CN114181916B - Artificially modified enzyme based on glucose oxidase and expression application thereof - Google Patents

Abstract

The invention provides an artificially modified enzyme based on glucose oxidase and expression application thereof, wherein a glucose oxidase M8 gene from Aspergillus niger is modified through rational design and site-directed mutagenesis technology, and finally a mutant with remarkably improved thermal stability is obtained and is applied to a eukaryotic cell expression system. Therefore, the problem of excessive loss of the enzyme activity of the glucose oxidase in the high-temperature granulation process of the complete feed is solved.

Description

Artificially modified enzyme based on glucose oxidase and expression application thereof

Technical Field

The invention mainly relates to a rational protein engineering evolution means, which can modify the existing protein structure to enhance the catalytic performance. The invention also relates to application of the glucose oxidase gene subjected to site-directed mutagenesis in secretion production of glucose oxidase, belonging to the fields of protein engineering and production of glucose oxidase.

Background

Glucose Oxidase (e.c. 1.1.3.4, abbreviated as GOD) can specifically catalyze beta-D-Glucose to generate gluconic acid and hydrogen peroxide in the presence of oxygen, and is an important industrial enzyme in the food industry. The method is widely applied to deoxidation of foods such as wine, beer, fruit juice, milk powder and the like, flour improvement, food browning prevention and the like, and also widely applied to rapid detection of foods and biosensors. Meanwhile, the method is also used for improving the process of wheat flour and the like.

The microorganism has fast growth and reproduction and wide source, is the main source for producing glucose oxidase, and the main producing strains are aspergillus niger and penicillium.

However, the fermentation of aspergillus niger is mainly used for producing gluconic acid or gluconate, such as sodium gluconate and calcium gluconate, so that glucose oxidase mainly appears as a byproduct, and the enzyme activity of the glucose oxidase is not high, and most of the glucose oxidase has single-digit enzyme activity.

With the development of molecular biology techniques, researchers try to express exogenous genes through a microbial expression system, so as to achieve the purpose of efficiently expressing a certain protein. Exogenous gene expression systems that have been developed include E.coli, bacillus, streptomyces, aspergillus, yeast, insect, mammalian cells, and the like. Generally, an expression system using prokaryotic cells as foreign genes is easy to operate, various technologies are mature, but limited by the prokaryotic system, proteins from eukaryotic sources cannot be folded correctly during expression or essential modification after transcription and translation is absent, so that the proteins are expressed, but the proteins have no biological activity and no further utilization value, and toxic proteins or proteins with antigen action in the prokaryotic system are likely to be mixed in final products, so that the results are interfered.

However, prokaryotic expression still has important significance, and can be used as construction and amplification of target genes and plasmids before expression of eukaryotic systems.

While eukaryotic mammalian cells can be ideally modified after protein expression, this method has not become a widely used method because of the complexity and cost of the procedures and the possibility of viral infection in later cultured cells.

The predominant protein expression system is therefore the yeast system.

The yeast system has become a model operation platform through decades of development, and various operations are improved. In the aspect of protein expression, the protein expression has an intracellular environment suitable for the correct folding of eukaryotic gene products, and particularly, some key glycosylation sites of the protein can be modified, so that some proteins with the glycosylation requirements can express activity.

After the expression system is selected, the improvement of the performance of the exogenous glucose oxidase is only realized by modifying the gene sequence of the glucose oxidase at the gene level.

As early as 1998, the structure of glucose oxidase was analyzed by scholars.

Glucose oxidase is one of the preferred alternatives to antibiotics in the large context of overall resistance banning in feed production after 7 months of 2020. However, the complete feed production needs to be carried out by high-temperature granulation at 85 ℃ for 3 minutes, and although many feed enterprises accept the efficacy of the glucose oxidase, the enzyme is considered to be added into the feed and subjected to high-temperature granulation at 85 ℃ for 3 minutes, so that the enzyme activity is almost remained, and the improvement of the tolerance of the glucose oxidase to high temperature (at 85 ℃ for 3 minutes) is of great significance.

Tu and the like in 2019 have studied on the thermal stability of the modified enzyme, glucose oxidase with improved thermal stability under the condition of not damaging enzyme activity is obtained for the first time through four-round mutation, in 2020, on the basis of 2019, mutant M8 is obtained through four-round mutation, the nucleotide sequence of the mutant M8 is shown as SEQ ID No.1, the thermal stability of the mutant M8 is further improved, but the thermal stability of the mutant M8 still cannot meet the requirements of high-temperature granulation and other processes on the glucose oxidase, and the mutant M8 is still insufficient for commercial application.

Therefore, under the condition of not damaging specific enzyme activity, further improving the thermal stability of the enzyme is one of the research and development directions of the technicians in the field, and further modification on the basis of the artificially designed and modified enzyme is more difficult and challenging.

Disclosure of Invention

The invention aims to solve the problems and provide an artificially modified enzyme based on glucose oxidase and expression application thereof, wherein a glucose oxidase M8 gene from Aspergillus niger is modified through rational design and site-directed mutagenesis technology, and finally a mutant with remarkably improved thermal stability is obtained.

The purpose of the invention is realized as follows:

the invention provides an artificially modified enzyme based on glucose oxidase, and the amino acid sequence of the artificially modified enzyme is shown as SEQID NO. 2.

A gene encoding the above-mentioned glucose oxidase-based artificially modified enzyme.

The nucleotide sequence of the gene is shown in SEQ ID NO. 3.

A recombinant expression vector comprising the nucleotide sequence described above.

A host cell comprising the recombinant expression vector described above.

The invention also provides application of the artificial modified enzyme gene based on the glucose oxidase in secretion expression of the glucose oxidase.

Connecting the new artificially modified glucose oxidase gene obtained by rational design and site-directed mutagenesis in prokaryotic cells with shuttle expression plasmids used for genetic manipulation of the prokaryotic cells to form recombinant expression plasmids; transforming the recombinant expression plasmid into eukaryotic cell expression protein and integrating the eukaryotic cell expression protein into a eukaryotic cell genome to obtain recombinant yeast; culturing the recombinant yeast, and inducing the secretory expression of the glucose oxidase.

In the application, the prokaryotic cell is escherichia coli, and the eukaryotic cell expression protein is pichia pastoris GS115 expression protein.

On the basis of the obtained modified high-thermal-stability enzyme (mutant M8), the invention further improves the thermal stability of the enzyme under the condition of not damaging the specific enzyme activity, replaces valine at the 402 th site of the amino acid sequence of the glucose oxidase M8 with phenylalanine and increases a His6 label at the N end.

The invention establishes a system biological analysis and new element function prediction model by analyzing high-throughput omics data of target compound biosynthesis, optimizing a mathematical model and an algorithm tool of protein design, constructing intelligent algorithms such as deep learning and the like. Analyzing the high-precision structure of the protein, exploring molecular mechanisms such as substrate identification, catalytic activity, structural stability and the like, and realizing rational design of mutation points. Aiming at the designed mutation, a site-directed mutagenesis kit is adopted to construct a plasmid containing the target protein, and then the plasmid is transformed into yeast for induced expression. The obtained target protein is purified, and the gene is verified to have higher thermal stability on the basis of not damaging the specific enzyme activity.

Drawings

FIG. 1 is a nucleic acid electrophoresis verification of the plasmid of mutant pPIC 9K-V402F in example 1;

FIG. 2 is a photograph showing the electrophoretic verification of the protein expression of the mutant V402F of example 1 after purification;

FIG. 3 is a standard curve of ABTS color reaction in example 3;

FIG. 4 is a graph showing the measurement of thermal stability of V402F and M8 in example 4.

Detailed Description

The present invention will be further described with reference to the following examples, but is not limited thereto. The raw materials of the reagents described in the following examples are commercially available raw materials except for the source, and the reagents are prepared by a conventional method. The methods not detailed in the examples are all conventional in the art.

Biological material:

a wild-type glucose oxidase gene sequence (taking M8 reported in literature as the standard, and named as 'glucose oxidase M8 gene' below) is synthesized by adopting a total artificial synthesis mode, the nucleotide sequence is shown as SEQ ID NO.1, and a pPIC9K plasmid containing the glucose oxidase M8 gene, namely the pPIC9K-M8 plasmid, is constructed.

Example 1 construction of plasmid containing mutant V402F Gene

The V402F mutant is obtained by replacing valine at the 402 th amino acid sequence of glucose oxidase M8 with phenylalanine and adding a His6 tag at the N terminal.

The target gene of the mutant is constructed by taking a glucose oxidase M8 gene sequence as a template and adopting a PCR method, and the specific primer design is shown in the following table:

the method comprises the following steps of taking a glucose oxidase M8 gene sequence as a template, and carrying out site-directed mutagenesis on the glucose oxidase M8 gene sequence by the method to obtain a mutant target gene V402F, wherein the method comprises the following specific steps:

amplification of 1 pPIC9K-M8 plasmid

1.1 transformation

The synthesized pPIC9K-M8 plasmid was transformed into E.coli DMT chemical complex Cell and cultured.

(1) Add 1ul 100ng/ul pPIC9K-M8 plasmid to 50ul competent cells (plasmid added just after the competent cells had thawed), flick and mix well, ice bath for 20-30 min.

(2) The water bath was heat-shocked at 42 ℃ for 45 seconds and immediately placed on ice for 2 minutes.

(3) 250ul of LB medium equilibrated to room temperature was added thereto, and the mixture was cultured at 37 ℃ for 1 hour at 200 rpm.

(4) AMP-resistant plates were pre-warmed in a 37 ℃ incubator.

(5) 200ul of the bacterial suspension was applied evenly to the plate and cultured overnight in an incubator at 37 ℃.

1.2 Flat sheet picking

Single colonies growing round and large on the plate were picked out, added to a 50ml centrifuge tube containing 10ml of LB medium, and cultured at 37 ℃ for 16 hours.

1.3 plasmid extraction

(1) Adding the grown 2ml of bacterial liquid into a centrifuge tube, centrifuging at 11000Xg for 1min, discarding the supernatant, repeating for 5 times, and collecting 10ml of escherichia coli in each tube.

(2) 400ul FAPD1 buffer was added and the E.coli was resuspended by pipetting with a pipette. Before adding FAPD1, it is checked that RNase has been added to FAPD1 and that granular cells are not visible,

(3) 400ul FAPD2 buffer was added and the tube was gently spun 8 times, vigorously spinning to break the plasmid. To lyse the cells, it was necessary to stand at room temperature for 3 minutes

(4) 600ul FAPD3 buffer was added and the tube gently rotated 8 times to immediately neutralize the lysate

(5) 18000Xg centrifuging for 5min to separate lysate, and placing FAPD tube in the kit into a collection tube during centrifuging

(6) Taking the supernatant, adding the supernatant into a FAPD tube, centrifuging at 11000Xg for 30s, discarding the effluent liquid, and repeating until the supernatant is completely collected into the FAPD tube.

(7) Adding 800ul W1 buffer into FAPD tube, centrifuging at 11000Xg for 30s, and discarding the effluent liquid

(8) Adding 700ul Wash buffer,11000xg, centrifuging for 30s, discarding the effluent liquid, repeating once

(9) Placing FAPD tube into collecting tube, centrifuging at 18000Xg for 3 min, air drying for 5min to dry FAPD tube

(10) Placing FAPD tube into 1.5ml ep tube, adding 50ul of Elution Buffer to the membrane of FAPD tube, taking care not to puncture the membrane, and standing FAPD tube for 1min

(11) 18000Xg, centrifuging for 1min to elute pPIC9K-M8 plasmid DNA, and storing the DNA at-20 ℃.

2 acquisition of the Gene of interest V402F and amplification of the pPIC 9K-V402F plasmid

2.1 construction of site-directed mutagenesis V402F plasmid

The system is as follows:

conditions are as follows:

2.2 transformation of the cultured bacteria and selection of the culture

Repeat 1.1

Selecting a bill: single colonies growing round and large on the plate were picked out, added to a 1.5ml centrifuge tube containing 1ml LB medium, and cultured at 37 ℃ for 10 hours.

2.3 validation of seed-preserving sequencing

And (3) verification: and (3) verifying whether the electrotransformation is successful or not by nucleic acid gel electrophoresis, and obtaining a target gene fragment by using a PCR amplification technology, wherein the nucleic acid electrophoresis is verified as shown in figure 1.

Seed preservation: adding 500ul of glycerol 50%

Sequencing: plasmid sequencing was performed by Kirschner

Thus, a pPIC 9K-V402F plasmid containing the mutant V402F gene was obtained.

2.4 plasmid extraction

The single colonies which were confirmed were picked up, added to a 50ml centrifuge tube containing 10ml of LB medium, and cultured at 37 ℃ for 16 hours.

Repeat step 1.3 to obtain pPIC 9K-V402F plasmid DNA, store DNA at-20 ℃.

Example 2 acquisition of mutant V402F protein of interest

The extracted pPIC 9K-V402F plasmid is electrically transferred into GS115 Pichia pastoris competent cells, and the expression is induced by methanol.

The GS115 expressing glucose oxidase was centrifuged, and the supernatant was precipitated with ammonium sulfate and dialyzed overnight. The obtained target protein is purified by a HiTrap Q column, and finally the mutant V402F target protein is obtained, and the protein electrophoresis verification is shown in figure 2.

Thus, the mutant V402F target protein was finally expressed and purified in this example.

Example 3 determination of enzymatic Activity of V402F target protein

In this example, a V402F target protein and glucose oxidase M8 (prepared as in example 2) were reacted with glucose to generate hydrogen peroxide, which was then reacted with ABTS to develop a color, and the absorbance at 415nm was measured, thereby calculating the activity of glucose oxidase, specifically as follows:

1. drawing of ABTS color reaction standard curve

The determination system comprises: except for ensuring that the system is far in excess except for ABTS, the enzyme is changed into hydrogen peroxide

HRP (horse radish peroxidase), the specific enzyme activity of which is more than or equal to 250u/mg

The determination system comprises:

wherein ABTS was diluted to 5nmol/ml, 10nmol/ml, 25nmol/ml, 50nmol/ml, 80nmol/ml, 100nmol/ml in gradient.

100ul was taken, so the abscissa should be 0.5nmol, 1nmol, 2.5nmol, 5nmol, 8nmol, 10nmol.

From this, the corresponding absorbance was measured, and a standard curve was plotted as shown in FIG. 3, and the slope of the linear curve was 0.2276 by fitting.

2. Enzyme activity assay for V402F target protein

The determination system comprises:

the concentration of the V402F target protein prepared in example 2 was 0.16mg/ml as measured by Nano microspectrophotometer, and the concentration of glucose oxidase M8 was 0.642mg/ml as measured by Nano microspectrophotometer

Diluting two target protease liquids of 0.16mg/ml V402F and 0.642mg/ml M8 by five hundred times, adding glucose, HRP and ABTS into a 96-well plate, placing the plate into an enzyme labeling instrument, shaking and uniformly mixing for 5min at 30 ℃, adding 40ul of diluted enzyme liquid into a reaction system, shaking and reacting for 10min, and simultaneously measuring a light absorption value.

The glucose oxidase reacts with glucose to generate hydrogen peroxide, the hydrogen peroxide then reacts with ABTS for color development, the light absorption value can be measured at the wavelength of 415nm, and the activity of the glucose oxidase is calculated according to the following formula:

wherein 0.2276 is the slope of ABTS color reaction standard curve.

Glucose oxidase M8 was also measured for enzyme activity by the above-described method as a control, and the results of the measurement were as follows:

specific enzyme activity = enzyme activity/concentration

From the above assay results, it can be seen that the specific enzyme activity of the mutant V402F is slightly higher than that of the M8 protein.

Example 4 thermostability assay of V402F protein of interest

The determination method comprises the following steps: the V402F target protein and the M8 protein are incubated at 80 ℃ for different times (0 s, 30s, 60s, 90s, and 120s, respectively) and enzyme activity determination is performed (using the determination method in example 3), and the obtained results are shown in fig. 4, where% = the currently determined enzyme activity/enzyme activity at 0s is 100%, and it can be seen from the graph that at 90s, the% of the remaining enzyme activity of the M8 protein is 23.2%, and the% of the remaining enzyme activity of the V402F target protein is as high as 46.7%, which indicates that the thermal stability of the mutant V402F is significantly improved relative to that of M8.

The above embodiments are provided only for illustrating the present invention and not for limiting the present invention, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and therefore all equivalent technical solutions should also fall within the scope of the present invention, and should be defined by the claims.

SEQUENCE LISTING

<110> university of east China; ningxia Xisheng industry group Co., ltd; cangzhou Xia Chengmei Biotechnology Ltd

<120> artificially modified enzyme based on glucose oxidase and expression application thereof

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<170> PatentIn version 3.3

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Claims (5)

1. An artificially modified enzyme based on glucose oxidase is characterized in that the amino acid sequence of the enzyme is shown in SEQ ID NO. 2.

2. A gene encoding the glucose oxidase-based engineered enzyme of claim 1.

3. A recombinant expression vector, which comprises a nucleotide sequence shown as SEQ ID NO. 3.

4. A host cell comprising the recombinant expression vector as claimed in claim 3.

5. Use of the gene of claim 2 for the secretory expression of glucose oxidase.

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