CN109022403A

CN109022403A - A kind of aspergillus nidulans chitin deacetylase and its preparation method and application

Info

Publication number: CN109022403A
Application number: CN201810757956.4A
Authority: CN
Inventors: 杜昱光; 焦思明; 程功; 张毓宸; 冯翠; 任立世; 李建军; 未金花
Original assignee: Institute of Process Engineering of CAS
Current assignee: Institute of Process Engineering of CAS
Priority date: 2018-07-11
Filing date: 2018-07-11
Publication date: 2018-12-18

Abstract

The invention discloses a kind of aspergillus nidulans chitin deacetylases and its preparation method and application.The present invention obtains the chitin deacetylase coding gene sequence in aspergillus nidulans (Aspergillus nidulans) according to Pichia pastoris codon-bias, using full genome synthetic method, and the nucleic acid sequence after optimization is as shown in SEQ ID NO.2.Secreting, expressing further is carried out using chitin deacetylase encoding gene of the pichia yeast expression system the optimization, obtains aspergillus nidulans chitin deacetylase, amino acid sequence is as shown in SEQ ID NO.1.The aspergillus nidulans chitin deacetylase that the present invention obtains can remove the acetyl group in chitosan and chitosan oligosaccharide, to obtain the chitosan or chitosan oligosaccharide of specific structure.Chitosan or chitosan oligosaccharide after these are regulated can have new or higher bioactivity compared with before regulation.Therefore, the enzyme and its deacetylated product all have good prospects for commercial application.

Description

A kind of aspergillus nidulans chitin deacetylase and its preparation method and application

Technical field

The invention belongs to chitosan or chitosan oligosaccharide preparation technical fields, and in particular to a kind of aspergillus nidulans (Bradyrhizobium diazoefficiens) chitin deacetylase and its preparation method and application.

Background technique

A series of bioactivity such as chitosan has antibacterial and excitation animals and plants are immune.Currently, mainly being made by chemical method The deacetylated acquisition of chemistry is carried out to chitin with highly basic, be easy to cause environmental pollution.Directly using chitin deacetylase to several Fourth matter carries out the deacetylated acquisition acetyl group site that is possible to and is distributed controllable chitosan, and environmentally friendly.Chitosan oligosaccharide is crust The oligomer that element or its deacetylated product chitosan are generated through the degradation of the methods of physics, chemistry or zymetology, by Glucosamine (Glucosamine, GlcN), N-acetylglucosamine (N-Acetylglucosamine, GlcNAc) or combination It is formed by β-Isosorbide-5-Nitrae glucosides key connection, the usual degree of polymerization (degree of polymerization, DP) < 20.Chitosan oligosaccharide has A series of bioactivity such as anti-inflammatory, antitumor and activated plant is immune.The study found that the performance of chitosan oligosaccharide bioactivity is tied with it Structure, as DP and deacetylation (Degree of deacetylation, DDA) even distribution of the acetyl group on oligonucleotide chain etc. are close Cut phase is closed.Therefore, the controllable chitosan oligosaccharide of preparation structure is that the field is multi-functional and the new direction of high activity new-product development.Using not Chitin deacetylase with substrate identification types is deacetylated to the progress of chitin, chitosan or chitosan oligosaccharide, can obtain specific Deacetylation and the chitosan or chitosan oligosaccharide of the distribution of acetyl group site, may have higher or new bioactivity.Currently, still lacking Weary industrial chitin deacetylase preparation, limits the development in the field.

Summary of the invention

It is an object of the present invention to provide a kind of aspergillus nidulans chitin deacetylases and its preparation method and application；It is intended to A kind of chitin deacetylase of economical and efficient is provided.

Present invention technical solution used for the above purpose is as follows:

The present invention by being optimized to the chitin deacetylase coding gene sequence in aspergillus nidulans, in favor of its Secreting, expressing is realized in Pichia pastoris, to efficiently quickly obtain the high chitin deacetylase of deacetylase activity.

A kind of aspergillus nidulans chitin deacetylase provided by the invention, amino acid sequence is as shown in SEQ ID NO.1:

SEQ ID NO.1:

LEKREAEATPLPLVRRVPTGQVITQCTTPNTIALTFDDGPSEYTPQLLDLLSRYSARATFFVLGDAAAQNPGLLQRM RDEGHQVGAHTYDHVSLPSLGYDGIASQMTRLEEVIRPALGVAPAYMRPPYLETNELVLQVMRDLDYRVISASVDTK DYENQDADAIINTSFQLFLDQLDAGGNIVLAHDIHYWTVASLAERMLQEVNARGLIATTVGDCLGDGEIAWYH。

The present invention also provides a kind of aspergillus nidulans chitin deacetylase genes, encode above-mentioned aspergillus nidulans chitin Deacetylase.Preferably, the nucleotide sequence of the aspergillus nidulans chitin deacetylase gene is as shown in SEQ ID NO.2:

SEQ ID NO.2:

Ctcgagaagagagaggctgaggctactccattgccattggttagaagagttccaactggtcaagttatcactcaatg tactactccaaacactatcgctttgactttcgacgacggtccatccgagtacactccacaattgttggacttgttgt ccagatactccgctagagctactttcttcgttttgggtgacgctgctgctcaaaacccaggtttgttgcaaagaatg agagacgagggtcaccaagttggtgctcacacttacgaccacgtttccttgccatccttgggttacgacggtatcgc ttcccaaatgactagattggaggaggttatcagaccagctttgggtgttgctccagcttacatgagaccaccatact tggagactaacgagttggttttgcaagttatgagagacttggactacagagttatctccgcttccgttgacactaag gactacgagaaccaagacgctgacgctatcatcaacacttccttccaattgttcttggaccaattggacgctggtgg taacatcgttttggctcacgacatccactactggactgttgcttccttggctgagagaatgttgcaagaggttaacg ctagaggtttgatcgctactactgttggtgactgtttgggtgacggtgagatcgcttggtaccactaagcggccgcg 。

The present invention also provides the recombinant expression carriers comprising above-mentioned aspergillus nidulans chitin deacetylase gene.It is preferred that Ground, the expression vector are pGBG1, and expression vector pGBG1 is obtained in the following way:

By carrying out codon optimization to the signal peptide sequence (being detailed in shown in SEQ ID NO.4) in expression vector pPIC9, The new signal peptide sequence (being detailed in shown in SEQ ID NO.5) for being suitble to express in Pichia pastoris is obtained, and utilizes Nsi I/Xho Signal peptide sequence shown in SEQ ID NO.5 is substituted signal peptide shown in original SEQ ID NO.4 by I double digestion, is expressed Carrier pGBG1.The secreting, expressing that expression vector pGBG1 can make destination protein more efficient in Pichia pastoris.

Wherein, expression vector pPIC9 and Pichia pastoris GS115 is commercially produced product.

Aspergillus nidulans chitin deacetylase of the present invention the preparation method comprises the following steps: by above-mentioned recombinant expression carrier (for example, nucleotide sequence is as gene constructed into expression vector such as chitin deacetylase shown in SEQ ID NO.2), which imports, finishes Red yeast cells induces and obtains the chitin deacetylase of secreting, expressing.Preferably, the Pichia pastoris is GS115.

Further, the specific preparation step of aspergillus nidulans chitin deacetylase of the present invention includes: (1) according to complete The Preference that red yeast codons use carries out password to the chitin deacetylase gene original series from aspergillus nidulans Son optimization；(2) gene after optimization is carried out fully synthetic and constructed into expression vector pGBG1；(3) to the expression vector of building After carrying out linearization for enzyme restriction, give up the segment containing resistant gene, recycle the segment containing chitin deacetylase gene and leads Enter in Pichia pastoris GS115, induces and obtain the chitin deacetylase of secreting, expressing.

Chitosan oligosaccharide substrate is hydrolyzed using the thick enzyme supernatant of inducing expression and uses MALDI-TOF mass spectrometry method to production The composition of object is analyzed.The result shows that aspergillus nidulans chitin deacetylase of the present invention can be individually used for chitosan Or chitosan oligosaccharide is deacetylated；Or the aspergillus nidulans chitin deacetylase and other enzymes, such as chitin deacetylase, several Fourth matter enzyme or chitosan etc. are used in mixed way, and coordinate system is for specific structure chitosan or chitosan oligosaccharide.

The aspergillus nidulans chitin deacetylase that the present invention obtains can take off the acetyl group in chitosan and chitosan oligosaccharide It removes, to obtain the chitosan or chitosan oligosaccharide of specific structure.Chitosan or chitosan oligosaccharide after these are regulated compared with before regulation, There can be new or higher bioactivity.Therefore, the enzyme and its deacetylated product all have good prospects for commercial application.

Compared with prior art, the invention has the benefit that

1. chitin deacetylase gene source of the invention uses pichia pastoris yeast expression system in aspergillus nidulans Secreting, expressing.Aspergillus nidulans sheet is as non-pathogenic bacteria, and pichia pastoris yeast (Pichia pastoris) expression system is also It is used for the expression of the food-grade enzyme preparations such as lactase and phospholipase C, its own is also used for the production of zytase (GB2760-2014).Therefore, using the chitin deacetylase gene in aspergillus nidulans source in Pichia pastoris secreting, expressing, Its product chitin deacetylase can be used for the production of food-grade chitosan or chitosan oligosaccharide.

2. there is no the chitin deacetylase of commercialization at present, the present invention may fill up this blank, poly- in new construction shell Sugar and chitosan oligosaccharide preparation field obtain wide application.

Detailed description of the invention

Fig. 1 is the recombinant expression carrier ancda1-pGBG1 and its digestion products gel electrophoresis figure in the embodiment of the present invention Spectrum；

Fig. 2 is the Pichia yeast engineering fermentation liquid of the gene of chitin deacetylase containing aspergillus nidulans in the embodiment of the present invention The SDS-PAGE map of supernatant；

Fig. 3 is the MALDI-TOF mass spectrogram of low deacetylation chitosan oligosaccharide (chitin oligo saccharide, NACOS) in the embodiment of the present invention；

Fig. 4 is low deacetylation chitosan oligosaccharide (chitin oligo saccharide, NACOS) in the embodiment of the present invention through chitin deacetylase The MALDI-TOF mass spectrogram of product is obtained after ANCDA1 hydrolysis；

Fig. 5 is low deacetylation chitosan oligosaccharide (chitin oligo saccharide, NACOS) in the embodiment of the present invention¹H NMR identifies map；

Fig. 6 is low deacetylation chitosan oligosaccharide (chitin oligo saccharide, NACOS) in the embodiment of the present invention¹³C NMR identifies map；

Fig. 7 is low deacetylation chitosan oligosaccharide (chitin oligo saccharide, NACOS) in the embodiment of the present invention through chitin deacetylase Product is obtained after ANCDA1 hydrolysis¹H NMR identifies map；

Fig. 8 is low deacetylation chitosan oligosaccharide (chitin oligo saccharide, NACOS) in the embodiment of the present invention through chitin deacetylase Product is obtained after ANCDA1 hydrolysis¹³C NMR identifies map.

Specific embodiment

Technical solution of the present invention is described in detail below with reference to embodiment.The reagent and biomaterial used below It if not otherwise specified, is commercially produced product.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer suggests Condition carry out.

The codon optimization of 1 chitin deacetylase gene of embodiment and full genome synthesis

Under the premise of not changing amino acid sequence, using the preference codon of Pichia pastoris, engineer's structure nest is bent Mould chitin deacetylase (as shown in sequence SEQ ID NO.1, GenBank accession number:XP_ 682649.1) encoding gene, specific nucleotide sequence are shown in SEQ ID NO.2.Nucleotide sequence and original coding after optimization Gene order (as shown in sequence SEQ ID NO.3, GenBank accession number:XM_677557.1) homology is 72%.It is fully synthetic that gene order after optimization entrusts Beijing Qing Kexin industry Bioisystech Co., Ltd to carry out, and synthesizes the base of acquisition Because sequence designations are chitin deacetylase gene ancdal.

The expression vector establishment of 2 chitin deacetylase Gene A NCDA1 of embodiment

Using Nsi I/Xho I double digestion, by the signal peptide sequence as shown in SEQ ID NO.4 in expression vector pPIC9 The signal peptide sequence as shown in SEQ ID NO.5 is replaced with, expression vector pGBG1 is obtained.Using restriction enzyme Xho I and Not I carries out double digestion to the cloning vector containing chitin deacetylase gene ancda1, obtains target gene fragment, simultaneously Double digestion is carried out to expression vector pGBG1 using identical restriction endonuclease, recycles large fragment.Two recovery products are attached, and are obtained Recombinant vector is obtained, ancda1-pGBG1 is named as.To determine that target chitin deacetylase gene has been built up into carrier, I Double digestion and single endonuclease digestion are carried out to the recombinant vector using Xho I/Not I and Bgl II respectively, and agar is carried out to product There is target gene fragment as a result as shown in Figure 1: after double digestion between 500bp-750bp in sugared gel electrophoresis, with The segment 693bp of ancda1 is consistent；After BglII digestion, there are expected two segments, be followed successively by containing target gene Large fragment and small fragment containing resistant gene.

The screening of 3 chitin deacetylase Pichia yeast engineering of embodiment and chitin deacetylase preparation

By the recombinant plasmid ancda1-pGBG1 of acquisition after restriction enzyme Bgl II linearisation, gel electrophoresis separation And the nucleotide fragments (biggish segment as shown in Figure 1) containing target gene are cut, electric shock imports Pichia pastoris GS115 In, by the recon by being screened on histidine auxotrophy MD plate.6 single colonies of picking are inoculated in 200mL respectively It in BMGY culture medium, is cultivated 48 hours under 30 DEG C and 250rpm, supernatant is abandoned in centrifugation, and the BMMY that equivalent is added carries out inducing expression. Add afterwards for 24 hours methanol to its final concentration of 1%, it is later primary every adding for 24 hours, be centrifuged after total induction 120h, use SDS- PAGE detects the target protein expression in albumen supernatant.Fermentation supernatant containing target protein is denoted as chitin and takes off second Acyl enzyme ANCDA1 (Fig. 2), the deacetylated and product structure for later period chitosan oligosaccharide are identified.Above-mentioned MD agar plate, BMMY fine jade Rouge plate, BMGY culture medium, BMMY culture medium are the common culture medium of yeast expression system, can directly be bought or according to existing Technical literature is prepared.

4 chitin deacetylase ANCDA1 of embodiment hydrolyzes low deacetylation chitosan oligosaccharide and its product mass spectra analysis

The low deacetylation chitosan oligosaccharide (NACOS) for passing through chemical acetylation preparation 10g early period is weighed, be dissolved in water simultaneously constant volume To 100mL, it is adjusted to pH 8.5.The ANCDA1 crude enzyme liquid of 10mL fermentation is added, is stirred to react 48 hours at 40 DEG C.Reaction terminates Afterwards, centrifugation removes a small amount of insoluble substance, and supernatant is concentrated and after being lyophilized, and is denoted as NACOS-ANCDA1.First use MALDI-TOF mass spectrum Method analyzes NACOS and its deacetylated product NACOS-ANCDA1.Method particularly includes: a certain amount of sample is weighed, is prepared The aqueous solution for being 2mg/mL at concentration, draws after filtering on 1 μ L point sample to sample panel, and after it is spontaneously dried, 1 μ L matrix is added 2,5-dihydroxybenzoic acid (DHB) solution uses autoflex III smartbeam type MALDI-TOF mass spectrum after its drying Instrument (Bruker company) is detected (cation reflective-mode).The testing result of NACOS and NACOS-ANCDA1 is successively such as Fig. 3 And shown in Fig. 4.N-acetylglucosamine is represented with A, D represents Glucosamine, and subsequent number, which represents, contains the monosaccharide Number, the two adduction are the degree of polymerization of oligosaccharides.From the results of view, NACOS is mainly degree of polymerization 4-8, full acetyl and lacks 1-2 The chitosan oligosaccharide of acetyl group；In NACOS-ANCDA1 after ANCDA1 enzymatic treatment, full acetylated chitooligose is obviously less, further Deacetylated chitosan oligosaccharide product specific gravity increases, and prompts that deacetylation has occurred.

5 low deacetylation chitosan oligosaccharide of embodiment and its ANCDA1 hydrolysate liquor NMR analysis

To further confirm that deacetylation has occurred under the action of chitin deacetylase ANCDA1 really in NACOS, We are analyzed using sample of the liquid NMR method to deacetylated front and back.Method particularly includes: 25mg sample is weighed respectively NACOS and NACOS-ANCDA1, it is each that 0.50mL D is added₂O is loaded detection to it after completely dissolution.With 3- (trimethyl silyl Base) propionic acid-d4 sodium salt be internal standard, sample is detected using AVANCE III 600MHz Nuclear Magnetic Resonance, wherein¹H NMR inhibits water peak when detecting to reduce its interference.The initial data of acquisition is opened using analysis software MestReNova Afterwards, after using Internal standard correction methods shift value, privileged site monosaccharide is marked, specific reference displacement values include¹In H NMR Reducing end D sugared (- D, 5.19ppm), reducing end A sugared (- A, 5.43ppm) and¹³The non-reducing end D of 5 carbon (C5) in C NMR (D-, 79.0ppm) and non-reducing end A sugared (A-, 78.5ppm) etc..Sample NACOS's¹H NMR analyzes result as shown in figure 5, can Know that the deacetylation 16% of the chitosan oligosaccharide sample, reducing end are mainly A sugar；¹³It is as shown in Figure 6 that C NMR analyzes result, it is known that non-to go back Former end is mainly A sugar, and simultaneously containing a small amount of D sugar, the two ratio is 1:0.26.NACOS-ANCDA1's¹H NMR analyzes result such as Shown in Fig. 7, it is known that the deacetylation 50% of the sample, reducing end are mainly A sugar；¹³It is as shown in Figure 8 that C NMR analyzes result, it is known that Non-reducing end contains A sugar and D sugar simultaneously, and the two ratio is 1:0.19.The above NMR analysis result explanation, ANCDA1 have really Chitin deacetylase activity, the deacetylation of NACOS is from 16% 50% (Fig. 5 and the Fig. 7) that become that treated；The chitin is de- Acetyl enzyme without apparent deacetylation, prompts deacetylated possible main the non-reducing end and reducing end acetyl group of NACOS Occur at the intermediate position of sugar chain.

It should be noted last that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting.Although ginseng It is described the invention in detail according to embodiment, it will be apparent to an ordinarily skilled person in the art that technical side of the invention Case is modified or replaced equivalently, and without departure from the spirit and scope of technical solution of the present invention, should all be covered in the present invention Scope of the claims in.

Sequence table

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tccgagtaca ctccacaatt gttggacttg ttgtccagat actccgctag agctactttc 180

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Claims

1. a kind of aspergillus nidulans chitin deacetylase, it is characterised in that: the amino of the aspergillus nidulans chitin deacetylase Acid sequence is as shown in SEQ ID NO.1.

2. a kind of aspergillus nidulans chitin deacetylase gene, it is characterised in that: it is several to encode aspergillus nidulans described in claim 1 Fourth matter deacetylase.

3. aspergillus nidulans chitin deacetylase gene according to claim 2, it is characterised in that: the nucleotide of the gene Sequence is as shown in SEQ ID NO.2.

4. the recombinant expression carrier comprising aspergillus nidulans chitin deacetylase 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 suitble to finishing The signal peptide sequence as shown in SEQ ID NO.5 expressed in red yeast；

Using Nsi I/Xho I double digestion, the signal peptide sequence as shown in SEQ ID NO.4 in expression vector pPIC9 is replaced For the signal peptide sequence as shown in SEQ ID NO.5, expression vector pGBG1 is obtained.

6. a kind of preparation method of aspergillus nidulans chitin deacetylase 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 Aspergillus nidulans chitin deacetylase.

7. preparation method according to claim 6, it is characterised in that: the Pichia pastoris is GS115.

8. aspergillus nidulans chitin deacetylase described in claim 1 chitin, chitosan or chitosan oligosaccharide it is deacetylated in Using.