CN118325879B - Algin lyase mutant PL31PtM and application thereof - Google Patents
Algin lyase mutant PL31PtM and application thereof Download PDFInfo
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- C12N9/88—Lyases (4.)
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- C12N15/09—Recombinant DNA-technology
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
- C12Y402/02011—Poly(alpha-L-guluronate) lyase (4.2.2.11), i.e. alginase II
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Abstract
The invention belongs to the technical fields of molecular biology and protein engineering, and particularly relates to an algin lyase mutant PL31PtM and application thereof. The invention obtains an alginate lyase gene sequence PL31Pt through codon optimization, the nucleotide sequence information is shown as SEQ ID NO.1, the sequence is used for the construction process of recombinant alginate lyase PL31Pt engineering bacteria, recombinant PL31Pt is further used as a template, and a mutant PL31PtM with improved thermal stability is obtained through proteolytic design, the nucleotide sequence is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4; the efficient preparation of the mutant PL31PtM is realized through high-density fermentation. The mutant PL31PtM obtained by the invention can be used in the preparation process of alginate oligosaccharides, and has wide application range.
Description
Technical Field
The invention belongs to the technical fields of molecular biology and protein engineering, and particularly relates to an algin lyase mutant PL31PtM and application thereof.
Background
Algin is mainly derived from brown algae such as kelp, kelp and gulfweed, and is formed by randomly arranging beta-D-mannuronic acid and alpha-L-guluronic acid through alpha/beta-1, 4 glycosidic bonds. The algin is further degraded to form algin oligosaccharides with different polymerization degrees. Compared with algin, the algin oligosaccharide has good water solubility, biological activity and biological utilization rate, so that the algin oligosaccharide has better application value. Research shows that the algin oligosaccharide has the activities of antioxidation, bacteriostasis, immunoregulation and the like, and can be applied to the field of food and medicine. In addition, research shows that in the field of agricultural planting, the algin oligosaccharide can be used as a biological stimulus to promote plant growth, improve fruit and vegetable quality and the like. Heretofore, the preparation method of alginate oligosaccharides mainly comprises a chemical method and an enzymatic method, wherein the chemical method is a mainstream method due to rapidness and low cost. Compared with a chemical method, the enzymatic process has the advantages of mild reaction conditions, environmental protection, controllable processing process, perfect product structure and the like. However, the enzyme method has high processing cost and limits the large-scale application of the enzyme method. Algin lyase plays an important role in the enzymatic process and is also a main factor causing high cost of the enzymatic process. Therefore, the development of the efficient and low-cost algin lyase is of great significance.
The algin lyase degrades algin into algin oligosaccharide by beta-elimination. Algin lyase is widely available and can be divided into several families based on amino acid similarity. The current research on algin lyase is mainly focused on the PL7 and PL17 families, and other family sources of algin lyase are reported to be less. Therefore, further development of other family sources of algin lyase and expansion of a series of studies have been conducted, which is important for expanding the application range of algin lyase.
Disclosure of Invention
Aiming at the problems, the invention takes paenibacillus algin lyase PL31Pt derived from PL31 family as a research object, provides an algin lyase mutant PL31PtM and application thereof, effectively improves the enzyme activity and stability, and lays a solid foundation for the next industrialized application.
In order to achieve the above purpose, the specific technical scheme adopted by the invention is as follows:
an algin lyase mutant PL31PtM, wherein the amino acid sequence of the algin lyase mutant PL31PtM is shown as SEQ ID NO. 3.
ASVTCSTASCLTNALAQAAPGDVITLAAGVTFNGKFVAAANGTPTGKITLQSASSSNKAELNGGGTGSGYTLHVTGDHWVIKDLKITNAKKGIMLDPANYTLIDGAEVYQIGEEGVHYRDGFSYNTIRNSYFHDIGTVNPAFGEAIYVGSDKGKWGTFNAATNHNTIANNTIGPNVAAEHIDIKEGSTGTLVENNTFDGTGMSGANAADSFIDVKGNNDVIRGNIGYRNGNSNIKDAFQVHQRAAGWGQNAIFTNNTVYLPNTTAYVVNAASGTTASASGNTRYPAGNMYTGSVTGGAKTGHSSASSMP（SEQ ID NO.3）;
Preferably, the coding amino acid sequence is a polynucleotide sequence as shown in SEQ ID NO. 4.
GCTTCTGTTACTTGTTCTACTGCTTCTTGTTTGACTAACGCTTTGGCTCAAGCTGCTCCTGGTGACGTTATTACTTTGGCTGCTGGTGTTACTTTTAATGGTAAATTTGTTGCTGCTGCTAATGGTACTCCAACTGGTAAAATTACTTTGCAATCTGCTTCTTCTTCTAATAAGGCTGAATTGAACGGTGGTGGTACTGGTTCTGGTTATACTTTGCATGTTACTGGTGACCATTGGGTTATTAAGGATTTGAAAATTACCAACGCTAAGAAGGGTATTATGTTGGATCCTGCTAACTATACTTTGATTGATGGTGCTGAAGTTTACCAAATTGGTGAAGAAGGTGTTCATTACAGAGATGGTTTTTCTTATAATACTATCAGAAACTCTTACTTCCACGATATTGGTACTGTTAACCCAGCTTTTGGTGAAGCTATCTATGTTGGTTCTGATAAGGGTAAATGGGGTACTTTTAATGCTGCTACTAACCATAATACTATCGCTAACAATACTATCGGTCCAAATGTTGCTGCTGAACATATTGATATTAAGGAAGGTTCTACTGGTACTTTGGTTGAAAACAATACTTTTGATGGTACTGGTATGTCTGGTGCTAACGCTGCTGATTCTTTTATTGATGTTAAGGGTAACAACGATGTTATTAGAGGTAACATTGGTTACAGAAACGGTAATTCTAACATTAAGGATGCTTTTCAAGTCCATCAAAGAGCTGCTGGTTGGGGTCAAAACGCTATTTTCACTAATAATACTGTCTACTTGCCCAATACTACTGCTTACGTTGTTAACGCTGCTTCTGGTACTACTGCTTCTGCTTCTGGTAACACTAGATACCCTGCTGGTAATATGTACACTGGTTCTGTTACTGGTGGTGCTAAGACTGGTCATTCTTCTGCTTCTTCCATGCCA（SEQ ID NO.4）;
The invention also provides a recombinant expression vector comprising the nucleotide sequence of the algin lyase mutant PL31 PtM.
The invention also provides a recombinant engineering bacterium which comprises the recombinant expression vector.
Preferably, the recombinant engineering bacteria take pichia pastoris engineering bacteria as hosts.
Preferably, the pichia pastoris engineering bacteria comprise pichia pastoris X33.
The invention also provides an application of the alginate lyase mutant PL31PtM in preparing alginate oligosaccharides.
Preferably, the alginate oligosaccharides are prepared by hydrolyzing alginate using an alginate lyase mutant PL31 PtM.
The invention also provides application of the recombinant engineering bacterium in preparing a mutant PL31PtM with high enzyme specific activity.
The invention takes Paenibacillus algin lyase PL31Pt from PL31 family as a research object. Firstly, recombining and expressing the alginate lyase PL31Pt derived from the paenibacillus, secondly, obtaining a mutant PL31PtM with improved thermal stability through rational design, and finally, efficiently preparing the mutant PL31PtM and optimizing an enzymatic process to prepare the alginate oligosaccharides.
The invention is mainly realized by the following technology: (1) According to the codon preference of pichia pastoris, an algin lyase gene sequence PL31Pt is synthesized; (2) Constructing and screening recombinant algin lyase PL31Pt engineering bacteria; (3) Analyzing and measuring the Pt enzymology characteristics of the recombinant algin lyase PL 31; (4) Recombinant PL31Pt is used as a template, and a mutant PL31PtM with improved thermal stability is obtained through a proteolytic design; (5) The efficient preparation of the mutant PL31PtM is realized through high-density fermentation; (6) mutant PL31PtM enzymatic method for preparing alginate oligosaccharides.
Compared with the prior art, the algin lyase mutant PL31PtM provided by the invention has the following advantages: the invention provides an algin lyase mutant PL31PtM, which effectively improves the thermal stability, can be applied to enzymatic preparation of algin oligosaccharide, effectively expands the application range of algin and lays a foundation for the next industrialized application.
Drawings
FIG. 1 is a graph showing pH and temperature characteristics of recombinant algin lyase PL31 Pt;
FIG. 2 is a three-dimensional conformation and structure evaluation chart of alginate lyase PL31 Pt;
FIG. 3 is a graph showing the optimal reaction temperature and thermal stability of the mutant PL31 PtM;
FIG. 4 shows a high-density fermentation curve and a protein electrophoresis diagram of the mutant PL31 PtM;
FIG. 5 is a diagram showing the optimization of the process for preparing alginate oligosaccharides by using the mutant PL31PtM enzyme method.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) j. Sambrook, or according to the kit and product instructions; the reagents and biological materials, unless otherwise specified, are commercially available.
The experimental materials and reagents involved in the invention are as follows:
1. strains and vectors:
Coli strain Top10 was routinely stored in the laboratory and Pichia pastoris X33 (cat# C18000) was purchased from Invitrogen corporation.
2. Enzyme and kit
Q5 Hi-Fi Taq enzyme MIX (cat# M0492S) was purchased from NEB company; plasmid extraction, gel purification kit (cat# DP 209-02) was purchased from Tiangen Biochemical technology (Beijing); restriction enzymes were purchased from baori doctor materials technology (beijing) limited; zeocin is available from Invitrogen.
3. Culture medium
The escherichia coli culture medium is LB liquid culture medium: (1% (w/v) peptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl, pH 7.0), the remainder being water. LBZ was LB medium plus 25. Mu.g/mL Zeocin (bleomycin).
The yeast culture medium is YPD culture medium: (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose, the balance being water. Yeast selection medium was YPDZ (YPD+100 mg/L zeocin).
The yeast induction medium is BMGY medium: (1% (w/V) yeast extract, 2% (w/V) peptone, 1.34% (w/V) YNB, 0.00004% (w/V) Biotin, 1% glycerol (V/V)), and the balance water. And (3) injection: YNB is a yeast nitrogen source foundation (Yeast Nitrogen Base); biotin is Biotin.
4. Reagent for measuring algin lyase activity
The activity of alginate lyase is measured by DNS method, and the main reagents comprise substrate sodium alginate (concentration is 0.5%, m/v) and color reagent DNS reagent (6.3%o (w/v) 3, 5-dinitrosalicylic acid; 18.2% (w/v) potassium sodium tartrate tetrahydrate; 5%o (w/v) phenol; 5%o (w/v) anhydrous sodium sulfite, and the balance water).
EXAMPLE 1 construction and screening of recombinant algin lyase PL31Pt engineering bacteria
The gene sequence of Paenibacillus (Paenibacillus tyrfis) algin lyase (Gene accession number: BSDJ 01000004.1) was obtained by analyzing NCBI database, and its full length was 1023bp. After translation by bioinformatics software dnamann 6.0, it was found that the algin lyase PL31Pt corresponding to this sequence consisted of 340 amino acids. The first 31 amino acids of algin lyase PL31Pt were found to be the signal peptide sequence by analysis of the on-line signal peptide software SignalP-5.0 Server (https:// services. Healthcare. Dtu. Dk/services/SignalP-5.0 /). Furthermore, the theoretical molecular weight and isoelectric point of the algin lyase PL31Pt from which the signal peptide was removed were found to be 31.8kDa and 6.13, respectively, by computational analysis of computer pI/MW (https:// www.expasy.org/resources/computer-pI-MW) in the on-line bioinformatics website expasy.
Since pichia pastoris is used as a recombinant expression host in the present invention, it is necessary to optimize the paenibacillus (Paenibacillus tyrfis) algin lyase gene sequence according to the preference of pichia pastoris codons. In addition, the signal peptide used in recombinant expression is alpha signal peptide, so that the signal peptide coding sequence of alginate lyase PL31Pt needs to be removed in gene optimization, and the alginate lyase gene PL31Pt is finally obtained through codon optimization, and the sequence is shown as SEQ ID NO.1, and the corresponding amino acid sequence is shown as SEQ ID NO. 2. The optimized algin lyase gene PL31Pt sequence is synthesized by Anhui general biological Co., ltd, and the algin lyase gene PL31Pt is directly connected to the vector pPICZ alpha A in the synthesis process, so that the expression vector pPICZ alpha A-PL31Pt is obtained.
The construction of the recombinant yeast engineering bacteria is approximately as follows: (1) Linearizing the expression vector pPICZ alpha A-PL31Pt with restriction enzyme SacI, purifying and recovering with reference to Tiangen biochemical technology (Beijing) Limited glue purification kit (product number: DP 209-02), and measuring the concentration; (2) Placing pichia pastoris competent cells on ice for 30min, transferring the purified linearization expression vector pPICZ alpha A-PL31Pt into the pichia pastoris competent cells, and placing on ice for 20min; (3) Transferring the yeast competent cells containing the linearization expression vector pPICZ alpha A-PL31Pt into an electrocuvette, and performing electric shock transformation (the transformation parameters are 1500 kilovolts and 400 ohms) by an electroporation apparatus; (4) Transferring the converted product into a 2mL centrifuge tube, adding 0.5mL of 1M sorbitol solution, standing at 30 ℃ for 2 hours, and uniformly coating on a YPDZ plate; (5) And (3) standing and culturing the coated flat plate at 30 ℃ for 3 to 5 days to obtain the yeast transformant.
Screening experiments were performed on yeast transformants obtained by culture, and the experimental procedures were approximately as follows: (1) The individual yeast transformants were individually picked into 50mL centrifuge tubes containing 5mL BMGY, cultured at 30℃and 200rpm for 24 hours, and then induction culture was started. Methanol is added into the culture medium according to the proportion of 0.75% (volume ratio, v/v) every 24 hours in the induction culture process to carry out induction culture, and algin lyase activity is measured after 48 hours of culture.
The algin lyase activity assay method is as follows: (1) Preheating sodium alginate and diluted enzyme solution at 50deg.C for 5min respectively; (2) Respectively taking 50 mu L of preheated enzyme solution, adding the preheated enzyme solution into a 2mL centrifuge tube, then adding 350 mu L of sodium alginate solution (the concentration is 0.5%, m/v), and reacting for 30min at 50 ℃; (3) After 600. Mu.L of DNS color developing agent is added into the reaction solution, the reaction solution is subjected to color development in boiling water bath at 100 ℃ for 10 min; (4) After the developed solution was cooled to room temperature for 20min, the solution was centrifuged, and the supernatant was taken and absorbance was measured at 540nm, with inactivated enzyme solution as a control throughout the measurement. Algin lyase activity is defined as: the amount of enzyme required to produce 1. Mu. Mol glucose equivalent of alginate oligosaccharides per 30min was defined as one activity unit.
Through screening 50 recombinant transformants, an enzyme activity dominant bacterium (named P13) is finally obtained, and the fermentation enzyme activity is 12U/mL.
EXAMPLE 2 characterization of the enzymatic Properties of recombinant algin lyase PL31Pt
To further analyze recombinant algin lyase PL31Pt (abbreviated as recombinant PL31 Pt), it was first purified. The purification process is approximately as follows: (1) Inoculating recombinant engineering bacteria P13 into a 500mL shake flask containing 100mL BMGY culture medium, performing induction culture for 120h, centrifuging at 6 ℃ and 8000rpm for 10min, and collecting supernatant enzyme solution; (2) Ultrafiltering and concentrating the supernatant enzyme solution by a 10kDa ultrafilter tube, and purifying the ultrafiltered enzyme solution by using a Ni-IDA protein purification kit (product number C600292) of Shanghai biological engineering Co., ltd; (3) The purified recombinant PL31Pt enzymatic properties including enzyme reaction kinetic parameters, pH properties, temperature properties and metal ion stability were determined.
The kinetic parameters of the recombinant PL31Pt enzyme reaction after purification were determined as follows: (1) Preparing sodium alginate (1-10 mg/mL) with different concentrations as a substrate for standby; (2) Respectively measuring hydrolysis reaction speeds of recombinant PL31Pt on sodium alginate with different concentrations; (3) And carrying out fitting analysis by software GRAPHPAD PRISM by taking sodium alginate with different concentrations as an abscissa and taking hydrolysis reaction speeds of the recombinant PL31Pt on substrates with different concentrations as an ordinate to obtain the Michaelis constant and the maximum reaction speed of the recombinant PL31 Pt. The specific activity of the recombinant PL31Pt enzyme, the Michaelis constant Km and the maximum reaction speed Vmax are 1152U/mg, 1.23mg/mL and 1906.8 mu M/min/mg respectively.
The pH profile of recombinant PL31Pt was determined to include optimal reaction pH and pH stability. The pH of the recombinant PL31Pt optimum reaction was determined as follows: the enzyme activity of recombinant PL31Pt at pH5.0-9.0 was measured at 50deg.C to determine the highest pH of the enzyme activity as 100% and the relative enzyme activities at other pH were calculated.
As can be seen from FIG. 1B, the optimal reaction pH of recombinant PL31Pt is 7.0, and the relative enzyme activity is more than 70% in the pH range of 6.0 to 9.0.
The pH stability of recombinant PL31Pt was determined as follows: the remaining enzyme activity was measured after the recombinant PL31Pt was left at pH5.0 to 9.0 for 4 hours at 25 ℃, and the remaining enzyme activity at other pH was calculated with the enzyme activity of the untreated sample being 100%.
As can be seen from FIG. 1B, recombinant PL31Pt had good stability after 4 hours of standing at pH5.0 to 9.0, and the residual enzyme activity of all samples was more than 90%.
The recombinant PL31Pt temperature profile assay included an optimal reaction temperature and thermal stability. Wherein the optimum reaction temperature is determined as follows: the enzyme activities of recombinant PL31Pt at different temperatures of 30-70 ℃ were measured at pH7.0 to determine the enzyme activity at the highest temperature of the enzyme activity as 100%, and the relative enzyme activities at the other temperatures were calculated.
As can be seen from FIG. 1A, the optimal reaction of recombinant PL31Pt is 50 ℃, and the relative enzyme activity is more than 60% in the range of 40-60 ℃.
Recombinant PL31Pt thermostability was determined as follows: the residual enzyme activity was measured after heat treatment of the diluted enzyme solution (between 1U/mL and 2U/mL) at different temperatures (30 ℃ C. And 70 ℃ C.) for 30min, and the residual enzyme activity at different treatment temperatures was calculated with the enzyme activity of the sample without heat treatment set to 100%.
The experimental results are shown in FIG. 1A. The recombinant PL31Pt has good thermal stability in the range of 30-40 ℃, and the residual enzyme activities are all more than 90%. When the heat treatment temperature is higher than 40 ℃, the residual enzyme activity is reduced sharply, and after the heat treatment for 30 minutes at 60 ℃ and 70 ℃, the relative enzyme activity and the residual enzyme activity are only 36.2% and 12.5%, respectively.
The effect of different metal ions on recombinant PL31Pt stability was determined as follows: recombinant PL31Pt was added to buffers containing 1mM and 5mM of different metal ions, respectively, and the activity was measured after standing at room temperature for 4 hours, and the remaining enzyme activities were calculated using the untreated sample as a control.
As can be seen from Table 1, the metal ions Na +、K+、Ca+ and Mg 2+ have an activating effect on recombinant PL31Pt, and the residual enzyme activities are all more than 100%; the metal ions Mn 2+、Cu2+ and Zn 2+ have an inhibitory effect on the recombinant PL31 Pt.
TABLE 1 influence of different metal ions on recombinant PL31Pt stability
EXAMPLE 3 rational design to promote recombinant PL31Pt thermal stability
From the results of the temperature measurement section of example 2, it is known that recombinant PL31Pt has poor thermal stability, thereby limiting its industrial application, and thus improving its thermal stability is of great importance. The thermal stability of the recombinant PL31Pt is improved through the protein rational design.
The PL31Pt was first modeled three-dimensionally by the on-line bioinformatics software SWISS-MODEL (https:// swissmodel. Expasy. Org /). By predictive analysis, the three-dimensional conformation of the PL31Pt protein was obtained (figure 2A). As can be seen from fig. 2A, PL31Pt is mainly composed of β -sheet and random coil. The accuracy of the three-dimensional conformation of recombinant PL31Pt was assessed by on-line bioinformatics software SAVES v 6.0.0 (https:// saves. Mbi. Ucla. Edu /), and the experimental results are shown in FIG. 2B. As can be seen from FIG. 2B, the results of the pull-up plot show that 94.4% of the amino acids are in the optimal region and 5.6% of the amino acids are in the other allowed regions, indicating that the obtained model conformation is correct. And finally determining the three-dimensional conformation of the recombinant PL31Pt target through modeling and model evaluation.
And then, calculating and analyzing the three-dimensional conformation of the recombinant PL31Pt by three thermal stability prediction analysis line software, and searching a target amino acid mutation site. These three bioinformatics online software are PROSS (https:// pros. Weizmann. Ac. Il/step/pross-terms /), consensus filter (https:// kazlab. Umu /) and Fireprot (https:// loschmidt. Chemi. Muni. Cz/fireprotweb/.
And selecting a target mutation site according to a prediction analysis result, wherein the selection is based on the fact that at least two bioinformatics software are required to predict the same mutant. The final positions for mutation experiments were 9, S9T, G34P, T43S, S68F, H97P, S122F, F158T, K235G and D261P, respectively.
TABLE 2 predictive analysis results for different bioinformatics software
Expression vectors corresponding to the different mutants (S9T, G P, T43S, S F, H97P, S122F, F158T, K235G and D261P) were constructed by site-directed mutagenesis. Corresponding amplification primers are designed according to different single-point mutants. The primer sequences are S9T-fw and S9T-rev respectively; G34P-fw and G34P-rev; T43S-fw and T43S-rev; S68F-fw and S68F-rev; H97P-fw and H97P-rev; S122F-fw and S122F-rev; F158T-fw and F158T-rev; K235G-fw and K235G-rev; D261P-fw and D261P-rev.
The construction and screening methods of the expression vectors corresponding to the different mutants are approximately as follows (taking S9T as an example): (1) PCR amplification was performed using the expression vector pPICZ alpha A-PL31Pt as a template and the primers S9T-fw and S9T-rev, wherein the PCR amplification system is shown in Table 3, and the amplification conditions are as follows: pre-denaturation at 98 ℃ for 15s; denaturation at 98℃for 5s, annealing at 54℃for 20s, extension at 72℃for 30s, amplification for 30 cycles; (2) Carrying out enzymolysis on the amplified product for 5 hours by using restriction enzyme DpnI, so as to decompose a template pPICZalpha A-PL31Pt (the existence of the template can cause a plurality of false positives when large intestine is transformed); (3) Transferring the enzymatic hydrolysis product into escherichia coli Top10 by adopting a heat shock method; (4) Screening a large intestine transformant by adopting a bacterial liquid PCR method, firstly, respectively picking the recombinant transformant into an LB culture medium in a single colony mode, culturing for 4 hours at a temperature of 200rpm and 37 ℃, taking 2 mu L of bacterial liquid as a template, carrying out PCR amplification by using primers (5 'AOX-fw: GACTGGTTCCAATTGACAAGC and 3' AOX-rev: GGCACCTGGCATTCTGACAT CC), changing an amplification primer into 5'AOX-fw and 3' AOX according to an amplification system in Table 3, changing an amplification template into the bacterial liquid of the recombinant transformant, and carrying out pre-denaturation for 60 seconds at the amplification condition of 94 ℃; denaturation at 94℃for 10s, annealing at 50℃for 30s, extension at 72℃for 60s, amplification for 33 cycles, sequencing of the correct product, and determining mutation sites based on the sequencing results.
TABLE 3 Gene amplification reaction System
Finally obtaining different single-point mutant expression vectors through experiments, wherein the single-point mutant expression vectors are respectively as follows: pPICZ αA-1 (corresponding to S9T), pPICZ αA-2 (corresponding to G34P), pPICZ αA-3 (corresponding to T43S), pPICZ αA-4 (corresponding to S68F), pPICZ αA-5 (corresponding to H97P), pPICZ αA-6 (corresponding to S122F), pPICZ αA-7 (corresponding to F158T), pPICZ αA-8 (corresponding to K235G), and pPICZ αA-9 (corresponding to D261P).
The constructed mutant expression vector was transferred into Pichia X33, and the cultivation process of recombinant Pichia was the same as in example 1. And (3) carrying out heat stability measurement on fermentation enzyme solutions of different mutants so as to judge whether the mutants are effective mutants. The thermal stability test method is as follows: the diluted enzyme solution was incubated in a water bath at 60℃for 30min, and then the residual enzyme activity was measured, and a sample without heat treatment was used as a control.
The results of the thermal stability of the different single point mutations are shown in Table 4, and it is clear from Table 4 that the mutants G34P, H, 97P, S, F and D261P can improve the thermal stability, and the residual enzyme activities are 45.6%, 51.3%, 48.3% and 62.1% respectively, which are 1.25 times, 1.40 times, 1.33 times and 1.71 times of the starting template PL31Pt after the heat preservation in a water bath at 60 ℃ for 30 min.
TABLE 4 Single point burst thermal stability
Example 4 combination of mutations further improves thermostability
Based on the 4 effective mutants G34P, H97P, S F and D261P obtained in example 3, combinatorial mutation was performed to further improve thermostability. Since the mutant D261P has the most remarkable lifting effect in example 3, double mutants D261P-G34P, D P-H97P and D261P-S122F were constructed using it as a template. The construction of the double mutant was identical to that of the single point mutant in example 3. The construction process of the double mutant D261P-G34P is as follows: the expression vector pPICZ alpha A-9 corresponding to the mutant D261P is used as a template, the amplification is carried out through G34P-fw and G34P-rev, the amplified product is subjected to enzymolysis by DpnI, the product after enzymolysis is transferred into escherichia coli Top10, and whether the construction of the expression vector corresponding to the double mutant D261P-G34P is successful is determined through bacterial liquid PCR and sequencing. Finally obtaining the expression vectors pPICZ alpha A-9-2 (corresponding to mutant D261P-G34P), pPICZ alpha A-9-5 (corresponding to mutant D261P-H97P) and pPICZ alpha A-9-6 (corresponding to mutant D261P-S122F) corresponding to different double mutants through experiments.
The constructed double mutant expression vectors are respectively transferred into Pichia X33, and the cultivation process of the recombinant Pichia is consistent with that of the example 1. And carrying out heat stability measurement on fermentation enzyme solutions of different double mutants so as to judge whether the mutants are effective mutants. The thermal stability test method is as follows: the diluted enzyme solution was incubated in a water bath at 60℃for 30 minutes, and then the residual enzyme activity was measured, whereby a sample without heat treatment was used as a control.
The results of the thermal stability of the different double mutants are shown in Table 5, and as can be seen from Table 5, the improvement of the thermal stability of the double mutant D261P-G34P is not obvious. The other two double mutants D261P-H97P and D261P-S122F, after being incubated in a water bath at 60 ℃ for 30 minutes, had residual enzyme activities of 80.2% and 73.6% respectively, which were 1.29 times and 1.18 times that of the starting template D261P respectively.
TABLE 5 Single point burst thermal stability
On the basis of double mutation, multi-point combined mutation is carried out, and the multi-combined mutant comprises D261P-H97P-G34P, D P-H97P-S122F and D261P-H97P-G34P-S122F. The construction process of the multiple point combinatorial mutation was identical to that of the single point mutant in example 3. Finally obtaining the expression vectors pPICZ alpha A-9-5-2 (corresponding to D261P-H97P-G34P), pPICZ alpha A-9-5-6 (corresponding to D261P-H97P-S122F) and pPICZ alpha A-9-5-2-6 (corresponding to D261P-H97P-G34P-S122F) corresponding to different multi-combination mutants through experiments.
The constructed multiple combination mutant expression vectors are respectively transferred into Pichia X33, and the culture process of the recombinant Pichia is consistent with that of the example 1. And (3) carrying out thermal stability measurement on fermentation enzyme solutions of different multi-combination mutants, wherein the thermal stability measurement method comprises the following steps: the diluted enzyme solution was incubated in a water bath at 60℃for 30min, and then the residual enzyme activity was measured, and a sample without heat treatment was used as a control.
As shown in Table 6, the results of the thermal stability of the various combinations are shown in Table 6, and it is clear from Table 6 that the combination mutants D261P-H97P-S122F are excellent in the improvement effect, and the residual enzyme activities after 30 minutes of water bath incubation at 60℃are 91.2 times, 1.47 times and 1.14 times that of PL31Pt, D261P and D261P-H97P, respectively. For convenience of writing, the combination mutant D261P-H97P-S122F was named PL31PtM, and its corresponding expression vector pPICZαA-9-5-6 was named pPICZαA-PL31PtM.
TABLE 6 Single point mutation thermostability
Example 5 isolation, purification and characterization of mutant PL31PtM
Mutant PL31PtM was isolated and purified by the method provided in example 2. Purified recombinant PL31PtM was obtained experimentally. The recombinant PL31PtM was subjected to enzymatic property measurements (including enzyme reaction kinetic parameters, temperature properties, pH properties and metal ion stability) with PL31Pt as a control throughout the experiment.
The kinetic parameters of the mutant PL31PtM enzyme reaction were measured by the same experimental method as in example 2, and the measurement results are shown in Table 7. Compared with the starting template PL31Pt, the mutant PL31PtM enzyme reaction kinetic parameters are improved to a certain extent, so that the mutant PL31PtM enzyme reaction kinetic parameters have better catalytic efficiency, and therefore the mutant PL31PtM enzyme reaction kinetic parameters have stronger competitiveness in the downstream application field.
TABLE 7 kinetic parameters of the mutant PL31PtM enzyme reaction
The measurement of the temperature characteristics of the mutant PL31PtM includes the optimum reaction temperature and the thermal stability, and the optimum reaction temperature of the mutant PL31PtM is measured by the same experimental method as in example 2, and the PL31Pt is used as a control in the whole experimental process. The experimental results are shown in FIG. 3A. The optimal reaction temperature of the mutant PL31PtM is 60 ℃, and is improved by 10 ℃ compared with PL31Pt, and the mutant PL31PtM has better enzyme activity under high temperature conditions.
Mutant PL31PtM thermostability was determined as follows: the mutant PL31PtM was treated in water bath at different temperatures of 40℃to 70℃for 30min, the remaining enzyme activity was measured, and the enzyme activity of the untreated sample was set to 100%, and the whole experimental procedure was conducted with PL31Pt as a control, and the experimental results are shown in FIG. 3B. Compared with the starting template PL31Pt, the thermal stability of the mutant PL31PtM is effectively improved, and after water bath treatment for 60min at 60 ℃ and 70 ℃, the residual enzyme activities of the mutant PL31PtM are 91.2% and 65.3%, respectively, which are 2.53 times and 5.41 times that of the starting template PL31 Pt.
Mutant PL31PtM optimum reaction pH measurement was performed by the same experimental method as in example 2, with PL31Pt as a control throughout the experiment. The experiment shows that the optimal reaction pH of the mutant PL31PtM is similar to that of the starting template PL31Pt, the optimal reaction pH is 7.0, and the mutant PL31PtM has good hydrolytic activity within the pH range of 6.0 to 9.0. The pH profile measurement showed that the temperature elevation of the mutant PL31PtM did not change its pH profile.
The pH stability of the mutant PL31PtM was determined by the same experimental method as in example 2, with PL31Pt as a control throughout the experiment. The mutant PL31PtM has good stability after being left for 4 hours at pH5.0 to 9.0, and the residual enzyme activity of all samples is more than 90 percent.
The effect of different metal ions (1 mM) on the stability of the mutant PL31PtM was determined by the same experimental method as in example 2, with PL31Pt as a control throughout the experiment, and the experimental results are shown in Table 8. As can be seen from Table 8, the metal ions Na +、K+、Ca+ and Mg 2+ have an activating effect on the mutant PL31PtM, and the residual enzyme activities are all more than 100%; the metal ions Mn 2+、Cu2+ and Zn 2+ have an inhibitory effect on the mutant PL31 PtM.
TABLE 8 influence of different metal ions on stability of recombinant PL31PtM
EXAMPLE 6 efficient preparation of mutant PL31PtM
In the part, pichia pastoris is used as a host, and the efficient preparation of the mutant PL31PtM is realized by constructing high-copy recombinant engineering bacteria and high-density fermentation. Many documents reported earlier indicate that the copy number of the target gene and the solid plate zeocin concentration are positively correlated. Therefore, the part screens high copy recombinant engineering bacteria by increasing the concentration of YPDZ solid plate zeocin. Recombinant Pichia pastoris engineering bacteria were constructed by the same experimental method as in example 1, and transformants were plated on solid plates with zeocin concentration of 400 mg/L. Through screening, an enzyme activity dominant bacterium named X33-PtM is obtained.
The screened enzyme activity dominant bacterium X33-PtM is taken as a research object, and high-density fermentation is carried out in a 7L fermentation tank. The experimental procedure was approximately as follows: the single colony engineering bacteria are inoculated into a 250mL triangular flask containing 50mL YPG culture medium, and are cultured at 30 ℃ under shaking at 200rpm for overnight. The recombinant yeast engineering bacteria cultured overnight are inoculated into a 500 mL triangular flask containing 100mL YPG culture medium according to the inoculum size of 1% (v/v), and are cultured overnight under shaking at 30 ℃ and 200rpm until the OD 600 is more than 10. The recombinant yeast engineering bacteria cultured overnight for the second time were inoculated into a 7L fermenter containing 3L of BSM medium at an inoculum size of 10% (v/v). The culture conditions of the recombinant yeast engineering bacteria in a 7L fermentation tank are as follows: the temperature was 30℃and the pH was 5.0, the stirring speed was 500rpm, and the air flow rate was 40L/min. In the initial stage of culture, glycerol was used as a carbon source for cell growth. When the wet weight of the thalli reaches a certain amount (about 180 g/L), stopping feeding glycerol, and after the glycerol is absorbed by the thalli, starting to induce with methanol (the dissolved oxygen rises rapidly). The addition amount of methanol is adjusted according to dissolved oxygen (the dissolved oxygen in the fermentation process is not less than 10%). During the culture, samples were taken every 24 hours to determine the enzyme activity.
The high-density fermentation result of the recombinant engineering bacteria X33-PtM is shown in figure 4. As shown in FIG. 4A, the activity of alginate lyase increases gradually with increasing fermentation time, and the activity of X33-PtM fermentation enzyme reaches 1725U/mL when the culture is induced for 168 hours. In addition, as can be seen from the results of the protein electrophoresis analysis of FIG. 4B, the protein band of the supernatant fermentation broth is single, which indicates that the supernatant is basically mutant PL31PtM, and the protein electrophoresis shows that the molecular weight of the mutant PL31PtM is about 47kDa and is larger than the predicted theoretical molecular weight in example 1, which indicates that glycosylation modification exists in the mutant PL31PtM during the recombinant expression process of Pichia pastoris.
EXAMPLE 7 mutant PL31PtM enzymatic preparation of alginate oligosaccharides
Sodium alginate is used as a substrate, and alginate oligosaccharides are prepared by a mutant PL31PtM enzymatic method. First, process optimization was performed, including substrate concentration (1% to 3%, m/v), reaction time (1 hour to 4 hours), and enzyme addition amount (2U/mL, 4U/mL, and 6U/mL). The experimental procedure was approximately as follows: (1) The total reaction system is 50mL, sodium alginate and enzyme solution with different concentrations are added according to the experimental requirements, the reaction conditions are 60 ℃, pH7.0, the rotating speed is 150rpm, and hydrolysis experiments are respectively carried out at different times according to the experimental requirements; (2) Centrifuging the hydrolyzed reaction solution at 10 ℃ for 30min, and taking a supernatant; (3) Filtering the obtained supernatant through a 0.22 micron filter membrane, and freeze-drying the filtered liquid to finally obtain the alginate oligosaccharide sample.
The hydrolysis rate is calculated as follows: hydrolysis rate = (alginate oligosaccharide sample/initial sodium alginate weight) 100%
The process of optimizing the concentration of different substrates is as follows: the sodium alginate content was set to 1%, 2% and 3%, the enzyme addition was 4U/mL, the reaction temperature was 60 ℃, the reaction pH was 7.0, 150rpm, and the reaction time was 2 hours. As shown in FIG. 5A, the higher the substrate content, the lower the hydrolysis rate, and the hydrolysis rates corresponding to 1%, 2% and 3% substrate contents were 75.2%, 65.8% and 42.6%, respectively, as can be seen from FIG. 5A. Considering the analysis together, the final substrate content was chosen to be 2% for the next experiment.
And optimizing the enzyme addition amount based on the experimental result of the substrate content, wherein the enzyme addition amount is respectively set to be 2U/mL, 4U/mL and 6U/mL. The substrate content was 2%, the reaction temperature was 60 ℃, the reaction pH was 7.0, 150rpm, the reaction time was 2 hours, and the experimental results were shown in FIG. 5B. As can be seen from FIG. 5B, the hydrolysis rates gradually increased with increasing enzyme addition amounts, and the hydrolysis rates of enzyme addition amounts of 2U/mL, 4U/mL and 6U/mL were 55.6%, 65.9% and 78.6%, respectively. The final enzyme addition was chosen to be 6U/mL for further experiments.
The reaction time was optimized based on the substrate content and the amount of enzyme used, and the reaction time was set to 1 hour, 2 hours, 3 hours and 4 hours. The substrate content was 2%, the enzyme addition amount was 6U/mL, the reaction pH7.0, the reaction temperature was 60℃and 150rpm, and the experimental results were shown in FIG. 5C. As is clear from FIG. 5C, the reaction time of 4 hours is preferably 90.1% and the hydrolysis rate of the next 3 hours is 87.2%. The final hydrolysis time was set to 3 hours in combination.
Through optimizing the concentration of the reaction substrate, the addition amount of the enzyme and the reaction time, the optimal process conditions of enzymolysis are finally determined as follows: substrate concentration 2%, enzyme addition 6U/mL, reaction time 3 hours, reaction temperature 60 ℃, reaction pH 7.0, rotational speed 150rpm.
Under the condition of the optimal process, the hydrolysis reaction system is enlarged to 5 liters, and the reaction is carried out in a 7 liter reaction tank, wherein the process is as follows: (1) Weighing 100g of sodium alginate, placing in a 7-liter reaction tank, adding 5 liters of 0.1mol/L phosphate buffer solution with pH7.0, setting the rotating speed at 150rpm and the temperature at 60 ℃, and stirring until the sodium alginate is dissolved into colloid; (2) Adding 18mL of enzyme solution (total enzyme activity is about 30000U) to a 7-liter reaction tank for reaction; (3) When the reaction time reached 3 hours, the reaction temperature was raised to 90 ℃, the mutant PL31PtM was inactivated, the reaction was stopped, and the hydrolysate was transferred to a 5 liter beaker; (4) And (3) referring to a method of a process optimization process, centrifuging, filtering and freeze-drying the hydrolysate.
Through experiments and analysis, the hydrolysis rate of the 5L reaction system reaches 89.1% under the optimal process condition.
Finally, it should be noted that the above-mentioned embodiments are merely illustrative of the principles, performances and effects of the present invention, and are not meant to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (9)
1. The algin lyase mutant PL31PtM is characterized in that the amino acid sequence of the algin lyase mutant PL31PtM is shown as SEQ ID NO. 3.
2. The algin lyase mutant PL31PtM according to claim 1, wherein the coding of the amino acid sequence is a polynucleotide sequence as shown in SEQ ID No. 4.
3. A recombinant expression vector comprising the nucleotide sequence of the algin lyase mutant PL31PtM of claim 2.
4. A recombinant engineering bacterium comprising the recombinant expression vector of claim 3.
5. The recombinant engineering bacterium according to claim 4, wherein the recombinant engineering bacterium is a Pichia pastoris engineering bacterium.
6. The recombinant engineering bacterium of claim 5, wherein the pichia pastoris engineering bacterium comprises pichia pastoris X33.
7. The use of the alginate lyase mutant PL31PtM of claim 1 in the preparation of alginate oligosaccharides.
8. The use according to claim 7, wherein the alginate oligosaccharides are obtained by hydrolyzing algin using an alginate lyase mutant PL31 PtM.
9. Use of a recombinant engineering bacterium according to any one of claims 4-6 in the preparation of a mutant PL31PtM having high specific enzymatic activity.
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| CN101423854A (en) * | 2008-12-02 | 2009-05-06 | 中国海洋大学 | Method for preparing algin oligosacchride by using algin lyase |
| CN109852601A (en) * | 2019-03-04 | 2019-06-07 | 江南大学 | It is a kind of can efficient application N- glycosylation algin catenase mutant and construction of genetic engineering method |
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| CN112646807B (en) * | 2020-12-25 | 2022-07-05 | 北京雷力海洋生物新产业股份有限公司 | Genetic engineering strain for high yield of alginate lyase and fermentation method thereof |
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