A rhizopus oryzae lipase mutant and use thereof

Abstract

The application discloses a rhizopus oryzae lipase mutant and application thereof. By mutating threonine at the 326th position of the rhizopus oryzae lipase into valine and mutating glutamic acid at the 287th position into alanine or valine, the heat stability of the obtained mutant is significantly improved compared with that of a wild type, and the mutant can better meet the requirements of industrial production. In combination with the high Sn-1, 3 selectivity of the mutant, the mutant has higher industrial application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, in particular to a Rhizopus oryzae lipase mutant and application thereof. BACKGROUND

[0002] Lipase (triacylglycerol hydrolase, E.C. 3.1.1.3) catalyzes the hydrolysis, ester synthesis and ester exchange reaction of long-chain triglycerides, and is the most widely used enzyme in industry, such as in food, petroleum, pharmaceutical, paper, leather, detergent and cosmetic industries. However, lipase has some shortcomings, which hinder its application in industrial fields, such as poor thermal stability. High thermal stability of enzymes has always been a key factor for successful industrial biological processes, because the reaction rate increases exponentially with temperature until the temperature at which the enzyme is denatured. In addition, the improvement of thermal stability is also related to the improvement of long-term survival rate under mild conditions, the enhancement of the ability to maintain activity in non-aqueous solvents, and longer half-life under harsh industrial process conditions. Therefore, enzymes with excellent thermal stability have always been the goal pursued by people, because even a slight improvement can save huge costs in the manufacturing process.

[0003] At present, many technical means have been successfully used to improve the thermal stability of lipase, such as immobilization by using solid carriers, modification of enzymes by chemical methods, and addition of additives. However, in recent years, with the development of genetic engineering and protein engineering technology, more and more attention has been paid to the modification of enzymes at the molecular level, and great success has been achieved. SUMMARY

[0004] The present application aims to overcome the shortcomings and deficiencies of the prior art, and provides a Rhizopus oryzae lipase mutant.

[0005] Another object of the present application is to provide the application of the above-mentioned Rhizopus oryzae lipase mutant.

[0006] The object of the present application is achieved by the following technical solutions:

[0007] A Rhizopus oryzae lipase mutant is at least one of the following:

[0008] ROL-T326V, which has a T326V mutation relative to the wild-type ROL;

[0009] ROL-T326V / E287A, which has a T326V / E287A mutation relative to the wild-type ROL;

[0010] ROL-T326V / E287V, which has a T326V / E287V mutation relative to the wild-type ROL;

[0011] The amino acid sequence of the wild-type ROL is shown as SEQ ID NO. 1.

[0012] The Rhizopus oryzae lipase mutant, wherein:

[0013] The amino acid sequence of ROL-T326V is shown as SEQ ID NO. 3;

[0014] The amino acid sequence of ROL-T326V / E287A is shown as SEQ ID NO. 5;

[0015] The amino acid sequence of ROL-T326V / E287V is shown as SEQ ID NO. 7.

[0016] The coding gene of the Rhizopus oryzae lipase mutant, wherein the nucleotide sequence is obtained according to the codon coding rule; preferably, the nucleotide sequence of the coding gene of ROL-T326V is shown as SEQ ID NO. 4, the nucleotide sequence of the coding gene of ROL-T326V / E287A is shown as SEQ ID NO. 6, and the nucleotide sequence of the coding gene of ROL-T326V / E287V is shown as SEQ ID NO. 8.

[0017] The Rhizopus oryzae lipase mutant, wherein the thermal stability is significantly improved compared with the wild type.

[0018] An expression vector comprising the coding gene.

[0019] An engineering bacterium comprising the coding gene in the genome; preferably, the cell comprises the expression vector.

[0020] The Rhizopus oryzae lipase mutant, the expression vector or the engineering bacterium is applied to decompose oil and fat.

[0021] The Rhizopus oryzae lipase mutant, the expression vector or the engineering bacterium is applied to oil and fat processing or biodiesel production.

[0022] The present application has the following advantages and effects compared with the prior art:

[0023] The present application mutates the threonine at the 326th position of the Rhizopus oryzae lipase into valine and the glutamic acid at the 287th position into alanine or valine, so that the thermal stability of the obtained mutant is significantly improved compared with the wild type, which can better meet the industrial production requirements, and the high Sn-1,3 selectivity of the mutant itself makes it more valuable for industrial application. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1SDS-PAGE protein gel map of Rhizopus oryzae lipase and its mutants in Example 1 of the present application; wherein, M: protein marker; 1: ROL pure enzyme; 2: ROL-T326V pure enzyme; 3: ROL-T326V / E287A pure enzyme; 4: ROL-T326V / E287V pure enzyme.

[0025] Figure 2 Optimum temperature curve of Rhizopus oryzae lipase and its mutants.

[0026] Figure 3 Half-life of Rhizopus oryzae lipase and its mutants at 45℃.

[0027] Figure 4 Residual activity curve of Rhizopus oryzae lipase and its mutants under the action of different temperatures. DETAILED DESCRIPTION

[0028] The present application will be further described in conjunction with the examples and the accompanying drawings, but the embodiments of the present application are not limited thereto.

[0029] In the following examples, if the specific test conditions are not specified, the general test conditions or the test conditions recommended by the reagent companies are usually followed. The materials, reagents, etc. used are reagents and materials obtained from commercial channels if not specifically stated.

[0030] Example 1 Secretory expression and purification of Rhizopus oryzae lipase mutants

[0031] 1.1 Construction of mutant Pichia pastoris secretory expression strain

[0032] The insertion fragments of the mutants ROL-T326V, ROL-T326V / E287A and ROL-T326V / E287V and the wild type ROL were constructed on the basis of the ROL gene, the amino acid sequences and the corresponding nucleotide sequences of the encoding genes are shown in SEQ ID NO. 1-8, the insertion fragments of the mutants were synthesized by a gene company, the insertion fragments were ligated after the starting plasmid pPICZαA was digested with EcoRI and XbaI endonuclease to obtain plasmids pPICZαA-ROL, pPICZαA-ROL-T326V, pPICZαA-ROL-T326V / E287A, pPICZαA-ROL-T326V / E287V, the plasmids were linearized by using restriction endonuclease Sac I and transformed into Pichia pastoris X33, the transformation liquid was spread on YPD plates containing 100 μg / mL Zeocin, and cultured at 30℃ for 3 days, the single colonies grown were the expression strains.

[0033] The single colony of the selected Rhizopus oryzae lipase mutant was inoculated into 50 mL YPD liquid medium (250 mL) and cultured at 30°C and 240 r / min for 24 h. The fermentation liquid was inoculated into 50 mL BMGY medium (100 mM pH 6.0 potassium phosphate buffer, 1% yeast powder, 2% peptone, 0.34% amino acid-free yeast nitrogen source (YNB), 1% ammonium sulfate, 4 x 10 -5 -5 The bacterial body was resuspended in 300 mL BMMY liquid medium (100 mM pH 6.0 potassium phosphate buffer, 1% yeast powder, 2% peptone, 0.34% amino acid-free yeast nitrogen source (YNB), 1% ammonium sulfate, 4 x 10

[0034] 1.2 Purification of mutant protein

[0035] The prepared crude enzyme liquid was loaded onto the Ni column equilibrated with 3 volumes of PBS buffer. The purification system was equilibrated with PBS buffer, and then the impure proteins were eluted with PBS buffer containing 20 mM imidazole. The eluate buffer (300 mM imidazole and 500 mM NaCl, PBS buffer with pH 7.0) was used for elution, and the elution peak was collected, which was the purified enzyme. Figure 1 The SDS-PAGE gel map of wild-type Rhizopus oryzae lipase ROL and its mutants is shown in the experimental results, which shows that the target proteins of wild-type Rhizopus oryzae lipase ROL and three Rhizopus mutants were expressed and purified.

[0036] 1.3 Detection of mutant protein concentration

[0037] ​Mutant protein concentration was determined by using Brandford protein assay kit. A series of BSA concentration was prepared as protein concentration standard, and a standard curve was obtained. 10 μl of different concentration of standard protein was added into 96 well plate, and 200 μl of Brandford reagent was added. After 5 min of reaction at room temperature, the absorbance was measured at 595 nm. The standard curve of protein concentration was obtained according to the known protein concentration and the corresponding absorbance. The absorbance of mutant protein at 595 nm was measured under the same reaction condition, and the protein concentration of mutant protein was obtained by comparing with the standard curve. The results showed that the purity of all proteins was more than 75%.

[0038] Example 2 Optimum reaction temperature and thermal stability determination of Rhizopus oryzae lipase mutants

[0039] The method for determining lipase activity was as follows: the hydrolysis activity of lipase was determined by acid-base titration method. 4 wt% PVA aqueous solution and olive oil were mixed in a volume ratio of 3:1, and an olive oil emulsion was formed by homogenization under a high-pressure homogenizer, which was used as a substrate.

[0040] 4 g of prepared olive oil emulsion was added to 5 ml of PBS buffer (20 mM, pH 7), and 15 ml of industrial alcohol was added to the blank control. After preheating at 150 rpm and 40°C for 5 min, 1 mL of enzyme solution (ROL wild type and mutant proteins prepared in Example 1, 1.2, at a concentration of 200 μM) was added, and then the reaction was carried out for 10 min. After the reaction was completed, 15 ml of ethanol was immediately added to terminate the reaction. Titration was performed with 0.05 mol / L sodium hydroxide solution, and a few drops of phenolphthalein were added as an indicator until the liquid changed from milky white to reddish.

[0041] The enzyme activity was defined as the amount of enzyme required to hydrolyze olive oil to release 1 μmol of fatty acid per minute.

[0042] The lipase activity was determined in the corresponding buffer system at pH 7.0 using olive oil emulsion as a substrate at 25°C, 30°C, 35°C, 40°C, 45°C, 50°C and 55°C, respectively, with three parallel experimental groups. The highest enzyme activity at each temperature was taken as 100%, and the relative enzyme activity of the remaining experimental groups was calculated to obtain the temperature-relative enzyme activity curve, as shown in Figure 2 The results showed that the optimum reaction temperature of mutant T326V was the same as that of the wild type, which was still 35°C, while the optimum reaction temperature of T326V / E287A and T326V / E287V was increased to 45°C.

[0043] Example 3 Half-life determination of Rhizopus oryzae lipase mutants

[0044] The method for determining the half-life of lipase at 45°C was as follows:

[0045] The enzyme solution (ROL wild-type and mutant proteins prepared in 1.2 of Example 1, concentration 200 μM) was incubated at 45 °C. Samples were taken at different treatment times, and lipase activity was determined by acid-base titration according to Example 2. The residual activity percentage was calculated based on the enzyme activity of the untreated sample. A graph of the residual activity percentage (ln) against time (min) was plotted, and the slope of the straight line represents the inactivation constant K. inact , by t 1 / 2 =ln2 / K inact The half-life of the lipase at this temperature was obtained. 1 / 2 It is the time required for a protein to lose 50% of its activity, and it is a parameter characterizing the kinetic stability of a protein.

[0046] The results of the half-life determination are as follows Figure 3 As shown, after incubation at 45℃ for 30 min, the wild-type ROL retained 62.69% of its initial enzyme activity, while T326V, T326V / E287A, and T326V / E287V retained 78.89%, 84.92%, and 92.40%, respectively. This indicates that the thermostability of these three mutants was improved compared to the wild type. The half-life of the wild-type ROL was 38.51 min, while the half-lives of T326V, T326V / E287A, and T326V / E287V were 92.42, 126.03, and 315.07 min, respectively, which are 2.40, 3.27, and 8.18 times that of the wild type, demonstrating a significant improvement. This proves that the above mutations enhanced the kinetic stability of Rhizopus oryzae lipase.

[0047] Example 4: Determination of T15 50 in Rhizopus oryzae lipase mutant

[0048] When an enzyme is incubated at a certain temperature for 15 minutes, and its residual activity is half that of the untreated enzyme, this temperature is defined as the T15 50 value of the enzyme. The temperature was precisely controlled using a constant-temperature water bath shaker. The enzyme was incubated at different temperatures for 15 minutes. The activity of the untreated enzyme solution was defined as 100%, and the residual activity after incubation at each temperature was measured.

[0049] The method for determining the T15 50 of lipase is as follows:

[0050] The enzyme solution (ROL wild-type and mutant proteins prepared in 1.2 of Example 1, concentration 200 μM) was aliquoted into PCR tubes. The tubes were incubated at different temperatures for 15 min using a PCR instrument, followed by 5 min on ice and 10 min at room temperature. The residual enzyme activity in each tube was measured using the method described in Example 2. A graph was plotted between relative residual activity and temperature, with the activity of the untreated enzyme solution defined as 100%. The residual activity was plotted against temperature, and the graph was fitted using the sigmoidal Boltzmann fit function in Origin 8.0 software. The inflection point was T15 50°C.

[0051] The residual activity of wild-type and mutant enzymes was plotted against temperature, and the result was fitted using the sigmoidal Boltzmann fit function in Origin 8.0 software. The inflection point of the fit was taken as the T15 50 value of the enzyme. The results are shown below. Figure 4 As shown, the curves are obtained by fitting with sigmoidal Boltzmann fit. Below 60℃, both wild-type and mutant maintained good activity. Wild-type showed inactivation starting at 55℃, while the mutant T326V / E287V maintained nearly 100% residual activity below 55℃. The T15 50 values ​​for WT, T326V, T326V / E287A, and T326V / E287V were 61.94, 63.57, 64.94, and 66.44℃, respectively. The T15 50 values ​​for T326V, T326V / E287A, and T326V / E287V were increased by 1.63, 3.00, and 4.50℃ compared to the wild-type, respectively.

[0052] Example 5: Determination of kinetic parameters of Rhizopus oryzae lipase mutant

[0053] Enzymatic reaction kinetics parameter determination method: under the conditions of pH 7.0, temperature 30℃, different concentrations (50-3000 μM) of p-nitrophenyl caprylate (pNPC) as substrate, the hydrolysis activity of wild type and mutant was determined respectively. The total volume of enzyme activity determination reaction system was 1 mL: including 900 μL phosphate buffer solution (50 mM, pH 7.0), 50 μL pNPC (10 mM, acetonitrile solution) and 50 μL enzyme solution (ROL wild type and mutant protein prepared in example 1, 1.2, concentration was 200 μM). After mixing the reaction system, it was preheated at 30℃, 150 rpm for 5 min, then the substrate was added for reaction, 1 mL anhydrous ethanol was added to terminate the reaction after 10 min, and centrifuged at 12000 rpm, room temperature for 1 min. 200 μL supernatant was detected at 405 nm, and the enzyme activity was calculated according to the standard curve. Three groups of parallel experiments were set, one group was blank control. For the blank control, 50 μL PBS buffer (50 mM, pH 7.0) was used instead of cell suspension.

[0054] The enzyme activity of ROL was calculated according to the following formula:

[0055] Hydrolysis activity (U / mL) = n * c * v1 / t * v2

[0056] Wherein, n represents the dilution multiple of enzyme solution; c represents the concentration of pNP generated in the above enzyme activity determination system, μM; v1 represents the volume of the above enzyme activity determination system, L; t represents the reaction time, min; v2 represents the volume of enzyme solution added in the above enzyme activity determination system, mL.

[0057] Establishment of standard curve: different concentrations (0, 1, 2, 5, 10, 15, 20 μM, acetonitrile solution) of 4-nitrophenol (p-nitrophenyl, pNP) were prepared respectively. 950 μL phosphate buffer solution (50 mM, pH 7.0), 50 μL pNP (0, 1, 2, 5, 10, 15, 20 μM, acetonitrile solution) and 1 mL anhydrous ethanol were added in a 2 mL centrifuge tube, and the absorbance value was determined at 405 nm. Three groups of parallel experiments were set, and the average value of A 405 was taken as the ordinate, and the corresponding pNP concentration was taken as the abscissa to draw the standard curve.

[0058] Using the hydrolysis activity data of ROL on different concentrations of pNPC, the Michaelis-Menten nonlinear fitting (3-5) was carried out by GraphPad Prism software, so as to obtain K m and V max , and then according to the function equation of maximum rate and enzyme concentration V max = kcat X [E], the kinetic constant K m , k cat and k cat / K m .

[0059] In order to explore the difference in catalytic level between the wild type ROL and the mutants, the enzyme kinetic parameters of the purified wild type ROL and the three mutants were determined. Different concentrations of pNPC were used to react with the enzyme to determine the reaction rate, and the Michaelis-Menten nonlinear fitting was performed by using GraphPad Prism software to calculate the V m , K M , k cat and k cat / K M of the wild type ROL and the three mutants. The experimental results are shown in Table 1, and the k cat / K M of the mutants T326V and T326V / E287V is slightly lower than that of the wild type, indicating that after mutation, the stability of the protein is improved, but the catalytic efficiency of the enzyme is negatively affected to a certain extent. However, the k cat / K M of T326V / E287A is basically the same as that of the wild type, proving that the catalytic performance of the mutant is not affected.

[0060] Table 1 Kinetic parameters of ROL and its mutants

[0061]

[0062] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, which are all included in the protection scope of the present application.

Claims

1. A lipase mutant of Rhizopus oryzae, characterized in that: the lipase mutant of Rhizopus oryzae is ROL-T326V / E287A or ROL-T326V / E287V; the amino acid sequence of ROL-T326V / E287A is shown in SEQ ID NO.5; the amino acid sequence of ROL-T326V / E287V is shown in SEQ ID NO.

7. 2.A gene encoding the lipase mutant of Rhizopus oryzae according to claim 1, characterized in that: the nucleotide sequence is obtained according to the codon usage. 3.An expression vector, characterized in that: the expression vector comprises the gene according to claim 2. 4.An engineered bacterium, characterized in that: the genome comprises the expression vector according to claim 3. 5.The lipase mutant of Rhizopus oryzae according to claim 1, the expression vector according to claim 3, or the engineered bacterium according to claim 4 are used for decomposing oil and fat. 6.The lipase mutant of Rhizopus oryzae according to claim 1, the expression vector according to claim 3, or the engineered bacterium according to claim 4 are used for oil and fat processing or biodiesel production.

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