CN106554953B

CN106554953B - Paenibacillus L-asparaginase and coding gene and application thereof

Info

Publication number: CN106554953B
Application number: CN201610892344.7A
Authority: CN
Inventors: 江正强; 杨绍青; 史然; 刘瑜; 穆青
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2016-10-12
Filing date: 2016-10-12
Publication date: 2020-06-02
Anticipated expiration: 2036-10-12
Also published as: CN106554953A

Abstract

The invention relates to a Paenibacillus L-asparaginase, and a coding gene and application thereof. The L-asparaginase is Rhizobium etli type L-asparaginase derived from paenibacillus, and specifically is protein consisting of amino acid sequences shown in a sequence 2 or 4 in a sequence table. The protein provided by the invention has the following enzymological properties as L-asparaginase: the specific enzyme activity is 35.2U mg^‑1The optimum reaction pH is 8.5, and the reaction is stable within the pH range of 5.5-10.0; the optimal reaction temperature is 45 ℃, and the reaction temperature is stable within 55 ℃, and the thermal stability is better. The protein is applied to potato chips and moon cakes, so that the content of acrylamide in a final product is remarkably reduced by 86% and 52%, respectively. The protein provided by the invention can effectively reduce the acrylamide content in the baked food, ensures the safety of the food, and has important social significance and economic benefit. The protein provided by the invention can also meet the special requirements on the reaction pH and the thermal stability of the L-asparaginase in industrial applications such as food, medicine and the like, and has great application potential.

Description

Paenibacillus L-asparaginase and coding gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a Paenibacillus L-asparaginase and a coding gene and application thereof.

Background

L-asparaginase (EC 3.5.1.1, L-asparaginase), also known as L-asparaginyl amidohydrolase, can specifically hydrolyze asparagine to produce L-aspartic acid and ammonia, and has important application value in the fields of food, medicine and the like.

In the food industry, L-asparaginase can be used as a high-efficiency food additive, L-asparagine and reducing sugar contained in a baking food raw material can generate carcinogenic substance acrylamide through Maillard reaction in a high-temperature baking process, and the L-asparagine can be hydrolyzed after the L-asparaginase is added into the baking food raw material, so that the generation amount of acrylamide in the baking food is reduced, and the safety of the food is improved.

In medicine, L-asparaginase, a nutrient substance required for cell proliferation, is used as a drug for treating cancer, and normal cells have L-asparagine synthesis ability, while cancer cells cannot synthesize itself due to lack of L-asparagine synthesis mechanism, so that if L-asparaginase is introduced during the proliferation of cancer cells to hydrolyze L-asparagine, it lacks the nutrient supply of L-asparagine and stops proliferation until death (Husain I, Sharma A, Kumar S, Malik F.2016.purification and culture of glutamine front anaerobas otitids. biochemicals: indec human apoptosis in human molmo-4 cells. Biochimie 121: 38-51). Because of its application in food processing and medicine fields, L-asparaginase has become one of the hot spots in research and product development.

L-asparaginase is widely present in plants, animals and microorganisms and was initially valued by researchers based on its medicinal efficacy. In 1953, Kidd found that guinea pig serum has anticancer effect for the first time, and further proved by Broom in 1961An anti-tumor factor in guinea pig serum is L-asparaginase (Broome JD.1981.L-asparaginase: discovery and maintenance as a tumor-inhibiting agent. cancer Treat Rep 65: 111-114). Later, as L-asparaginase was studied in depth, researchers succeeded in preparing L-asparaginase from Escherichia coli (Escherichia coli) and Erwinia carotovora (Erwinia carotovora) and applying it to the Treatment of acute lymphocytic Leukemia, Hopkinson disease, lymphosarcoma, melanosarcoma and pancreatic Cancer in Children, and achieved good therapeutic effects (Duval M, Suciu S, Ferster A, Rialland X, Nelken B, Lutz P, Benoit Y, Robert A, Manel AM, Vilmer E, Otten J, Philippo N.2002. company of Escherichia coli-asparagine with Erwinia in the same effort of the molecular family of childhood polypeptides: stress of transformed European Cancer research and emission 2734. the inventor et al. To date, relatively few studies on L-asparaginase have been made in China, and the focus has been mainly on the preparation of bacterial L-asparaginase, such as E.coli (patent No.: CN 101748094) and B.subtilis (patent No.: CN102864163, CN 103243063). However, there are few reports of L-asparaginase derived from Paenibacillus, and only Wakil et al isolated L-asparaginase-producing Paenibacillus polymyxa (Paenibacillus validus) from soil with 6.2U mL of enzyme activity^-1And no relevant experiments were carried out for gene expression and use in food (Wakil SM, AdeleganAA. screening, production and optimization of L-aspartic from soil bacteria isolated in Ibadan, south-western Nigeria. journal of Basic and applied sciences,2015,11: 39-51). In addition, Rhizobium etli-type L-asparaginase is a novel L-asparaginase, first discovered by Borek and Jask Lo lski from Rhizobium CFN42 (Borek D, Jaskolski M. sequence analysis of enzymes with Asparaginase activity. acta Biochimica Polonica,2001,48(4): 893). Few reports have been made on Rhizobium etli type L-asparaginase, and only one L-asparaginase gene derived from Rhizobium etli is currently subjected to homologous expression and enzymatic properties are determined (Moreno-Enriquez A. Biochemical characteristics)a member of the creation of a creative, sterile, type of a type of l-asparagase (ansa) from Rhizobium etli, journal of microbiological and Biotechnology,2012,22(3):292-300), but there is no relevant application study.

The enzyme is an ecological high-efficiency catalyst and has strong specificity, namely, one enzyme can only catalyze and convert one substance. At present, the total yield of enzymes for global industry breaks through 100 million tons, and the annual yield of the enzymes in China accounts for about 1/3 of the total yield in the world. It is estimated that the global market for enzymes in 2017 has sold a total of 50-60 billion dollars.

The most important uses of enzymes are concentrated in the food industry, and in addition, they are widely used in the pharmaceutical industry, feed and other industries. If calculated as consumed quantities, the food industry consumes approximately 2/3 of the total world enzyme production, and the other industries consume enzymes totaling the remaining 1/3. In recent years, some therapeutic enzyme preparations such as antibacterial enzyme, cellolytic enzyme, mucolytic enzyme, analgesic enzyme, antitumor enzyme, immunoactivator enzyme and the like have been newly developed abroad. The enzyme preparations for medical use have become a new variety of therapeutic agents in the world, and with the increasing appearance of new enzyme preparations, the enzymes for medical use will become a new growth point in the international pharmaceutical market. Compared with the advanced level at abroad, the enzyme produced in China is mainly concentrated on the enzyme for food industry, which accounts for about 96% of the domestic enzyme preparation production value, while the variety for treatment is very few, and at present, only a few varieties such as multienzyme tablets, lumbrokinase and the like exist. It is believed that the development of novel medical enzyme production in China has wide market prospect under the drive of the new trend of international drug administration.

Acrylamide has been one of the major concerns in the international food industry since its first discovery in 2002. Acrylamide can be produced by thermal conductivity reactions (maillard reactions) during various cooking processes. The research shows that the acrylamide has carcinogenic effect on animals and has potential carcinogenic risk on human beings. Therefore, reducing acrylamide production in food applications/products is one of the concerns of manufacturers. A great deal of research work has been carried out by food safety authorities, academic research institutions and food manufacturers, elucidating the process of acrylamide formation, the potential risks to the users and how measures can be taken to suppress its production in food.

Since the discovery that certain foods contain acrylamide, various studies have provided manufacturers with a number of acrylamide solutions. Nowadays, L-asparaginase has been shown to be effective in inhibiting acrylamide, and asparaginase can effectively inhibit acrylamide formation in certain foods (up to 90%), while the nutritional characteristics, color and taste of the food are not affected. Asparaginase works on the principle of converting the acrylamide precursor "asparagine" to another natural amino acid, "aspartic acid", so that asparagine does not react chemically to form acrylamide when processing carbohydrate foods such as bread, cookies, crackers, potato products, and grains.

At present, the research on asparaginase in China is less, and particularly, the research on the L-asparaginase which has excellent enzymology and is suitable for food safety is less in report.

Disclosure of Invention

The invention aims to provide a paenibacillus L-asparaginase, a coding gene and an application thereof, wherein the L-asparaginase is Rhizobium etli type L-asparaginase derived from paenibacillus, and the specific enzyme activity is 35.2U mg^-1The optimum reaction temperature is 45 ℃ and the optimum reaction pH is 8.5, and the method has strict substrate specificity and almost no L-glutaminase activity. These excellent properties indicate that the L-asparaginase is suitable to be used as a food additive in baked goods to reduce the generation of carcinogenic acrylamide in the product.

The L-asparaginase provided by the invention is named as PbAsnase in English, is derived from Paenibacillus latens (Paenibacillus barenggoltzii) CAU904 (the strain is preserved in China center for culture Collection of microorganisms (CGMCC for short, address: Shangguan road in Beijing city), has the strain number of No.9530), and is protein of the following (1) or (2) or (3):

(1) protein consisting of amino acid sequences shown in sequence 2 of a sequence table;

(2) protein consisting of amino acid sequences shown in sequence 4 of a sequence table;

(3) and (3) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the (1) or (2) and has the same function (such as L-asparaginase activity) and is derived from the (1) or (2).

In order to facilitate the purification of the protein in (1), a tag as shown in Table 1 may be attached to the amino terminus or the carboxyl terminus of the protein consisting of the amino acid sequence shown in sequence 2 in the sequence listing.

TABLE 1 sequences of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Streep-tag II	8	WSHPQFEK
			c-Myc	10	EQKLISEEDL

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

The coding gene of the protein can be obtained by deleting one or more codons of amino acid residues from a DNA sequence shown in a sequence 1 in a sequence table, and/or carrying out missense mutation of one or more base pairs, and/or connecting a coding sequence of a label shown in the table 1 at the 5 'end and/or the 3' end of the coding sequence.

The gene encoding the protein also belongs to the protection scope of the invention.

The gene can be a DNA molecule of the following (a) or (b) or (c) or (d):

(a) DNA molecules shown in sequence 1 of a sequence table;

(b) DNA molecules shown in sequence 3 of a sequence table;

(c) a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (a) or (b) and which encodes the protein (having L-asparaginase activity);

(d) a DNA molecule having at least 75% sequence identity to the DNA molecule defined in (a) or (b) or (c) and encoding said protein (having L-asparaginase activity).

The stringent conditions can be hybridization and washing with 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS solution at 65 ℃ in DNA or RNA hybridization experiments.

The recombinant vector (such as recombinant expression vector), expression cassette, transgenic cell line or recombinant bacterium containing the gene all belong to the protection scope of the invention.

The recombinant expression vector containing the gene can be constructed by using the existing expression vector. When the gene is used to construct a recombinant expression vector, any one of an enhanced promoter and a constitutive promoter may be added before the transcription initiation nucleotide, and they may be used alone or in combination with other promoters. In addition, when the gene of the present invention is used to construct a recombinant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of recombinants, the recombinant expression vector used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound which can produce a color change, a gene for a resistant antibiotic marker or a chemical-resistant marker, or the like.

The recombinant expression vector may specifically be as follows (I):

(I) inserting the gene into a multiple cloning site of a pET-28a (+) vector to obtain a recombinant plasmid;

(II) introducing the recombinant expression vector of the (I) into escherichia coli to obtain recombinant bacteria;

the Escherichia coli is preferably Escherichia coli BL21(DE3) or Rosetta (DE 3).

The invention protects the application of the protein as L-asparaginase.

The pH of the reaction is 5.5 to 10.0, specifically 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0;

it may be 10.0 to 11.0, specifically 10.0, 10.5, or 11.0

The reaction temperature for L-asparaginase is less than 55 deg.C, such as 20-55 deg.C, 30-50 deg.C, 20-30 deg.C, 30-40 deg.C, 40-45 deg.C, 45-50 deg.C, or 50-55 deg.C, specifically 20 deg.C, 30 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, or 55 deg.C.

The invention protects the application of the protein in food processing or as a food additive.

The invention protects the use of said proteins for reducing the amount of acrylamide formed in baked goods.

The invention protects the application of the protein, the gene, the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium in the preparation of the L-asparaginase.

The invention has the beneficial effects that:

the protein provided by the invention has the following enzymological properties as L-asparaginase: the specific enzyme activity is 35.2Umg^-1The optimum reaction pH value is 8.5, and the reaction is stable within the pH range of 5.5-10.0; the optimal reaction temperature is 45 ℃, the stability is within 55 ℃, the residual enzyme activity reaches more than 90 percent after the heat preservation is carried out for 30min at 55 ℃, and the better thermal stability is shown. Experiments prove that the content of acrylamide in the final product is remarkably reduced by 86% and 52% respectively by applying the protein to potato chips and moon cakes. The protein provided by the invention can effectively reduce the acrylamide content in the baked food, ensures the safety of the food, and has important social significance and economic benefit. The protein provided by the invention can meet the special requirements on the reaction pH and the thermal stability of the L-asparaginase in industrial applications such as food, medicine and the like, and has great application potential.

Drawings

FIG. 1 is an agarose gel electrophoresis of the PCR amplification product of example 1.

FIG. 2 is an SDS-PAGE pattern.

FIG. 3 shows the optimum reaction pH for Paenibacillus L-asparaginase.

FIG. 4 shows the pH stability of Paenibacillus L-asparaginase.

FIG. 5 shows the optimum reaction temperature for Paenibacillus L-asparaginase.

FIG. 6 shows the temperature stability of Paenibacillus L-asparaginase.

FIG. 7 shows the half-life of Paenibacillus L-asparaginase.

Figure 8 is a graph of paenibacillus L-asparaginase reducing acrylamide levels in potato chips.

FIG. 9 is a graph of Paenibacillus L-asparaginase reducing acrylamide levels in mooncakes.

Detailed Description

The experimental procedures used in the following examples are conventional ones unless otherwise specified.

Materials, reagents such as molecular reagents, cloning expression vectors, strains, fermentation raw materials and the like used in the following examples are commercially available unless otherwise specified.

The method for measuring the enzyme activity of L-asparaginase in the following examples and definitions are as follows:

taking 100 mu L of 0.04mol L ^-150 μ L of 0.05mol L of L-asparagine^-1CHES (2- (cyclohexylamino) ethanesulfonic acid) (pH 8.5) buffer, and 100. mu.L of enzyme solution to be assayed were reacted at 45 ℃ for 10min, and 50. mu.L of trichloroacetic acid (TCA) (1.5mol L) was added^-1) The reaction was terminated. After centrifugation, 100. mu.L of the supernatant was collected, and then 3.7mL of water and 200. mu.L of Nessler reagent were added to develop color, and absorbance was measured at 450 nm. Ammonium sulfate was plotted as a standard curve.

Definition of L-asparaginase enzyme activity: the amount of enzyme required to catalyze the hydrolysis of L-asparagine to 1. mu. mol ammonium ion per minute under the above conditions.

Example 1 obtaining of Gene PbAsnase and protein PbAsnase

1. Extracting the genomic DNA of Paenibacillus latens (Paenibacillus barenggiltzii) CAU904, taking the genomic DNA as a template, and performing PCR (polymerase chain reaction) on the genomic DNA by using artificially synthesized PbAsnaseFNheF:

5′-ctcagGCTAGCATGAACTATAACGAAGCTTTGCTGG-3' (restriction sites for the restriction enzyme NheI are underlined) and PbAsnaseRHOR:

5′-gtcatCTCGAGTTAGCGTTCCAGCTGGAACAC-3' (underlined is the restriction site of restriction enzyme XhoI) as a primer, and PCR was carried out to obtain a DNA fragment. The amplified products were detected by 1% agarose gel electrophoresis, and the results are shown in FIG. 1, where M represents different molecular weight standards, and 1 represents the DNA fragment obtained by PCR amplification. As a result, the DNA fragment obtained by PCR amplification was about 1kb in size.

The PCR reaction procedure is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 30s, extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 5 min.

2. And (3) carrying out double digestion on the DNA fragment obtained in the step 1 by using restriction endonucleases NheI and XhoI, and recovering a digestion product.

3. The vector pET-28a (+) (Novagen, catalog No. 69864-3CN) was double-digested with the restriction enzymes NheI and XhoI, and the vector backbone of about 5300bp was recovered.

4. the enzyme digestion product is connected with a vector framework, and escherichia coli DH5 α is transformed to obtain a recombinant plasmid pET-28a (+) -PbAsnase.

According to the sequencing result, the structure of the recombinant plasmid pET-28a (+) -PbAsnase is described as follows: the small DNA fragment between the NheI recognition sequence and the XhoI recognition sequence of the vector pET-28a (+) is replaced by a DNA molecule with the nucleotide sequence shown in the sequence 1 of the sequence table. In the recombinant plasmid pET-28a (+) -PbAsnase, a DNA molecule shown in a sequence 1 of a sequence table is fused with a coding sequence of a His-tag label (consisting of 6 histidine residues) on a vector skeleton to form a fusion gene shown in a sequence 3 of the sequence table, and a recombinant protein PbAsnase with the His-tag label shown in a sequence 4 of the sequence table is expressed.

A gene shown in 1 st to 1011 th positions in a sequence 1 of a sequence table is named as a gene PbAsnase, a protein coded by the gene is named as a protein PbAsnase, and the amino acid sequence of the protein is shown as a sequence 2 of the sequence table.

Example 2 expression of PbAsnase Gene and purification of recombinant protein

One, construction of recombinant strains

The recombinant plasmid pET-28a (+) -PbAsnase is transformed into Escherichia coli BL21(DE3) to obtain a recombinant bacterium, and the recombinant bacterium is named as BL21(DE3) -pET-28a (+) -PbAsnase.

Expression of PbAsnase Gene

The recombinant strain BL21(DE3) -pET-28a (+) -PbAsnase is inoculated into a seed culture medium, and kanamycin (50 mu g mL) is used for seed culture^-1) The LB medium of (1.5% (w/v) agar was added to prepare an LB solid medium plate. Selecting positive transformant from solid culture medium plate, culturing at 37 deg.C for 15 hr, transferring to 200mL LB medium at 2%, culturing at 37 deg.C, and culturing in OD₆₀₀When reaching 0.6-0.8, IPTG was added to a final concentration of 1mmol L^-1And performing induction culture at 20 ℃ for 15h, and centrifuging to collect cells. Then resuspending the cells, breaking the cell wall by ultrasonic wave and centrifuging to obtainThe supernatant is the crude enzyme solution.

Thirdly, purifying the recombinant protein PbAsnase

Based on the fact that pET-28a (+) plasmid contains a sequence for coding His-Tag label protein, the Ni-IDA affinity column is selected to be used for purifying the recombinant protein:

first with equilibration buffer (50mmol L)^-1pH 8.0Tris-HCl buffer, 0.5mol L^-1NaCl，20mmol L^-1Imidazole) at 1.0mL min^-1 Flow rate elution 10 column volumes (5-10 column volumes can all);

adding the crude enzyme solution in the second step for 0.5mL min^-1Sampling at a flow rate;

then, the mixture was washed with an equilibration buffer and an eluent A (50mmol L)^-1pH 8.0Tris-HCl buffer, 0.5mol L^- ¹NaCl，50mmol L^-1Imidazole) at 1.0mL min^-1Eluting at flow rate to OD₂₈₀Less than 0.05, washing off foreign protein;

finally, eluent B (50mmol L)^-1pH 8.0Tris-HCl buffer, 0.5mol L^-1NaCl，200mmol L^-1Imidazole), and collecting the solution of the eluent B passing through the column, namely the purified recombinant protein PbAsnase solution.

The purity of the purified recombinant protein PbAsnase solution was examined by SDS-PAGE (Laemmli UK.1970. clearage of structural proteins reducing the assembly of the head of bacterial genes T4.Nature 227:680-685), and the results are shown in FIG. 2, wherein M is a low molecular weight standard protein; 1 is crude enzyme solution; 2 is purified recombinant protein PbAsnase solution. The result shows that after purification, an obvious single band is obtained, and the molecular weight is 41.1 kDa.

Determining the total protein amount, the L-asparaginase enzyme activity and the specific enzyme activity in the purified recombinant protein PbAsnase solution and the corresponding unpurified crude enzyme solution; calculating the purification recovery rate by taking the total enzyme activity of the crude enzyme solution as 100 percent; the fold purification was calculated with the fold purification of the crude enzyme solution being 1, and the results are shown in Table 2. The protein content was determined by the method of Lowry et AL (Lowry OH, Rosebrough NJ, Farr AL, Randall RJ.1951.protein measurement with the folinophenol reagent. J Biol Chem 193:265-275), bovine serum albumin was used as a standard protein, and the method of measuring L-asparaginase enzyme activity was the same as the first step in example 3 described below.

TABLE 2 purification Table

Example 3L-asparaginase enzymatic Properties of the recombinant protein PbAsnase

The solution of the recombinant protein PbAsnase used in this example was a solution of purified recombinant protein PbAsnase.

1. Optimum reaction pH

The solution of the recombinant protein PbAsnase is used as enzyme solution to be detected, the enzyme activity determination method is the same as the above method, and the difference is that the following different buffer solutions (the concentration is 50mmol L all) are respectively adopted^-1): MOPS (3-morpholine propanesulfonic acid) buffer solution with the pH value of 6.0-8.0; Tris-HCl (3- (hydroxymethyl) aminomethane-hydrochloric acid) buffer, pH 7.0-8.5; CHES (2- (cyclohexylamino) ethanesulfonic acid) buffer, pH 8.0-10.0; Glycine-NaOH (Glycine-sodium hydroxide) buffer, pH 8.5-10.5; CAPS (3- (cyclohexylamino) -1-propanesulfonic acid) buffer, pH 10.0-11.0. L-asparaginase was dissolved in 5 buffer systems at different pH values. Then, the enzyme activity of the L-asparaginase is measured at 45 ℃, and the relative enzyme activity (%) is calculated by taking the highest point of the enzyme activity as 100%.

2. Stability of pH

Respectively diluting the solution of the recombinant protein PbAsnase by using different pH value buffer solutions and HAc-NaAc buffer solutions (pH 4.0-6.0) in the step 1, then respectively treating the solution in a water bath kettle at 35 ℃ for 30min, then rapidly placing the solution in ice water for cooling for 30min, then measuring the activity of residual L-asparaginase, taking the untreated diluted solution of the recombinant protein PbAsnase as a control, and finally calculating the relative enzyme activity (%), namely: the residual L-asparaginase enzyme activity accounts for the percentage of the control L-asparaginase enzyme activity.

As a result:

as shown in FIG. 3, the optimal reaction pH of the recombinant protein PbAsnase is 8.5, and the buffer used is CHES buffer, wherein "△" is MOPS buffer, "▲" is Tris-HCl buffer, "□" is CHES buffer, "tangle-solidup" is Glycine-NaOH buffer, and "O" is CAPS buffer.

As shown in FIG. 4, the recombinant protein PbAsnase was stable at pH 5.5-10.0, had a residual L-asparaginase enzyme activity of more than 80% and exhibited good pH stability, and in FIG. 4, "■" was HAc-NaAc buffer, ". ▲" was MOPS buffer, ". ●" was Tris-HCl buffer, "△" is MOPS buffer, ". tangle-solidup" was Glycine-NaOH buffer, and ". smal" was CAPS buffer.

3. Optimum reaction temperature

Diluting the solution of the recombinant protein PbAsnase by a proper multiple to 50mmol L^-1Measuring the enzyme activity of L-asparaginase in CHES buffer solution with pH of 8.5 at 20-70 deg.C (specifically 20 deg.C, 30 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 60 deg.C, 70 deg.C), and calculating relative enzyme activity (%) with the highest point of enzyme activity as 100%.

4. Temperature stability

Diluting the solution of the recombinant protein PbAsnase by a proper multiple to 50mmol L^-1Treating the protein in a CHES buffer solution with the pH of 8.5 at different temperatures of 20-70 ℃ (specifically 20 ℃,30 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ and 70 ℃) for 30min, then placing the treated protein in an ice water bath for cooling for 30min, finally measuring the activity of residual L-asparaginase, and calculating the relative enzyme activity (%) by taking the activity of the L-asparaginase in the diluted recombinant protein PbAsnase solution which is not treated as a reference.

5. Half life

Diluting the solution of the recombinant protein PbAsnase by a proper multiple to 50mmol L^-1And (2) respectively placing the recombinant protein PbAsnase into CHES buffer solution with the pH of 8.5, treating the solution at different temperatures of 45 ℃, 50 ℃, 55 ℃ and 60 ℃ for 0-4h, sampling at different time intervals, determining the activity of residual L-asparaginase by taking the solution of the diluted recombinant protein PbAsnase which is not subjected to the treatment as a reference, and calculating the percentage of the activity of the residual enzyme in the reference enzyme to obtain the time for the L-asparaginase enzyme activity of the recombinant protein PbAsnase to decay to 50% at different temperatures.

As a result:

as shown in FIG. 5, the optimal reaction temperature of the recombinant protein PbAsnase is 45 ℃.

As shown in FIG. 6, the recombinant protein PbAsnase is kept relatively stable at a temperature below 55 ℃ (20-55 ℃), the enzyme activity of L-asparaginase can be kept above 90%, and the enzyme activity of L-asparaginase is rapidly reduced after the temperature exceeds 55 ℃.

As shown in FIG. 7, the black diamonds represent 45 ℃, the white squares represent 50 ℃, the black triangles represent 55 ℃, the black circles represent 60 ℃, and the half-lives of the recombinant protein PbAsnase at 45 ℃, 50 ℃, 55 ℃ and 60 ℃ are 3462, 853, 309 and 26min, respectively.

Example 4 use of recombinant protein PbAsnase to reduce acrylamide levels in moon cakes and potato chips

First, preparation of moon cake and potato chips

Moon cake production (Cantonese style): firstly weighing 65g of invert syrup, then weighing 20g of edible oil, and stirring by using a rubber scraper while adding. Then adding 3g of kansui and 100g of flour, uniformly stirring, and adding solutions (enzyme solutions) of recombinant protein PbAsnase with different concentrations in the process to ensure that the enzyme activity of L-asparaginase in each gram of flour is 0, 1, 5, 10, 40 and 80U respectively. Covering the stirred dough with a preservative film, placing the dough in a proofing box, standing and proofing for 1h (the temperature is 45 ℃ and the humidity is 85%), and preparing the moon cake skin. Then, the moon cake wrapper and the stuffing are uniformly rolled into balls, the stuffing is wrapped in the wrapper, the ratio of the wrapper to the stuffing is 3:1, and the moon cake wrapper and the stuffing are pressed and molded by a mold. And finally, baking the moon cake blank in an oven, wherein the upper fire and the lower fire are both 180 ℃, and baking for 10min to obtain the moon cake.

Potato chip preparation: after peeling the potatoes, the potatoes are washed clean by clear water, and the slices are 2.0mm in thickness and 40mm in diameter. Immediately after slicing, the slices were rinsed with deionized water for 1min to remove starch from the surface. Then blanching in deionized water at 85 deg.C for 3.5 min. Then placing the potato slices in solutions of recombinant protein PbAsnase with different concentrations (the enzyme activities of L-asparaginase are 0, 5, 10, 20, 40 and 80U mL respectively)^-1) Soaking in water at 45 deg.C with potato/water ratio of 1/2(w/w). After treatment, the potato pieces were dried at room temperature for 30min and then fried in an oil pan at 170 ℃ (peanut oil) for about 5min until the moisture content of the final potato pieces was 2%.

(II) extracting and detecting acrylamide in moon cakes and potato chips

Extraction of acrylamide in the sample: mashing potato chips and baked moon cake peels respectively in a mortar, weighing 1g of the mashed potato chips and baked moon cake peels into a 50mL test tube, adding 5mL of n-hexane, carrying out oscillation extraction for 1min, then centrifuging at 5,000rpm for 10min, removing an upper liquid n-hexane layer, adding 5mL of n-hexane, extracting according to the method, and repeating the steps for 2 times (the n-hexane needs to be sucked and removed completely). Then, 10mL of ultrapure water was added thereto and the mixture was vigorously shaken for 1min, and then 10mL of acetonitrile, 4g of anhydrous magnesium sulfate and 1g of sodium chloride were added thereto and the mixture was shaken for 1 min. Centrifuging at 10,000rpm at 4 deg.C for 10min, collecting acetonitrile layer liquid 6mL, rotary evaporating at 35 deg.C for oven drying, adding 1mL ultrapure water for redissolving, and filtering with 0.45 μm filter membrane to obtain final extract, and detecting acrylamide content by the following chromatography and mass spectrometry:

and (3) chromatographic detection conditions: the column was Atlantis TM d C18(5 μm, 150 mm. times.2.1 mm); the mobile phase consists of a solution A and a solution B, wherein the solution A is a formic acid aqueous solution with the volume percentage content of 0.1 percent, the solution B is 100 percent methanol, the volume ratio of the solution A to the solution B is 90:10, and the flow rate is 0.2mL min^-1The column temperature was 30 ℃ and the amount of the extract to be sampled was 10. mu.L. The detection wavelength was 205 nm.

Mass spectrum detection conditions: capillary voltage 3.5 kV; the sheath gas temperature is 250 deg.C, and the flow rate is 11L min^-1(ii) a The cone hole voltage is 35V, the temperature of the drying gas is 250 ℃, and the flow rate is 5L min^-1ESI + mode, quantitative MRM mode, acrylamide qualitative ion (72/55), capillary exit voltage 135eV, and collision energy 6 eV.

The chromatographic and mass spectrometric measurement results show that: when the enzyme acts on the potato chips, the addition amount of the L-asparaginase is 80U mL^-1At this time, the amount of acrylamide produced was reduced by 86% (see FIG. 8). When the addition amount of the L-asparaginase is 80U g^-1Flour, which can reduce the acrylamide content in the baked moon cake skin by 52% (see figure 9).

Those not described in detail in this specification are within the skill of the art.

Claims

1. An application of protein as L-asparaginase,

the protein is the protein of the following (1), 2 or 3):

the pH value of the reaction is 5.5-10.0 when the L-asparaginase is used;

the reaction temperature for the L-asparaginase is 55 ℃ or lower.

2. The protein of claim 1, wherein the gene encoding the protein is a DNA molecule of the following (a) or (b):

(a) DNA molecules shown in sequence 1 of a sequence table;

(b) DNA molecules shown in sequence 3 of the sequence table.

3. Use of the protein of claim 1 in food processing.

4. Use of a protein as claimed in claim 1 for reducing the amount of acrylamide formed in a baked good.