CN109750014B - Fusion type rhizopus chinensis lipase with improved heat stability and application thereof - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and discloses a fusion-type Rhizopus sinensis lipase with improved thermal stability and its application. The amino acid sequence of the fusion-type lipase is as shown in SEQ ID NO.4 or SEQ ID NO.5. shows that the fusion type lipase is encoded by the N-terminus of the amino acid sequence of Rhizopus sinensis lipase fused with a short amphipathic peptide consisting of 30 to 32 amino acids, and the amino acid sequence of the amphipathic short peptide is SEQ ID NO.2 or SEQ ID NO.2. As shown in ID NO.3; the two fusion-type lipases have relatively ideal heat-resistant properties, so they are particularly suitable for industrialized large-scale production.

Description

A kind of fusion type rhizopus sinolina lipase and its application that a kind of thermostability improves

technical field

The invention belongs to the technical field of bioengineering, and relates to a fusion type lipase, in particular to a fusion type Rhizopus sinensis lipase with improved thermal stability and application thereof.

Background technique

Lipase can gradually hydrolyze triglycerides into fatty acids, diglycerides, monoglycerides and glycerol; at the same time, it can also catalyze the reactions of acidolysis, alcoholysis, aminolysis, transesterification and ester synthesis. Because of its unique catalytic activity, it is widely used in food processing, oil processing, leather processing, bioenergy and feed addition, and lipase also plays a vital role in the development of renewable energy and environmental protection effect. Although many kinds of lipases derived from Rhizopus have been available in industrial production, most of the natural Rhizopus lipases are mesophilic lipases with low thermal stability and easy inactivation under high temperature conditions, while the oil processing reaction is generally All need to be carried out at a higher temperature, and the defect of the high temperature resistance of the natural rhizopus lipase has greatly increased the cost of industrial production, and also limited the application of lipase in industrial production.

The thermal stability of Rhizopus sinica lipase is relatively poor, and the industrial production loss is relatively large, so the production cost will also be greatly increased. If the thermal stability of Rhizopus sinica lipase can be improved, its production cost will also decrease thereupon. Therefore, the present invention makes it a lipase with relatively high heat resistance by modifying the Rhizopus chinensis lipase gene.

Contents of the invention

The object of the present invention is to provide a fusion type lipase and its application, aiming at solving the problems of poor heat resistance and unsatisfactory industrial production of Rhizopus sinensis lipase.

The present invention is specifically realized through the following technical solutions:

A fusion-type Rhizopus sinensis lipase with improved thermostability is composed of an amphipathic short peptide composed of 30-32 amino acids fused to the amino acid sequence of Rhizopus sinensis lipase at the N-terminus, wherein the Rhizopus sinensis lipase The amino acid sequence is shown in SEQID NO.1.

Specifically, the parental short peptide is parental short peptide 1 or parental short peptide 2, and the fusion type lipase is fused with a segment consisting of 30 Amphiparental short peptide 1 and amphiparental short peptide 2 composed of ~32 amino acids were obtained.

The amino acid sequences of the amphipathic short peptide 1 and the amphipathic short peptide 2 are respectively shown in SEQ ID NO.2 and SEQ ID NO.3.

The amino acid sequence of the fusion lipase of the present invention is shown in SEQ ID NO.4 or SEQ ID NO.5.

The optimum pH value of the enzymatic reaction of the fusion lipase is 9.0; the optimum temperature is 40°C; under the conditions of pH4-pH10, 37°C, the pH can be tolerated for 1 hour, and the residual activity is still 50%. -Under the conditions of pH 10 and 37°C, the pH is tolerated for 1 hour, and the residual activity is still more than 80%. The lipase-fused parent short peptide 1 has a residual activity of more than 50% at 60°C for 45 minutes; the lipase-fused parent short peptide 2 At 60°C for 30 minutes, the residual activity was above 50%.

In another aspect of the present invention, the amino acid sequence of SEQ ID NO.4 or SEQ ID NO.5 is modified, deleted or added with one or several amino acids to obtain an amino acid sequence that maintains only 90% homology Also within the protection scope of the present invention.

The present invention also provides a method for constructing the above-mentioned fusion lipase, comprising the following steps:

1) Design primers F1 and R1, F2 and R2 for template-free PCR to obtain parental short peptide 1 and parental short peptide 2; then design primers F3 and R3, F3 and R4 to carry out parental short peptide 1 and parental short peptide 2 respectively PCR amplification;

2) Using the recombinant plasmid in which the Rhizopus sinensis lipase gene is connected to the carrier as a template, design primers F4 and R5, F5 and R5 to amplify lipase 1 and lipase 2 by PCR respectively;

3) F3 and R5 were used as primers, and the parental short peptide 1 and lipase 1, and the parental short peptide 2 and lipase 2 were used as templates for fusion PCR amplification, respectively.

4) recombining the fusion PCR amplification product with the vector;

5) Put 5 μL of the recombinant product in 50 μL DH5α competent cells, cool in an ice bath, heat shock, add 500 μL LB medium after cooling, and culture at 180°C at 37°C for 1 hour, centrifuge to retain part of the supernatant to suspend the precipitate, and take all the bacterial liquid to smear plates, cultured overnight at 37°C;

6) Carry out screening and verification of positive clones, pick a single colony in the corresponding resistant LB medium, culture for 2-3 hours and then identify by PCR, send the screened positive clones for sequencing, and compare the sequencing results with the original sequence.

The nucleotide sequences of the above primers F1, R1, F2, R2, F3, R3, F4, R4, F5 and R5 are respectively shown in SEQ ID NO.8-17.

The expression vectors of the encoding genes SEQ ID NO.4 and SEQ ID NO.5 of the present invention are selected from pPIC9K, pPIC9, pPICZaA\B\C, pPICZA\B\C or PGAPZaA\B\C.

In another aspect of the present invention, the application of the two fusion lipases provided by the present invention in feed additives is also within the protection scope of the present invention.

The beneficial effects of the present invention are:

The fusion lipase provided by the present invention, in addition to constructing recombinant plasmids with pPIC9K, the fused gene can also construct recombinant plasmids with expression vectors such as pPIC9, pPICZaA\B\C, pPICZA\B\C, PGAPZaA\B\C, In transforming the corresponding host bacteria, antibiotics such as G418 and Zeocin are added to the plate to screen and obtain lipase genetically engineered bacteria, and then new lipase is obtained by fermentation. This makes it a lipase with high heat resistance. These two lipases are resistant to high temperatures compared with wild-type lipase. It has been proved by experiments that the relative enzyme activity of lipase fusion parent short peptide 1 is about 67.98% after 60°C tolerance for 20 minutes. , the relative enzymatic activity of the lipase fusion parent short peptide 2 is about 59.65%, while that of the wild-type lipase is only 50.68%. At 60°C, the remaining half of the relative enzymatic activity of wild-type lipase is about 20 minutes, while the remaining half of the relative enzymatic activity of lipase fusion parent short peptide 1 is about 45 minutes, and the relative enzymatic activity of lipase fusion parent short peptide 2 is about 20 minutes. The time for the remaining half is about 30 minutes. The heat resistance of these two lipases has been significantly improved, and the loss rate in industrial production has been reduced to a certain extent.

Description of drawings

Fig. 1 is the flow chart of the construction method of fusion type lipase provided by the embodiment of the present invention;

Fig. 2 is the determination curve figure of optimum pH that the embodiment of the present invention provides;

Fig. 3 is the optimal temperature curve figure that the embodiment of the present invention provides;

Fig. 4 is the pH tolerance curve figure that the embodiment of the present invention provides;

Fig. 5 is a 60°C tolerance curve diagram provided by an embodiment of the present invention.

detailed description

The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention. Apparently, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Through the simulation analysis of the protein spatial structure of Rhizopus sinensis lipase, it was fused with the parental short peptide 1 and the parental short peptide 2 respectively. The specific embodiment is to use the Rhizopus sinorifica lipase gene as a template, and carry out the fusion of the Rhizopus sinensis lipase gene and the parental short peptide by the fusion PCR method of the gene, obtain two kinds of fusion type lipase genes, and combine the two kinds of fusion type fat The enzyme gene was connected with pPIC9, pPICZaA\B\C, pPICZA\B\C, PGAPZaA\B\C and other vectors to construct recombinant plasmids, which were transferred into corresponding host bacteria (GS115 or X33, SMD1168, PICHIAPINK) for heterologous expression, and fermentation Two lipases are available. The two lipases can function well in an acidic environment, and have relatively ideal heat-resistant properties, are suitable for high-temperature-resistant granulation, and are therefore suitable for industrial production.

The fused amino acid sequence of the lipase in the embodiment of the present invention is shown in SEQ ID NO.4 or SEQ ID NO.5.

1. Experimental materials and reagents:

Gene source strain: Rhizopus chinensis CCTCCM201021 screened and preserved by our laboratory; expression host bacteria and vectors: GS115 and pPIC9K were purchased from Novagen; lipase recombinant plasmid was constructed by the laboratory; host bacteria: DH5α competent cells were purchased from In Beijing Quanshijin Company.

Main reagents: DNA Marker, Protein Marker (TaKaRa Company); Plasmid Mini Kit I (Omega Company). Agarose was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; nucleic acid dyes were purchased from Biotech Biological Company; restriction endonucleases and PCR amplification enzymes were purchased from TaKaRa Company.

Experimental equipment: centrifuge (Eppendorf); PCR amplification instrument (Bio-Rad); nucleic acid electrophoresis instrument (Bio-Rad); protein electrophoresis instrument (Amersham Bioscience); gel imager (Bio-Rad).

Main media: YPD, LB, and yeast fermentation media (FA and FB) were all prepared in accordance with the recommended methods in the "Invitrogen Company Operation Manual".

2. Determination of lipase enzyme activity

Under certain conditions, the amount of enzyme needed to hydrolyze the p-NP substrate to generate 1 μmoL of p-nitrophenol per minute, that is, one enzyme activity unit, expressed in U.

1) Experimental instruments: constant temperature water bath; pH instrument; microplate reader (Bio-Rad), etc.

2) Experimental material: p-nitrophenol palmitate (p-NPC16) (Sigma Company).

3) Solution preparation:

pH buffer: 0.1mol/L citrate monohydrate buffer and 0.1mol/L phosphate buffer (pH2-7);

0.1mol/L Tris-HCl buffer (pH7-9);

0.1mol/L glycine-NaOH buffer (pH9-12).

Substrate solution: 10 mmol/L p-nitrophenol palmitate (p-NPC16).

4) Using the p-nitrophenol method (p-nitrophenol): the total system is 500 μL, which contains 420 μL of 50 mmol/L buffer solution, 30 μL of 10 mmol/L substrate p-NP and 50 μL of diluted enzyme solution. After mixing the substrate and buffer, preheat at the reaction temperature for 2 minutes, add diluted enzyme solution and mix well, add 50 μL 1.0 mol/L SDS for 5 minutes to stop the reaction, and add 500 μL 1.0 mol/L Na ₂ CO ₃ for color development; The OD value was measured at a wavelength of 405 nm.

The preparation of embodiment 1 lipase fusion parent short

As shown in Figure 1, the obtaining method of two kinds of lipases of the embodiment of the present invention comprises the following steps:

(1) RCR amplification: Design primers (F1, R1) and (F2, R2) for template-free PCR to obtain parental short peptide 1 and parental short peptide 2; then design primers to synthesize parental short peptide 1 (F3, R3 ), parental short peptide 2 (F3, R4) for PCR amplification; using the recombinant plasmid of Rhizopus chinensis lipase gene connected to the carrier as a template, design primers (F4, R5) (F5, R5) for PCR amplification of fat Enzyme gene 1, lipase gene 2;

(2) Using F3 and R5 as primers, respectively use parental short peptide 1 and Rhizopus sinensis lipase gene 1, parental short peptide 2 and Rhizopus sinensis lipase gene 2 as templates to carry out fusion PCR amplification respectively;

(3) Recombinant plasmid construction: recombine the fusion PCR product and vector at 37°C for 30 minutes;

(4) Transformation: add 5 μL of the mutated product to 50 μL DH5α competent cells, flick and mix well, and place on ice for 30 min; heat shock at 42°C for 45 s and immediately cool on ice for 10 min; add 500 μL LB medium, 180 Transform and incubate at 37°C for 1 hour; centrifuge at 7,000rpm for 3 minutes, discard the supernatant, retain 100-150 μL of the supernatant and lightly flick the suspended bacteria, take all the bacterial liquid to spread on the plate, and culture overnight at 37°C;

(5) Verification of positive clones: Pick a single colony in 500 μL of correspondingly resistant LB medium, culture it at 200 rpm for 2-3 hours, and then identify it by PCR; send the screened positive clones for sequencing, and compare the sequencing results with the original sequence;

(6) Find the recombinant plasmid with correct fusion; transfer the mutant plasmid into Pichia pastoris GS115 or X33, SMD1168, PICHIAPINK for expression, ferment and compare enzyme activity to study enzymology and application characteristics.

Wherein, the primers designed in the above method are specifically as follows:

The amino acid sequences of the amphipathic short peptide 1 and the amphipathic short peptide 2 are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

The lipase gene of Rhizopus sinica was fused with parental short peptides 1 and 2, and the fusion was carried out according to the above experimental method, and then sent to Huada Gene Company for sequencing. The results were as shown in the sequences of SEQ ID NO.4 and SEQ ID NO.5, and the transformed yeast strain had lipase Activity, one strain with high fermentation enzyme activity unit was selected for fermentation to obtain enzyme liquid for enzymatic property determination.

Embodiment 2 lipase optimum pH is measured

Adjust the pH of the buffer solution to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, dilute the enzyme solution to an appropriate multiple, and measure the optimum pH at 37°C according to the lipase activity assay method. After the pH, add half points on both sides of the maximum value and continue to detect the optimum value (for example, if the optimum pH is 9, then take pH8, 8.5, 9, 9.5 and 10 to measure according to the lipase activity assay method).

The results of the optimal pH value of the lipase enzymatic reaction are shown in Figure 2. The optimal value of lipase fusion parent short peptide 1, 2 and wild-type lipase was 9, and there was no significant change.

Embodiment 3 lipase optimum temperature is measured

According to the assay method of lipase activity, under the conditions of the above-mentioned optimum pH, the reactants were placed at different temperatures to react at 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, after measuring the optimum temperature, add half points on both sides of the maximum value (for example, if the optimum temperature is 40°C, add 30°C, 35°C, 40°C, 45°C, 50°C to determine the lipase activity method) .

The optimum temperature for the enzymatic reaction of lipase is shown in Figure 3. The optimum temperature for lipase gene fusion parent short peptide 1 and 2 and wild-type lipase is 40°C, respectively.

Example 4 Lipase pH Tolerance Determination

Adjust the buffer to different pH: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, dilute the enzyme solution with these different buffer solutions, and start timing from the time the enzyme solution is put in, The diluted enzyme solution was placed in a water bath at 37°C for 1 hour, then placed on ice, and immediately reacted at the optimum pH and temperature according to the lipase activity assay method. The enzyme solution of the control group is the enzyme solution that has not been tolerated.

It can be seen from Figure 4 that the resistance curves of the three lipases have the same tendency, and the relative enzyme activity of the three lipases is significantly increased when the pH is between 4 and 9 at 37°C for 1 hour.

Embodiment 5 lipase temperature tolerance assay

Dilute the enzyme solution to the corresponding multiple, and then put it into different temperatures: 60°C for 1min, 3min, 5min, 7min, 10min, 15min, 20min, 25min, 30min, 35min, 45min, 60min, then follow the lipase activity assay method React at optimum pH and optimum temperature. The enzyme solution of the control experiment group is the enzyme solution without temperature tolerance.

The temperature tolerance of lipase at high temperature is shown in Figure 5. As the temperature increases, the relative enzyme activity decreases continuously, and as time increases, the relative enzyme activity also gradually decreases. No matter at any temperature and time, the relative enzyme activity after fusion is higher than that before mutation, and the relative enzyme activity of fusion lipase 1 is about 67.98%, and the relative activity of fusion lipase 2 is about 67.98%. 59.65%, while only 50.68% of wild-type lipase remained. At 60°C, the remaining half of the relative enzymatic activity of wild-type lipase is about 20 minutes, while the remaining half of the relative enzymatic activity of fusion-type lipase 1 is about 45 minutes, and the remaining half of the relative enzymatic activity of fusion-type lipase 2 is about 20 minutes. The time is about 30 minutes.

Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variants, the scope of the invention is defined by the appended claims and their equivalents.

sequence listing

<110> Yunnan Normal University

<120> A fusion-type Rhizopus chinensis lipase with improved thermostability and its application

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tttggttatta acgaaggaag ctgtttg 1167

<210> 8

<211> 59

<212>DNA

<213> Artificial Sequence

<400> 8

gctgaagctg aagctaaagc taaagctgaa gctgaagcta aagctaaacc aactccacc 59

<210> 9

<211> 59

<212>DNA

<213> Artificial Sequence

<400> 9

agttggagtt ggagtagttg gtggagttgg agtagttggt ggagttggtt tagctttag 59

<210> 10

<211> 59

<212>DNA

<213> Artificial Sequence

<400> 10

gctgaagctg aagctaaagc taaagctgaa gctgaaccga aagtcagccc ggaggctgt 59

<210> 11

<211> 59

<212>DNA

<213> Artificial Sequence

<400> 11

gagctcagcc tccttcttaa cagcctccgg gctgactttc ggttcagctt cagctttag 59

<210> 12

<211> 44

<212>DNA

<213> Artificial Sequence

<400> 12

gaggctgaag cttacgtaga attcgctgaa gctgaagcta aagc 44

<210> 13

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 13

accagcaaca ggaacagttg gagttggagt 30

<210> 14

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 14

accagcaaca ggaacgagct cagcctcctt 30

<210> 15

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 15

actccaactc caactgttcc tcttgctggt 30

<210> 16

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 16

gttcctgttg ctggtcataa aggttcagtc 30

<210> 17

<211> 44

<212>DNA

<213> Artificial Sequence

<400> 17

tctaaggcga attaattcgc ggccgcctac aaacagcttc cttc 44

Claims (2)

1. Fusion type rhizopus chinensis (with improved heat stability)Rhizopus chinensis ) The lipase is characterized in that the amino acid sequence of the fusion type rhizopus chinensis lipase is shown as SEQ ID NO. 5.

2. Use of the fused Rhizopus chinensis lipase of claim 1 in feed additives.

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