CN112175974A - Chitin deacetylase gene, chitin deacetylase and preparation method and application thereof - Google Patents
Chitin deacetylase gene, chitin deacetylase and preparation method and application thereof Download PDFInfo
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- CN112175974A CN112175974A CN202011011189.6A CN202011011189A CN112175974A CN 112175974 A CN112175974 A CN 112175974A CN 202011011189 A CN202011011189 A CN 202011011189A CN 112175974 A CN112175974 A CN 112175974A
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- cdaba
- chitin deacetylase
- chitin
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Abstract
The invention belongs to the technical field of genetic engineering, and particularly relates to a chitin deacetylase gene, a chitin deacetylase and a preparation method and application thereof. The nucleotide sequence of the chitin deacetylase gene cdaba is shown in SEQ ID NO 1, and the chitin deacetylase gene is obtained by comprehensively optimizing the sequence of the chitin deacetylase derived from Bacillus atrophaeus. The amino acid sequence of the chitin deacetylase CdaBa is shown in SEQ ID NO. 2, the chitin deacetylase CdaBa not only enriches the variety and source ways of the chitin deacetylase, but also has the specific activity of 352U/mg, shows good stability in a wider temperature range and a wider pH range, and has good application prospect and industrial value.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a chitin deacetylase gene CdaBa, a corresponding recombinant expression vector and a recombinant expression strain, as well as a chitin deacetylase CdaBa, and a preparation method and application thereof.
Background
Chitin is a linear polymer formed by randomly connecting N-acetyl-beta-D-glucosamine through beta-1, 4 glycosidic bonds, has a structure similar to cellulose, and is a renewable polysaccharide second to cellulose in nature. Chitin is poorly soluble due to its dense crystalline structure and is almost insoluble in water, dilute acids, dilute bases, concentrated bases, and common organic solvents, thereby limiting its commercial application. The research shows that the derivative of chitin, namely chitosan (the product of chitosan after deacetylation) can be well dissolved in inorganic acids such as dilute nitric acid, hydrochloric acid, acetic acid and the like and most organic acids, and the research shows that the chitosan has biological activities such as bacteriostasis, cancer resistance, blood pressure reduction and the like, and can be applied to industries such as medicine, food and the like.
The predominant current method for converting chitin to chitosan is chemical, which is the removal of the acetyl groups of chitin by the application of large amounts of concentrated alkali to obtain chitosan. This method, although efficient, has a number of disadvantages such as: the structure of chitin is damaged, the deacetylation process is difficult to control accurately, the environmental pollution caused by reaction emissions is serious, and the like, so that the method has important significance for finding a green and efficient processing method. The chitin deacetylase can catalyze chitin deacetylation to form chitosan, belongs to enzymatic conversion, and has the advantages of high deacetylation degree, consistent deacetylation degree, mild reaction conditions, complete product structure, easy process control, no environmental pollution and the like. At present, the main problems of limiting the industrialized application of chitosan method deacetylation are that the fermentation activity of chitin deacetylase is low, the production cost is high, and the overall production cost of the enzymatic method process is high. Therefore, the improvement of the fermentation enzyme activity of the chitin deacetylase and the reduction of the production cost are the basis of the wide application of the chitosan deacetylation.
Disclosure of Invention
The invention aims to provide a chitin deacetylase gene CdaBa, a corresponding recombinant expression vector, a recombinant expression strain, a chitin deacetylase CdaBa, a preparation method and an application thereof, and aims to solve the technical problem of low fermentation activity of the existing chitin deacetylase.
In order to achieve the above object, in one aspect of the present invention, there is provided a chitin deacetylase gene cdaba, whose nucleotide sequence is the nucleotide sequence shown in SEQ ID No. 1, or a nucleotide sequence with the same function obtained by deletion, insertion or substitution of the nucleotide sequence shown in SEQ ID No. 1.
In another aspect of the present invention, a chitin deacetylase CdaBa is a protein encoded by the chitin deacetylase gene CdaBa of the present invention, and the amino acid sequence of the protein is the amino acid sequence shown in SEQ ID NO. 2, or the amino acid sequence with the same function obtained by deletion, insertion or substitution of the amino acid sequence shown in SEQ ID NO. 2.
In another aspect of the invention, a recombinant expression vector is provided, which comprises the chitin deacetylase gene cdaba provided by the invention.
In another aspect of the present invention, a recombinant expression strain is provided, which comprises the recombinant expression vector provided by the present invention.
In another aspect of the present invention, a method for preparing a chitin deacetylase, CdaBa, is provided, which comprises the following steps:
providing a chitin deacetylase gene cdaba, an expression vector and an expression strain;
amplifying the chitin deacetylase gene cdaba, and connecting an amplification product with the expression vector to obtain a recombinant expression vector;
transferring the recombinant expression vector into an expression strain to obtain a recombinant expression strain;
culturing the recombinant expression strain to obtain chitin deacetylase CdaBa;
wherein the nucleotide sequence of the chitin deacetylase gene cdaba is the nucleotide sequence shown as SEQ ID NO. 1, or the nucleotide sequence with the same function obtained by deletion, insertion or substitution of the nucleotide sequence shown as SEQ ID NO. 1; the amino acid sequence of the chitin deacetylase CdaBa is the amino acid sequence shown in SEQ ID NO. 2, or the amino acid sequence shown in SEQ ID NO. 2 is subjected to deletion, insertion or substitution to obtain the amino acid sequence with the same function.
In the last aspect of the invention, the invention provides the application of the chitin deacetylase CdaBa or the chitin deacetylase CdaBa prepared by the preparation method of the chitin deacetylase CdaBa in chitin hydrolysis.
The natural bacteria of Bacillus atrophaeus have the defects of difficult control of culture process, easy degradation of strain activity, low yield of chitin deacetylase and the like when the chitin deacetylase is produced by fermentation, and the defects prevent the application of the chitin deacetylase in catalyzing the chitin deacetylation. According to the codon preference of pichia pastoris, the expression adaptation index of a sequence derived from Bacillus atrophaeus (GenBank: CP024051.1) chitin deacetylase is increased to 0.75 from the original 0.51, and the GC content is increased to 41.2% from 36.7%, so that the chitin deacetylase gene cdaba is obtained. Through the comprehensive optimization, the sequence stability and codon adaptability of the obtained chitin deacetylase gene CdaBa are remarkably improved, high-efficiency expression can be realized in a pichia pastoris expression system, and the production cost of the chitin deacetylase gene CdaBa can be reduced.
The chitin deacetylase CdaBa provided by the invention not only enriches the species and source ways of the chitin deacetylase, but also has the enzyme specific activity of 352U/mg, shows good stability in a wider temperature range and a wider pH range, and has good application prospect and industrial value.
The recombinant expression vector provided by the invention comprises the chitin deacetylase gene cdaba, and the chitin deacetylase gene cdaba is subjected to codon optimization according to the preference of pichia pastoris, so that the stability and codon adaptability are better, and correspondingly, the recombinant expression vector comprising the chitin deacetylase gene cdaba also has better stability, is favorable for realizing the stable and efficient expression of the chitin deacetylase gene cdaba, and plays an important role in reducing the production cost of the chitin deacetylase gene cdaba.
The recombinant expression strain provided by the invention comprises the recombinant expression vector provided by the invention, so that the recombinant expression strain can be used for expressing chitin deacetylase CdaBa encoded by a chitin deacetylase gene CdaBa. Meanwhile, the sequence of the chitin deacetylase gene CdaBa has good stability and codon adaptability, so that the obtained recombinant expression strain can stably and efficiently express the chitin deacetylase CdaBa.
In the preparation method of the chitin deacetylase CdaBa, a heterogenous recombination method is adopted, the chitin deacetylase gene CdaBa is amplified to obtain a corresponding recombinant expression vector and a recombinant expression strain, and the corresponding recombinant expression vector and the recombinant expression strain are cultured to obtain the chitin deacetylase CdaBa. The preparation method provided by the invention has the advantages of simple steps, easily controlled process, capability of producing the chitin deacetylase CdaBa stably, efficiently and at low cost, and good industrial prospect. Meanwhile, the chitin deacetylase CdaBa prepared by the preparation method of the chitin deacetylase CdaBa also has the advantages of high enzyme activity and stable enzymological characteristics.
The chitin deacetylase CdaBa provided by the invention or the chitin deacetylase CdaBa prepared by the preparation method can be used for catalyzing chitin deacetylation. The chitin deacetylase CdaBa has high enzyme activity and good stability in a wide temperature range and a wide pH range, so that when the chitin deacetylase CdaBa is used for catalyzing chitin deacetylation, a catalytic reaction can be efficiently and stably carried out.
Drawings
FIG. 1 is a graph showing high-density fermentation culture of recombinant expression strains according to example 5 of the present invention;
FIG. 2 is an SDS-PAGE protein electrophoresis chart of the chitin deacetylase CdaBa obtained in example 6 of the present invention;
FIG. 3 is a graph showing the optimal reaction temperature and thermal stability of the chitin deacetylase CdaBa obtained in example 6 of the present invention;
FIG. 4 is a graph showing the pH optimum and pH stability of the chitin deacetylase CdaBa obtained in example 6 of the present invention;
FIG. 5 is a graph showing the results of the hydrolytic activities of CdaBa, a chitin deacetylase obtained in example 6 according to the present invention, on different substrates.
Detailed Description
In order to make the objects, technical solutions and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described, and the embodiments described below are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments obtained by a person of ordinary skill in the art without any inventive step in connection with the embodiments of the present invention shall fall within the scope of protection of the present invention. Those whose specific conditions are not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In the description of the present invention, the term "and/or" describing an association relationship of associated objects means that there may be three relationships, for example, a and/or B, may mean: a is present alone, A and B are present simultaneously, and B is present alone. Wherein A and B can be singular or plural. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
It should be understood that the weight of the related components mentioned in the embodiments of the present invention may not only refer to the specific content of each component, but also represent the proportional relationship of the weight among the components, and therefore, it is within the scope of the disclosure that the content of the related components is scaled up or down according to the embodiments of the present invention. Specifically, the weight described in the embodiments of the present invention may be a unit of mass known in the chemical field such as μ g, mg, g, kg, etc.
In addition, unless the context clearly uses otherwise, an expression of a word in the singular is to be understood as including the plural of the word. The terms "comprises" or "comprising" are intended to specify the presence of stated features, quantities, steps, operations, elements, portions, or combinations thereof, but are not intended to preclude the presence or addition of one or more other features, quantities, steps, operations, elements, portions, or combinations thereof.
It should be noted that the molecular biology experimental methods not specifically described in the examples of the present invention are performed by referring to the specific methods listed in the molecular cloning experimental manual (third edition) j. sambrook, or according to the kit and the product specification; related reagents and biomaterials, if not specifically stated, are commercially available.
The embodiment of the invention provides a chitin deacetylase gene cdaba, the nucleotide sequence of which is the nucleotide sequence shown in SEQ ID NO. 1, or the nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO. 1.
Nucleotide sequence of chitin deacetylase gene cdaba (SEQ ID NO: 1):
atgtctaactctccaagaagatacagaaagatgaagcaaatcggtaagatcgctttcggtatcttgat gatcttgggtgttactttgttctgtatctcttggaagactttggctggtccaagattggaaccaatcaaggaatctgcttctttgtctgaacaaatgcaaaaggaaaagaacaagaactctgctaagaagcaagacgaacaaaagacttctccagaaatcggtaaggttgtttacttgactttcgacgacggtccacacccagctgcttctgaagaaatcatgaacttgttgcacaagtacaacgctaagggtactttcttcatgttgaagccaaacatcgttcaaaacccagacatcgttaagaagatggttgaatctggtcactctgttggttctcacggtgttactcacaaggtttctgaaatctacaagtctccagactctttcgctactgaaatgaacgacactttggacttcatcaaggaaactactaaggttaacactcacttgatcagagctccatacggttctaagccatacatcactggtccattcagagaagttgttaagagaaaccaattcaacttgtgggactggactgttgactctgaagactggaagtacactcacggtgaattcatcaagaacactatccaacaagttaacaacttggttggtaaggaaccattggttgttttgatgcacgaaaagccaactactgctgcttacttgggtgaattgttgaagtacttcagagaatctggttacgaaatgaaggctatcgacgactctatcaagccagttcaattcagattcaactaa
in the previous research, the activity of the chitin deacetylase in Bacillus atrophaeus fermentation liquor is compared and analyzed, and the chitin deacetylase in the Bacillus atrophaeus fermentation liquor has the advantages of good thermal stability, high catalytic activity, wide action pH and the like. However, the natural bacteria of Bacillus atrophaeus have many disadvantages in the fermentative production of chitin deacetylases: (1) the culture process is not easy to control; (2) the activity of the strain is easy to degrade, and the strain needs to be repeatedly activated and rejuvenated; (3) the enzyme components in the fermentation liquor are complex, the yield of the chitin deacetylase is low, and the main enzyme components are protease and amylase, so that the processing and the like at the later stage are not facilitated. These disadvantages have hindered the use of chitin deacetylases to catalyze chitin deacetylation. According to the codon preference of pichia pastoris, the expression adaptive index of a sequence derived from Bacillus atrophaeus (GenBank: CP024051.1) chitin deacetylase is increased to 0.75 from the original 0.51, and the GC content is increased to 41.2% from 36.7%, so that the chitin deacetylase gene cdabaa is obtained. Through the comprehensive optimization, the sequence stability and codon adaptability of the obtained chitin deacetylase gene CdaBa are remarkably improved, high-efficiency expression can be realized in a pichia pastoris expression system, and the production cost of the chitin deacetylase gene CdaBa can be reduced.
Correspondingly, the embodiment of the invention also provides a chitin deacetylase CdaBa, which is a protein encoded by the chitin deacetylase gene CdaBa, and the amino acid sequence of the chitin deacetylase CdaBa is the amino acid sequence shown in SEQ ID NO. 2, or the amino acid sequence with the same function is obtained by deletion, insertion or substitution of the amino acid sequence shown in SEQ ID NO. 2.
Amino acid sequence of chitin deacetylase CdaBa (SEQ ID NO: 2):
MSNSPRRYRKMKQIGKIAFGILMILGVTLFCISWKTLAGPRLEPIKESASLSEQMQKEKNKNSAKKQDEQKTSPEIGKVVYLTFDDGPHPAASEEIMNLLHKYNAKGTFFMLKPNIVQNPDIVKKMVESGHSVGSHGVTHKVSEIYKSPDSFATEMNDTLDFIKETTKVNTHLIRAPYGSKPYITGPFREVVKRNQFNLWDWTVDSEDWKYTHGEFIKNTIQQVNNLVGKEPLVVLMHEKPTTAAYLGELLKYFRESGYEMKAIDDSIKPVQFRFN
the chitin deacetylase CdaBa provided by the embodiment of the invention not only enriches the species and source ways of the chitin deacetylase, but also has the specific activity of 352U/mg, shows good stability in a wider temperature range and a wider pH range, and has good application prospect and industrial value.
Correspondingly, the embodiment of the invention also provides a recombinant expression vector which comprises the chitin deacetylase gene cdaba provided by the embodiment of the invention.
The recombinant expression vector provided by the embodiment of the invention comprises the chitin deacetylase gene cdaba, and the chitin deacetylase gene cdaba is subjected to codon optimization according to the preference of pichia pastoris, so that the stability and codon adaptability are better, and correspondingly, the recombinant expression vector comprising the chitin deacetylase gene cdaba provided by the embodiment of the invention also has better stability, is favorable for realizing the stable and efficient expression of the chitin deacetylase gene cdaba, and plays an important role in reducing the production cost of the chitin deacetylase gene cdaba.
Correspondingly, the embodiment of the invention also provides a recombinant expression strain, which comprises the recombinant expression vector provided by the embodiment of the invention.
The recombinant expression strain provided by the embodiment of the invention comprises the recombinant expression vector provided by the embodiment of the invention, so that the recombinant expression strain can be used for expressing the chitin deacetylase CdaBa encoded by the chitin deacetylase gene CdaBa. Meanwhile, the sequence of the chitin deacetylase gene cdaba has good stability and codon adaptability, so that the obtained recombinant expression strain can stably and efficiently express the chitin deacetylase CdaBam.
Correspondingly, the embodiment of the invention also provides a preparation method of the chitin deacetylase CdaBa, which comprises the following steps:
s1, providing a chitin deacetylase gene cdaba, an expression vector and an expression strain;
s2, amplifying the chitin deacetylase gene cdaba, and connecting the amplification product with an expression vector to obtain a recombinant expression vector;
s3, transferring the recombinant expression vector into an expression strain to obtain a recombinant expression strain;
s4, culturing the recombinant expression strain to obtain chitin deacetylase CdaBa;
wherein, the nucleotide sequence of the chitin deacetylase gene cdaba is the nucleotide sequence shown in SEQ ID NO. 1, or the nucleotide sequence with the same function obtained by deletion, insertion or substitution of the nucleotide sequence shown in SEQ ID NO. 1; the amino acid sequence of the chitin deacetylase CdaBa is the amino acid sequence shown in SEQ ID NO. 2, or the amino acid sequence shown in SEQ ID NO. 2 is obtained by deletion, insertion or substitution, and has the same function.
In the preparation method of the chitin deacetylase CdaBa provided by the embodiment of the invention, a heterologous recombination method is adopted, the chitin deacetylase gene CdaBa is amplified to obtain a corresponding recombinant expression vector and a recombinant expression strain, and the corresponding recombinant expression vector and the recombinant expression strain are cultured to obtain the chitin deacetylase CdaBa. The preparation method provided by the embodiment of the invention has the advantages of simple steps, easily controlled process, capability of stably and efficiently producing the chitin deacetylase CdaBa at low cost and good industrial prospect. Meanwhile, the chitin deacetylase CdaBa prepared by the preparation method of the chitin deacetylase CdaBa provided by the embodiment of the invention also has the advantages of high enzyme activity and stable enzymological characteristics.
Specifically, in S1, the nucleotide sequence of the chitin deacetylase gene cdaba is shown in SEQ ID NO:1, and the sequence is obtained by comprehensively optimizing a sequence (GenBank: CP024051.1) derived from Bacillus atrophaeus chitin deacetylase, and the specific optimization method and the advantages of the optimized gene are as described above and are not repeated herein.
The expression vector is used for constructing a recombinant expression vector for expressing the chitin deacetylase gene cdaba shown in SEQ ID NO. 1 in the embodiment of the invention. In some embodiments, a pichia pastoris expression vector is selected. The reason is that the chitin deacetylase gene cdaba provided by the embodiment of the invention is optimized according to the codon preference of pichia pastoris, so that the expression stability and the expression quantity of the chitin deacetylase gene cdaba can be further improved by selecting a pichia pastoris expression vector. Specifically, a pichia pastoris expression vector pPICZ α A can be selected for construction of a recombinant expression vector, and accordingly, the resulting recombinant expression vector is named pPICZ α A-cdabaa.
Expression strains, used in the examples of the present invention to construct recombinant expression strains. In some embodiments, a pichia pastoris expression strain is selected. The chitin deacetylase gene CdaBa provided by the embodiment of the invention is optimized according to codon preference of pichia pastoris, and a pichia pastoris expression system is a mature expression system in the field, so that the chitin deacetylase gene CdaBa has the advantages of easiness in genetic engineering operation, capability of realizing high-density fermentation, high expression level, easiness in protein separation and purification, suitability for industrial large-scale production and the like, and is favorable for obtaining the chitin deacetylase CdaBa with high enzyme activity and lower production cost. Specifically, pichia pastoris X33 can be selected as the expression strain.
In S2, the chitin deacetylase gene cdaba is amplified, and the amplification product is connected with an expression vector to obtain a recombinant expression vector. In some embodiments, the primers used to amplify the chitin deacetylase gene, cdaba, include a forward primer, cdaba-F, having the nucleotide sequence shown in SEQ ID NO. 3, and a reverse primer, cdaba-R, having the nucleotide sequence shown in SEQ ID NO. 4.
The nucleotide sequence of cdaba-F (SEQ ID NO: 3):
agtcgaattcggtccaagattggaaccaatca
the nucleotide sequence of cdaba-R (SEQ ID NO: 4):
ttctctagagttgaatctgaattgaact
in S3, the obtained recombinant expression vector is transferred into an expression strain to obtain a recombinant expression strain which can express corresponding protein. The embodiment of the invention has no special requirements on the specific construction method of the recombinant expression strain, and the conventional method in the field can be adopted.
In S4, the recombinant expression strain is cultured to make the nucleotide sequence of the chitin deacetylase gene CdaBa replicated along with the reproduction of the recombinant expression strain, and the culture of the recombinant expression strain is collected, separated and purified to obtain the chitin deacetylase CdaBa. In some embodiments, the recombinant expression strain is cultured by a high density fermentation process comprising: the recombinant expression strain was inoculated into a 250mL Erlenmeyer flask containing YPG medium and cultured overnight at 30 ℃ and 200rpm, and then inoculated into a 500mL Erlenmeyer flask containing YPG medium and cultured overnight with shaking at 30 ℃ and 200 rpm. Inoculating the recombinant expression strain obtained by twice overnight culture into a fermentation tank containing BSM culture medium, and performing fermentation culture at 30 deg.C, pH5.0, stirring speed of 500rpm, and air flow rate of 40L/min to obtain fermentation broth. Purifying the fermentation liquid obtained by high-density fermentation culture to obtain the chitin deacetylase CdaBa.
Correspondingly, the embodiment of the invention also provides application of the chitin deacetylase CdaBa provided by the embodiment of the invention or the chitin deacetylase CdaBa prepared by the preparation method of the chitin deacetylase CdaBa in catalysis of chitin deacetylation.
The chitin deacetylase CdaBa provided by the embodiment of the invention or the chitin deacetylase CdaBa prepared by the preparation method of the invention can be used for catalyzing chitin deacetylation. The chitin deacetylase CdaBa has high enzyme activity and good stability in a wide temperature range and a wide pH range, so that when the chitin deacetylase CdaBa is used for catalyzing chitin deacetylation, a catalytic reaction can be efficiently and stably carried out.
In some embodiments, the reaction temperature of the chitin deacetylase CdaBa provided by the embodiments of the present invention in the reaction for catalyzing the deacetylation of chitin is 30 ℃ to 60 ℃, and the optimal reaction temperature is 50 ℃.
In some embodiments, the chitin deacetylase CdaBa provided by the embodiments of the present invention catalyzes the reaction of chitin deacetylation at a reaction pH of 6.0-10.0, and an optimal reaction pH of 7.0.
In order to make the details and operation of the above-mentioned embodiments of the present invention clearly understood by those skilled in the art and to make the progress of the chitin deacetylase gene, the chitin deacetylase and the methods and applications thereof apparent, the above-mentioned technical solutions are illustrated by the following examples.
Experimental materials and reagents referred to in the following examples:
strain and carrier: coli strain Top10, Pichia pastoris X33, expression vector pPICZ alpha A were all purchased from commercial sources.
Q5 high fidelity Taq enzyme MIX was purchased from NEB; the plasmid extraction and gel purification kit is purchased from Tiangen Biotechnology (Beijing) Co., Ltd; restriction enzymes were purchased from daisies technologies (beijing) ltd; bleomycin (Zeocin) was purchased from Invitrogen corporation.
Culture medium: the E.coli medium was LB (1% (w/v) peptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl, pH 7.0). LBA for LB medium add 25 u g/mL bleomycin.
The yeast medium was YPD (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose). The yeast screening culture medium is YPDZ (YPD +100mg/L bleomycin);
the Yeast induction medium was BMGY (1% (w/v) Yeast extract, 2% (w/v) peptone, 1.34% (w/v) YNB, 0.00004% (w/v) biotin, 1% glycerol (v/v)), wherein YNB was the Yeast Nitrogen source Base (Yeast Nitrogen Base).
BMMY medium: the composition was the same as BMGY, except that 0.5% (v/v) methanol was used instead of glycerin.
Reagent for measuring chitin deacetylase activity: 200mg/L of p-nitroacetanilide aqueous solution; 0.05mol/L of phosphate buffer pH 7.0.
Example 1
This example provides a process for analyzing the sequence of a chitin deacetylase gene and optimizing the codon, comprising the following steps:
(11) based on the defects of the natural bacterium Bacillus atrophaeus, the expression efficiency is improved by a heterologous recombinant expression mode. Wherein, a pichia pastoris expression system is selected as a heterologous expression host. The first step in heterologous recombinant expression is to find the Bacillus atrophaeus chitin deacetylase gene. Its whole genome sequence was submitted to NCBI database in 2017 with accession number CP 024051.1. Through analyzing the genome data, the nucleotide in the genome 4031023-4031853 interval is found to be the coding gene of the chitin deacetylase CdaBa. The total length of the gene is 831bp, and 276 amino acids are coded. The first 38 amino acids of the chitin deacetylase CdaBa were found to be the signal peptide by signal peptide prediction software SignalP-5.0Server prediction analysis.
(12) Due to differences in gene coding between Bacillus atrophaeus and pichia pastoris, the chitin deacetylase CdaBa needs to be optimized according to pichia pastoris codon bias. The optimized encoding gene of chitin deacetylase CdaBa is named as CdaBa (the nucleotide sequence is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO: 2). Compared with the original gene, the GC content of cdaba is 41.2 percent, and is closer to the Pichia pastoris coding preference (the GC content of the original gene is 36.7 percent); the expression adaptation index (CAI) of cdaba was increased from 0.51 to 0.75.
Example 2
This example provides a process for constructing a recombinant expression vector containing the chitinase gene cdaba obtained in example 1, wherein the recombinant expression vector uses pPICZ alpha A as an expression vector, and the signal peptide is an alpha signal peptide carried by pPICZ alpha A, so that the cdaba signal peptide needs to be removed in the construction process. The method comprises the following specific steps:
(13) designing a pair of primers cdaba-F and cdaba-R according to the sequence of the synthetic gene cdaba, wherein the nucleotide sequences are respectively shown as SEQ ID NO. 3-4, and the primers are used for amplifying the cdaba gene without signal peptide;
(14) obtaining a gene cdabas with a signal peptide removed by PCR amplification, respectively carrying out enzyme digestion on an expression vector pPICZ alpha A and the gene cdabas overnight by using restriction endonucleases EcoRI and XbaI, purifying and recovering the overnight enzyme digested expression vector pPICZ alpha A and the gene cdabas, and then carrying out a ligation reaction;
(15) transferring the ligation reaction product into escherichia coli Top10 by adopting a heat shock method, and verifying a recombinant transformant by adopting a bacterial liquid PCR;
(16) and inoculating the successfully verified transformant into an LBZ liquid culture medium, extracting plasmids, performing sequencing verification, and finally obtaining an expression vector pPICZ alpha A-cdabas.
Example 3
This example provides a process for constructing a recombinant pichia pastoris engineering strain containing the recombinant expression vector obtained in example 2, which includes the following steps:
(17) after the vector pPICZ alpha A-cdabas is linearly expressed by using restriction endonuclease SacI, the vector is transferred into Pichia pastoris X33 by adopting an electrical transformation method, so that different positive transformants are obtained. The recombinant pichia pastoris is screened by a 24-pore plate method, and the method comprises the following specific steps: the recombinant transformants on the YPDZ plates were picked up one by one with a toothpick into 24-well plates containing 2mL of BMGY medium per well, incubated overnight at 30 ℃ and 200rpm for 24 hours, then centrifuged at 4000rpm to remove the supernatant, 2mL of BMMY medium was added, incubated at 30 ℃ and 200rpm for 24 hours, and the chitinase activity of the recombinant transformants was determined.
(18) The method for measuring the activity of chitin deacetylase is as follows: adding 600 μ L of 0.05mol/L pH7.0 phosphate buffer solution with 50 deg.C pre-incubation, 200 μ L of 200mg/L paranitroacetanilide aqueous solution and 200 μ L enzyme solution into 2mL centrifuge tube, reacting in 50 deg.C water bath for 10min, terminating the reaction in boiling water bath, centrifuging for 10min, and measuring absorbance of supernatant at 410 nm. The control was 200. mu.L of enzyme solution inactivated in a boiling water bath of the same concentration for 10 min. Definition of enzyme activity unit (U): the amount of enzyme required to produce 1. mu.g of p-nitroaniline per hour under the above reaction conditions was one unit of enzyme activity. Through screening 72 positive transformants, finally obtaining 3 strains with enzyme activity superiority which are respectively named as Cd3(15U/mL), Cd71(12U/mL) and Cd11 (9U/mL).
Example 4
The present embodiment provides a process of performing shake flask fermentation culture on 3 strains with superior enzyme activity obtained in example 3, specifically as follows:
(19) firstly, inoculating the corresponding recombinant engineering strain into a 50mL centrifuge tube containing 5mL of BMGY culture medium, culturing at 30 ℃ and 220rpm for about 24 hours, and inoculating the cultured recombinant yeast engineering strain into a 250mL triangular flask containing 50mL of BMMY culture medium according to the inoculation amount of 1% (v/v). The shake flask culture condition is 30 ℃, 220rpm, 1% (v/v) methanol is added every 24 hours for induction, simultaneously, sampling is carried out for measuring the activity of chitin deacetylase, and the enzyme activity measurement shows that the enzyme activities of Cd3, Cd71 and Cd11 after 96 hours of induction culture are 43U/mL, 35U/mL and 31U/mL respectively.
Example 5
The embodiment provides a process for performing high-density fermentation culture on the recombinant engineering bacteria Cd3 obtained in the embodiment 4 by shake-flask fermentation culture, which comprises the following specific steps:
(20) the single colony recombinant engineered yeast strain was inoculated into a 250mL Erlenmeyer flask containing 50mL YPG medium, and cultured overnight at 30 ℃ with shaking at 200 rpm. The overnight cultured recombinant engineered yeast was inoculated into a 500mL Erlenmeyer flask containing 100mL YPG medium at an inoculum size of 1% (v/v), and cultured overnight at 30 ℃ with shaking at 200rpm until OD 600 was more than 10. The recombinant engineered yeast strain obtained by two overnight cultures was inoculated into a 5L fermentor containing 2L of BSM medium at an inoculum size of 10% (v/v). The culture conditions of the recombinant yeast engineering bacteria in a 5L fermentation tank are as follows: the temperature was 30 ℃, the pH was 5.0, the stirring speed was 500rpm, and the air flow rate was 40L/min. In the initial stage of culture, cells were grown using glycerol as a carbon source. When the wet weight of the cells reaches about 185g/L, the glycerol feeding is stopped, and the induction with methanol is started after the glycerol is completely absorbed by the cells (the dissolved oxygen rises rapidly). The amount of methanol added was adjusted according to the dissolved oxygen. In the culture process, samples are taken every 24 hours to determine the wet weight, the enzyme activity and the total protein concentration of the thalli. The resulting high density fermentation culture is shown in FIG. 1.
As can be seen from FIG. 1, the fermentation enzyme activity reached a maximum (1352U/mL) when the induction culture was carried out for 144 hours, the protein concentration was 4.23g/L at the maximum, and the wet weight of the cells reached a maximum (452g/L) after the induction for 168 hours.
Example 6
This example provides a process of purifying the fermentation broth obtained in example 5 to obtain chitin deacetylase CdaBa, specifically as follows:
(21) centrifuging fermentation liquor of a 5L fermentation tank, taking supernatant, purifying and recovering;
(22) carrying out ultrafiltration concentration on the supernatant enzyme solution by using a 10kDa ultrafiltration tube;
(23) purifying by using a Ni-IDA protein purification kit to obtain the chitin deacetylase CdaBa.
The specific activity of the purified chitin deacetylase CdaBa enzyme is 352U/mg through experimental determination. The SDS-PAGE protein after purification showed the results shown in FIG. 2, and it is clear from FIG. 2 that the recombinant chitin deacetylase CdaBa after purification showed two bands (about 35kDa and 27kDa, respectively). The online software NetNGlyc 1.0Server prediction analysis finds that CdABa has N glycosylation sites at 157 th amino acid, so that the CdABa is subjected to glycosylation modification during pichia pastoris recombinant expression, and recombinant CdABa with two sizes is presented. The recombinant CdABa of 35kDa is found to be a glycosylation modification through a deglycosylation experiment, and only a band with the size of about 27kDa is left after deglycosylation.
Experimental example 1
The enzyme activities of the chitin deacetylase CdaBa obtained in example 6 at different temperatures of 30-90 ℃ were measured at pH7.0, and the enzyme activities at the highest temperature were measured as 100%, and the relative enzyme activities at other temperatures were calculated. The results of the detection are shown in FIG. 3. As can be seen from FIG. 3, when the temperature is in the range of 30-60 ℃, the relative enzyme activities of the chitin deacetylase CdaBa are all greater than 65%, and the optimal reaction temperature is 50 ℃.
The thermal stability was determined as follows: the chitin deacetylase CdaBa is subjected to water bath heat treatment for 10 minutes at different temperatures of 30-90 ℃, the residual enzyme activity is measured, and the relative residual enzyme activity at other temperatures is calculated by taking the enzyme activity of a sample which is not subjected to heat treatment as 100%. The results of the experiment are shown in FIG. 3. As shown in FIG. 3, the chitin deacetylase CdaBa has good stability at the temperature of 30-80 ℃, and the residual enzyme activity is greater than 62% after heat treatment for 10 minutes.
Experimental example 2
The enzyme activities of the chitin deacetylase CdaBa obtained in example 6 at pH 3.0-10.0 were measured at 50 ℃ respectively, the enzyme activity at the highest pH was measured as 100%, the relative enzyme activities at other pH were calculated, and the results are shown in FIG. 4. As can be seen from FIG. 4, the optimum reaction pH value of the chitin deacetylase CdaBa is 7.0, and the relative enzyme activity is more than 65% in the range of pH 6.0-10.0.
The effect of different pH on the stability of chitin deacetylase CdaBa was determined as follows:
the chitin deacetylase CdaBa is stored at room temperature for 6 hours under the condition of pH 3.0-10.0 to determine the residual enzyme activity, the enzyme activity of a sample which is not treated is taken as 100%, the relative residual enzyme activity at other temperatures is calculated, and the detection result is shown in figure 4. As can be seen from FIG. 4, the residual enzyme activities of the chitin deacetylase CdaBa are all greater than 70% in the pH range of 5.0-10.0 after being stored for 6 hours at room temperature, which indicates that the chitin deacetylase CdaBa has good stability in the pH range.
Experimental example 3
This example examined the effect of different metal ions on the stability of the chitin deacetylase CdaBa obtained in example 6.
In the environment containing different metal ions of 1mmol/L, the chitin deacetylase CdaBa is preserved for 5h at the temperature of 30 ℃, and the enzyme activity without adding the metal ion enzyme solution is taken as 100 percent, and the residual enzyme activity under other conditions is calculated. The results of the experiment are shown in table 1.
TABLE 1 Effect of different Metal ions on the stability of recombinant chitin deacetylase CdaBa
As can be seen from Table 1: ca2+And Co2+Activating chitin deacetylase CdaBa, wherein the residual enzyme activities are respectively 105% and 110%; mg (magnesium)2+And Cu2+Inhibiting chitin deacetylase CdaBa, wherein the residual enzyme activities are 71% and 75% respectively; the residual metal ions have little influence on the chitin deacetylase CdaBa, and the residual enzyme activities are all more than 90 percent.
Experimental example 4
This example examined the hydrolytic activity of the chitin deacetylase CdaBa obtained in example 6 on different substrates.
The hydrolytic activity of the recombinant chitin deacetylase CdaBa on different substrates was evaluated using the kit method (acetic acid assay kit, from R-Biopharm AG, Germany). The substrates used in the experiments included chitin powder (Sigma C7170), colloidal chitin (from laboratory) and ethyleneglycol chitin (from laboratory). The experimental procedure was as follows:
(24) 0.05g of each substrate was weighed out and dissolved in 10ml of 25mM phosphate buffer (pH7.0), and 300U of recombinant chitin deacetylase CdaBa was added;
(25) placing the enzyme and substrate mixture on a shaking table, and reacting for 6 hours at 40 ℃ and 200 rpm;
(26) centrifuging to obtain supernatant, performing boiling water bath on the supernatant for 5 minutes, cooling to room temperature, performing activity determination, and calculating relative enzyme activities under other substrates according to the enzyme activity under the substrate with the highest determined activity as 100%, wherein the result is shown in FIG. 5.
As can be seen from FIG. 5, the recombinant chitin deacetylase CdaBa is most active on ethanediol chitin, followed by colloidal chitin (relative activity 40%) and chitin powder (relative activity 23%). Therefore, the chitin deacetylase CdaBa obtained in the embodiment of the invention can effectively hydrolyze acetyl of chitin, and has great application potential in the field of chitin deacetylation.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Shenzhen Runkang ecological environment, Inc.; Shenzhen Nuopuxin agro-chemical, Inc
<120> chitin deacetylase gene, chitin deacetylase and preparation method and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 831
<212> DNA
<213> Bacillus atrophaeus
<400> 1
atgtctaact ctccaagaag atacagaaag atgaagcaaa tcggtaagat cgctttcggt 60
atcttgatga tcttgggtgt tactttgttc tgtatctctt ggaagacttt ggctggtcca 120
agattggaac caatcaagga atctgcttct ttgtctgaac aaatgcaaaa ggaaaagaac 180
aagaactctg ctaagaagca agacgaacaa aagacttctc cagaaatcgg taaggttgtt 240
tacttgactt tcgacgacgg tccacaccca gctgcttctg aagaaatcat gaacttgttg 300
cacaagtaca acgctaaggg tactttcttc atgttgaagc caaacatcgt tcaaaaccca 360
gacatcgtta agaagatggt tgaatctggt cactctgttg gttctcacgg tgttactcac 420
aaggtttctg aaatctacaa gtctccagac tctttcgcta ctgaaatgaa cgacactttg 480
gacttcatca aggaaactac taaggttaac actcacttga tcagagctcc atacggttct 540
aagccataca tcactggtcc attcagagaa gttgttaaga gaaaccaatt caacttgtgg 600
gactggactg ttgactctga agactggaag tacactcacg gtgaattcat caagaacact 660
atccaacaag ttaacaactt ggttggtaag gaaccattgg ttgttttgat gcacgaaaag 720
ccaactactg ctgcttactt gggtgaattg ttgaagtact tcagagaatc tggttacgaa 780
atgaaggcta tcgacgactc tatcaagcca gttcaattca gattcaacta a 831
<210> 2
<211> 276
<212> PRT
<213> Bacillus atrophaeus
<400> 2
Met Ser Asn Ser Pro Arg Arg Tyr Arg Lys Met Lys Gln Ile Gly Lys
1 5 10 15
Ile Ala Phe Gly Ile Leu Met Ile Leu Gly Val Thr Leu Phe Cys Ile
20 25 30
Ser Trp Lys Thr Leu Ala Gly Pro Arg Leu Glu Pro Ile Lys Glu Ser
35 40 45
Ala Ser Leu Ser Glu Gln Met Gln Lys Glu Lys Asn Lys Asn Ser Ala
50 55 60
Lys Lys Gln Asp Glu Gln Lys Thr Ser Pro Glu Ile Gly Lys Val Val
65 70 75 80
Tyr Leu Thr Phe Asp Asp Gly Pro His Pro Ala Ala Ser Glu Glu Ile
85 90 95
Met Asn Leu Leu His Lys Tyr Asn Ala Lys Gly Thr Phe Phe Met Leu
100 105 110
Lys Pro Asn Ile Val Gln Asn Pro Asp Ile Val Lys Lys Met Val Glu
115 120 125
Ser Gly His Ser Val Gly Ser His Gly Val Thr His Lys Val Ser Glu
130 135 140
Ile Tyr Lys Ser Pro Asp Ser Phe Ala Thr Glu Met Asn Asp Thr Leu
145 150 155 160
Asp Phe Ile Lys Glu Thr Thr Lys Val Asn Thr His Leu Ile Arg Ala
165 170 175
Pro Tyr Gly Ser Lys Pro Tyr Ile Thr Gly Pro Phe Arg Glu Val Val
180 185 190
Lys Arg Asn Gln Phe Asn Leu Trp Asp Trp Thr Val Asp Ser Glu Asp
195 200 205
Trp Lys Tyr Thr His Gly Glu Phe Ile Lys Asn Thr Ile Gln Gln Val
210 215 220
Asn Asn Leu Val Gly Lys Glu Pro Leu Val Val Leu Met His Glu Lys
225 230 235 240
Pro Thr Thr Ala Ala Tyr Leu Gly Glu Leu Leu Lys Tyr Phe Arg Glu
245 250 255
Ser Gly Tyr Glu Met Lys Ala Ile Asp Asp Ser Ile Lys Pro Val Gln
260 265 270
Phe Arg Phe Asn
275
<210> 3
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
agtcgaattc ggtccaagat tggaaccaat ca 32
<210> 4
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ttctctagag ttgaatctga attgaact 28
Claims (10)
1. A chitin deacetylase gene cdaba is characterized in that the nucleotide sequence is shown in SEQ ID NO. 1, or the nucleotide sequence with the same function is obtained by deletion, insertion or substitution of the nucleotide sequence shown in SEQ ID NO. 1.
2. A chitin deacetylase CdaBa, which is the protein encoded by the chitin deacetylase gene CdaBa of claim 1, having an amino acid sequence represented by SEQ ID NO. 2, or an amino acid sequence having the same function obtained by deletion, insertion or substitution of the amino acid sequence represented by SEQ ID NO. 2.
3. A recombinant expression vector comprising the chitin deacetylase gene cdaba according to claim 1.
4. A recombinant expression strain comprising the recombinant expression vector of claim 3.
5. A preparation method of chitin deacetylase CdaBa is characterized by comprising the following steps:
providing a chitin deacetylase gene cdaba, an expression vector and an expression strain;
amplifying the chitin deacetylase gene cdaba, and connecting an amplification product with the expression vector to obtain a recombinant expression vector;
transferring the recombinant expression vector into an expression strain to obtain a recombinant expression strain;
culturing the recombinant expression strain to obtain chitin deacetylase CdaBa;
wherein the nucleotide sequence of the chitin deacetylase gene cdaba is the nucleotide sequence shown as SEQ ID NO. 1, or the nucleotide sequence with the same function obtained by deletion, insertion or substitution of the nucleotide sequence shown as SEQ ID NO. 1; the amino acid sequence of the chitin deacetylase CdaBa is the amino acid sequence shown in SEQ ID NO. 2, or the amino acid sequence shown in SEQ ID NO. 2 is subjected to deletion, insertion or substitution to obtain the amino acid sequence with the same function.
6. The method for preparing the chitin deacetylase CdaBa according to claim 5, wherein in the step of amplifying the chitin deacetylase gene CdaBa, primers used for amplification comprise a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown as SEQ ID NO. 3, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 4.
7. The method for preparing the chitin deacetylase CdaBa of claim 5, wherein the expression vector is a Pichia pastoris expression vector.
8. The method of claim 5, wherein the expression strain is a Pichia pastoris expression strain.
9. Use of the chitin deacetylase CdaBa of claim 2 or of any one of claims 5 to 8, prepared by a process for its preparation, for catalysing the deacetylation of chitin.
10. The use according to claim 9, wherein in the catalyzed reaction of chitin deacetylation, the temperature of the reaction is between 30 ℃ and 60 ℃; and/or
The pH of the reaction is 6.0-10.0.
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Cited By (1)
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| CN111996205A (en) * | 2020-08-10 | 2020-11-27 | 深圳润康生态环境股份有限公司 | Chitinase gene, chitinase and preparation method and application thereof |
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