CN112574973A - Lipase with improved heat resistance - Google Patents

Abstract

The application relates to a lipase with improved heat resistance, wherein the amino acid sequence of the lipase is a sequence obtained by replacing and modifying glutamic acid at 292 th position of SEQ ID No.2 with phenylalanine or isoleucine.

Description

Lipase with improved heat resistance

Technical Field

The present application relates to a lipase, and more particularly, to a lipase with improved thermostability.

Background

Lipase (EC 3.1.1.3) is a serine hydrolase that hydrolyzes triglycerides into fatty acids, diglycerides, monoglycerides and glycerol; meanwhile, the catalyst can also catalyze the reactions of acidolysis, alcoholysis, ammonolysis, transesterification, ester synthesis and the like of the ester. Lipases are widely used in processes of food processing, oil processing, leather processing, bioenergy, feed addition, etc. because of their unique catalytic activities. Although a plurality of lipases from rhizopus are available in industrial production, most of natural rhizopus lipases are mesophilic lipases, so that the thermal stability is not high, the industrial production cost is greatly increased, and the application of the lipases in industrial production is also limited.

Therefore, the present application intends to improve the heat resistance of the lipase by genetic modification, thereby increasing the economic value of the lipase in industrial application.

Disclosure of Invention

The purpose of this application lies in reforming transform current lipase, utilizes structural analysis and point mutation technique to effectively promote lipase's heat resistance, and then increase lipase's industrial application value.

To achieve the above object, one of the broader embodiments of the present application provides a lipase having an amino acid sequence modified by substitution of glutamic acid at position 292 of SEQ ID No.2 with phenylalanine or isoleucine.

In one embodiment, the gene encoding the SEQ ID No.2 is the RCL gene isolated from Rhizopus chinensis.

In one embodiment, the lipase has the amino acid sequence shown in SEQ ID No. 6.

In one embodiment, the lipase has the amino acid sequence shown in SEQ ID No. 8.

Another broader embodiment of the present application is to provide a nucleic acid molecule encoding the aforementioned lipase.

Yet another broader embodiment of the present application is to provide a recombinant plasmid comprising the aforementioned nucleic acid molecule.

Drawings

FIG. 1 shows the nucleotide sequence as well as the amino acid sequence of the wild-type lipase RCL.

FIG. 2 shows the primer sequences used in the point mutation technique.

FIG. 3 shows the nucleotide sequence and amino acid sequence of the mutant lipase RCL of E292F.

FIG. 4 shows the nucleotide sequence and amino acid sequence of the mutant lipase RCL of E292I.

FIG. 5 shows the lipase thermotolerance analysis of wild-type and mutant proteins E292F and E292I.

Detailed Description

Some exemplary embodiments that embody features and advantages of the present application will be described in detail in the description that follows. It is understood that the present application is capable of various modifications in various embodiments without departing from the scope of the application and that the description and drawings are to be taken as illustrative in nature and not as limiting the application.

The lipase of the present application is a lipase (RCL) secreted from Rhizopus chinensis (Rhizopus chinensis) which is a filamentous fungus, and the optimum activity of the lipase is at 40 ℃ and pH 7.5. The lipase gene RCL is separated from rhizopus chinensis and is constructed in a vector, and is introduced into industrial common Pichia pastoris (Pichia pastoris) to express the protein. In order to improve the heat resistance of the lipase RCL, the protein structure analysis (PDB:4L3W) is used for finding out the unstable part in the structure to carry out mutation transformation so as to increase the value of the lipase in industrial application and reduce the production cost.

Through structural analysis, glutamic acid (glutamic acid) which is near an active region and close to a C end and is positioned at a 292 th position on an amino acid sequence is selected, and is singly mutated into phenylalanine (phenylalanine) and isoleucine (isoleucine) respectively by using a site-directed mutagenesis technology, so that the structure is stabilized by increasing hydrophobic acting force (hydrophic interaction), and the mutant proteins can be found to successfully improve the heat resistance of the lipase, and the industrial application value of the lipase is further increased.

The method for modifying lipase and the modified lipase obtained by the method are described in detail below.

FIG. 1 shows the nucleotide sequence as well as the amino acid sequence of the wild-type lipase RCL. As shown in FIG. 1, the wild-type lipase gene RCL comprises 888 bases (nucleotide sequence is shown by SEQ ID No. 1) and 296 amino acids (amino acid sequence is shown by SEQ ID No. 2). First, the RCL gene was constructed on pPICZ α a vector. Then, the linearized plasmid DNA is transformed into pichia pastoris, the transformed bacterial liquid is smeared on YPD plates containing 100 mu g/ml zeocin antibiotic, the YPD plates are cultured for 2 days at the temperature of 30 ℃, single bacterial colonies with target genes are screened out and are respectively cultured in YPD culture media for 1 day. In order to amplify the bacterial quantity, the bacterial strain is inoculated into BMGY culture medium for every other day, then the bacterial strain is changed into BMMY culture medium for induction production of target protein, and in 5 days, sampling is carried out every 24 hours, 0.5% methanol is supplemented, and the growth of the bacterial quantity and the protein expression condition are confirmed.

Two mutant genes of the lipase gene RCL are obtained by using a point mutation technology, a wild-type gene RCL is used as a template for carrying out polymerase chain reaction, and mutation primers used in the method are shown in FIG. 2, wherein E292F means that amino acid at the 292 th position of the lipase RCL is mutated into phenylalanine (phenylalanine) from glutamic acid (glutamic acid), and the sequence of the E292F mutation primer is marked by SEQ ID No. 3; E292I means that the amino acid at position 292 of lipase RCL is mutated from glutamic acid (glutamic acid) to isoleucine (isoleucine), and the E292I mutation primer sequence is indicated as SEQ ID No. 4. Therefore, the two mutant genes of lipase RCL obtained by the point mutation technique of the present application are E292F and E292I, respectively.

FIGS. 3 to 4 show the nucleotide and amino acid sequences of two mutants constructed according to the present invention. FIG. 3 shows the nucleotide sequence and amino acid sequence of the mutant lipase RCL of E292F, wherein the nucleotide sequence is shown as SEQ ID No.5, the amino acid sequence is shown as SEQ ID No.6, and the amino acid at position 292 is mutated from glutamic acid (glutamic acid) to phenylalanine (phenylalanine). FIG. 4 shows the nucleotide sequence and amino acid sequence of the mutant lipase RCL of E292I, wherein the nucleotide sequence is shown as SEQ ID No.7, the amino acid sequence is shown as SEQ ID No.8, and the amino acid at position 292 is mutated from glutamic acid (glutamic acid) to isoleucine (isoleucine).

After the mutation reaction is completed, DpnI is added to remove the original template, and then the plasmid DNA is introduced into Escherichia coli for replication and amplification. The success or failure of the mutation was confirmed by DNA sequencing. Finally, as described in the protein expression step, the successfully mutated genes are respectively introduced into pichia pastoris to express the mutant proteins, and lipase activity determination and heat resistance analysis are carried out.

The lipase can hydrolyze triglyceride into fatty acid, diglyceride and glycerol under certain conditions, and the fatty acid generated by hydrolysis can be titrated with standard alkali solution, and the titer value represents the enzyme activity, and the reaction formula is RCOOH + NaOH → RCOONa + H₂And O. Therefore, the activity of lipase can be calculated by measuring the amount of fatty acid released during the reaction. The reaction mixture containing 1ml of the enzyme protein diluted appropriately, and a mixture of 4% polyvinyl alcohol and olive oil as substrates was allowed to act at 40 ℃ for 15 minutes in a phosphate buffer solution atmosphere of pH 7.5. Then, 15ml of 95% ethanol was added to terminate the reaction. After 2 drops of 1% phenolphthalein indicator solution are added, 0.05M sodium hydroxide solution is used for neutralization titration until the reaction end point, and the enzyme activity is calculated according to the amount of consumed alkali solution.

For the analysis of heat resistance, the wild protein and mutant protein at 70 ℃ heat treatment for 2 minutes, then placed on ice for 5 minutes to reduce the temperature, and then placed at room temperature for 5 minutes to recover. And finally, detecting the residual activity of the sample after temperature treatment.

FIG. 5 shows the lipase thermotolerance analysis of wild-type and mutant proteins E292F and E292I, where the lipase activity of the non-heat treated samples was set to 100%. As shown in FIG. 5, the activity of the wild-type protein (Wt) was only about 4% remained after the high temperature treatment at 70 ℃ for 2 minutes, while the residual activities of the E292F and E292I mutant proteins were 30% and 10%, respectively. The above results show that both mutant proteins E292F and E292I have higher thermotolerance than the wild protein, and also indicate industrial application value with greater potential.

In summary, in order to improve the thermostability of the lipase RCL, the present application further analyzes the protein structure thereof, selects glutamic acid (Glu292) at the 292 th position of the amino acid sequence for modification, and performs single mutation to phenylalanine (E292F) and isoleucine (E292I), respectively. The results show that both of the engineered muteins E292F and E292I are more thermostable than the wild-type protein. Therefore, the lipase RCL mutant protein for improving heat resistance is successfully designed, and the value of the lipase in the wide range of industries can be further improved.

While the present invention has been described in detail with respect to the above embodiments, it will be apparent to those skilled in the art that various modifications can be made without departing from the scope of the invention as defined in the appended claims.

Sequence listing

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Claims (6)

1. A lipase, the amino acid sequence of which is a sequence obtained by replacing glutamic acid at position 292 of SEQ ID No.2 with phenylalanine or isoleucine.

2. The lipase according to claim 1, wherein the gene encoding the SEQ ID No.2 is the RCL gene isolated from Rhizopus chinensis (Rhizopus chinensis).

3. The lipase of claim 1, having an amino acid sequence shown in SEQ ID No. 6.

4. The lipase of claim 1, having an amino acid sequence shown in SEQ ID No. 8.

5. A nucleic acid molecule encoding the lipase of claim 1.

6. A recombinant plasmid comprising the nucleic acid molecule of claim 5.

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