention；
Fig. 4 is the MALDI-TOF mass spectrograms of chitosan oligosaccharide COS-62-BACSN in the embodiment of the present invention.
Embodiment
Technical scheme is described in detail with reference to embodiment.The reagent and biomaterial used below
If not otherwise specified, it is commercially produced product.Unreceipted actual conditions person in embodiment, suggest according to normal condition or manufacturer
Condition carry out.
The codon optimization of the chitosanase gene of embodiment 1 and full genome synthesis
On the premise of amino acid sequence is not changed, using the preference codon of Pichia pastoris, artificial optimization understands starch
Bacillus chitosan enzyme (GH46 families) (as shown in sequence SEQ ID NO.1, GenBank accession number:
ABS75305.1 encoding gene), specific nucleotide sequence are shown in SEQ ID NO.2.Nucleotide sequence after optimization is dived with original
In chitosan enzyme coding gene sequence, as shown in sequence SEQ ID NO.3, GenBank accession number:
CP000560.1 (3093420-3094256), homology highest, it is 74%.Gene order student on commission work after optimization carries out complete
Synthesis, the gene order for synthesizing acquisition are named as chitosanase gene bacsn.
The chitosanase gene bacsn of embodiment 2 expression vector establishment
The cloning vector containing chitosanase gene bacsn is carried out first by restriction enzyme Xho I and Not I
Double digestion, target gene fragment is obtained, while double digestion is carried out to expression vector pGBG1 using identical restriction endonuclease, recovery is big
Fragment.Two recovery products are attached, and are obtained recombinant vector, are named as bacsn-pGBG1.To determine target chitosan enzyme base
Because having been built up into carrier, we carry out double digestion and list using Xho I/Not I and Bgl II to the recombinant vector respectively
Digestion, and row agarose gel electrophoresis are entered to product, as a result as shown in Figure 1：After double digestion, between 750bp and 1000bp
There is target gene fragment, be consistent with bacsn fragment 762bp；After Bgl II digestions, there are expected two pieces
Section, is followed successively by the large fragment containing target gene and the small fragment containing resistant gene.
It is prepared by the screening of the chitosan enzyme Pichia yeast engineering of embodiment 3 and chitosan enzyme
By the recombinant plasmid bacsn-pGBG1 of acquisition after restriction enzyme BglII linearisations, gel electrophoresis separates simultaneously
The nucleotide fragments (larger fragment as shown in Figure 1) containing target gene are cut, electric shock is imported in Pichia pastoris GS115,
BMMY containing colloid chitosan (0.5%) will be laid in by the recon for screening to obtain on histidine auxotrophy MD flat boards
Cultivated on agar plate, therefrom filter out the maximum monoclonal bacterial strain of hydrolysis circle.By the single bacterium of the monoclonal bacterial strain of screening
Fall to be inoculated in 200mL BMGY culture mediums, cultivated 48 hours under 30 DEG C and 250rpm, supernatant is abandoned in centrifugation, adds the BMMY of equivalent
Carry out induced expression.Added after 24h methanol to its final concentration of 1%, added once every 24h later, altogether induce 120h after
Centrifugation, supernatant are the crude enzyme liquid containing chitosan enzyme (being designated as chitosan enzyme BACSN).Albumen table is detected using SDS-PAGE
It is as shown in Figure 2 up to situation, its result.Protein concentration in Bradford methods measure crude enzyme liquid is 0.23mg/mL；DNS methods are surveyed
Its fixed specific enzyme activity is 52.2U/mL.Above-mentioned MD agar plates, BMMY agar plates, BMGY culture mediums, BMMY culture mediums are ferment
The conventional culture medium of female expression system, it can directly buy or be prepared according to existing literature technology.
The chitosan enzyme BACSN of embodiment 4 hydrolysis chitosan with high deacetylation degree prepares chitosan oligosaccharide
Weigh 50g chitosan (deacetylations：94%), add in the acetic acid aqueous solutions of 1000mL 1.5%, pH 5-6.Fully
After dissolving, the chitosan enzyme BACSN crude enzyme liquids of 10mL fermentations are added, stirring reaction 48 hours at 40 DEG C.After reaction terminates, centrifugation
Not tolerant is removed, the rotated evaporimeter of supernatant is concentrated into about 300mL at 40 DEG C, by finished product chitosan oligosaccharide is freeze-dried to obtain, orders
Entitled COS-94-BACSN.The chitosan oligosaccharide COS-94-BACSN of a certain amount of preparation is weighed, it is the water-soluble of 20mg/mL to be configured to concentration
Liquid, efficient liquid phase chromatographic analysis to be used for after filtering.High performance liquid chromatograph connection EISD is used for chitosan oligosaccharide
Signal detection, chitosan oligosaccharide is separated using XAmide chromatographic columns (Hua Puxinchuan Science and Technology Ltd.s), acetonitrile concentration successively decreases
(70%-50%) mode elutes, and column temperature is 30 DEG C, detector air pressure 23psi, flow velocity：1mL/min.Mobile phase is 0.1M formic acid
Amine (pH3.2), acetonitrile and the aqueous solution.Elution time：40min.As a result as shown in figure 3, from figure 3, it can be seen that obtained shell is few
Sugared product is degree of polymerization 2-7 chitosan oligosaccharide.
The chitosan enzyme BACSN of embodiment 5 hydrolysis low deacetylation chitosans prepare chitosan oligosaccharide
Weigh 50g chitosan (deacetylations：62%), add in the acetic acid aqueous solutions of 1000mL 1.5%, (pH 5-6).Fill
Divide after dissolving, the chitosan enzyme BACSN crude enzyme liquids of addition 10mL fermentations, stirring reaction 48 hours at 40 DEG C.After reaction terminates, from
Heart removal not tolerant, the rotated evaporimeter of supernatant are concentrated into about 300mL at 40 DEG C, and finished product chitosan oligosaccharide life is obtained by being freeze-dried
Entitled COS-62-BACSN.Because the product component is complex, it is difficult to be effectively separated, therefore adopted by liquid relative composition
Its component is analyzed with MALDI-TOF mass spectrometry methods.Specific method is：The COS-62-BACSN of a certain amount of preparation is weighed,
The aqueous solution that concentration is 2mg/mL is configured to, is drawn after filtering in 1 μ L point samples to sample panel, after its natural drying, adds 1 μ L
Matrix DHB (DHB) solution, the smartbeam type MALDI-TOF matter of autoflex III is used after its drying
Spectrometer (Bruker companies) is detected (cation reflective-mode).Mass Spectrometer Method result is as shown in Figure 4：For ease of distinguishing, with A
Represent 2-Acetamido-2-deoxy-D-glucose, D represents Glucosamine, and subsequent digitized representation contains the number of the monose, the two add and
For the degree of polymerization of oligosaccharides.From the point of view of Fig. 4 result, low deacetylation chitosan oligosaccharide COS-62-BACSN components are more complicated：Mass spectrum side
Method can detect degree of polymerization 2-15 oligosaccharides, and the chitosan oligosaccharide of different acetyl degree under the same degree of polymerization be present.
It should be noted last that the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted.Although ginseng
The present invention is described in detail according to embodiment, it will be apparent to an ordinarily skilled person in the art that the technical side to the present invention
Case is modified or equivalent substitution, and without departure from the spirit and scope of technical solution of the present invention, it all should cover in the present invention
Right among.
Sequence table
<110>Chinese Academy Of Sciences Process Engineering Research Institute
<120>A kind of bacillus amyloliquefaciens chitosan enzyme and its preparation method and application
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180 185 190
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ttggacgacg gtagaggtta cacttgtggt agagctggtt tcactactgc tactggtgac 180
gctttggagg ttgttgaggt ttacactaag gctgttccaa acaacaagtt gaagaagtac 240
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ttcgcttccg cttggagatc cttgggtaac gacaaggctt tcagagctgc tcaagacaag 360
gttaacgact ccttgtacta ccaaccagct atggagagat ccgagaacgc tggtttgaag 420
actgctttgg ctaaggctgt tatgtacgac actgttatcc aacacggtga cggtgacgac 480
ccagactcct tctacgcttt gatcaagaga actaacaaga agatgggtgg ttccccaaag 540
gacggtactg acgagaagaa gtggttgaac aagttcttgg acgttagata cgacgacttg 600
atgaacccat ccgacgagga cactcaagac gagtggagag agtccgttgc tagagttgac 660
gttttcagag acatcgttaa ggagaagaac tacaacttga acggtccaat ccacgttaga 720
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aaggatcaga agcgccgggc ggaacagctg accagcatct ttgaaaacgg aaagacggaa 180
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Claims (9)
- A kind of 1. bacillus amyloliquefaciens chitosan enzyme, it is characterised in that：The amino of the bacillus amyloliquefaciens chitosan enzyme Acid sequence is as shown in SEQ ID NO.1.
- A kind of 2. bacillus amyloliquefaciens chitosanase gene, it is characterised in that：Encode the solution starch gemma described in claim 1 Bacillus chitosan enzyme.
- 3. bacillus amyloliquefaciens chitosanase gene according to claim 2, it is characterised in that：The nucleotides of the gene Sequence is as shown in SEQ ID NO.2.
- 4. include the recombinant expression carrier of bacillus amyloliquefaciens chitosanase gene described in Claims 2 or 3.
- 5. recombinant expression carrier according to claim 4, it is characterised in that：The expression vector is pGBG1, expression vector PGBG1 is obtained in the following way：The signal peptide sequence as shown in SEQ ID NO.4 in expression vector pPIC9 is subjected to codon optimization, acquisition is adapted to finishing The signal peptide sequence as shown in SEQ ID NO.5 expressed in red yeast；Using Nsi I/Xho I double digestions, the signal peptide sequence as shown in SEQ ID NO.4 in expression vector pPIC9 is replaced The signal peptide sequence as shown in SEQ ID NO.5 is changed to, obtains expression vector pGBG1.
- 6. a kind of preparation method of the bacillus amyloliquefaciens chitosan enzyme described in claim 1, the preparation method include with Lower step：Recombinant expression carrier described in claim 4 or 5 is imported into Pichia pastoris, induces and obtains secreting, expressing Bacillus amyloliquefaciens chitosan enzyme.
- 7. preparation method according to claim 6, it is characterised in that：The Pichia pastoris is GS115.
- 8. application of the bacillus amyloliquefaciens chitosan enzyme in chitosan or chitin degrading described in claim 1.
- 9. application according to claim 8, it is characterised in that：The bacillus amyloliquefaciens chitosan enzyme is individually used for shell Polyose degradation prepares chitosan oligosaccharide；Or the bacillus amyloliquefaciens chitosan enzyme and other chitinases or chitosan enzyme It is used in mixed way, Synergistic degradation chitosan or chitin.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108977418A (en) * | 2018-07-11 | 2018-12-11 | 中国科学院过程工程研究所 | A kind of high-efficiency nitrogen-fixing raw rhizobium chitosan oligosaccharide deacetylase and its preparation method and application slowly |
| CN114807190A (en) * | 2021-01-27 | 2022-07-29 | 中国科学院过程工程研究所 | Antarctic lichen streptomyces chitosanase gene and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108977418A (en) * | 2018-07-11 | 2018-12-11 | 中国科学院过程工程研究所 | A kind of high-efficiency nitrogen-fixing raw rhizobium chitosan oligosaccharide deacetylase and its preparation method and application slowly |
| CN114807190A (en) * | 2021-01-27 | 2022-07-29 | 中国科学院过程工程研究所 | Antarctic lichen streptomyces chitosanase gene and application thereof |
| CN114807190B (en) * | 2021-01-27 | 2024-06-04 | 中国科学院过程工程研究所 | Streptomyces antarcticus chitosanase gene and application thereof |
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