CN111154745B

CN111154745B - Chitin deacetylase, coding gene and application

Info

Publication number: CN111154745B
Application number: CN201811325613.7A
Authority: CN
Inventors: 尹恒; 朱先玉
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2018-11-08
Filing date: 2018-11-08
Publication date: 2022-09-20
Anticipated expiration: 2038-11-08
Also published as: CN111154745A

Abstract

The invention relates to chitin deacetylase and a coding gene, and preparation and application thereof. The invention also provides a method for preparing the chitin deacetylase, namely, a genetic engineering technical method is utilized to clone the gene of the chitin deacetylase onto an escherichia coli expression vector PET-22b to obtain an escherichia coli recombinant strain capable of heterologously expressing the chitin deacetylase, and the prepared chitin deacetylase PsCDA can enable specific deacetylation of chitotetraose (A4) to generate chitin oligosaccharide (A3D1) with 75% acetyl degree and chitosan oligosaccharide (A2D2) with 50% acetyl degree by heterologously expressing the strain. Can act on chitin and chitosan oligosaccharide from different sources such as shrimp and crab shells. The chitin deacetylase provided by the invention can be widely applied to the aspects of food science, biomedicine, chemical materials and the like.

Description

Chitin deacetylase, coding gene and application

Technical Field

The invention relates to a chitin deacetylase coding gene in Penicillium (American strain classification No. Penicillium oxalicum 114-2, Taxinomy ID:933388) and a preparation and application patent specification. The invention also provides a recombinant plasmid and a recombinant genetic engineering strain of the chitin deacetylase. The chitin deacetylase PsCDA can be widely applied to the aspects of food science, biomedicine, chemical materials and the like.

Background

Chitin is the second most renewable natural resource in nature next to cellulose, and is widely present in the ocean. (Chen Sha Bo, Wu Gen Fu. science and technology bulletin, 2004, (03): 258) 262) chitosan is a deacetylation product of chitin, is a unique basic polysaccharide in the nature, has the molecular formula of (C6H11NO4) n, has the characteristics of broad-spectrum antibacterial property, water retention property, reproducibility, good biocompatibility, degradability, higher drug loading property, NO toxic or side effect and the like, and is widely applied to the fields of medicine, environmental protection, food, agriculture, paper making, cosmetics and the like after being modified by a plurality of chemical and physical methods such as glutaraldehyde crosslinking method, grafting, acetylation and the like. The traditional chemical method for producing chitosan comprises the following steps: selecting clean shrimp and crab shells, soaking the shrimp and crab shells in 4-6% hydrochloric acid to remove inorganic salts, boiling the shrimp and crab shells with 10% sodium hydroxide to elute proteins, bleaching, cleaning and drying the shrimp and crab shells to obtain chitin, and preserving the chitin for 1-3 hours at 100-180 ℃ with 40-60% concentrated sodium hydroxide to remove acetyl groups, clean and dry the chitin to obtain chitosan. The method has the technical defects of strict requirements on reaction conditions, generation of a large amount of byproducts, serious environmental pollution, difficult separation and the like. The reaction condition for producing chitosan by the biological enzyme method is mild, pollution-free and stable in deacetylation degree, and a new method is provided in the aspect of producing chitosan. It has now been found that chitin deacetylases, which are widely present in eukaryotes and prokaryotes, can be deacetylated directly from chitin to chitosan [ Zhao y. journal of Biotechnology,2008,136:290 ]. Research shows that the chitin deacetylase has realized heterologous expression in Pichia pastoris or Escherichia coli to obtain chitin deacetylase with high expression amount, high stability and high activity, and some chitin deacetylases have been applied in production practice.

Blair, d.e. et al show by multiple sequence alignment of the Chitin Deacetylase (CDA) family that the CDA family of sequences contains five conserved sequences with several conserved aspartic acid and histidine residues that constitute the active site of CDA. These five motifs were designated as motif 1(TFDD), motif 2(HSWSHP), motif 3(RPPY), motif 4(DSLDW), and motif 5(GSIVLMH), respectively. Motif 1(TFDD) includes two aspartic acid residues; one aspartate residue interacts with zinc or cobalt and the second aspartate residue binds to the substrate, allowing the reaction to form acetic acid. Motif 2(HSWSHP) contains two histidines, one serine or one threonine, where histidine can bind to metal ions to stabilize the structure of the enzyme and serine or threonine can bind to histidine to form hydrogen bonds. Motif 3(RPPY) forms one side of the notch of the active site, which can coordinate the catalytic activity of binding acetate, zinc and aspartate, tyrosine can form hydrogen bonds with acetate, and when tyrosine is mutated to alanine in peptidoglycan deacetylase from streptococcus pneumoniae, the enzyme is inactivated. Motif 4(DSLDW) constitutes the other side of the active site groove, and tryptophan is a key amino acid of this motif. Motif 5(GSIVLMH) contains leucine from the hydrophobic pocket and histidine from the product acetic acid binding (Blair D E, Proceedings of the National Academy of Sciences of the United States of America,2005,102(43): 15429) 15434).

CDA was first discovered by Hearaki et al in 1974 from Mucorarixii, a genus of Mucorarouxii of the class Zygomycota, and some of the enzymatic properties of the enzyme were studied. CDA is also produced in fungi including Mucor, Rhizopus, Penicillium, Aspergillus, and anthracnose of cucumber (Cucumber anthracnose, Puccinia striiformis (Puccinia striformis)), and it is also found in Alcaligenes bacteria of the genus Bacillus, and Srinivasan isolated this basic bacterium that secretes CDA in large quantities extracellularly in 1998.

TABLE 1 partial CDA-producing strains and enzymatic Properties

As can be seen from the table, the molecular weight of CDA is mostly between 30-80KD, the isoelectric points are all within the acid range, the pH value is not more than 4, the optimum temperature of enzyme is mostly 50 ℃, but the optimum pH value range of CDA from different sources is from acid pH to alkaline pH. CDA is also found in the midgut peritrophic membranes of many crustacean insects, such as Drosophila melanogaster, Anophelesgamuae, Apis melifera, Mamestra consortia, Helicoverpa armigera, Trichoplusia ni, Tribolium castaneum, and the like, and CDA is present in the body of the insects.

The chitin deacetylase is used for replacing the concentrated alkali pyrolysis method which is commonly adopted at present to produce high-quality chitosan, so that the problem of environmental pollution in the existing chitosan production can be solved, and high-quality chitosan products such as chitosan products with uniform acetylation degree and narrow molecular weight distribution range, chitosan oligosaccharide with specific acetylation positions and the like can be produced, and therefore, the chitin deacetylase has important application value in the fields of industrial application, biomedicine and the like. The prepared chitin deacetylase (PsCDA) has high expression level, can produce chitosan oligosaccharide with specific deacetylation degree, and can be applied to the production of chitosan oligosaccharide or chitosan with specific deacetylation degree and specific deacetylation site.

Disclosure of Invention

The first purpose of the invention is to find a recombinant chitin deacetylase, the coding gene of the enzyme is SEQ ID NO.1, the amino acid sequence of the chitin deacetylase produced by the coding is SEQ ID NO.2, and the chitin deacetylase is secreted and expressed in a dimer form.

The second objective of the invention is that the chitin deacetylase PsCDA can specifically act on chitotetraose. Specific deacetylation of chitotetraose (A4) produced chitooligosaccharides with 75% acetyl degree (A3D1) and chitooligosaccharides with 50% acetyl degree (A2D2), respectively.

The third purpose of the invention is the application of the chitin deacetylase PsCDA produced by heterologous expression in the degradation of chitin and chitin oligosaccharide:

1) used for producing deacetylation products such as chitosan, chitosan oligosaccharide, glucosamine, etc. from shells of crustaceans such as shrimps, crabs, squid, oysters, cuttlefish, etc., cell walls of algae, exoskeletons of invertebrates, and chitin in epidermis.

2) Removing acetyl of chitin or chitosan oligosaccharide to obtain chitin deacetylation product such as chitosan, chitosan oligosaccharide, glucosamine, etc

In addition, the chitin deacetylase PsCDA can be mixed with other chitin degrading enzymes or deacetylases and is applied to the aspect of synergistically breaking glycosidic bonds of chitin and chitosan oligosaccharide.

The chitin deacetylase PsCDA is used to produce products of chitosan oligosaccharides or chitosan of a specific degree of deacetylation, a specific deacetylation site.

The invention also provides a method for preparing the chitin deacetylase, namely, a genetic engineering technical method is utilized to clone the gene of the chitin deacetylase onto an escherichia coli expression vector PET-22b to obtain an escherichia coli recombinant strain capable of heterologously expressing the chitin deacetylase, and the prepared chitin deacetylase PsCDA can enable specific deacetylation of chitotetraose (A4) to generate chitin oligosaccharide (A3D1) with 75% acetyl degree and chitosan oligosaccharide (A2D2) with 50% acetyl degree by heterologously expressing the strain. The gene sequence of the chitin deacetylase PsCDA is obtained by cloning Penicillium (American strain classification number Penicillium oxalicum 114-2, Taxomomy ID:933388) genome through a PCR technology.

The gene coding region is 927bp in length, 309 amino acids are coded, the molecular weight is 35.5kDa, the dimer molecular weight is 71kDa, and the gene belongs to the 4 th family of polysaccharide lyase. Heterogeneously expressing the prepared chitin deacetylase PsCDA, which can specifically act on the chitotetraose. Specific deacetylation of chitotetraose (A4) produced chitooligosaccharides with 75% acetyl degree (A3D1) and 50% acetyl degree (A2D2), respectively. Can act on chitin and chitosan oligosaccharide from different sources such as shrimp and crab shells.

The chitin deacetylase PsCDA can be widely applied to the fields of agriculture, food, feed additives, medicine, food science and the like.

Drawings

FIG. 1: and (3) constructing a phylogenetic tree of Penicillium sp.1805.

FIG. 2: and (3) carrying out electrophoretic detection on the chitin deacetylase PsCDA gene.

FIG. 3: PAGE patterns of recombinant chitin deacetylase.

FIG. 4: and (3) identifying the enzyme activity of PsCDA.

Detailed Description

Example 1: phylogenetic tree construction

Penicillium (American strain classification number Penicillium oxalicum 114-2, Taxomo ID:933388) Penicillium sp.1805 is separated and purified by a fungal genome extraction kit (manufacturer: manufacturer, model: B518229-0050), genome is extracted, and ITS1 and ITS4 primers are cloned for fungal identification, the sequencing result is subjected to sequence comparison on NCBI, and the sequence with close relationship is downloaded to construct a phylogenetic tree through MEGA 7.

Example 2: cloning chitin deacetylase gene in full length.

Extracting RNA from Penicillium (American strain classification number Penicillium oxalicum 114-2, Taxinomy ID:933388), reverse transcribing to form cDNA, and designing primer CDA-F: 5'-CTAGTCTAGAATGGCCCCCAAGAGAGTATTG-3'; CDA-R5 '-CCGCTCGAGCTACTTCTTGAGCATCTGACC-3'. PCR amplification was performed using the reverse transcribed cDNA as a template. The PCR reaction conditions are as follows: 5min at 94 ℃ for 1 cycle; 30 cycles of 94 ℃ for 30s, 45 ℃ for 30s, and 72 ℃ for 1min for 30 s; 10min at 72 ℃ for 1 cycle. After the PCR product is analyzed by agarose gel electrophoresis, the target fragment is recovered by cutting gel and is connected to a pMD19-T vector for sequencing.

A sequence table:

information of SEQ ID No.1

(a) Sequence characterization

Length: 927 nucleotide

Type (2): nucleotide(s)

Chain type: single strand

(b) Molecular type: DNA

Description of the sequence: a gene encoding chitin deacetylase derived from Penicillium sp (U.S. Strain accession No. Penicillium oxalicum 114-2, Taxinomy ID: 933388).

SEQ ID NO.1CDA target gene sequence

ATGGCCCCCAAGAGAGTATTGATCACCTACGGTGTTGACGTCGATGCTGTTGCCGGTTGGCTCGGTTCCTATGGGGGTGAAGATTCGACAAACGACATTAGCCGAGGATACTGGGCTGGAACTGTGGGTACACGAAGGCTTCTAAAGCTCTTTGACAAGTATCAGATCAAGACAACATGGTTCATTCCTGGTCATTCACTGGAGACCTTTCCCGAAGACATGGCAGCAGTCCGCGATGCGGGGCATGAAATTGGACTGCACGGTTATTCGCATGAAAACCCAACAGACATGACTATTGAGCAGCAGCGGGATGTCCTAGACAAGACATATCGCATGCTGACTGAGTTCACCGGTAAACCACCACGGGGAAGTGTCGCGCCTTGGTGGGAGACCAGCAAAGAAGGCGCCCAATTACTCCTTGACTACGGCATTGAATATGATCACAGCATGAGTCACGAGGATTGTCAGGCGTACTATCTTCGTGCAGGTGACACGTGGACCAAGATTGACTACACTCAAAAGGCAGAAACCTGGATGAAGCCACTGGAGCACGGCACGCAGACTGGTCTGGTGGAGATCCCATCCAATTGGTACATTGATGATCTGCCACCCATGATGTTCATCAAGAACGCCCCGAACAGTCATGGCTTTGTCAACGCCCGAGACGTGGAGGACATCTGGCGCGATCACTTTGACTATTTCTACCGCGAGTATGACGAGTTTATTTTTCCAATTACTATTCATCCGGATGTGTCGGGAAGACCACATGTCCTTTTGATGCATGAGCGCCTCATTGAGCATTTCAAGAAGCACGAGGGCGTTGAATTCGTGACAATGGAGCAAGTTTGTGACGAGTTCAAGAAGAAGAACCCTGTCCCGGAGGGCGCTTTGATGCCTGCGCCCGTTGGTCAGATGCTCAAGAAGTAG

Information of SEQ ID No.2

(a) Sequence characterization

Length: 308 amino acid

Type (2): amino acids

Chain type: single strand

(b) Molecular type: protein

Description of the sequence: amino acid sequence of chitin deacetylase from Penicillium sp (U.S. strain Classification No. Penicillium oxyalicum 114-2, Taxinomy ID: 933388). The molecular weight of the chitin deacetylase monomer is 35.552 kDa.

SEQ ID NO.2CDA amino acid sequence

MAPKRVLITYGVDVDAVAGWLGSYGGEDSTNDISRGYWAGTVGTRRLLKLFDKYQIKTTWFIPGHSLETFPEDMAAVRDAGHEIGLHGYSHENPTDMTIEQQRDVLDKTYRMLTEFTGKPPRGSVAPWWETSKEGAQLLLDYGIEYDHSMSHEDCQAYYLRAGDTWTKIDYTQKAETWMKPLEHGTQTGLVEIPSNWYIDDLPPMMFIKNAPNSHGFVNARDVEDIWRDHFDYFYREYDEFIFPITIHPDVSGRPHVLLMHERLIEHFKKHEGVEFVTMEQVCDEFKKKNPVPEGALMPAPVGQMLKK

Example 3 heterologous expression of genes of interest

The PCR amplification product and pET-22b vector plasmid are subjected to double restriction enzyme digestion by BamHI and HindIII, the digestion is carried out for 12h at 37 ℃, the fragment is connected to the pET-22b vector plasmid, and the connection is carried out for 18h at 16 ℃. Transferring 5 mu L of the ligation product into E.coli DH5 alpha, selecting a monoclonal, and carrying out PCR detection and sequencing verification on bacterial liquid to obtain the recombinant plasmid psckda-PET-22 b.

The recombinant plasmid with correct sequencing is extracted and transferred into an expression competent cell of escherichia coli BL21(DE3), and monoclonal PCR verification is picked. The single clones verified to be correct were cultured in 10mL of LB medium containing ampicillin at a final concentration of 100. mu.g/mL for 12 hours as seed solutions. Adding the seed solution into 1L fermentation medium at a volume ratio of 1:100, performing induced culture at 16 deg.C for 12h, centrifuging at 12000r for 10min, collecting thallus, and extracting crude enzyme by osmotic shock method. The crude enzyme was purified by gradient elution using nickel column, the buffer solution was phosphate buffer solution with different concentration gradient of imidazole at pH 7.4, and the target protein was eluted and purified at 250mM of imidazole.

Example 4: mass spectrometric identification of enzyme reaction products

0.25mg/ml chitotetraose substrate, 0.75. mu.M recombinase solution, and 50mmol/L (Tris-cl, pH8.0, 5mM CoCl2) buffer (pH8.0) were reacted at 37 ℃ for 10 hours, and the enzyme reaction product was detected by mass spectrometry. As shown in fig. 4.

Sequence listing

<110> institute of chemistry and physics, large connection of Chinese academy of sciences

Chitin deacetylase and coding gene and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 927

<212> DNA

<213> Penicillium oxalicum)

<400> 1

atggccccca agagagtatt gatcacctac ggtgttgacg tcgatgctgt tgccggttgg 60

ctcggttcct atgggggtga agattcgaca aacgacatta gccgaggata ctgggctgga 120

actgtgggta cacgaaggct tctaaagctc tttgacaagt atcagatcaa gacaacatgg 180

ttcattcctg gtcattcact ggagaccttt cccgaagaca tggcagcagt ccgcgatgcg 240

gggcatgaaa ttggactgca cggttattcg catgaaaacc caacagacat gactattgag 300

cagcagcggg atgtcctaga caagacatat cgcatgctga ctgagttcac cggtaaacca 360

ccacggggaa gtgtcgcgcc ttggtgggag accagcaaag aaggcgccca attactcctt 420

gactacggca ttgaatatga tcacagcatg agtcacgagg attgtcaggc gtactatctt 480

cgtgcaggtg acacgtggac caagattgac tacactcaaa aggcagaaac ctggatgaag 540

ccactggagc acggcacgca gactggtctg gtggagatcc catccaattg gtacattgat 600

gatctgccac ccatgatgtt catcaagaac gccccgaaca gtcatggctt tgtcaacgcc 660

cgagacgtgg aggacatctg gcgcgatcac tttgactatt tctaccgcga gtatgacgag 720

tttatttttc caattactat tcatccggat gtgtcgggaa gaccacatgt ccttttgatg 780

catgagcgcc tcattgagca tttcaagaag cacgagggcg ttgaattcgt gacaatggag 840

caagtttgtg acgagttcaa gaagaagaac cctgtcccgg agggcgcttt gatgcctgcg 900

cccgttggtc agatgctcaa gaagtag 927

<210> 2

<211> 308

<212> PRT

<213> Penicillium oxalicum)

<400> 2

Met Ala Pro Lys Arg Val Leu Ile Thr Tyr Gly Val Asp Val Asp Ala

1 5 10 15

Val Ala Gly Trp Leu Gly Ser Tyr Gly Gly Glu Asp Ser Thr Asn Asp

20 25 30

Ile Ser Arg Gly Tyr Trp Ala Gly Thr Val Gly Thr Arg Arg Leu Leu

35 40 45

Lys Leu Phe Asp Lys Tyr Gln Ile Lys Thr Thr Trp Phe Ile Pro Gly

50 55 60

His Ser Leu Glu Thr Phe Pro Glu Asp Met Ala Ala Val Arg Asp Ala

65 70 75 80

Gly His Glu Ile Gly Leu His Gly Tyr Ser His Glu Asn Pro Thr Asp

85 90 95

Met Thr Ile Glu Gln Gln Arg Asp Val Leu Asp Lys Thr Tyr Arg Met

100 105 110

Leu Thr Glu Phe Thr Gly Lys Pro Pro Arg Gly Ser Val Ala Pro Trp

115 120 125

Trp Glu Thr Ser Lys Glu Gly Ala Gln Leu Leu Leu Asp Tyr Gly Ile

130 135 140

Glu Tyr Asp His Ser Met Ser His Glu Asp Cys Gln Ala Tyr Tyr Leu

145 150 155 160

Arg Ala Gly Asp Thr Trp Thr Lys Ile Asp Tyr Thr Gln Lys Ala Glu

165 170 175

Thr Trp Met Lys Pro Leu Glu His Gly Thr Gln Thr Gly Leu Val Glu

180 185 190

Ile Pro Ser Asn Trp Tyr Ile Asp Asp Leu Pro Pro Met Met Phe Ile

195 200 205

Lys Asn Ala Pro Asn Ser His Gly Phe Val Asn Ala Arg Asp Val Glu

210 215 220

Asp Ile Trp Arg Asp His Phe Asp Tyr Phe Tyr Arg Glu Tyr Asp Glu

225 230 235 240

Phe Ile Phe Pro Ile Thr Ile His Pro Asp Val Ser Gly Arg Pro His

245 250 255

Val Leu Leu Met His Glu Arg Leu Ile Glu His Phe Lys Lys His Glu

260 265 270

Gly Val Glu Phe Val Thr Met Glu Gln Val Cys Asp Glu Phe Lys Lys

275 280 285

Lys Asn Pro Val Pro Glu Gly Ala Leu Met Pro Ala Pro Val Gly Gln

290 295 300

Met Leu Lys Lys

305

Claims

1. Chitin deacetylasePsThe application of CDA in deacetylation of chitotetraose is characterized in that: the coding gene of the enzyme is SEQ ID NO.1, and the amino acid sequence of the chitin deacetylase generated by the coding of the coding gene is SEQ ID NO. 2;

the chitin deacetylase is secreted and expressed in a form of dimer,PsCDA is capable of acting specifically on chitotetraose; specific deacetylation of chitotetraose (A4) resulted in chitooligosaccharide with 75% acetyl degree (A3D1) and chitooligosaccharide with 50% acetyl degree (A2D2), respectively.