CN112522246A - Method for artificially constructing chitin corpuscle multienzyme complex scaford-ChiC-ChiA-sg and application - Google Patents

Method for artificially constructing chitin corpuscle multienzyme complex scaford-ChiC-ChiA-sg and application Download PDF

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CN112522246A
CN112522246A CN202011398844.8A CN202011398844A CN112522246A CN 112522246 A CN112522246 A CN 112522246A CN 202011398844 A CN202011398844 A CN 202011398844A CN 112522246 A CN112522246 A CN 112522246A
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chitin
chic
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chia
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陈可泉
周宁
张阿磊
陈雪曼
陈燕
王子豪
刘鑫
欧阳平凯
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Nanjing Tech University
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Abstract

The invention provides a method for artificially constructing a chitin corpuscle multienzyme complex scaford-chi A-sg and application thereof. The multienzyme complex utilizes an interaction protein pair existing in a cellulosome from the nature, the adhesion protein (cohesins) in the multienzyme complex is artificially fused and expressed to construct a protein scaffold, and the anchoring protein is fused and expressed on corresponding chitinase, chitinase exonuclease and N-acetylglucosaminidase, so that the chitinase multienzyme complex is obtained by extracellular self-assembly; then the multienzyme complex is applied to the degradation of chitin to prepare the N-acetylglucosamine. The multienzyme complex not only realizes the extracellular self-assembly of free chitinase, but also fixes the distance between the enzymes, promotes the synergistic effect of enzyme elements in the system and forms a substrate channel, thereby improving the degradation efficiency of chitin, obtaining a single monosaccharide product and reducing the production and subsequent separation costs.

Description

Method for artificially constructing chitin corpuscle multienzyme complex scaford-ChiC-ChiA-sg and application
Technical Field
The invention belongs to the technical field of biology, and relates to an enzyme preparation, a method for artificially constructing a small chitin body multienzyme complex scaford-chiC-chiA-sg and application thereof.
Background
According to the statistics of the Chinese fishery association, the total yield of the crayfish breeding in 2018 nationwide is 141.5 ten thousand tons, and the hairy crabs are 80 ten thousand tons, and still show a rapid growth trend. As one of crayfish and hairy crab consumption origins, Jiangsu province only consumes at least 45 tons of crayfish every day in Nanjing City. The leftovers such as heads, shells and the like generated in the processing of the shrimps and the crabs account for about 30 to 40 percent of the body weight. At present, the selling price of the shrimp and crab shell dry powder is only 500-. The value of the shrimp and crab shells can bring ecological and economic benefits. However, to date there has been no satisfactory solution. Most of the shrimp and crab shells are directly discarded or buried, which not only causes resource waste, but also causes serious environmental problems.
The shrimp and crab shells contain a plurality of high value-added substances, such as chitin, protein, lipid, astaxanthin and the like. Wherein, chitin is polysaccharide polymerized by N-acetylglucosamine, exists in three configurations of alpha, beta and gamma in nature, and the alpha-chitin is the most main configuration. Chitin plays an important role in many fields. For example, it can be used as medical material such as artificial skin, molecular sieve, surgical suture, bacteriostatic fabric, hemostatic material, etc.; can be used as antistaling agent, antiseptic and health agent in food industry; in addition to being used as a humectant and a tackifier … … in the cosmetic industry, chitin can also be used as a raw material for preparing functional sugar N-acetylglucosamine (GlcNAc).
GlcNAc has many biological activities, and can be used as health product additive, and can be used for synthesizing important nitrogen-containing compounds such as ceramide, ethanolamine and furan amine derivatives. Therefore, efficient degradation of chitin to the monosaccharide GlcNAc is critical to its utilization. The traditional method for preparing GlcNAc by using chitin as a substrate is a chemical method, the biological activity of the product is poor, the generation of byproducts is accompanied, and meanwhile, the use of strong acid and strong base is involved. Therefore, with the increase of environmental protection pressure, the search for a green and efficient preparation method of GlcNAc is urgent.
The method for preparing GlcNAc by degrading chitin with a biological enzyme method has the characteristics of green process, strong specificity and high biological activity of products, and is considered as an ideal path for replacing the traditional chemical method. Many wild-type bacteria in nature can secrete chitin degrading enzyme systems, and can be used for degrading chitin. For example, Donzelli et al, using the strain Bacillus licheniformis SK-1 chitinase system, degraded crystalline chitin, producing 78% GlcNAc and 10% chitobiose in 6 days. (Donzelli OG, Harman GE. enhanced enzymic hydrolyisis of langoustine shell peptides with mixtures of enzymes from bacteria and fungal sources carbohydrate Research,2003,338(18): 1823) 1833)8 ]; kudan et al degraded 200mg of crystalline chitin using the Trichoderma sp.G and Bacillus sp.W chitinase systems yielding 36mg GlcNAc and 28mg chitobiose over 8 days. (Kudan S, Eksittikul T, Pichia nkura R, Park RD. preparation of N-acetyl-D-glucosamine and N, N' -diacetylchitobiose by enzymic hydrolysides of a chip with its yield chips. journal of Biotechnology 2010,150: 89-89.). These studies indicate that most wild-type chitinase strains have problems such as low degradation efficiency and accumulation of chitobiose when they degrade crystalline chitin. In EP2013355, the authors, by virtue of the action of cellulosome, form a ternary enzyme complex that degrades cellulose, greatly increasing the saccharification efficiency of lignocellulose.
According to the comprehensive literature and patent reports, the distance between enzymes is not fixed in the traditional free multi-enzyme catalytic system, and the intermediate product cannot be captured by the enzyme of the next step in time, so that the inhibition effect on other enzymes is generated, which is a general problem in the multi-enzyme catalytic system. In the chitinase system, the phenomenon that disaccharide accumulation negative feedback inhibits the whole enzyme activity also exists. Therefore, there is a need in the art for a method for increasing the rate of degradation of chitin to produce GlcNAc by increasing the spatial distance between enzymes without inhibiting the enzymes, by fully exploiting the physical space synergy between enzymes and enzymes, and by removing the inhibition of the intermediate product, chitobiose.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for artificially constructing a small chitinase complex scaffold-chiC-chiA-sg and application thereof. The method simulates a cellulosome multienzyme molecular machine in cellulase degrading bacteria in nature on the basis of a previously researched chitinase system, utilizes interaction proteins in the cellulosome to construct a protein scaffold, draws the space distance between chitinase and enzyme, fully exerts the synergistic action between the enzyme and the enzyme, further removes the inhibition effect of intermediate product chitobiose, and greatly improves the efficiency of degrading chitin by the chitinase system to synthesize GlcNAc.
In order to solve the problems of the prior art, the invention adopts the technical scheme that:
a method for artificially constructing a chitinous-chitinous multienzyme complex, scaffold-chiC-chiA-sg, comprising the steps of:
step 1, construction of protein scaffold scaford-ChBD
Fusing and expressing three different fibronectin I-1, cohesinII and cohesinI-3 in a cellulosome and a chitin binding domain ChBD to construct a protein scaffold scaford-ChBD;
step 2, construction of chitinase-dockerins
Three enzymes that play key roles in chitin degradation, respectively: c-terminal fusion anchoring proteins of chitin endonuclease chiC, chitin exonuclease chiA and N-acetylglucosaminidase sg Dockerin1, Dockerin2 and Dockerin3 respectively form chiC-doc1, chiA-doc2 and sg-doc 3;
step 3, extracellular self-Assembly of the chitinous bodies scaford-ChiC-ChiA-sg
The purified protein scaffold scaford-ChBD and three types of chitinases with dockerins are self-assembled in vitro to form a multienzyme complex scaford-chiC-chiA-sg.
As an improvement, the cohesin I-1, cohesin II, cohesin I-3 in the protein scaffold scaford-ChBD in step 1 are respectively from the strains Clostridium cellulovorans, Clostridium cellulolyticum, and Clostridium thermocellum, and the chitin binding domain gene ChBD is from the strain chitin bacteria meiyuanesi SYBC-H1.
As an improvement, the dockerin proteins dockerin-I-1, dockerin-II, dockerin-I-3 described in step 2 are derived from the strains Clostridium cellulovorans, Clostridium cellulolyticum, and Clostridium thermocellum, respectively; chitinase ChiA and chitinase ChiC are from the strain Serratia marcescens, and chitinase sg is from the strain Chitinolyticbacter meiyuanesi SYBC-H1.
As a refinement, the conditions for extracellular self-assembly of the chitinous bodies, scaffold-chiC-chiA-sg, described in step 3 are the protein scaffold, scaffold-ChBD: chiC-doc 1: chiA-doc 2: the protein molar ratio of sg-doc3 was 1: 1: 1: 1, incubation at 4 ℃,200 rpm, pH7.0 for 2 h.
In a further improvement, the protein concentration of the added protein scaffold scaford-ChBD, chiC-doc1, chiA-doc2 and sg-doc3 is 0.2 mu M or 0.4 mu M.
The improvement is that the chitin is one or more of granular chitin, colloidal chitin or high-pressure homogenized chitin.
The application of the multi-enzyme complex scaford-chiC-chiA-sg in preparing N-acetylglucosamine by degrading chitin.
The improvement is that the chitin corpuscle and free chitinase are added into a 50mL centrifuge tube to the final concentration of 0.2 mu M, and the pH value is 7.0TBS to supplement to a 20mL reaction system, wherein the concentration of chitin is 10 g/L; the reaction system is carried out at 37 ℃ and 200 rpm; and 20 mu.L of the conversion solution is taken out at intervals of 0.5h, 1h, 2h, 4h, 8h, 12h and 20h, and the GlcNAc concentration in the conversion solution is measured by using high performance liquid chromatography after dilution.
In a further improvement, the concentration of the substrate chitin in the step 4 is 10-30 g/L.
Has the advantages that:
in the prior art, chitin is degraded by single enzyme degradation or wild bacteria enzyme production degradation. Compared with the prior art, the method for artificially constructing the small chitin body multienzyme complex scaffold-chiC-chiA-sg and the application thereof provided by the invention have the advantages that the artificial construction of the small chitin body degrades the chitin, the method is environment-friendly, high in degradation efficiency, high in monosaccharide yield, reusable and the like, the downstream separation process is simplified, the production cost is reduced, and a certain promotion effect is realized on enzymatic degradation of the chitin to prepare GlcNAc. Has the following advantages:
(1) the chitin corpuscle constructed by the invention fixes the space distance between chitinases, fully exerts the proximity and synergistic effects between the enzymes and realizes the efficient degradation of the chitin.
(2) The chitin body can be added with a second batch of chitin to continue reaction after the complete degradation of the chitin of one batch is finished, the chitin and ChBD on the chitin body are subjected to affinity adsorption, the chitin adsorbed with the multienzyme complex is centrifugally collected, and a proper amount of buffer is added to carry out repeated degradation experiments; the supernatant is collected and used for the subsequent purification work of GlcNAc, and the coupled process of degradation, adsorption and re-degradation is realized.
Drawings
FIG. 1 is a multienzyme assembly scheme according to the present invention;
FIG. 2 is a SDS-PAGE of the scaffold protein and each of the chitinases;
FIG. 3 is a liquid phase analysis of chitin hydrolysate, A being a chitinase hydrolysate; b is a mixed sugar standard product; 1-GlcNAc; 2-chitobiose; 3-chitotriose;
FIG. 4 is a graph showing the progress of chitin degradation by chitin bodies.
Detailed description of the preferred embodiments
Example 1
The following examples are presented to enable one of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way.
Example 1 construction of protein scaffold scaford-ChBD
The fibronectin coenosinI-1, coenosinII and coenosinI-3 are respectively derived from strain Clostridium cellulovorans (ATCC 35296)T),Clostridium cellulolyticum,(ATCC 35319T) And Clostridium thermocellum (ATCC 27405)T) The genome of (a). The chitin binding domain gene ChBD is derived from the laboratory collection strain Chitinolyticbacter meiyuanansis SYBC-H1(ATCC BAA-2140)T) The genome of (a). The genes cohesinI-1, cohesinII and cohesinI-3 were obtained by whole-gene synthesis of the general biosystems (Anhui) Co., Ltd.
The plasmid construction of the protein scaffold scaford-ChBD adopts a one-step cloning method, homologous arms are designed in upper and lower primers of genes cohesinI-1, cohesinII, cohesinI-3 and ChBD, and the plasmid is constructed with a linearized vector pETDuet under the action of a homologous recombinase (Vazyme CE 113). The specific implementation steps are as follows: 1. using plasmid containing cohesin I-1 gene as template, using primer F-cohesin I-1 and R-cohesin I-1 to amplify to obtain cohesin I-1 gene fragment with homologous arms at two ends, and using DNA purification kit (TiangGen) to purify cohesin I-1 gene fragment for later use; the gene fragments of cohesin II and cohesin I-3 were obtained by amplifying with the primers F-cohesin II/R-cohesin II and F-cohesin I-3/R-cohesin I-3 respectively in the same manner, and purified for use. Taking the total DNA of the Chitinolyticbacter meiyuannesi SYBC-H1 as a template, carrying out PCR amplification by using primers F-ChBD and R-ChBD to obtain a target gene ChBD, and purifying for later use. 2. The pETDuet is subjected to double digestion by using restriction enzyme BamHI/XhoI, and a target gene is recovered by using a gel recovery kit (Tianggen) for later use. 3. The purified coensinI-1, coensinII, coensinI-3 and ChBD gene fragments and linearized pETDuet are unloaded, added into a recombination system according to the instruction proportion of a homologous recombinase (Vazyme CE113), and subjected to metal bath at 37 ℃ for 30min to obtain pETDuet-coensinI-1-coensinII-coensinI-3-ChBD. After positive transposons are obtained through transformation and sequencing, the positive transposons are introduced into escherichia coli BL21(DE3) to complete the construction of the protein scaffold scaford-ChBD.
EXAMPLE 2 construction of chitinase-dockerins
The dockerin-I-1, dockerin-II, dockerin-I-3 proteins were derived from the strain Clostridium cellulovorans (ATCC 35296), respectivelyT),Clostridium cellulolyticum,(ATCC 35319T) And Clostridium thermocellum (ATCC 27405)T). The genes dockerin-I-1, dockerin-II, and dockerin-I-3 were obtained by whole-gene synthesis by general biosystems (Anhui) Ltd. The chitin endonuclease chiC and the chitin exonuclease chiA are derived from a laboratory collection strain Serratiamarcescens (ATCC 13880)T) N-acetylglucosaminidase sg is derived from the laboratory collection strain Chitinolyticbacter meiyuanansis SYBC-H1(ATCC BAA-2140)T) The genome of (a).
The plasmid construction of the chiC-doc1 adopts a one-step cloning method, homologous arms are designed and added into upper and lower primers of genes chiC and dockerin-I-1, and the plasmid pET22b-chiC-doc1 is constructed with a linearized vector pET22b under the action of a homologous recombinase (Vazyme CE 113). The specific implementation steps are as follows: 1. taking an empty plasmid containing a gene dockerin-I-1 as a template, carrying out PCR amplification on the empty plasmid by using a primer F-dockerin I-1/R-dockerin I-1 to obtain a target gene dockerin-I-1 with homologous arms at two ends, and purifying a cohesinI-1 gene fragment by using a DNA purification kit (TianGen) for later use; the target gene chiC is obtained by using the genome of Serratia marcocens as a template and amplifying by using a primer F-chiC/R-chiC, and is purified for later use. 2. EcoRI/XhoI double digestion pET22b empty plasmid, glue recovery for use. 3. The purified target genes cohesinI-1, chiC and the linearized vector pET22b are added into a recombination system according to the instruction proportion of homologous recombinase (Vazyme CE113), and the recombinant plasmid pET22b-chiC-doc1 is obtained after 30min of metal bath at 37 ℃. After positive transposons are obtained through transformation and sequencing, the positive transposons are introduced into escherichia coli BL21(DE3), and the construction of a chitinase endonuclease element pET22b-chiC-doc1 is completed. The construction of the chitin exonuclease element pET28a-chiA-doc2 and the N-acetylglucosaminidase element pET28a-sg-doc3 was accomplished in the same manner.
Table 1 shows the primers used in the construction of examples 1 and 2
Figure BDA0002811780780000061
Figure BDA0002811780780000071
Example 3 SDS-PAGE analysis of the various enzymatic elements in the chitinous bodies
Preparing seed liquid of the constructed protein scaffold scaford-ChBD, exonuclease chiC-doc1, endonuclease chiA-doc2, N-acetylglucosaminidase sg-doc3 and chitinase (chiC, chiA and sg) of non-esterified dockerin, culturing for 6-7h at 37 ℃ and 200rpm by taking LB culture medium as culture medium, inoculating the seed liquid into fermentation culture medium filled with 100ml of LB by volume fraction of 2%, culturing to OD of 0.6 at 37 ℃ and 200rpm, adding inducer IPTG by adding amount of 1 per thousand, inducing for 20h at 18 ℃ and 200rpm, and then finishing fermentation and collecting fermentation liquid. 8000g of the fermentation liquor is centrifuged, then the thalli are collected, washed twice by distilled water, resuspended in 25mL TBS (50mM pH7.0), and centrifuged at 8000g after ultrasonication, and the supernatant is collected, namely the crude enzyme solution of each enzyme element and stored at 4 ℃ for later use. As each enzyme element is provided with a His label, each enzyme element is purified on an AKTA protein purification instrument by using a nickel column to obtain pure enzymes of protein scaffold scaford-ChBD, exonuclease chiC-doc1, endonuclease chiA-doc2, N-acetylglucosaminidase sg-doc3 and chitinases (chiC, chiA and sg) without dockerins. Each purified enzyme (15. mu.L) was subjected to heat denaturation with 4X protein SDS loading buffer (TaKARa), and then subjected to SDS-PAGE gel electrophoresis analysis, the results of which are shown in FIG. 2.
EXAMPLE 4 extracellular self-Assembly of the chitinous bodies scaford-ChiC-ChiA-sg
After determining the protein concentration of each enzyme element by the coomassie brilliant blue method, the protein scaffold: chiC-doc 1: chiA-doc 2: the protein molar ratio of sg-doc3 was 1: 1: 1: 1 to 50mM TBS (pH7.0) buffer solution to a final concentration of 0.2. mu.M for each enzyme element, and incubation at 4 ℃ and 200rpm for 2h completed the extracellular self-assembly of the chitinase body scaford-chip-ChiA-sg.
Example 5 validation of Assembly of the Chitosan bodies scaford-ChiC-ChiA-sg
The chitin bodies, scaford-chi a-sg, were validated for assembly using an AKTA protein purifier equipped with a GE gel exclusion column. The unassembled free protein (protein scaffold scaford-ChBD, exonuclease chiC-doc1, endonuclease chiA-doc2 and N-acetylglucosaminidase sg-doc3) and the assembled chitin corpuscle are respectively loaded, and the molecular weight of the chitin corpuscle is far larger than that of a single favorable protein, so that a larger assembly peak is generated compared with the peak generating later stage of the free protein of a control group, and the chitin corpuscle can be verified to finish self-assembly in vitro to form a relatively compact multi-enzyme molecular machine.
EXAMPLE 6 degradation of chitin by Multi-enzyme Complex to prepare N-acetylglucosamine
The chitin degradation system was performed in a 50mL centrifuge tube, and the chitinous bodies obtained in example 4 and free chitinase were added to the 50mL centrifuge tube to a final concentration of 0.2. mu.M, respectively, and supplemented with TBS (pH7.0) to 20mL of the reaction system, wherein the chitin concentration was 10 g/L. The reaction was carried out at 37 ℃ and 200 rpm. And 20 mu.L of the conversion solution is taken out at intervals of 0.5h, 1h, 2h, 4h, 8h, 12h and 20h, and the GlcNAc concentration in the conversion solution is measured by using high performance liquid chromatography after dilution.
The detection conditions for GlcNAc were liquid phase analysis on an Agilent 1290Infinity UHPLC column, Altima HP HILIC column (4.6X 250mm), mobile phase 85% acetonitrile by volume; the flow rate is 0.8mL/min, the detection wavelength of the ultraviolet detector is 210nm, the sample injection amount is 10 mu L, and the column temperature is 40 ℃.
The results are shown in FIGS. 3-4, and it can be seen from FIG. 3 that the chitin bodies degrade the chitin products into single monosaccharides, and the conversion rate of the products reaches 93%. FIG. 4 clearly shows that the degradation activity of assembled chitinous bodies is significantly improved compared with that of free chitinase, and 1.39g/L and 3.05g/L of products are obtained in the 20h degradation process, and the yield is improved by 1.19 times, which fully embodies the proximity effect and the substrate channel effect of the artificial chitinous bodies.
Sequence listing
<110> Nanjing university of industry
<120> method for artificially constructing chitin corpuscle multienzyme complex scaford-chiC-chiA-sg and application thereof
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cacagccagg atccgccggt ggaagccgtt acc 33
<210> 2
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cgagctcgaa ttcggaataa tggtcacggt acc 33
<210> 3
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ccgaattcga gctcgatgga agcaaccccg agt 33
<210> 4
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tgcggccgca agcttggccg catctttcag cgg 33
<210> 5
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
aagcttgcgg ccgcagaact gggcgtggtg gtg 33
<210> 6
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gagatctgcc atatgaccca cattcacacc acc 33
<210> 7
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
catatggcag atctcatgta cccggtatgg cag 33
<210> 8
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tttaccagac tcgaggtggt gatgatggtg atggccgact acgcgccata c 51
<210> 9
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cagccggcga tggccatgga tatgagcaca aataacatta ttaatgccg 49
<210> 10
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gtggtggtgg tggtgctcga gggcgatgag ctgccacagg 40
<210> 11
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tggcagctca tcgcccgtgt tgatctgaaa ggcgat 36
<210> 12
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gtggtggtgc tcgagtgcca gcagtgcttt ttt 33
<210> 13
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
tgccgcgcgg cagccatgcg caaatttaat aaaccgctg 39
<210> 14
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cggtttattc tgcagttgaa cgccggcgct gtt 33
<210> 15
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
agcgccggcg ttcaactgca gaataaaccg gtg 33
<210> 16
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gagtgcggcc gcaagcttct gtgcatcata atcgct 36
<210> 17
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gtgccgcgcg gcagccatat gagccgtccc gccggatcg 39
<210> 18
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ccgggaagtc cacacgggcg cccacctgca ccgcgcg 37
<210> 19
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gtgcaggtgg gcgcccgtgt ggacttcccg gat 33
<210> 20
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gtggtggtgg tggtggtgct cgagtatttt tatacggcag ggt 43

Claims (9)

1. A method for artificially constructing a chitinous-chitinous multienzyme complex, scaffold-chiC-chiA-sg, comprising the steps of:
step 1, construction of protein scaffold scaford-ChBD
Fusing and expressing three different fibronectin I-1, cohesinII and cohesinI-3 in a cellulosome and a chitin binding domain ChBD to construct a protein scaffold scaford-ChBD;
step 2, construction of chitinase-dockerins
Three enzymes that play key roles in chitin degradation, respectively: c-terminal fusion anchoring proteins of chitin endonuclease chiC, chitin exonuclease chiA and N-acetylglucosaminidase sg Dockerin1, Dockerin2 and Dockerin3 respectively form chiC-doc1, chiA-doc2 and sg-doc 3;
step 3, extracellular self-Assembly of the chitinous bodies scaford-ChiC-ChiA-sg
The purified protein scaffold scaford-ChBD and three types of chitinases with dockerins are self-assembled in vitro to form a multienzyme complex scaford-chiC-chiA-sg.
2. The method of claim 1, wherein the adhesion proteins cohesinI-1, cohesinII of the protein scaffold scaford-ChBD in step 1,cohesinI-3are respectively derived from strainsClostridium cellulovorans, Clostridium cellulolyticum, and Clostridium thermocellumChitin binding domain gene ChBD from bacterial strainsChitinolyticbacter meiyuanensi SYBC-H1。
3. The method of claim 1, wherein the dockerin proteins dockerin-I-1, dockerin-II, and dockerin-I-3 of step 2 are derived from the respective strainsClostridium cellulovorans, Clostridium cellulolyticum, and Clostridium thermocellum(ii) a Chitinase chiA and chitinase chiC are from bacterial strainsSerratia marcescensChitinase sg is derived from a strainChitinolyticbacter meiyuanensi SYBC-H1。
4. The method of claim 1, wherein the conditions for extracellular self-assembly of the chitinous body scadord-chiC-chiA-sg in step 3 are as follows: protein scaffold scaford-ChBD: chiC-doc 1: chiA-doc 2: the protein molar ratio of sg-doc3 was 1: 1: 1: 1, incubation at 4 ℃,200 rpm, pH7.0 for 2 h.
5. The method of claim 4, wherein the protein scaffold scaford-ChBD, chiC-doc1, chiA-doc2 and sg-doc3 have a protein concentration of 0.2 μ M or 0.4 μ M.
6. Use of the multi-enzyme complex scaford-chi A-sg according to claim 1 for the degradation of chitin to produce N-acetylglucosamine.
7. The use of claim 6, wherein the chitin is one or more of a granular chitin or a colloidal chitin or a high pressure homogenized chitin.
8. The use of claim 6, wherein the chitinous bodies and free chitinase are added to a 50mL centrifuge tube to a final concentration of 0.2. mu.M, and supplemented with pH7.0 TBS to 20mL reaction system, wherein the chitin concentration is 10 g/L; the reaction system is carried out at 37 ℃ and 200 rpm; and 20 mu.L of the conversion solution is taken out at intervals of 0.5h, 1h, 2h, 4h, 8h, 12h and 20h, and the GlcNAc concentration in the conversion solution is measured by using high performance liquid chromatography after dilution.
9. The use of claim 6, wherein the substrate chitin is present at a concentration of 10-30 g/L.
CN202011398844.8A 2020-12-02 2020-12-02 Method for artificially constructing chitin corpuscle multienzyme complex scaford-ChiC-ChiA-sg and application Pending CN112522246A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109810966A (en) * 2019-03-28 2019-05-28 南京工业大学 A kind of chitinase CmChi6 gene and its clonal expression and application
CN109880859A (en) * 2019-04-01 2019-06-14 南京工业大学 A kind of method of immobilised lysine decarboxylase production pentanediamine

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109810966A (en) * 2019-03-28 2019-05-28 南京工业大学 A kind of chitinase CmChi6 gene and its clonal expression and application
CN109880859A (en) * 2019-04-01 2019-06-14 南京工业大学 A kind of method of immobilised lysine decarboxylase production pentanediamine

Non-Patent Citations (3)

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Title
ALEI ZHANG， 等: "A novel bacterial β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis Activities", BIOTECHNOL BIOFUELS *
JIE ZHOU 等: "Immobilization and Purification of Enzymes With the Novel Affinity Tag ChBD-AB From Chitinolyticbacter meiyuanensis SYBC-H1", FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY *
刘妍池: "设计人工"几丁质小体"降解几丁质", 中国优秀硕士学位论文全文数据库，工程科技1辑 *

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Application publication date: 20210319