CN107586768A - A kind of Bacillus circulans chitosan enzyme and its preparation method and application - Google Patents
A kind of Bacillus circulans chitosan enzyme and its preparation method and application Download PDFInfo
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- CN107586768A CN107586768A CN201711018444.8A CN201711018444A CN107586768A CN 107586768 A CN107586768 A CN 107586768A CN 201711018444 A CN201711018444 A CN 201711018444A CN 107586768 A CN107586768 A CN 107586768A
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- chitosan
- bacillus circulans
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- chitosan enzyme
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Abstract
The invention discloses a kind of Bacillus circulans chitosan enzyme and its preparation method and application.The present invention obtains the chitosan enzyme coding gene sequence in Bacillus circulans (Bacillus circulans), the nucleotide sequence after optimization is as shown in SEQ ID NO.2 according to Pichia pastoris codon-bias using full genome synthetic method.Efficient secretory expression further is carried out to the chitosan enzyme encoding gene of optimization this described using pichia yeast expression system, obtains Bacillus circulans 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 0.77mg) degraded 5g chitosans, and same amount of chitosan of degrading about needs the neutral proteinase 150mg from bacillus subtilis, efficiency improves nearly 200 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 circulans (Bacillus
Circulans) 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 circulans chitosan enzyme and its preparation method and application；It is intended to carry
For a kind of specific chitosan enzyme of economical and efficient.
Present invention technical scheme used to achieve the above object is as follows：
The present invention by being optimized to the codon of Bacillus circulans chitosan enzyme (GH46 families) encoding gene, with
Secreting, expressing is realized in Pichia pastoris beneficial to it, is gathered so as to efficiently quickly obtain the high Bacillus circulans shell of hydrolysing activity
Carbohydrase.
A kind of Bacillus circulans chitosan enzyme provided by the invention, its amino acid sequence is as shown in SEQ ID NO.1：
SEQ ID NO.1：
LEKREAEAASPDDNFSPETLQFLRNNTGLDGEQWNNIMKLINKPEQDDLNWIKYYGYCEDIEDERGYTI
GLFGATTGGSR
DTHPDGPDLFKAYDAAKGASNPSADGALKRLGINGKMKGSILEIKDSEKVFCGKIKKLQNDAAWRKAMW
ETFYNVYIRYS
VEQARQRGFTSAVTIGSFVDTALNQGATGGSDTLQGLLARSGSSSNEKTFMKNFHAKRTLVVDTNKYNK
PPNGKNRVKQW
DTLVDMGKMNLKNVDSEIAQVTDWEMK。
Present invention also offers a kind of Bacillus circulans chitosanase gene, encodes above-mentioned Bacillus circulans shell and gathers
Carbohydrase.Preferably, the nucleotide sequence of the Bacillus circulans chitosanase gene is as shown in SEQ ID NO.2：
SEQ ID NO.2：
ctcgagaagagagaggctgaggctgcttccccagacgacaacttctccccagagactttgcaattcttgagaaacaa
cactggtttggacggtgagcaatggaacaacatcatgaagttgatcaacaagccagagcaagacgacttgaactgga
tcaagtactacggttactgtgaggacatcgaggacgagagaggttacactatcggtttgttcggtgctactactggt
ggttccagagacactcacccagacggtccagacttgttcaaggcttacgacgctgctaagggtgcttccaacccatc
cgctgacggtgctttgaagagattgggtatcaacggtaagatgaagggttccatcttggagatcaaggactccgaga
aggttttctgtggtaagatcaagaagttgcaaaacgacgctgcttggagaaaggctatgtgggagactttctacaac
gtttacatcagatactccgttgagcaagctagacaaagaggtttcacttccgctgttactatcggttccttcgttga
cactgctttgaaccaaggtgctactggtggttccgacactttgcaaggtttgttggctagatccggttcctcctcca
acgagaagactttcatgaagaacttccacgctaagagaactttggttgttgacactaacaagtacaacaagccacca
aacggtaagaacagagttaagcaatgggacactttggttgacatgggtaagatgaacttgaagaacgttgactccga
gatcgctcaagttactgactgggagatgaagtaagcggccgcg。
Present invention also offers the recombinant expression carrier for including above-mentioned Bacillus circulans 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 circulans chitosan enzyme of the present invention comprises the following steps：By above-mentioned restructuring table
Import and finish up to carrier (for example, chitosanase gene of the nucleotide sequence as shown in SEQ ID NO.2 is built into expression vector)
Red yeast cells, induce and obtain the chitosan enzyme of secreting, expressing.
Preferably, the Pichia pastoris is GS115.
Further, the specific preparation process of Bacillus circulans chitosan enzyme of the present invention includes：(1) it is red according to finishing
The Preference that yeast codons use, it is excellent to carrying out codon from the chitosanase gene original series of Bacillus circulans
Change；(2) gene after optimization is carried out fully synthetic and built into expression vector pGBG1；(3) expression vector of structure is carried out
After linearization for enzyme restriction, give up the fragment containing resistant gene, reclaim the fragment containing chitosanase gene and import Pichia pastoris
In cell 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 circulans chitosan enzyme of the present invention can
The degraded for being individually used for chitosan prepares chitosan oligosaccharide；Or the Bacillus circulans chitosan enzyme is with removing Bacillus circulans shell
Other chitosan enzymes or chitinase outside dextranase are used in mixed way, Synergistic degradation chitosan or chitin.
Present invention also offers application of the above-mentioned Bacillus circulans chitosan enzyme in chitosan or chitin degrading.It is excellent
Selection of land, Bacillus circulans chitosan enzyme are individually used for degradation of chitosan and prepare chitosan oligosaccharide；Or the ring-type gemma bar
Bacterium chitosan enzyme is used in mixed way with other chitinases in addition to Bacillus circulans chitosan enzyme 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, having preferable chitosan hydrolyzate activity from the neutral proteinase of bacillus subtilis, (the thick enzyme dry powder of 1g is about
30g chitosans can thoroughly be hydrolyzed).It is presumed that produce chitosan enzyme present in the strain of the hydrolysing activity.Existing hair
Chitosan enzyme caused by fermenting process is accessory substance, and enzyme activity is too low.Therefore, it is necessary to chitosan enzyme therein carry out high efficient expression with
Increase its enzyme activity.Chitosan enzyme encoding gene is widely present in bacillus.In the present invention, we choose one
From the chitosan enzyme of the family of Bacillus circulans glycoside hydrolase 46, according to the Preference of Pichia pastoris codon, pass through
Full genome synthetic method obtains its encoding gene and secreting, expressing is carried out in Pichia pastoris.The chitosan enzyme of expression can substitute
Existing commodity neutral proteinase etc. is used for the large-scale production of 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 circulans, pichia pastoris phaff expression system point is used
Secrete expression.Bacillus circulans sheet is as nonpathogenic bacteria, and the enzyme security in its source is secure, and pichia pastoris phaff
(Pichia pastoris) expression system also has been used for the expression of the food-grade enzyme preparations such as lactase and phospholipase C, its own
Also it is used for the production (GB2760-2014) of zytase.Therefore, existed using the chitosanase gene in Bacillus circulans source
Secreting, expressing in Pichia pastoris, its product chitosan enzyme can turn into the production enzyme preparation of food-grade chitosan oligosaccharide or chitin oligo saccharide.
2. chitosanase gene in the present invention, can be real because the codon-bias according to Pichia pastoris is optimized
Efficient secretory expression in present Pichia pastoris.Through determination of activity and conversion, 1mL crude enzyme liquids (containing 0.77mg zymoproteins) can be complete
5g chitosans are hydrolyzed, and that commodity neutral proteinase is then 150mg to application amount.Therefore, compared with commodity neutral proteinase, this
The chitosan enzyme efficiency of invention secreting, expressing improves nearly 200 times, and there is replacement existing goods enzyme to prepare chitosan oligosaccharide for scale
Huge applications potentiality.
Brief description of the drawings
Fig. 1 is the recombinant expression carrier bccsn-pGBG1 and its digestion products gel electrophoresis spectrum in the embodiment of the present invention；
On Pichia yeast engineering zymotic fluids of the Fig. 2 for the chitosanase gene containing Bacillus circulans in the embodiment of the present invention
Clear SDS-PAGE collection of illustrative plates, wherein, arrow meaning is target product；
Fig. 3 is the high-efficient liquid phase chromatogram of chitosan oligosaccharide COS-94-BCCSN in the embodiment of the present invention；
Fig. 4 is the MALDI-TOF mass spectrograms of chitosan oligosaccharide COS-62-BCCSN 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's ring-type bud
Spore bacillus chitosan enzyme (GH46 families) (as shown in sequence SEQ ID NO.1, GenBank accession number:
BAA01474.2 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:
D10624.2 (569-1474), homology highest, it is 76%.Gene order student on commission work after optimization carries out fully synthetic, synthesis
The gene order of acquisition is named as chitosanase gene bccsn.
The chitosanase gene bccsn of embodiment 2 expression vector establishment
The cloning vector containing chitosanase gene bccsn 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 bccsn-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 bccsn fragment 813bp；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 bccsn-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 BCCSN).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 0.77mg/mL；DNS methods are surveyed
Its fixed specific enzyme activity is 49.1U/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 BCCSN 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 BCCSN 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-BCCSN.The chitosan oligosaccharide COS-94-BCCSN 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 BCCSN 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 BCCSN 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-BCCSN.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-BCCSN 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-BCCSN 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 circulans chitosan enzyme and its preparation method and application
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Leu Glu Lys Arg Glu Ala Glu Ala Ala Ser Pro Asp Asp Asn Phe Ser
1 5 10 15
Pro Glu Thr Leu Gln Phe Leu Arg Asn Asn Thr Gly Leu Asp Gly Glu
20 25 30
Gln Trp Asn Asn Ile Met Lys Leu Ile Asn Lys Pro Glu Gln Asp Asp
35 40 45
Leu Asn Trp Ile Lys Tyr Tyr Gly Tyr Cys Glu Asp Ile Glu Asp Glu
50 55 60
Arg Gly Tyr Thr Ile Gly Leu Phe Gly Ala Thr Thr Gly Gly Ser Arg
65 70 75 80
Asp Thr His Pro Asp Gly Pro Asp Leu Phe Lys Ala Tyr Asp Ala Ala
85 90 95
Lys Gly Ala Ser Asn Pro Ser Ala Asp Gly Ala Leu Lys Arg Leu Gly
100 105 110
Ile Asn Gly Lys Met Lys Gly Ser Ile Leu Glu Ile Lys Asp Ser Glu
115 120 125
Lys Val Phe Cys Gly Lys Ile Lys Lys Leu Gln Asn Asp Ala Ala Trp
130 135 140
Arg Lys Ala Met Trp Glu Thr Phe Tyr Asn Val Tyr Ile Arg Tyr Ser
145 150 155 160
Val Glu Gln Ala Arg Gln Arg Gly Phe Thr Ser Ala Val Thr Ile Gly
165 170 175
Ser Phe Val Asp Thr Ala Leu Asn Gln Gly Ala Thr Gly Gly Ser Asp
180 185 190
Thr Leu Gln Gly Leu Leu Ala Arg Ser Gly Ser Ser Ser Asn Glu Lys
195 200 205
Thr Phe Met Lys Asn Phe His Ala Lys Arg Thr Leu Val Val Asp Thr
210 215 220
Asn Lys Tyr Asn Lys Pro Pro Asn Gly Lys Asn Arg Val Lys Gln Trp
225 230 235 240
Asp Thr Leu Val Asp Met Gly Lys Met Asn Leu Lys Asn Val Asp Ser
245 250 255
Glu Ile Ala Gln Val Thr Asp Trp Glu Met Lys
260 265
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ctcgagaaga gagaggctga ggctgcttcc ccagacgaca acttctcccc agagactttg 60
caattcttga gaaacaacac tggtttggac ggtgagcaat ggaacaacat catgaagttg 120
atcaacaagc cagagcaaga cgacttgaac tggatcaagt actacggtta ctgtgaggac 180
atcgaggacg agagaggtta cactatcggt ttgttcggtg ctactactgg tggttccaga 240
gacactcacc cagacggtcc agacttgttc aaggcttacg acgctgctaa gggtgcttcc 300
aacccatccg ctgacggtgc tttgaagaga ttgggtatca acggtaagat gaagggttcc 360
atcttggaga tcaaggactc cgagaaggtt ttctgtggta agatcaagaa gttgcaaaac 420
gacgctgctt ggagaaaggc tatgtgggag actttctaca acgtttacat cagatactcc 480
gttgagcaag ctagacaaag aggtttcact tccgctgtta ctatcggttc cttcgttgac 540
actgctttga accaaggtgc tactggtggt tccgacactt tgcaaggttt gttggctaga 600
tccggttcct cctccaacga gaagactttc atgaagaact tccacgctaa gagaactttg 660
gttgttgaca ctaacaagta caacaagcca ccaaacggta agaacagagt taagcaatgg 720
gacactttgg ttgacatggg taagatgaac ttgaagaacg ttgactccga gatcgctcaa 780
gttactgact gggagatgaa gtaagcggcc gcg 813
<210> 3
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gccgcagctt ctcctgacga caatttctcc ccagaaaccc tgcaatttct tcgcaataat 180
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gagaaaacct ttatgaagaa tttccatgcc aaacgtacct tggttgtgga taccaacaaa 780
tacaacaagc cacctaacgg taaaaaccgt gtaaaacaat gggacactct cgtggacatg 840
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aagtaa 906
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ggaagctgcc ctgtcttaaa cctttttttt tatcatcatt attagcttac tttcataatt 180
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ggaagctgcc ctgtcttaaa cctttttttt tatcatcatt attagcttac tttcataatt 180
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atcaaaaaac aactaattat tcgaaacgat ggctatccca agattcccat ccatcttcac 300
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Claims (9)
- A kind of 1. Bacillus circulans chitosan enzyme, it is characterised in that：The amino acid sequence of the Bacillus circulans chitosan enzyme Row are as shown in SEQ ID NO.1.
- A kind of 2. Bacillus circulans chitosanase gene, it is characterised in that：Encode the Bacillus circulans described in claim 1 Chitosan enzyme.
- 3. Bacillus circulans chitosanase gene according to claim 2, it is characterised in that：The nucleotides sequence of the gene Row are as shown in SEQ ID NO.2.
- 4. include the recombinant expression carrier of Bacillus circulans 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, by signal peptide sequence is replaced as shown in SEQ ID NO.4 in expression vector pPIC9 For the signal peptide sequence as shown in SEQ ID NO.5, expression vector pGBG1 is obtained.
- 6. a kind of preparation method of the Bacillus circulans chitosan enzyme described in claim 1, methods described comprise the following steps：Recombinant expression carrier described in claim 4 or 5 is imported into Pichia pastoris, induces and obtains the ring-type of secreting, expressing Bacillus chitosan enzyme.
- 7. preparation method according to claim 6, it is characterised in that：The Pichia pastoris is GS115.
- 8. application of the Bacillus circulans chitosan enzyme in chitosan or chitin degrading described in claim 1.
- 9. application according to claim 8, it is characterised in that：The Bacillus circulans chitosan enzyme is individually used for shell and gathered Sugar degraded prepares chitosan oligosaccharide；Or the Bacillus circulans chitosan enzyme mixes with other chitinases or chitosan enzyme Use, Synergistic degradation chitosan or chitin.
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| CN109097379A (en) * | 2018-09-13 | 2018-12-28 | 江南大学 | A method of improving chitinase expression quantity |
| TWI716702B (en) * | 2018-07-03 | 2021-01-21 | 國立高雄科技大學 | Engineered chitosanase and method of producing chitotriose |
| TWI721908B (en) * | 2018-07-03 | 2021-03-11 | 國立高雄科技大學 | Engineered chitosanase and method of producing chitobiose |
| CN112831510A (en) * | 2019-12-13 | 2021-05-25 | 中国科学院天津工业生物技术研究所 | Construction of recombinant bacterium for efficiently expressing chitinase and screening of high-enzyme-activity mutant |
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| TWI716702B (en) * | 2018-07-03 | 2021-01-21 | 國立高雄科技大學 | Engineered chitosanase and method of producing chitotriose |
| TWI721908B (en) * | 2018-07-03 | 2021-03-11 | 國立高雄科技大學 | Engineered chitosanase and method of producing chitobiose |
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| CN109097379B (en) * | 2018-09-13 | 2020-08-04 | 江南大学 | Method for improving expression quantity of chitinase |
| CN112831510A (en) * | 2019-12-13 | 2021-05-25 | 中国科学院天津工业生物技术研究所 | Construction of recombinant bacterium for efficiently expressing chitinase and screening of high-enzyme-activity mutant |
| CN112831510B (en) * | 2019-12-13 | 2022-04-22 | 中国科学院天津工业生物技术研究所 | Construction of recombinant bacterium for efficiently expressing chitinase and screening of high-enzyme-activity mutant |
| 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 |
| CN114480537A (en) * | 2021-12-21 | 2022-05-13 | 中国海洋大学 | Method for preparing chitosan oligosaccharide with high polymerization degree |
| CN114480537B (en) * | 2021-12-21 | 2023-06-20 | 中国海洋大学 | Method for preparing chitosan oligosaccharide with high polymerization degree |
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