CN111996205A - Chitinase gene, chitinase and preparation method and application thereof - Google Patents
Chitinase gene, chitinase and preparation method and application thereof Download PDFInfo
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- CN111996205A CN111996205A CN202010795529.2A CN202010795529A CN111996205A CN 111996205 A CN111996205 A CN 111996205A CN 202010795529 A CN202010795529 A CN 202010795529A CN 111996205 A CN111996205 A CN 111996205A
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- Prior art keywords
- chitinase
- chiam
- gene
- expression vector
- recombinant expression
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Abstract
The invention belongs to the technical field of genetic engineering, and particularly relates to a chitinase gene, chitinase and a preparation method and application thereof. The nucleotide sequence of the chitinase gene chiam is shown as SEQ ID NO.1, and is obtained by comprehensively optimizing the sequence of the chitinase derived from the Mediterranei Amycolatopsis mediterranei. The amino acid sequence of the chitinase ChiAm is shown as SEQ ID NO.2, the chitinase ChiAm not only enriches the types and the source approaches of the chitinase, but also has the specific activity of 48U/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 chitinase gene ChiAm, a corresponding recombinant expression vector and a recombinant expression strain thereof, a chitinase ChiAm, 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, and has a structure similar to that of cellulose. Chitin is a renewable polysaccharide second to cellulose in nature and is one of the most abundant natural high molecular compounds. Chitin hydrolysate chitooligosaccharide has good physiological function as a bioactive substance, and can be applied in the industries of biological medicine, food processing, agricultural planting, livestock breeding and the like. At present, the preparation of chitin oligosaccharide is mainly divided into a physical method, a chemical method and an enzymatic method. Compared with physical and chemical methods, enzymatic methods for preparing chitin oligosaccharides have many advantages, such as: mild reaction conditions, complete product structure, easy process control, no environmental pollution and the like.
Chitinase, a biological enzyme, specifically decomposes chitin to produce chitooligosaccharides with different molecular weights. As chitinase plays an important role in the enzymatic preparation process of chitosan oligosaccharide, many scientists at home and abroad develop research on the chitinase. The main problems of the prior art which limit the industrialized application of chitinase are low fermentation activity and high production cost. Therefore, the method reduces the production cost of the chitinase and is the basis for being widely applied to different fields.
Disclosure of Invention
The invention aims to provide a chitinase gene ChiAm, a corresponding recombinant expression vector and a recombinant expression strain, the chitinase Chiam, a preparation method and application thereof, and aims to solve the technical problem of low fermentation activity of the existing chitinase.
In order to achieve the above object, according to one aspect of the present invention, there is provided a chitinase gene chiam having a nucleotide sequence shown in SEQ ID NO. 1.
In another aspect of the invention, a chitinase ChiAm is provided, wherein the chitinase ChiAm is a protein coded by the chitinase gene Chiam provided by the invention, and the amino acid sequence of the chitinase Chiam is shown as SEQ ID NO. 2.
In still another aspect of the present invention, there is provided a recombinant expression vector comprising the chitinase gene chiam provided by the present 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 chitinase ChiAm is provided, which comprises the following steps:
providing chitinase gene chiam, an expression vector and an expression strain;
amplifying the chitinase gene chiam, 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 chitinase ChiAm;
wherein, the nucleotide sequence of the chitinase gene ChiAm is shown as SEQ ID NO.1, and the amino acid sequence of the chitinase Chiam is shown as SEQ ID NO. 2.
In the last aspect of the invention, the chitinase ChiAm or the application of the chitinase ChiAm prepared by the preparation method of the chitinase ChiAm in chitin hydrolysis is provided.
The fermentation broth of Amycolatopsis mediterranei (GenBank: CP003729.1) of Mediterranei has chitinase activity, but the activity is very low (the chitinase activity is about 0.8U/ml after 80 hours of culture), which is far lower than the standard of industrial production. According to the codon preference of pichia pastoris, the expression adaptation index CAI of the sequence derived from the Amycolatopsis mediterranei chitinase of the Mediterranei is improved from 0.42 to 0.78, and the GC content is adjusted from 68% to 46%, so that the chitinase gene chiam is obtained. Through the comprehensive optimization, the sequence stability and codon adaptability of the chitinase gene ChiAm are obviously improved, high-efficiency expression in a pichia pastoris expression system can be realized, and the production cost of the chitinase ChiAm can be reduced.
The chitinase ChiAm provided by the invention not only enriches the types and source ways of the chitinase, but also has the specific activity of 48U/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 chitinase gene chiam provided by the invention, and the chitinase gene chiam 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 chitinase gene chiam also has better stability, is favorable for realizing the stable and efficient expression of the chitinase gene chiam, and plays an important role in reducing the production cost of the chitinase gene chiam.
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 chitinase ChiAm encoded by chitinase gene Chiam. Meanwhile, the sequence of the chitinase gene ChiAm has better stability and codon adaptability, and the obtained recombinant expression strain can stably and efficiently express the chitinase ChiAm.
In the preparation method of the chitinase ChiAm, a heterogenous recombination method is adopted, the chitinase gene Chiam is amplified to obtain a corresponding recombinant expression vector and a corresponding recombinant expression strain, and the chitinase ChiAm is obtained by culturing. The preparation method provided by the invention has the advantages of simple steps, easily controlled process, capability of producing the chitinase ChiAmp stably, efficiently and at low cost, and good industrial prospect. Meanwhile, the chitinase ChiAm prepared by the preparation method of the chitinase ChiAm also has the advantages of high enzyme activity and stable enzymological characteristics.
The chitinase ChiAm provided by the invention or prepared by the preparation method of the chitinase ChiAm provided by the invention can be used for hydrolyzing chitin. The chitinase ChiAm has high enzyme activity and good stability in a wide temperature range and a wide pH range, so that the chitinase ChiAm can be used for hydrolyzing chitin efficiently and stably.
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 chitinase ChiAm obtained in example 6 of the present invention;
FIG. 3 is a graph showing the optimal reaction temperature and thermal stability of chitinase ChiAm obtained in example 6 of the present invention;
FIG. 4 is a graph showing the optimum reaction pH and pH stability of chitinase ChiAm obtained in example 6 of the present invention;
FIG. 5 is a graph showing the results of thin layer chromatography of the hydrolysate obtained after chitin was hydrolyzed by chitinase ChiAm obtained in example 6 of the present invention.
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 chitinase gene chiam, and the nucleotide sequence of the chitinase gene chiam is shown as SEQ ID NO. 1.
Nucleotide sequence of chitinase gene chiam (SEQ ID NO: 1):
atgagaagatctagattggctgctgttggtttggctgctgctactttcactgctactgctgtttcttt ggttttgggtgctccatctgctactgctgctttgtctaacaactggtacgctgctgctccatacttgatgccacaatctaacaacccaccagacccagttactgttatgaacgctactggtttgaaggctttccaattggctttcatcttggctccaaacggtggtggttgttctccaacttgggacggtacttctgctgtttcttctgacactgctgttgctggtgttatctctagaatcagaggtgctggtggtgacgtttctgtttctgttggtggttacggtggtactaagttgggtcaaacttgtggtactgttgctgctactgctgctgcttaccaacaagttatcactaagtactctttgaaggctatcgacttcgacttggaagaaccagaatacgaaaacactgctgctatcgctaacgaattgggtgctgctaagactttgcaagctaacaacccaggtttgttcgtttctgttactatgccaggtactgctgctggtactggttggttcggtactcaattgatcgaccaagctaagtctatcggtttctctccaaacaacttctctatcatgccattcgacggtggtttcaacggtggttcttctcaagtttctgctttggaagctttccacggtttgttgatgtctcacatgggttgggactctgctactgcttacgctcacgaaggtttctctggtatgaacggtaagtctgacgctgctgaaatgttctacacttctgacttccaaactgtttacgactacgctacttctcacggtttgggtagattcactttctggtctgttaacagagacagagcttgtgttggtactactgacaacggtgtttgttctaacgttccacaaaacgactgggacttcactaagttctctgttagattcgctggtgctactccaccacaaactactccaccagttactactactccaactactccaggtaacggttcttgtactgctgctgaatgggacagaactaaggtttacgttaaggacaacgttgtttctcacaactctcacaagtggactgctaagtggtggactcaaggtgaagaaccaggtactactggtgaatggggtgtttggcaagacaacggtgcttgttaa。
the fermentation broth of Amycolatopsis mediterranei (GenBank: CP003729.1) of Mediterranei has chitinase activity, but the activity is very low (the chitinase activity is about 0.8U/ml after 80 hours of culture), which is far lower than the standard of industrial production. According to the codon preference of pichia pastoris, the expression adaptation index CAI of the sequence of the chitinase derived from Amycolatopsis mediterranei in Mediterranei is increased from 0.42 to 0.78, and the GC content is adjusted from 68% to 46%, so that the chitinase gene chiam is obtained. Through the comprehensive optimization, the sequence stability and codon adaptability of the chitinase gene ChiAm are obviously improved, high-efficiency expression in a pichia pastoris expression system can be realized, and the production cost of the chitinase ChiAm can be reduced.
Correspondingly, the embodiment of the invention also provides chitinase ChiAm which is the protein coded by the chitinase gene Chiam provided by the embodiment of the invention, and the amino acid sequence of the chitinase gene Chiam is shown as SEQ ID NO. 2.
Amino acid sequence of chitinase ChiAm (SEQ ID NO: 2):
MRRSRLAAVGLAAATFTATAVSLVLGAPSATAALSNNWYAAAPYLMPQSNNPPDPVTVMNATGLKAFQLAFILAPNGGGCSPTWDGTSAVSSDTAVAGVISRIRGAGGDVSVSVGGYGGTKLGQTCGTVAATAAAYQQVITKYSLKAIDFDLEEPEYENTAAIANELGAAKTLQANNPGLFVSVTMPGTAAGTGWFGTQLIDQAKSIGFSPNNFSIMPFDGGFNGGSSQVSALEAFHGLLMSHMGWDSATAYAHEGFSGMNGKSDAAEMFYTSDFQTVYDYATSHGLGRFTFWSVNRDRACVGTTDNGVCSNVPQNDWDFTKFSVRFAGATPPQTTPPVTTTPTTPGNGSCTAAEWDRTKVYVKDNVVSHNSHKWTAKWWTQGEEPGTTGEWGVWQDNGAC。
the chitinase ChiAm provided by the embodiment of the invention not only enriches the types and source ways of the chitinase, but also has the specific activity of 48U/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 chitinase gene chiam provided by the embodiment of the invention.
The recombinant expression vector provided by the embodiment of the invention comprises the chitinase gene chiam provided by the embodiment of the invention, and the chitinase gene chiam is subjected to codon optimization according to the preference of pichia pastoris, so that the recombinant expression vector has better stability and codon adaptability, and accordingly, the recombinant expression vector comprising the chitinase gene chiam provided by the embodiment of the invention also has better stability, is favorable for realizing stable and efficient expression of the chitinase gene chiam, and plays an important role in reducing the production cost of the chitinase gene chiam.
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 invention, so that the recombinant expression strain can be used for expressing chitinase ChiAm encoded by chitinase gene Chiam. Meanwhile, the sequence of the chitinase gene ChiAm has better stability and codon adaptability, and the obtained recombinant expression strain can stably and efficiently express the chitinase ChiAm.
Correspondingly, the embodiment of the invention also provides a preparation method of the chitinase ChiAm, which comprises the following steps:
s1, providing chitinase gene chiam, an expression vector and an expression strain;
s2, amplifying the chitinase gene chiam, 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 chitinase ChiAm;
wherein, the nucleotide sequence of the chitinase gene ChiAm is shown as SEQ ID NO.1, and the amino acid sequence of the chitinase Chiam is shown as SEQ ID NO. 2.
In the preparation method of the chitinase ChiAm provided by the embodiment of the invention, a heterogenous recombination method is adopted, the chitinase gene Chiam is amplified to obtain a corresponding recombinant expression vector and a corresponding recombinant expression strain, and the chitinase ChiAm is obtained by culturing. The preparation method provided by the embodiment of the invention has simple steps, is easy to control the process, can stably and efficiently produce the chitinase ChiAmp at low cost, and has good industrial prospect. Meanwhile, the chitinase ChiAm prepared by the preparation method of the chitinase ChiAm 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 chitinase gene chiam is shown in SEQ ID NO:1, the sequence is obtained by comprehensively optimizing the sequence (GenBank: CP003729.1) derived from Amycolatopsis mediterranei chitinase of Mediterranei mycorrhiza fungi, 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 chitinase gene chiam shown as SEQ ID NO.1 in the embodiment of the invention. In some embodiments, a pichia pastoris expression vector is selected. The chitinase gene chiam 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 chitinase gene chiam can be further improved by selecting a pichia pastoris expression vector. Specifically, the 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-chiams.
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 chitinase gene ChiAm 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 chitinase gene ChiAm has the advantages of easiness in genetic engineering operation, capability of realizing high-density fermentation, higher expression level, easiness in protein separation and purification, suitability for industrial large-scale production and the like, and is favorable for obtaining the chitinase Chiam with high enzyme activity and lower production cost. Specifically, pichia pastoris X33 can be selected as the expression strain.
In S2, the chitinase gene chiam 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 chitinase gene chiam include a forward primer chiam-F and a reverse primer chiam-R, and the nucleotide sequence of the forward primer chiam-F is shown in SEQ ID NO. 3 and the nucleotide sequence of the reverse primer chiam-R is shown in SEQ ID NO. 4.
Nucleotide sequence of chiam-F (SEQ ID NO: 3):
agtcgaattcgctttgtctaacaactggtacgctg。
the nucleotide sequence of chiam-R (SEQ ID NO: 4):
ttctctagaacaagcaccgttgtcttgccaa。
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, the nucleotide sequence of chitinase gene ChiAm is replicated along with the reproduction of the recombinant expression strain, and then the culture of the recombinant expression strain is collected, separated and purified to obtain the chitinase ChiAm. In some embodiments, the recombinant expression strain is cultured by a high density fermentation process comprising: the recombinant expression strain was inoculated into a triangular flask containing BMGY medium and cultured overnight at 30 ℃ and 220rpm, and then inoculated into a triangular flask containing YPG medium and cultured overnight with shaking at 30 ℃ and 200 rpm. Inoculating the recombinant expression strain subjected to the twice overnight culture into a fermentation tank containing a BSM culture medium, and performing fermentation culture under the conditions of 30 ℃, pH5.0, stirring speed of 500rpm and air flow of 40L/min to obtain fermentation liquor. And purifying the fermentation liquor obtained by high-density fermentation culture to obtain the chitinase ChiAm.
Correspondingly, the embodiment of the invention also provides the application of the chitinase ChiAm provided by the embodiment of the invention or the chitinase ChiAm prepared by the preparation method of the chitinase ChiAm provided by the embodiment of the invention in chitin hydrolysis.
The chitinase ChiAm provided by the embodiment of the invention or prepared by the preparation method of the chitinase ChiAm provided by the embodiment of the invention can be used for hydrolyzing chitin. The chitinase ChiAm has high enzyme activity and good stability in a wide temperature range and a wide pH range, so that the chitinase ChiAm can be used for hydrolyzing chitin efficiently and stably.
In some embodiments, chitinase ChiAm provided by the embodiments of the invention has a reaction temperature of 30-60 ℃ in the reaction of hydrolyzing protein, and the optimal reaction temperature is 50 ℃.
In some embodiments, chitinase ChiAm provided by embodiments of the invention has a reaction pH of 3.0-8.0, and an optimal reaction pH of 5.0 in a reaction for hydrolyzing proteins.
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 chitinase gene, chitinase and its preparation and application obvious in the examples of the present invention, 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.
Example 1
This example provides a chitinase gene sequence analysis and codon optimization process, including the following steps:
(11) through analysis of Amycolatopsis mediterranei at NCBI genome data (GenBank: CP003729.1), the nucleotide in the 7823925-7825130 region of the genome is found to be the coding gene of chitinase ChiAm, the whole length of the gene is 1206bp, and the coding gene codes for 401 amino acids (the amino acid sequence is shown as SEQ ID NO. 2). The signal peptide prediction software SignalP-5.0Server prediction analysis finds that the first 32 amino acids of chitinase ChiAm are the signal peptide.
(12) Because of differences in gene coding between Amycolatopsis mediterranei and pichia pastoris, the chitinase ChiAm needs to be optimized according to pichia pastoris codon bias. The optimized chitinase ChiAm coding gene is named as ChiAm (the nucleotide sequence is shown as SEQ ID NO. 1). Compared with the original gene, the GC content of chiam is 46%, and the preference of pichia pastoris coding is closer to (the GC content of the original gene is 68%); the expression adaptation index of chiam is increased from 0.42 to 0.78, and the nucleotide similarity of the optimized chiam and the original gene is 78%.
Example 2
This example provides a construction process of a recombinant expression vector containing the chitinase gene chiam 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 signal peptide of the chiam itself needs to be removed in the construction process. The method comprises the following specific steps:
(13) designing a pair of primers, namely, chiam-F and chiam-R according to the sequence of a synthetic gene, wherein the nucleotide sequences of the primers are respectively shown as SEQ ID NO. 3-4, and the primers are used for amplifying the chiam gene from which signal peptide is removed;
(14) obtaining a gene chiams with a signal peptide removed by PCR amplification, respectively carrying out overnight enzyme digestion on an expression vector pPICZ alpha A and the gene chiams by using restriction enzymes EcoRI and XbaI, purifying and recovering the overnight enzyme digestion expression vector pPICZ alpha A and the overnight enzyme digestion expression vector chiams, 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 (3) inoculating the successfully verified transformant into an LBZ liquid culture medium, extracting plasmids, and performing sequencing verification to finally obtain an expression vector pPICZ alpha A-chiams.
Example 3
This example provides a process for constructing a recombinant pichia pastoris engineering strain containing the recombinant expression vector obtained in example 1, which includes the following steps:
(17) after the vector pPICZ alpha A-chiams 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 chitinase activity was determined as follows: firstly, preheating colloidal chitin and enzyme solution at 45 ℃; adding preheated 500 μ L enzyme solution into 10ml glass test tube, adding 500ml colloidal chitin solution, reacting at 45 deg.C for 30 min, adding 2ml DNS reagent to terminate reaction, developing in 100 deg.C boiling water bath for 10 min, cooling, centrifuging, collecting supernatant, and measuring light absorption value at 540 nm. The definition of the enzyme activity unit is as follows: the amount of enzyme used to produce 1. mu. mol of acetylglucosamine per minute was defined as one activity unit. By screening 96 positive transformants, 3 strains with dominant enzyme activity are finally obtained and named as Ch15(1.1U/mL), Ch66(0.8U/mL) and Ch23(0.5U/mL) respectively.
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, samples are taken for chitinase activity determination, and enzyme activity determination shows that enzyme activities of Ch15, Ch66 and Ch23 after 120 hours of induction culture are 3.1U/mL, 2.3U/mL and 2.1U/mL respectively.
Example 5
The embodiment provides a process for performing high-density fermentation culture on the recombinant engineering bacterium Ch15 obtained after shake flask fermentation culture in the embodiment 4, 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 bacteria reaches about 180g/L, the glycerol feeding is stopped, and the induction with methanol is started after the glycerol is completely absorbed by the bacteria (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 (63U/mL) when the induction culture was carried out for 120 hours, the total protein concentration was 1.92g/L at the maximum, and the wet weight of the cells reached a maximum (396g/L) after the induction for 144 hours.
Example 6
This example provides a process for purifying the fermentation broth obtained in example 5 to obtain chitinase ChiAm, which includes:
(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) and purifying by using a Ni-IDA protein purification kit to obtain the chitinase ChiAm.
The specific activity of the purified chitinase ChiAm enzyme is 48U/mg through experimental determination. The electrophoresis result of the purified SDS-PAGE protein is shown in FIG. 2, and it can be seen from FIG. 2 that the size of the recombinant chitinase ChiAm is about 45 kDa.
Experimental example 1
The enzyme activity of the chitinase ChiAm obtained in example 6 at different temperatures of 30-70 ℃ is measured under the condition of pH5.0, the enzyme activity at the highest temperature of the enzyme activity is measured to be 100%, and the relative enzyme activities at other temperatures are 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 chitinase ChiAm are all more than 60%, and the optimal reaction temperature is 50 ℃.
The thermal stability was determined as follows: and (3) carrying out water bath heat treatment on chitinase ChiAm at different temperatures of 30-60 ℃ for 60 minutes, then determining residual enzyme activity, and calculating the relative residual enzyme activity at other temperatures by taking the enzyme activity without a heat treatment sample as 100%. The results of the experiment are shown in FIG. 3. As can be seen from FIG. 3, the chitinase ChiAm has good stability within the temperature range of 30-50 ℃, and the residual enzyme activity is more than 80 percent after heat treatment for 60 minutes.
Experimental example 2
The enzyme activities of the chitinase ChiAm obtained in example 6 under the condition of pH 3.0-8.0 are respectively measured at 50 ℃, the enzyme activity under the highest pH value of the enzyme activity is measured to be 100%, the relative enzyme activities under other pH values are calculated, and the detection result is shown in figure 4. As can be seen from FIG. 4, the optimum reaction pH value of chitinase ChiAm is 5.0, the relative enzyme activity is more than 70% in the range of pH4.0-6.0, and when the pH is increased to 7.0 or decreased to 3.0, the relative enzyme activity is sharply decreased, respectively 25% and 8%.
The effect of different pH on chitinase ChiAm stability was determined as follows:
the chitinase ChiAm is stored for 6 hours at room temperature under the condition of pH 3.0-8.0 to determine the residual enzyme activity, the relative residual enzyme activity at other temperatures is calculated by taking the enzyme activity of a sample which is not processed as 100%, and the detection result is shown in figure 4. As can be seen from FIG. 4, the chitinase ChiAm has a residual enzyme activity of more than 85% in the pH range of 3.0-8.0 after being stored for 4 hours at room temperature, which indicates that the chitinase ChiAm has good stability in the pH range.
Experimental example 3
The chitinase ChiAm obtained in example 6 was used for hydrolyzing chitin as follows:
0.2g of colloidal chitin was dissolved in 50ml of sodium acetate buffer (pH5.0), 100U of chitinase ChiAm was added, hydrolysis was carried out at 50 ℃ and 120rpm, and samples were taken for 1 hour and 3 hours of hydrolysis respectively for thin layer chromatography. The thin layer chromatography method comprises the following steps: dispensing 5. mu.L of the hydrolysis reaction product and 5. mu.L of the chitosan oligosaccharide standard mixture onto a Silica gel plate (Silica gel 60, Merck), respectively; placing the well-spotted silica gel plate in an expansion cylinder for expansion, wherein the expansion buffer solution is a mixture of isopropanol, water and ammonia water (the volume ratio is 15:1: 7.5); taking the expanded silica gel plate out of the expansion cylinder, drying and spraying a display agent (the display agent is a mixture of acetone, aniline, diphenylamine and phosphoric acid, and the volume ratio is 100:2:2: 10); after drying, the silica gel plate was placed at 120 ℃ for high temperature development, and the results are shown in fig. 5.
As can be seen from FIG. 5, after the hydrolysis reaction proceeded for 1 hour, the hydrolysis product consisted mainly of acetylglucosamine, chitobiose and chitotriose, and when the hydrolysis reaction proceeded for 3 hours, the hydrolysis product consisted mainly of chitobiose and chitotriose.
When the chitinase ChiAm provided by the embodiment of the invention is used for hydrolyzing chitin, the obtained main products are chitobiose and chitotriose, and the chitobiose and the chitotriose are used as bioactive substances, so that the chitinase ChiAm has wide and good application effect and prospect in the industries of medicine, agriculture, food and the like. Therefore, the chitinase ChiAm provided by the embodiment of the invention has great application potential in the field of enzymatic hydrolysis preparation of chitosan oligosaccharide.
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 plant Nutrition technology Limited
<120> chitinase gene, chitinase, preparation method and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1206
<212> DNA
<213> Mediterranean Amycolatopsis mediterranei (Amycolatopsis mediterranei)
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atgagaagat ctagattggc tgctgttggt ttggctgctg ctactttcac tgctactgct 60
gtttctttgg ttttgggtgc tccatctgct actgctgctt tgtctaacaa ctggtacgct 120
gctgctccat acttgatgcc acaatctaac aacccaccag acccagttac tgttatgaac 180
gctactggtt tgaaggcttt ccaattggct ttcatcttgg ctccaaacgg tggtggttgt 240
tctccaactt gggacggtac ttctgctgtt tcttctgaca ctgctgttgc tggtgttatc 300
tctagaatca gaggtgctgg tggtgacgtt tctgtttctg ttggtggtta cggtggtact 360
aagttgggtc aaacttgtgg tactgttgct gctactgctg ctgcttacca acaagttatc 420
actaagtact ctttgaaggc tatcgacttc gacttggaag aaccagaata cgaaaacact 480
gctgctatcg ctaacgaatt gggtgctgct aagactttgc aagctaacaa cccaggtttg 540
ttcgtttctg ttactatgcc aggtactgct gctggtactg gttggttcgg tactcaattg 600
atcgaccaag ctaagtctat cggtttctct ccaaacaact tctctatcat gccattcgac 660
ggtggtttca acggtggttc ttctcaagtt tctgctttgg aagctttcca cggtttgttg 720
atgtctcaca tgggttggga ctctgctact gcttacgctc acgaaggttt ctctggtatg 780
aacggtaagt ctgacgctgc tgaaatgttc tacacttctg acttccaaac tgtttacgac 840
tacgctactt ctcacggttt gggtagattc actttctggt ctgttaacag agacagagct 900
tgtgttggta ctactgacaa cggtgtttgt tctaacgttc cacaaaacga ctgggacttc 960
actaagttct ctgttagatt cgctggtgct actccaccac aaactactcc accagttact 1020
actactccaa ctactccagg taacggttct tgtactgctg ctgaatggga cagaactaag 1080
gtttacgtta aggacaacgt tgtttctcac aactctcaca agtggactgc taagtggtgg 1140
actcaaggtg aagaaccagg tactactggt gaatggggtg tttggcaaga caacggtgct 1200
tgttaa 1206
<210> 2
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<213> Mediterranean Amycolatopsis mediterranei (Amycolatopsis mediterranei)
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Ser Asn Asn Pro Pro Asp Pro Val Thr Val Met Asn Ala Thr Gly Leu
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Lys Ala Phe Gln Leu Ala Phe Ile Leu Ala Pro Asn Gly Gly Gly Cys
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85 90 95
Ala Gly Val Ile Ser Arg Ile Arg Gly Ala Gly Gly Asp Val Ser Val
100 105 110
Ser Val Gly Gly Tyr Gly Gly Thr Lys Leu Gly Gln Thr Cys Gly Thr
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Val Ala Ala Thr Ala Ala Ala Tyr Gln Gln Val Ile Thr Lys Tyr Ser
130 135 140
Leu Lys Ala Ile Asp Phe Asp Leu Glu Glu Pro Glu Tyr Glu Asn Thr
145 150 155 160
Ala Ala Ile Ala Asn Glu Leu Gly Ala Ala Lys Thr Leu Gln Ala Asn
165 170 175
Asn Pro Gly Leu Phe Val Ser Val Thr Met Pro Gly Thr Ala Ala Gly
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Thr Gly Trp Phe Gly Thr Gln Leu Ile Asp Gln Ala Lys Ser Ile Gly
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Phe Ser Pro Asn Asn Phe Ser Ile Met Pro Phe Asp Gly Gly Phe Asn
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Gly Gly Ser Ser Gln Val Ser Ala Leu Glu Ala Phe His Gly Leu Leu
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Met Ser His Met Gly Trp Asp Ser Ala Thr Ala Tyr Ala His Glu Gly
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Phe Ser Gly Met Asn Gly Lys Ser Asp Ala Ala Glu Met Phe Tyr Thr
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Ser Asp Phe Gln Thr Val Tyr Asp Tyr Ala Thr Ser His Gly Leu Gly
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Arg Phe Thr Phe Trp Ser Val Asn Arg Asp Arg Ala Cys Val Gly Thr
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Thr Asp Asn Gly Val Cys Ser Asn Val Pro Gln Asn Asp Trp Asp Phe
305 310 315 320
Thr Lys Phe Ser Val Arg Phe Ala Gly Ala Thr Pro Pro Gln Thr Thr
325 330 335
Pro Pro Val Thr Thr Thr Pro Thr Thr Pro Gly Asn Gly Ser Cys Thr
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Ala Ala Glu Trp Asp Arg Thr Lys Val Tyr Val Lys Asp Asn Val Val
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Ser His Asn Ser His Lys Trp Thr Ala Lys Trp Trp Thr Gln Gly Glu
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Glu Pro Gly Thr Thr Gly Glu Trp Gly Val Trp Gln Asp Asn Gly Ala
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Cys
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<210> 4
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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ttctctagaa caagcaccgt tgtcttgcca a 31
Claims (10)
1. A chitinase gene chiam is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.
2. A chitinase ChiAm, wherein the chitinase ChiAm is a protein encoded by the chitinase gene Chiam of claim 1, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.
3. A recombinant expression vector comprising the chitinase gene chiam of claim 1.
4. A recombinant expression strain comprising the recombinant expression vector of claim 3.
5. A preparation method of chitinase ChiAm is characterized by comprising the following steps:
providing chitinase gene chiam, an expression vector and an expression strain;
amplifying the chitinase gene chiam, 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 chitinase ChiAm;
wherein, the nucleotide sequence of the chitinase gene ChiAm is shown as SEQ ID NO.1, and the amino acid sequence of the chitinase Chiam is shown as SEQ ID NO. 2.
6. The method for preparing the chitinase ChiAm according to claim 5, characterized in that in the step of amplifying the chitinase gene Chiam, primers for amplification comprise a forward primer and a reverse primer, wherein 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 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 chitinase ChiAm according to claim 2 or of the chitinase ChiAm prepared by the process according to any of claims 5 to 8 for hydrolyzing chitin.
10. The use according to claim 9, wherein in the reaction for hydrolyzing chitin, the temperature of the reaction is 30 ℃ to 60 ℃; and/or
The pH of the reaction is 3.0-8.0.
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| CN114736891A (en) * | 2022-06-15 | 2022-07-12 | 深圳润康生态环境股份有限公司 | Chitinase mutant ChiM-SS and application thereof |
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