CN117737038B - N-acetylglucosaminidase mutant De254P delta 5 and preparation and application thereof - Google Patents
N-acetylglucosaminidase mutant De254P delta 5 and preparation and application thereof Download PDFInfo
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses an N-acetylglucosaminidase mutant De254P delta 5, and preparation and application thereof, wherein the amino acid sequence of the mutant De254P delta 5 is shown as SEQ ID NO.1, and the nucleotide sequence of a coding gene of the mutant De254P delta 5 is shown as SEQ ID NO. 2. The invention solves the problems of low-temperature activity and poor thermal stability of the existing wild N-acetylglucosaminidase. The optimum temperature of the N-acetylglucosaminidase mutant De254P delta 5 is 35 ℃, and the residual enzyme activity is 52% and 25% after heat preservation for 1h at 37 ℃ and 50 ℃ respectively; compared with wild enzyme HJ5Nag, the optimal temperature is reduced by 10 ℃, the enzyme activity of the wild enzyme HJ5Nag is respectively improved by 41 percent and 25 percent when the wild enzyme HJ5Nag is preserved for 1 hour at 37 ℃, and the wild enzyme HJ5Nag has wide application prospect in the field of functional food production.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an N-acetylglucosaminidase mutant De254P delta 5 and preparation and application thereof.
Background
N-acetylglucosaminidase (GLCNACASES, N-acetyl-glucosaminidases, EC 3.2.1.52) is a glycoside hydrolase involved in the hydrolysis of N-acetyl-D-glucosamine (GlcNAc) at the non-reducing end of catalytic oligosaccharides, glycoproteins, glycolipids and other glycoconjugates. The hydrolysis product aminoglycoside and its derivative are widely used in agriculture, medicine, biotechnology and other fields. The prior literature 1（Ma et al. Categories and biomanufacturing methods of glucosamine, Appl Microbiol Biot, 2019,103(19):7883–7889） discloses that aminoglycosides and their derivatives are useful for wastewater treatment, drug delivery, wound healing promoters, food supplements, etc., and also for the production of sialic acid, bioethanol, single cell proteins, etc. Literature 2（Zhang et al. Enzymatic properties of β-N-acetylglucosaminidases, Appl Microbiol Biot, 2018, 102(1): 93–103） discloses that GH20 family GLCNACASES is the most industrially applicable enzyme among all families, and the characteristics of enzyme activity, low-temperature adaptability, thermal stability and the like directly influence the efficiency of producing functional acetylglucosaminide. HJ5Nag is used as a high-activity N-acetylglucosaminidase, but has poor thermal stability, only 11% of enzyme activity remains after being treated for 1h at 37 ℃, and the low-temperature activity is low, so that the application of the HJ5Nag in producing functional acetylglucosaminide is greatly limited.
Therefore, the low-temperature activity and the thermal stability of the N-acetylglucosaminidase are improved, and the application prospect in the field of functional food production is facilitated.
Disclosure of Invention
The invention aims to provide an N-acetylglucosaminidase mutant De254P delta 5 and preparation and application thereof, which solve the problems of low-temperature activity and poor thermal stability of the existing wild N-acetylglucosaminidase.
In order to achieve the aim, the invention provides an N-acetylglucosaminidase mutant De254P delta 5, and the amino acid sequence of the mutant is shown as SEQ ID NO. 1.
The invention provides a coding gene of the N-acetylglucosaminidase mutant De254P delta 5, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.
The invention provides a recombinant expression vector containing the coding gene of the N-acetylglucosaminidase mutant De254P delta 5.
Preferably, the recombinant expression vector is selected from pEASY-E2.
The invention provides a recombinant expression bacterium containing the coding gene of the N-acetylglucosaminidase mutant De254P delta 5.
Preferably, the recombinant expression bacterium is selected from the group consisting of E.coli BL21 (DE 3).
The invention provides an application of the N-acetylglucosaminidase mutant De254P delta 5 or the coding gene of the N-acetylglucosaminidase mutant De254P delta 5 in the field of functional food production.
The invention provides application of the recombinant expression vector in the field of functional food production.
The invention provides an application of the recombinant expression bacterium in the field of functional food production.
The N-acetylglucosaminidase mutant De254P delta 5 and the preparation and application thereof solve the problems of low-temperature activity and poor thermal stability of the existing wild N-acetylglucosaminidase and have the following advantages:
The invention utilizes the genetic engineering technology, the optimum temperature of the provided N-acetylglucosaminidase mutant De254P delta 5 with improved thermal stability is 35 ℃, and the residual enzyme activity is 52 percent and 25 percent after the temperature is respectively kept at 37 ℃ and 50 ℃ for 1 hour; compared with wild enzyme HJ5Nag, the optimal temperature of the mutant De254P delta 5 is reduced by 10 ℃, and the enzyme activity of the mutant De254P delta 5 is respectively improved by 41 percent and 25 percent when the mutant De254P delta 5 is subjected to heat preservation for 1h at 37 ℃. The N-acetylglucosaminidase mutant is beneficial to widening the application prospect in the field of functional food production.
Drawings
FIG. 1 shows the result of SDS-PAGE analysis of the wild-type enzyme HJ5Nag and mutant De254 P.DELTA.5 provided by the present invention.
FIG. 2 shows the results of the optimum temperature measurement of the wild-type enzyme HJ5Nag and the mutant De254 P.DELTA.5 provided by the present invention.
FIG. 3 shows the results of the measurement of the thermal stability at 37℃of the wild-type enzyme HJ5Nag and the mutant De254 P.DELTA.5 provided by the present invention.
FIG. 4 shows the results of 50℃thermal stability measurement of the wild-type enzyme HJ5Nag and mutant De254 P.DELTA.5 provided by the present invention.
FIG. 5 shows the results of the optimum pH measurement of the wild-type enzyme HJ5Nag and mutant De254 P.DELTA.5 provided by the present invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention uses some experimental materials and reagents used in the following examples:
1. Strains and vectors: coli (ESCHERICHIA COLI) BL21 (DE 3) was purchased from Beijing engine biotechnology Co., ltd, and the expression vector pEASY-E2 was purchased from Novagen.
2. Enzymes and other biochemical reagents: nickel-NTA-Agarose was purchased from QIAGEN, quickMutation TM Gene site-directed mutagenesis kit was purchased from Shanghai Biyun, p-nitrophenol-beta-N-acetylglucosaminide (PNPGLCNAC) was purchased from Shanghai leaf company, and the other was domestic reagents (all were purchased from general Biochemical reagent company).
3. Culture medium: LB medium: peptone 10g, yeast extract 5g, sodium chloride 10g, distilled water to 1000 mL, pH natural (about 7). The solid medium was supplemented with 2.0% (w/v) agar.
Description: the wild type N-acetylglucosaminidase HJ5Nag mentioned in the examples of the present invention is an expression product of a recombinant region inserted into the pEASY-E2 expression vector. The amino acid sequence is shown as SEQ ID NO.3, and GenBank accession number is ARJ33352; the gene for encoding the enzyme is N-acetylglucosaminidase gene hj5nag, the nucleotide sequence of which is shown in SEQ ID NO.4, and the GenBank accession number is KX400857.
The molecular biology experimental methods not specifically described in the present example were carried out with reference to the specific methods listed in the "molecular cloning experimental guidelines (third edition) j.
Example 1: construction and transformation of mutant De254 P.DELTA.5 expression vectors: the invention takes the nucleotide sequence of wild N-acetylglucosaminidase HJ5Nag as reference, the GenBank accession number is KX400857, the signal peptide is deleted (the nucleotide sequence of the peptide is AATCGTCGCCGAGGACGGGCCATCGCCGCCGCCACGGTGCTCGCCGCGTCGCTGGCG, the amino acid sequence is NRRRGRAIAAATVLAASLA), and the nucleotide sequence is shown as SEQ ID NO. 4.
(1) Based on the sequence of wild enzyme gene hj5nag and plasmid pEASY-E2-hj5nag with nucleotide sequence shown in SEQ ID NO.4, the recombinant primers F and R are designed by using CE Design software, and the specific sequence is as follows:
wherein, the sequence of the recombinant primer is as follows (5 '. Fwdarw.3'):
F（SEQ ID NO.5）：AGCCGGTCATCACCGACGCGGCGGCCACCGGCGGT；
R（SEQ ID NO.6）：CGTCGGTGATGACCGGCTCCTCCGCGAGCTCGG。
According to the manufacturer's instructions of QuickMutation TM gene site-directed mutagenesis kit, PCR amplification was performed using plasmid pEASY-E2-hj5nag as a template to obtain the PCR product of pEASY-E2-de254 P.DELTA.5. Wherein, the PCR amplification reaction program is as follows: pre-denaturation at 95 ℃ for 30min; then denaturation at 95 ℃ for 30sec, annealing at 60 ℃ for 30sec, and extension at 68 ℃ for 7min for 20 cycles; extending at 68 ℃ and complementing for 15min; temporarily storing at 4deg.C for 30min.
(2) After the PCR reaction, 1 mu L of DpnI is directly added into the PCR reaction system, and the mixture is evenly mixed and then incubated in a water bath kettle at 37 ℃ for 5min. After digestion, dpnI can be directly used for transformation or stored at-20 ℃ for standby.
(3) Transformation and identification: and (3) converting the PCR digestion product into competent cells of escherichia coli BL21 (DE 3) by a heat shock method, and selecting a single colony for culturing and preserving the strain to obtain recombinant expression bacteria containing an expression vector pEASY-E2-DE254P delta 5. Sequencing by the biological technology company of Beijing department further confirms that the nucleotide sequence of the mutant De254P delta 5 coding gene is shown as SEQ ID NO.2, and simultaneously, the amino acid sequence of the mutant De254P delta 5 is shown as SEQ ID NO.1, and the PMRET amino acid residue fragment at 254-258 is deleted compared with the amino acid sequence of wild enzyme HJ5Nag (SEQ ID NO. 3).
Example 2: preparation of wild-type enzyme HJ5Nag and mutant De254pΔ5: recombinant expression strains containing wild enzyme HJ5Nag and mutant De254P delta 5 coding genes are respectively inoculated into LB (containing 100 mug of mL −1 ampicillin) culture solution with an inoculum size of 0.1% (v/v), and are cultured at 37 ℃ and 180rpm overnight to obtain activated bacterial liquid.
Inoculating the activated bacterial liquid into fresh LB (containing 100 mu g of mL −1 ampicillin) culture liquid respectively according to the inoculation amount of 1% (v/v), carrying out rapid shaking culture for about 2-3 h by using a shaking table with a constant temperature of 37 ℃ and 180 rpm, adding 0.7-mM IPTG final concentration to induce when the OD 600 of the culture liquid reaches 0.6-1.0, and carrying out shaking culture at a constant temperature of 20 ℃ and 160 rpm for about 20 h. And (3) centrifuging the induced bacterial liquid in a low-temperature centrifuge at the temperature of 4 ℃ and the temperature of 6000 rpm for 10 min, and collecting bacterial bodies. After the cells were suspended at an appropriate ph=7.0 McIlvaine buffer, the cells were sonicated in an ice water bath. After the intracellular concentrated crude enzyme solution is centrifuged by 12000 rpm for 10 min, the supernatant is sucked and the target protein is respectively affinity eluted by using Nickel-NTA Agarose and 0-500 mM imidazole.
As shown in FIG. 1, the SDS-PAGE analysis results of the wild enzyme HJ5Nag and the mutant De254P delta 5 provided by the invention, wherein M is a protein Marker; the crude enzyme liquid of HJ5Nag is unpurified wild enzyme HJ5Nag; HJ5Nag is purified wild enzyme HJ5Nag; de254 P.DELTA.5 is a purified mutant De254 P.DELTA.5. As can be seen from FIG. 1, both the wild-type enzyme HJ5Nag and the mutant De254 P.DELTA.5 were purified, and the product was a single band.
Example 3: property determination of wild-type enzyme HJ5Nag and mutant De254pΔ5: (1) Activity analysis of purified wild-type enzyme HJ5Nag and mutant De254 P.DELTA.5
The activity determination method adopts a p-nitrophenol (pNP) method: dissolving a substrate PNPGLCNAC in a buffer to a final concentration of 2mM; the reaction system contains 50 mu L of proper enzyme solution and 450 mu L of substrate; after the substrate is preheated to 5min, enzyme solution is added to react 10 min, then 1M Na 2CO3 of 2mL is added to stop the reaction, and after the reaction is cooled to room temperature, OD value is measured at 405nm wavelength; 1 enzyme activity unit (U) is defined as the amount of enzyme required to cleave the substrate to produce 1. Mu. Mol of pNP per minute under the given conditions.
(2) Determination of optimum pH of purified wild-type enzyme HJ5Nag and mutant De254 P.DELTA.5
The two-step method is adopted to measure the optimal pH of the wild enzyme and the mutant, the activity is measured at1 pH unit point every interval for the first time, and the activity is measured at the highest pH unit point and the points of about 0.5 pH units, so that the measurement result can be accurately and verified under the condition that the measured sample size is basically unchanged. At 30 ℃, buffer solution is 0.1M McIlvaine buffer (pH=5.0-8.0) and 0.1M glycine-NaOH (pH=9.0), PNPGLCNAC is taken as a substrate, reaction is carried out for 10 min, and the enzyme activity of the purified N-acetylglucosaminidase is measured. The above conditional enzymatic reactions were first performed by placing the enzyme solution in a buffer containing 2mM substrate at ph=5.0, 6.0, 7.0, 8.0 and 9.0. The enzyme activities of the wild-type enzyme HJ5Nag and the mutant De254 P.DELTA.5 were highest at pH 6.0. The enzyme activities of the wild-type enzyme and the mutant were then determined in buffers containing 2mM substrate at ph=5.5, 6.0 and 6.5, respectively.
As shown in FIG. 5, the present invention provides the optimum pH measurement results of the wild-type enzyme HJ5Nag and mutant De254 P.DELTA.5. As can be seen from FIG. 5, the optimum pH of both the wild-type enzyme HJ5Nag and the mutant De254 P.DELTA.5 was 6.0.
(3) Thermal Activity and thermal stability assay of purified wild-type enzyme HJ5Nag and mutant De254 P.DELTA.5
Measurement of thermal Activity of enzyme: the two-step method is adopted to measure the optimal temperature of the wild enzyme and the mutant, the enzyme activity is measured at the temperature point of 10 ℃ at each interval for the first time, and the measurement is performed at the temperature point of the highest enzyme activity and the temperature points of 5 ℃ at the left and right sides of the highest enzyme activity for the second time, so that the measurement result can be accurately and verified under the condition that the measured sample size is basically unchanged. In a buffer solution with pH=6.0, enzymatic reaction is carried out at 0-60 ℃ first, and the enzyme activity is measured at intervals of 10 ℃ from 0 ℃ to obtain that the enzyme activity of wild enzyme HJ5Nag is highest at 40 ℃, and the enzyme activity of mutant De254P delta 5 is highest at 30 ℃. The enzyme activity of the wild-type enzyme was then determined at 35 ℃, 40 ℃ and 45 ℃ and the enzyme activity of the mutant at 25 ℃, 30 ℃ and 35 ℃ in a buffer at ph=6.0.
As shown in FIG. 2, the present invention provides the optimum temperature measurement results of the wild-type enzyme HJ5Nag and mutant De254 P.DELTA.5. As can be seen from FIG. 2, the optimum temperatures of the wild-type enzyme HJ5Nag and the mutant De254 P.DELTA.5 were 45℃and 35℃respectively.
Measurement of thermostability of enzyme: the enzyme solutions of the wild enzymes and the mutants with the same units are respectively treated under the water bath condition of 37 ℃ or 50 ℃, sampling is carried out at the time points of heat treatment for 10min, 20min, 30min and 60min, the residual enzyme activity of the taken samples is measured at the pH=6.0 and 30 ℃, and the enzyme activity of the enzyme solution which is not subjected to heat treatment is used as a control. PNPGLCNAC is used as a substrate, and the reaction is carried out for 10min, so that the enzyme activity of the purified N-acetylglucosaminidase is measured.
As shown in FIG. 3, the wild-type enzyme HJ5Nag and mutant De254 P.DELTA.5 provided by the invention have a thermal stability of 37 ℃. As shown in FIG. 4, the wild-type enzyme HJ5Nag and mutant De254 P.DELTA.5 provided by the present invention have a thermal stability of 50 ℃. From FIGS. 3 and 4, it can be seen that the mutant De254 P.DELTA.5 can still maintain the enzyme activities of 52% and 25% or more after being treated at 37℃and 50℃for 60min, respectively, while the remaining enzyme activity of the wild-type enzyme HJ5Nag is 11% after being treated at 37℃for 60min, and has been inactivated after being treated at 50℃for 60min, which indicates that the heat resistance of the mutant is significantly improved compared with that of the wild-type enzyme.
In conclusion, the invention deletes the PMRET amino acid residue fragments at 254-258 of the catalytic structure of the wild enzyme HJ5Nag to obtain the mutant De254P delta 5. The low-temperature activity of the mutant is improved, and the optimal temperature is reduced by 10 ℃ compared with the wild enzyme; the mutant can still keep the enzyme activity of more than 52 percent and 25 percent after being respectively kept at 37 ℃ and 50 ℃ for 60 minutes, and compared with wild enzyme, the enzyme activity of the mutant after being respectively kept at 37 ℃ and 50 ℃ for 60 minutes is improved by 41 percent and 25 percent, and the mutant can be used in industries such as functional food production and the like and has obvious application value.
While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.
Claims (9)
1. An N-acetylglucosaminidase mutant De254P delta 5 is characterized in that the amino acid sequence of the mutant is shown as SEQ ID NO. 1.
2. The coding gene of the N-acetylglucosaminidase mutant De254P delta 5 as set forth in claim 1, wherein the nucleotide sequence of the coding gene is shown in SEQ ID NO. 2.
3. A recombinant expression vector comprising the gene encoding the N-acetylglucosaminidase mutant De254pΔ5 of claim 2.
4. The recombinant expression vector of claim 3, wherein said recombinant expression vector is selected from the group consisting of pe asy-E2.
5. A recombinant expression bacterium comprising the gene encoding the N-acetylglucosaminidase mutant De254pΔ5 of claim 2.
6. The recombinant expression bacterium according to claim 5, wherein the recombinant expression bacterium is selected from the group consisting of E.coli BL21 (DE 3).
7. Use of the N-acetylglucosaminidase mutant De254pΔ5 of claim 1 or the coding gene of the N-acetylglucosaminidase mutant De254pΔ5 of claim 2 in the field of functional food production.
8. Use of the recombinant expression vector of claim 3 in the field of functional food production.
9. The use of the recombinant expression bacterium according to claim 5 in the field of functional food production.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN202410177836.2A CN117737038B (en) | 2024-02-08 | 2024-02-08 | N-acetylglucosaminidase mutant De254P delta 5 and preparation and application thereof |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1108790A2 (en) * | 1999-12-16 | 2001-06-20 | Kyowa Hakko Kogyo Co., Ltd. | Novel polynucleotides |
| CN105483102A (en) * | 2016-01-14 | 2016-04-13 | 云南师范大学 | Beta-N-acetylglucosamine enzyme hJ5nag resisting product inhibition and preparation method thereof |
| CN111983238A (en) * | 2020-08-19 | 2020-11-24 | 中南大学湘雅医院 | Probe for dynamically detecting protein O-GlcNAc glycosylation and preparation method and application thereof |
| CN116376875A (en) * | 2023-03-03 | 2023-07-04 | 云南师范大学 | N-acetylglucosaminidase mutant with improved heat resistance and application thereof |
| CN116555229A (en) * | 2023-05-25 | 2023-08-08 | 云南师范大学 | N-acetylglucosaminidase mutant, recombinant expression vector, bacterium and application |
-
2024
- 2024-02-08 CN CN202410177836.2A patent/CN117737038B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1108790A2 (en) * | 1999-12-16 | 2001-06-20 | Kyowa Hakko Kogyo Co., Ltd. | Novel polynucleotides |
| CN105483102A (en) * | 2016-01-14 | 2016-04-13 | 云南师范大学 | Beta-N-acetylglucosamine enzyme hJ5nag resisting product inhibition and preparation method thereof |
| CN111983238A (en) * | 2020-08-19 | 2020-11-24 | 中南大学湘雅医院 | Probe for dynamically detecting protein O-GlcNAc glycosylation and preparation method and application thereof |
| CN116376875A (en) * | 2023-03-03 | 2023-07-04 | 云南师范大学 | N-acetylglucosaminidase mutant with improved heat resistance and application thereof |
| CN116555229A (en) * | 2023-05-25 | 2023-08-08 | 云南师范大学 | N-acetylglucosaminidase mutant, recombinant expression vector, bacterium and application |
Non-Patent Citations (6)
| Title |
|---|
| The Role of Chitooligosaccharidolytic β-N-Acetylglucosamindase in the Molting and Wing Development of the Silkworm Bombyx mori;Zhang, BL等;INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES;20220430;第23卷(第7期);全文 * |
| Zhou,J.等.beta-N-acetylhexosaminidase [Microbacterium sp. HJ5].GenBank Database.2017,Accession NO.ARJ33352.1. * |
| Zhou,J.等.Microbacterium sp. HJ5 beta-N-acetylhexosaminidase gene, complete cds.GenBank Database.2017,Accession NO.KX400857.1. * |
| β-N-乙酰己糖胺酶的异源表达及酶学性质研究;宋志凤;中国优秀硕士学位论文全文数据库工程科技Ⅰ辑;20170215;B014-540 * |
| 粪肠球菌来源内切-β-N-乙酰氨基葡萄糖苷酶的重组表达及催化特性;黄云娜等;农业生物技术学报;20180423;第26卷(第04期);698-710 * |
| 糖苷内切酶法合成带有均一糖链的糖蛋白和糖肽;邹洋等;中国生物工程杂志;20120215;第32卷(第02期);107-116 * |
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