CN112359033B - Chitin binding domain and application thereof - Google Patents

Abstract

The invention discloses a chitin binding domain, which is constructed on a general expression vector and used for fusion expression of target proteins and realizing synchronous purification and immobilization of the target proteins. According to the invention, by utilizing the specific affinity of chitin and fusion protein, efficient immobilization of beta-glucosidase is realized, ginsenoside Rb1 is taken as a substrate, and ginsenoside Rd is obtained by utilizing efficient preparation of the immobilized enzyme. The immobilization method has the advantages of low cost, high enzyme immobilization efficiency, small enzyme activity loss, strong specificity and the like, and fundamentally solves the problems of irrecoverable free enzyme, poor stability, low repetition rate and the like of the traditional whole cell immobilization method.

Description

Chitin binding domain and application thereof

Technical Field

The invention belongs to the field of enzyme engineering, and particularly relates to an enzyme immobilization dominated by chitin binding domain and application thereof.

Background

Beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) has the effect of specifically hydrolyzing oligosaccharides containing glycosidic linkages and glycosyl analogues. In the aspect of energy industry, the beta-glucosidase can effectively hydrolyze oligosaccharides and cellobiose, so that the product inhibition of cellulose in the hydrolysis process of the cellulose can be eliminated, and the beta-glucosidase can be used together with other cellulose to effectively hydrolyze cellulose; in the aspect of food processing, beta-glucosidase can process some substances in green plants into spices, and the spices are used in fruit wine, fruit juice and tea juice to play a role in flavoring, so that the mouthfeel of the spices is enhanced, and the quality of products is improved; as a feed additive, the beta-glucosidase can hydrolyze glycosidic bonds in the feed to generate various nutritional components, so that the quality of the feed is improved; in the aspect of medicine, the beta-glucosidase can decompose saponins, ketones or phenols in fruits of some plants in the nature, and the produced aglycone and other substances have biological activity in human bodies and animal bodies, so that the application value of the beta-glucosidase has considerable potential. Currently, beta-glucosidase has been developed and utilized to some extent, but is mainly used as a free enzyme. Mainly has the defects of low stability, difficult separation from the substrate, repeated recycling and the like, and greatly increases the production cost. The enzyme immobilization design can realize the repeated recycling of the enzyme, is more beneficial to the separation of the enzyme from the substrate after the reaction, and is more suitable for industrial production and application due to the characteristics of stability and durability.

The enzyme immobilization is to bind or limit the enzyme to a certain area such as the surface or the inside of a solid material, and simultaneously maintain the catalytic activity of the enzyme, and after the catalysis is completed, the enzyme can be recycled through simple treatment steps such as centrifugation or filtration. After the enzyme is immobilized, the advantages of improved stability, easy recovery and repeated use, simplified process operation, reduced cost and the like are often presented. Currently, enzyme immobilization methods mainly include physical methods (physical adsorption methods, embedding methods, etc.) and chemical methods (binding methods, crosslinking methods, etc.). The two methods have the main problems in the enzyme immobilization process: the enzyme and the solid material often lack specificity, and pretreatment such as extraction and purification of the enzyme is needed in advance. The pretreatment processes not only can cause enzyme loss, but also can increase the investment in related reagents, equipment and the like, and can increase the use cost of the immobilized enzyme.

Therefore, development of an enzyme immobilization method having high specificity, high immobilization efficiency, low loss of enzyme activity and low cost has been demanded.

Disclosure of Invention

Problems to be solved by the invention

In view of the problems in the prior art, the chitin binding domain with chitin binding activity is obtained through excavation, and the purpose of synchronously completing enzyme purification and immobilization is achieved by utilizing the specific and efficient binding performance of the polypeptide and chitin.

Solution for solving the problem

(1) A chitin-binding domain having an amino acid sequence selected from any one of (i) - (iiI) as follows:

(i) And SEQ ID NO:1, a mutant polypeptide having at least 70%, at least 80%, or at least 90% sequence identity to the polypeptide of the amino acid sequence set forth in seq id no;

(ii) A polypeptide encoded by a polynucleotide that hybridizes under very high stringency conditions with a polynucleotide as set forth in (a) or (b):

(a) A polynucleotide encoding a polypeptide of the amino acid sequence shown in (i);

(b) The full-length complementary polynucleotide of (a);

(iii) Fragments of the polypeptides shown in (i), (ii) and which fragments still have chitin-binding activity.

(2) The chitin-binding domain of (1), wherein the amino acid sequence of the chitin-binding domain comprises a deletion or addition of at least one amino acid residue at the N-terminal or C-terminal position of the polypeptide of the sequence shown in (i).

(3) An expression vector comprising the chitin-binding domain of any one of (1) or (2).

(4) The expression vector of (3), further comprising a connecting peptide upstream of the chitin-binding domain for connecting a foreign gene to be immobilized.

(5) The chitin binding domain of any one of (1) - (2) or the expression vector of any one of (3) - (4) for enzyme immobilization.

(6) The use according to (5), wherein the immobilized enzyme is β -glucosidase.

(7) The preparation method of the immobilized enzyme is characterized by comprising the following steps:

1) Constructing a universal expression vector comprising the chitin-binding domain of any one of (1) - (2) and a connecting peptide;

2) Inserting exogenous genes to be immobilized into the universal expression vector in the step 1) to obtain a fusion protein expression vector;

3) Transforming host cells with the fusion protein expression vector constructed in the step 2) to obtain recombinant cells;

4) Culturing the recombinant cells in the step 3) to obtain fusion protein supernatant expressing the exogenous gene;

5) Mixing the supernatant obtained in the step 4) with chitin to realize enzyme immobilization and purification.

(8) The method for producing an immobilized enzyme according to (7), wherein the host cell is a bacterium or a fungus, preferably a bacterium, most preferably E.coli; the exogenous gene is preferably a gene encoding beta-glucosidase.

(9) A method for producing ginsenoside Rd by immobilized beta-glucosidase, which is characterized by comprising the following steps:

1) The immobilized beta-glucosidase prepared by the preparation method of (7) or (8);

2) And (3) taking ginsenoside Rb1 as a substrate, constructing a reaction system containing the immobilized beta-glucosidase in the step 1), and carrying out catalytic reaction to prepare the ginsenoside Rd.

(10) The method of (9), wherein the reaction system further comprises a buffer, preferably a phosphate buffer; the time of the catalytic reaction is preferably 2 hours.

Advantageous effects of the invention

1. The novel chitin binding domain is obtained by excavation, has stronger chitin specific binding capacity, uses low-cost renewable chitin or chitosan as an immobilization carrier, can realize the aim of synchronously completing enzyme purification and immobilization after specific binding, on one hand, greatly simplifies the enzyme immobilization process, on the other hand, the immobilization carrier is natural in origin, easy to obtain, low in cost and environment-friendly, and greatly reduces the enzyme immobilization cost;

2. the immobilized enzyme has the advantages of good adsorptivity, high immobilization efficiency, small enzyme activity loss and the like, can keep higher enzyme activity after being recycled for many times, solves the defects of irrecoverable free enzyme, poor stability and low repetition rate of the traditional whole cell immobilization method, has simple and convenient separation of products and enzymes, simplifies the separation process and reduces the production cost.

3. The invention firstly utilizes the chitin binding domain to immobilize the beta-glucosidase, and the beta-glucosidase fusion protein constructed by the invention can be specifically and affinity adsorbed on chitin to realize the immobilization of the beta-glucosidase, and is used for catalyzing ginsenoside Rb1 to efficiently convert Rd, and the conversion rate is 100%.

Drawings

FIG. 1 shows a plasmid map of a universal expression vector with chitin binding domain and a connecting peptide gene.

FIG. 2 shows a plasmid map of recombinant β -glucosidase.

Figure 3 shows the loading of recombinant β -glucosidase on chitin particles.

FIG. 4 shows the immobilized enzyme reuse lot and the remaining enzyme activities.

FIG. 5 shows a thin layer chromatography of a ginsenoside product converted using immobilized beta-glucosidase.

Detailed Description

Definition of the definition

As used in the present disclosure, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein and are polymers of amino acids of any length.

As used in this disclosure, the term "chitin-binding domain" refers to a polypeptide that specifically binds to chitin or chitosan, typically of small molecular weight (30-100 amino acids), while the chitin-binding domain has good stability, and can bind to chitin or chitosan in harsh environments, and thus has good utility in the purification and immobilization of a protein (enzyme) of interest.

As used in this disclosure, the term "sequence identity" or "percent identity" in the comparison of two polypeptides refers to that they are identical or have a specified percentage of identical sequences when compared and aligned for maximum correspondence using amino acid residue sequence comparison algorithms or by visual inspection.

Methods of determining "sequence identity" or "percent identity" to which the present disclosure relates include, but are not limited to: computer molecular biology (Computational Molecular Biology), lesk, a.m. editions, oxford university press, new york, 1988; biological calculation: informatics and genome project (Biocomputing: informatics and Genome Projects), smith, d.w. editions, academic press, new york, 1993; computer analysis of sequence Data (Computer Analysis of Sequence Data), first part, griffin, a.m. and Griffin, h.g. editions, humana Press, new jersey, 1994; sequence analysis in molecular biology (Sequence Analysis in Molecular Biology), von Heinje, g., academic Press, 1987 and sequence analysis primer (Sequence Analysis Primer), gribskov, m. and deveerux, j. Code MStockton Press, new york, 1991 and carllo, h. and Lipman, d., SIAM j.applied math.,48:1073 (1988). The preferred method of determining identity is to obtain the greatest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: GCG package (Devereux, J. Et al, 1984), BLASTP, BLASTN and FASTA (Altschul, S, F. Et al, 1990). BLASTX programs are available to the public from NCBI and other sources (BLAST handbook, altschul, S. Et al, NCBI NLM NIH Bethesda, md.20894; altschul, S. Et al, 1990). The well-known Smith Waterman algorithm can also be used to determine identity.

In some embodiments, a polypeptide of the disclosure having chitin binding activity comprises a polypeptide that hybridizes to SEQ ID NO:1 has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid residues. In other embodiments, a polynucleotide of the present disclosure encoding a polypeptide having chitin binding activity comprises a nucleotide sequence that encodes SEQ ID NO:2 has a "sequence identity" or "percent identity" of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide. The judgment/calculation of "sequence identity" or "percent identity" may be based on any suitable region of the sequence.

As used in the present disclosure, the term "fragment" means a polypeptide or a catalytic or carbohydrate binding module that lacks one or more (e.g., several) amino acids from the amino and/or carboxy terminus of a mature polypeptide or domain. In the technical scheme of the disclosure, the fragment has a chitin binding function.

As used in this disclosure, the term "mutant" refers to a polypeptide comprising an alteration (i.e., substitution, insertion, and/or deletion) at one or more (e.g., several) positions relative to the "wild-type", wherein a substitution refers to a substitution of an amino acid occupying one position with a different amino acid. Deletions refer to the removal of an amino acid occupying a position. Insertion refers to the addition of an amino acid immediately following the amino acid occupying the position.

In the present disclosure, "mutation" may also be comprised in the corresponding SEQ ID NO:1, and adding, deleting or substituting amino acids on the basis that the binding activity of the chitin binding domain is not affected at one or more positions of the sequence shown in 1. It is well known that altering a few amino acid residues in certain regions of a polypeptide, e.g., non-important regions, does not substantially alter biological activity. Illustratively, a "mutation" of the present disclosure is comprised in a sequence corresponding to SEQ ID NO:1 and at least one amino acid residue at least one of the C-terminal and N-terminal of the polypeptide of the sequence shown in fig. 1, and the polypeptide has chitin-binding activity. In some embodiments, a "mutation" of the disclosure corresponds to a sequence as set forth in SEQ ID NO:1 or C-terminal, from 1 to 5 amino acids, preferably from 1 to 3, most preferably 1, and having chitin binding activity.

Examples

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

The experimental techniques and methods used in this example are conventional techniques unless otherwise specified, such as those not specified in the following examples, and are generally performed under conventional conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Materials, reagents and the like used in the examples are all available from a regular commercial source unless otherwise specified.

Example 1: construction of general expression vectors with chitin binding domains

And (3) connecting the flexible connecting peptide with a chitin binding domain to construct a universal expression vector. Wherein the amino acid sequence of the chitin binding domain is shown as SEQ ID No.1, and the nucleotide sequence is shown as SEQ ID No. 2; the amino acid sequence of the flexible connecting peptide is shown as SEQ ID No.3, and the nucleotide sequence is shown as SEQ ID No. 4. Wherein the gene of the connecting peptide is positioned at the 5' end of the chitin binding domain gene and inserted into the pET22b vector to obtain the universal expression vector pLChBD. The plasmid map of the universal expression vector pLChBD is shown in fig. 1. Plasmid pLChBD was stored in E.coli DH 5. Alpha.

Example 2: recombinant beta-glucosidase construction with chitin binding domain

The genome of acidophilic heat-resisting bacterium Acidothermus cellulolyticus B (ATCC 43068) is used as a template, primers are designed according to the gene sequence of beta-glucosidase (the nucleotide sequence is SEQ ID No.5, the amino acid sequence is SEQ ID No. 6), ndeI and XhoI cleavage sites are selected, and primers P1 and P2 are designed as follows:

P1：5’-CGACTTCATATGATGACACAAATCGAAGAGCG-3’

P2：5’-ACCGCTCGAGTCAGGGCGCCGCGATCGTGTTC-3’

PCR amplification gave a fragment with the restriction enzymes NdeI and XhoI, and the universal expression vector pLChBD was also added with the restriction enzymes NdeI and XhoI and digested at 37℃for 3 hours. The double digested product was purified using a DNA purification kit. After mixing the two purified products, the whole gold company T4 DNA ligase was added and ligated at 25℃for 5 hours to give a recombinant plasmid. Coli DH 5. Alpha. Competent cells were transformed by CaCl2 transformation, plated on LB plates containing ampicillin, and cultured overnight at 37 ℃. And (3) picking positive growth colonies, and verifying by extracting recombinant plasmids from the kit, wherein the constructed recombinant plasmids are named as pLChBD-AcBg.

Example 3: preparation of recombinant beta-glucosidase with chitin binding domain

The recombinant plasmid pLChBD-AcBg obtained in example 2 was transformed into E.coli C43 (DE 3), the strain was spread on LB plate containing ampicillin, cultured overnight at 37℃to pick up colonies with good status, positive transformants were verified by PCR method, and cultured in liquid LB medium containing ampicillin to select recombinant E.coli strain meeting the requirements. The preserved strain is inoculated into 5mL of liquid LB culture medium containing ampicillin and cultured overnight at 37 ℃, 100mL of new LB liquid culture medium is inoculated with 1% (v/v) of inoculum size, when the strain is cultured until OD600 = 1.0, IPTG is added to a final concentration of 1mM/L, and the strain is induced and cultured for 24 hours at 25 ℃. The cells were collected by centrifugation at 8000rpm/min for 10min to induce expression, resuspended in 10mL of 20mM sodium phosphate buffer, pH6.0, containing 40mM/L NaCl, sonicated in an ice-water bath for 10min, and the cell wall-broken cells were centrifuged at 12000rpm/min for 10min, and the supernatant was used as a crude enzyme solution of recombinant beta-glucosidase for immobilization or lyophilization.

The LB culture medium comprises the following components: 10g/L of sodium chloride, 10g/L of peptone, 5g/L of yeast powder and pH7.0, and 1.5% of agar powder which is LB solid medium is added into the mixture, and the mixture is sterilized at 121 ℃ for 20min. When inoculating recombinant E.coli, filtered and sterilized ampicillin was added to the medium at a final concentration of 50. Mu.g/mL.

Example 4: preparation of immobilized beta-glucosidase

Mixing the recombinant beta-glucosidase obtained above with chitin powder: adding chitin particles with different weights into a crude enzyme solution containing 5 activity units (5U), adding sodium phosphate buffer (with final concentration of 20mM, pH of 6.0), keeping the total volume to be 1ml, and incubating at 800rpm for 30min at room temperature-40 ℃ to realize one-step immobilization purification of recombinant beta-glucosidase immobilized on the chitin particles. Separating the immobilized enzyme from the liquid by low-temperature high-speed centrifugation (10000 rpm,4 ℃ for 10 min), discarding the redundant supernatant, re-suspending the immobilized enzyme by using 20mM sodium phosphate buffer, centrifuging (10000 rpm,4 ℃ for 10 min) to remove the supernatant, repeating the operation for a plurality of times until no protein residue (detected by Coomassie brilliant blue solution) exists in the supernatant, removing non-specifically bound proteins, and removing chitin particles after the supernatant is the immobilized beta-glucosidase. The immobilized beta-glucosidase is subjected to freeze drying treatment to obtain the dried immobilized beta-glucosidase, which can be stored for long term use.

The enzyme activity determination method comprises the following steps: the reaction buffer containing 1mM/L of o-nitrophenyl-beta-D-glucoside (pNPG) in the final concentration was preheated at 60℃and 100. Mu.L of enzyme solution was added for reaction for 3min, the reaction was placed on ice water after the completion of the reaction, 500. Mu.L of 1M/L of Na2CO3 solution was added for stopping the reaction, and the absorbance was measured at 405nm by centrifugation. Definition of enzyme activity unit: the amount of enzyme that hydrolyzes pNPG (o-nitrophenyl-. Beta. -D-glucoside) to release 1. Mu.M o-nitrophenol (pNP) for 1 minute was taken as one enzyme activity unit (U). 1g of chitin particles were tested to bind up to 500U of beta-glucosidase. The results of the study are shown in FIG. 3.

Immobilized beta-glucosidase reusable batch test: the immobilized beta-glucosidase was tested for the first lot, defining the first lot enzyme activity as 100%. After the measurement, the reaction is washed by buffer solution, enzyme is fixed until no reaction liquid remains, the reaction is repeatedly carried out for 5 batches, and the ratio of the repeated use times to the residual enzyme activity is calculated. The test results showed that the residual activity of immobilized enzyme remained 42.8% of the initial activity after the continuous use of 5 batches. The test results are shown in FIG. 4.

Example 5: the immobilized beta-glucosidase is used for conversion of ginsenoside.

1U of immobilized beta-glucosidase is suspended in 20mM/L, pH6.0 sodium phosphate buffer solution, 200 mu L of 1mg/mL ginsenoside Rb1 solution is added, the reaction is carried out for different time at 60 ℃, and the immobilized enzyme and the reaction solution are separated by centrifugation at 12000rpm/min for 5 min. 100 mu l of water saturated n-butanol is added into the reaction solution for extraction for three times, the extract is collected, the n-butanol is completely volatilized at 80 ℃, the residue is dissolved by using methanol, the residue is filtered and filtered by a 0.22 mu m organic filter membrane, and the residue is stored in a penicillin bottle. The enzymatic hydrolysis products were analyzed using thin layer chromatography. Developing agent: chloroform, methanol, n-butanol and water are mixed according to the volume ratio of 13:10:10:8. The color-developing agent is as follows: 50ml of methanol and 10ml of concentrated sulfuric acid were added dropwise. Color development was performed at 105℃for 5 minutes.

The result of thin layer chromatography analysis shows that the immobilized beta-glucosidase converts ginsenoside Rb1 into Rd, 100% conversion can be realized in 2h, the reaction time is prolonged until the product is not changed any more in 24h, namely Rd is the final product of the immobilized beta-glucosidase converting ginsenoside Rb1 (figure 5).

Sequence listing

<110> Jilin university

<120> a chitin binding domain and uses thereof

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Pro Ala Tyr Asp Asn Ser Lys Val Tyr Val Gly Gly Asn Val Val Ser

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ccggcgtatg acaacagcaa ggtgtacgtg ggggggaatg tggtgagtta taacgggcac 60

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ggaggtggtg gatcaggagg tggtggatca ggaggtggtg gatcagaagc tgcagctaag 60

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<213> Acidothermus cellulolyticus

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Arg Ala Thr Ala Gln Arg Phe Ala Glu Tyr Ala Val Val Val Ala Arg

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Glu Leu Gly Asp Arg Val Asn Phe Trp Ile Thr Leu Asn Glu Pro Trp

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Cys Ala Ala Phe Leu Gly Tyr Gly Ala Gly Val His Ala Pro Gly His

180 185 190

Thr Asp Ser Ala Glu Ala Leu Thr Ala Ala His His Leu Leu Leu Ala

195 200 205

His Gly Leu Ala Val Gln Ala Leu Gly Ser Val Leu Pro Pro Asp Cys

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Gln Met Ala Ile Thr Leu Asn Pro Ala Val Ala Arg Pro Ala Ser Leu

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Ala Glu Glu Asp Val Ala Ala Ala Arg Lys Val Asp Gly Leu Gln Asn

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Arg Leu Trp Leu Asp Pro Leu Phe His Gly Thr Tyr Pro Gln Asp Val

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Val Asn Phe Thr Ser Lys Val Thr Asp Trp Ser Phe Val Arg Asp Asn

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Asp Leu Ala Val Ile Ala Thr Pro Phe Asp Ile Leu Gly Val Asn Tyr

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ctgaaccacg gcatcacgcc tgcactgacg ctttaccact gggacctccc gcaggcgttg 420

caagaccagg gcgggtggac gaatcgtgca actgcacagc gattcgctga atatgcggtc 480

gtcgtcgccc gcgaattggg tgatcgggtg aatttctgga ttactctcaa cgagccgtgg 540

tgcgcggcgt tcctcggtta cggggcgggc gttcatgcac ccggacacac cgacagtgcg 600

gaagccttga cggcggcgca tcacctgctc cttgcgcacg gcctggcagt ccaggccctg 660

ggctcggttc tgccgccgga ttgccagatg gcgatcacgt tgaatccagc ggtcgcgcga 720

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cggctctggc tggatccgct gtttcacggc acctatccgc aggatgtggt gaatttcacg 840

tcaaaagtca ccgactggtc gttcgtccgt gacaacgacc tcgcagtgat tgcgaccccc 900

ttcgacattc tgggggtcaa ttactataac ccggtcatcg tcggtcacta tgccggctcc 960

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tccggactct acgaactcct cattcggctg aaccgcgact atccacggcc gatcatgatt 1140

actgagaatg gcgccgcgtt cgatgatgtc gtcacggaca acaatcgggt gcgggatccg 1200

gcacgggcgg cgtacatcca ggaacatctt gccgccctcc accaagcgat tgccgacggc 1260

gtggacgttc gcggttatta cctctggtca ttgatcgaca actttgaatg ggcgtacgga 1320

tactcacgcc ggttcggcat cgtttatgtc gatttcgaga ctcaggagcg gatcatcaag 1380

gacagtgggt atttctactc gctggtcgca cggacgaaca cgatcgcggc gccctga 1437

Claims (9)

1. A polypeptide which is a chitin-binding domain, wherein the amino acid sequence of the polypeptide is selected from any one of (i) - (ii) as follows:

(i) SEQ ID NO:1, a polypeptide having an amino acid sequence as set forth in seq id no;

(ii) A polypeptide encoded by a polynucleotide encoding a polypeptide having an amino acid sequence as set forth in (i).

2. An expression vector for expressing the polypeptide of claim 1 as a chitin-binding domain.

3. The expression vector of claim 2, further comprising a linker peptide upstream of the polypeptide that acts as a chitin-binding domain for linking a foreign gene to be immobilized.

4. Use of a polypeptide according to claim 1 or an expression vector according to any one of claims 2-3 for immobilizing β -glucosidase.

5. The preparation method of the immobilized enzyme is characterized by comprising the following steps: 1) Constructing a universal expression vector comprising the polypeptide of claim 1 which is a chitin-binding domain and a linker peptide; 2) Inserting the beta-glucosidase coding gene to be immobilized into the general expression vector in the step 1) to obtain a fusion protein expression vector; 3) Transforming host cells with the fusion protein expression vector constructed in the step 2) to obtain recombinant cells; 4) Culturing the recombinant cells in the step 3) to obtain fusion protein supernatant expressing the exogenous gene; 5) Mixing the supernatant obtained in the step 4) with chitin to realize enzyme immobilization and purification.

6. The method for producing an immobilized enzyme according to claim 5, wherein the host cell is E.coli.

7. A method for producing ginsenoside Rd by immobilized beta-glucosidase, which is characterized by comprising the following steps: 1) The method according to any one of claims 5 or 6, wherein the immobilized beta-glucosidase is obtained; 2) And (3) taking ginsenoside Rb1 as a substrate, constructing a reaction system containing the immobilized beta-glucosidase in the step 1), and carrying out catalytic reaction to prepare the ginsenoside Rd.

8. The method of claim 7, the reaction system further comprising a buffer; the time of the catalytic reaction was 2 hours.

9. The method of claim 8, wherein the buffer is phosphate buffer.

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