Marinobacter lipase mnl and its coding gene

Abstract

The application discloses a marine bacillus lipase MNL and application of a coding gene thereof, the marine bacillus lipase MNL can remove oil stains, and the amino acid sequence is shown as SEQ ID NO.2.The marine bacillus lipase MNL from marine microorganism is found to have good stability under low temperature and alkaline conditions, has good surfactant tolerance, has good oil stain removal effect, has better effect after being compounded with a commercial detergent, can be used in a detergent product, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology. Specifically, this invention relates to the application of a marine bacillus lipase MNL and its encoding gene. Background Technology

[0002] The detergent industry is a very important application area for lipases. The washing environment is typically low-temperature and alkaline, therefore, lipases used in detergents must possess good activity and stability under these conditions.

[0003] Typical detergent formulations contain large amounts of anionic and nonionic surfactants, and sometimes also include cationic surfactants, anti-redeposition agents, and bleaching enhancers. Therefore, lipases used in detergents must possess excellent surfactant stability and reactivity unaffected by these surfactants.

[0004] Marine ecosystems possess abundant enzyme resources, but their industrial applications have not yet been widely developed. The harshness of marine ecosystems has led to the evolution of low-temperature resistant enzymes. Some low-temperature resistant lipases (EC3.1.1.3) can catalyze the hydrolysis of triglycerides at 0–30 °C, producing products including fatty acids and glycerol. These low-temperature, alkaline lipases have the potential to clean oil stains, making them valuable for applications in the detergent industry.

[0005] Current laundry detergent and / or fabric care compositions include surfactants, enzymes (proteases, amylases, lipases and / or cellulases), bleaching agents, and builder systems, among which the commercial application of lipases in detergents is not yet widespread. Summary of the Invention

[0006] Based on this, the purpose of this invention is to provide the application of marine bacillus lipase MNL and its encoding gene.

[0007] The specific technical solutions for achieving the above-mentioned objectives are as follows.

[0008] In a first aspect, the present invention provides the use of marine bacillus lipase MNL in removing oil stains or preparing detergents, wherein the amino acid sequence of marine bacillus lipase MNL is shown in SEQ ID NO.2.

[0009] In a second aspect, the present invention provides the application of the encoding gene of marine bacillus lipase MNL in the removal of oil stains or the preparation of detergents, wherein the nucleotide sequence of the encoding gene of marine bacillus lipase MNL is shown in SEQ ID NO.1.

[0010] A third aspect of the present invention provides the application of a recombinant expression vector containing the encoding gene of marine bacillus lipase MNL in the removal of oil stains or the preparation of detergents, wherein the nucleotide sequence of the encoding gene of marine bacillus lipase MNL is shown in SEQ ID NO.1.

[0011] In a fourth aspect, the present invention provides the application of engineered bacteria in the removal of oil stains or the preparation of detergents, wherein the engineered bacteria are transformed with a recombinant expression vector, the recombinant expression vector being an expression vector containing the coding gene of Marinebacterium lipase MNL, the nucleotide sequence of the coding gene of Marinebacterium lipase MNL being shown in SEQ ID NO.1.

[0012] In a fifth aspect, the present invention provides a detergent comprising Marinebacterium lipase MNL, the amino acid sequence of which is shown in SEQ ID NO.2.

[0013] A sixth aspect of the present invention provides a method for removing oil stains, comprising the steps of: using marine bacillus lipase MNL in a detergent, wherein the amino acid sequence of the marine bacillus lipase MNL is shown in SEQ ID NO.2.

[0014] This invention discovers that marine microbial lipase MNL has good stability under low temperature and alkaline conditions, and good surfactant tolerance. It has a good effect on removing oil stains, and its effect is even better when compounded with commercially available detergents. It can be used in detergent products and has broad application prospects. Attached Figure Description

[0015] Figure 1 SDS-PAGE analysis of the purity of Marine Bacillus lipase MNL in Example 2 of this invention; wherein, Lane M, standard molecular weight marker; Lane 1, total amount of cell lysate; Lane 2, supernatant of cell lysate after centrifugation; Lane 3, Sephadex G-25 eluted sample; Lane 4, Ni 2+ Affinity chromatography elution of samples.

[0016] Figure 2 The results show the enzyme activity of marine bacillus lipase MNL at different temperatures in Example 3 of this invention.

[0017] Figure 3 The results show the enzyme activity of marine bacillus lipase MNL after incubation at different temperatures for different times in Example 3 of this invention.

[0018] Figure 4 The results of the activity of marine bacillus lipase MNL at different pH values ​​are shown in Example 4 of this invention.

[0019] Figure 5 The results show the enzyme activity of marine bacillus lipase MNL under different pH conditions for 0-4 days in Example 4 of this invention. Detailed Implementation

[0020] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0021] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0022] In this invention, the inventors unexpectedly discovered that the marine bacillus lipase MNL exhibits good stability under low temperature and alkaline conditions (in its hydrolysis reaction of stearic acid-4-nitrobenzene, it exhibits high enzyme activity at pH 8.0~10.0 and 10~30℃, with optimal enzyme activity at pH 8.0 and 20℃; simultaneously, this lipase demonstrates good stability under low temperature and alkaline conditions, retaining 59% residual enzyme activity after 24 hours of treatment at 10~30℃, and at pH... Under 8.0 conditions, it retains 60% of its residual enzyme activity even after 4 days of treatment. It also exhibits good surfactant tolerance (especially enhanced by ionic surfactants, and good stability under the action of other detergent additives such as anti-redeposition agents and bleaching enhancers). Some surfactants, such as MES and STPP, can activate the recombinant expressed lipase, increasing its activity to 231% and 126% of the original enzyme activity, respectively). It effectively removes oil stains, and its effect is even better when combined with commercially available detergents (the experiment was conducted using JB-04 edible oil-stained cloth as specified in the national standard, with commercially available detergents as a control; the detergency value was 9.18 when MNL and commercially available detergents were used in combination; acid-base titration was performed on the washed wastewater to measure the free fatty acid content produced by the hydrolysis of oil by the lipase; the fatty acid content produced by the hydrolysis of oil by MNL and commercially available detergents was 176.83 μM). Therefore, Marine Bacillus lipase MNL can be used in detergent products and has good application prospects.

[0023] In some embodiments of the present invention, the application of a marine bacillus lipase MNL in the removal of oil stains is disclosed, the amino acid sequence of which is shown in SEQ ID NO.2.

[0024] In other embodiments of the present invention, the application of the encoding gene of Marinebacterium lipase MNL in the removal of oil stains is disclosed, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0025] Nucleotide sequence of Marine Bacillus lipase MNL (SEQ ID NO.1):

[0026] ATGACTCCGGGCGGTGATAGCCTGGCGGCGTACGCGGAAGATGGTCCGTTCGCAACCACCAGCCAGAGCGGCGGCTTCAGCTGCACCATCTACCGTCCGATCAGCCTGCAAGATGATCACCCGGTGATCCTGTGGGGCAACGGCACCGGTGCGAGCCCGTCTACCTACGGCAGCGGCCTGC GCCACTGGGCGAGCTGGGGTTTCGTTGTTGCGGCGGCGAACACCAGCAACGCGGGCTCTGGTGAAGAAATGCTGGATTGCCTGGATTACTTGCAGGGCACCAGCTACGCGGACCAGCTCGATTTCAGCAACGTTGGCGCGAGCGGTCACAGCCAGGTGGCGGTGGCACCATCATGGCGGCG CGTGATAACCGTATCACCGCGACCGCGCCGGTTCAGCCGTACATCCTGGGCCTGGGTCACGAAACCTCTAGCCAGTACCAGCAGACCGCGCCGATGCTGCTTCTGAGCGGTTCCGTTGATACCCTGGCGGGCCCGACCCTGAACCAGGCTCCGGTTTACCGTCGCGTTGATGTTCCGGTTT TCTGGGCGACCCTGCGTGGTGCAAGCCACTTTGAACCGGTTGGTAATATGGGTGATTTCCGTGGTATTACTACTGCGTGGTGGGTTGTACCAACTGACCGGTGACGCTGATGCTGCTGACCTGTTTACTGGTCCGTGCGAAGTGTGTGGTTTATCTGATTGGGATGTTGAACGTAAAGGTCTG

[0027] The amino acid sequence of marine bacillus lipase MNL (SEQ ID NO.2):

[0028] MTPGGDSLAAYAEDGPFATTSQSGGFSCTIYRPISLQDDHPVILWGNGTGASPSTYGSGLRHWASWGFVVAAANTSNAGSGEEMLDCLDYLQGTSYADQLDFSNVGASGHSQGGGGTIMAA RDNRITATAPVQPYILGLGHETSSQYQQTAPMLLLSGSVDTLAGPTLNQAPVYRRVDVPVFWATLRGASHFEPVGNMGDFRGITTAWWLYQLTGDADAADLFTGPCEVCGLSDWDVERKGL

[0029] In other embodiments of the present invention, the application of a marine bacillus lipase MNL in the preparation of detergents is disclosed, wherein the amino acid sequence of the marine bacillus lipase MNL is shown in SEQ ID NO.2.

[0030] In some embodiments, the washing product is a detergent.

[0031] In other embodiments of the present invention, the application of the encoding gene of Marinebacterium lipase MNL in the preparation of detergents is disclosed, the nucleotide sequence of the encoding gene of Marinebacterium lipase MNL is shown in SEQ ID NO.1.

[0032] In some embodiments, the washing product is a detergent.

[0033] In other embodiments of the present invention, the application of a recombinant expression vector containing the coding gene of Marinebacterium lipase MNL is disclosed in the removal of oil stains, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0034] In other embodiments of the present invention, the application of an engineered bacterium in the removal of oil stains is disclosed. The engineered bacterium is transformed with a recombinant expression vector, which is an expression vector containing the coding gene of Marinebacterium lipase MNL. The nucleotide sequence of the coding gene of Marinebacterium lipase MNL is shown in SEQ ID NO.1.

[0035] In other embodiments of the present invention, a method for removing oil stains is disclosed, comprising using the aforementioned Marine Bacillus lipase MNL in a detergent.

[0036] In other embodiments of the present invention, a detergent is disclosed comprising the aforementioned Marine Bacillus lipase MNL.

[0037] In the following embodiments, all raw materials not specified are commercially available.

[0038] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] Example 1: Construction of an expression vector for marine bacillus lipase MNL in Escherichia coli

[0040] Based on the *Marinobacter nanhaiticus* lipase MNL gene (GenBank accession number: WP_051079885.1) from GenBank, and after sequence optimization according to *E. coli* codon bias, the gene was synthesized by Shanghai Sangon Biotech Co., Ltd. The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The pET-28a-MNL plasmid was synthesized by Shanghai Sangon Biotech Co., Ltd.

[0041] The above plasmid was transformed into E. coli Top 10 competent cells, plated on LB plates (containing 100 ug / ml Kanamycin), and positive clones were selected for gene sequencing. The results showed that the pET-28a-MNL expression plasmid was obtained.

[0042] Example 2 Recombinant expression and purification of marine bacillus lipase MNL

[0043] The pET-28a-MNL expression plasmid constructed in Example 1 was heat-shocked and transformed into *E. coli* Shuffle T7 competent cells (purchased from Shanghai Vidi Biotechnology Co., Ltd.). The transformation solution was plated on LB agar (100 μg / ml Kanamycin) and incubated at 37°C for 10 hours. Single colonies from the agar were picked and incubated in 5 ml of LB medium at 37°C for 10 hours. Then, 5% of the colonies were inoculated into 100 ml of LB medium and fermented at 37°C for 3 hours. Finally, 10% of the colonies were inoculated into 500 ml of LB medium and incubated at 37°C for 4 hours. The temperature was then lowered to 18°C, 0.1 mM IPTG was added, and the cells were centrifuged after 18 hours of incubation to obtain the bacterial cells.

[0044] The bacterial cells were resuspended in 50 mM Tris-HCl (pH=8) buffer, and the lysate was obtained by sonication. The supernatant was obtained by centrifugation and purified by nickel affinity chromatography (elution buffer was Tris-HCl buffer containing 300 mM imidazole, pH 8.0) and G-25 desalting column to obtain the target protein. The purity of the purified recombinant protein was identified by SDS-PAGE electrophoresis.

[0045] The results are as follows Figure 1 As shown, from Figure 1As can be seen, a single electrophoretic band with a molecular weight of approximately 30 kD was obtained after nickel column affinity chromatography, indicating that purified marine bacillus lipase MNL was obtained.

[0046] Example 3: Optimal temperature and temperature stability test of marine bacillus lipase MNL

[0047] 1. Optimal reaction temperature

[0048] The enzyme activity of marine bacillus lipase MNL under different temperature conditions (5 ℃~70 ℃) was determined by colorimetry.

[0049] The reaction system consisted of: 50 mM Tris-HCl buffer (pH 8.0), 1 mM p-nitrophenol decanoate (p-NPd), and 10 μL enzyme solution.

[0050] The reaction was carried out at each temperature for 5 min, and the absorbance (OD) at 405 nm was measured. 405 The enzyme activity unit is U / mg. All experiments were repeated three times.

[0051] The results are as follows Figure 2 As shown, Marine Bacillus lipase MNL has high enzyme activity in the range of 10℃ to 30℃, with the highest enzyme activity at 20℃, indicating that the optimal reaction temperature for Marine Bacillus lipase MNL is 20℃.

[0052] 2. Temperature stability

[0053] First, *Marine Bacillus* lipase MNL was incubated at different temperatures (20℃ and 30℃), and samples were taken at 0 min, 60 min, 120 min, 240 min, 480 min, 960 min, and 1440 min, respectively. The remaining enzyme activity was determined by colorimetry. The effect of temperature on the stability of *Marine Bacillus* lipase MNL was expressed as relative enzyme activity, with the maximum measured enzyme activity defined as 100%. All experiments were repeated three times.

[0054] The results are as follows Figure 3 As shown in the figure. The results indicate that the marine bacillus lipase MNL can retain more than 60% of its lipase activity after treatment at 20 ℃~30 ℃ for 24 h.

[0055] The results of this embodiment show that marine bacillus lipase MNL has good stability at low temperatures.

[0056] Example 4 Optimal pH and pH stability test of marine bacillus lipase MNL

[0057] 1. Optimal reaction pH

[0058] The enzyme activity of marine bacillus lipase MNL under different pH conditions (5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0) was determined by colorimetry.

[0059] The reaction system is referenced in Example 3, wherein the various pH solutions used are: 50 mM citrate-phosphate buffer solution (pH 5.0), 50 mM Na2HPO4-NaH2PO4 solution (pH 6.0, pH 7.0), 50 mM Tris-HCl solution (pH 8.0, pH 9.0), and 50 mM glycine-sodium hydroxide buffer solution (pH 10.0, pH 11.0).

[0060] The reaction was carried out at each temperature for 5 min, and the absorbance (OD) at 405 nm was measured. 405 Enzyme activity was measured in U / mg. All experiments were repeated three times. The maximum enzyme activity was defined as 100%.

[0061] The results are as follows Figure 4 As shown, Marine Bacillus lipase MNL has high enzyme activity in the pH range of 8 to 10, with the highest enzyme activity at pH 8, indicating that the optimal reaction pH for Marine Bacillus lipase MNL is 8.

[0062] 2. pH stability

[0063] Marine bacteria lipase MNL was incubated at different pH values ​​(6.0, 7.0, 8.0, 9.0, 10) for 0–4 days, and its remaining enzyme activity was determined colorimetrically. The reaction system and enzyme activity determination method were the same as above. The effect of pH on the stability of Marine bacteria lipase MNL was expressed as relative enzyme activity, and the maximum enzyme activity was defined as 100%. All experiments were repeated three times.

[0064] The results are as follows Figure 5 As shown in the figure. The results indicate that the marine bacillus lipase MNL can maintain more than 50% of its lipase activity after treatment at pH 8-10 for 4 days, and can also maintain close to 30% of its enzyme activity after treatment at pH 7 for 4 days.

[0065] The results of this embodiment show that marine bacillus lipase MNL has good stability under alkaline conditions.

[0066] Example 5 Effect of surfactant on the activity of marine bacillus lipase MNL

[0067] Marine bacteria lipase MNL was incubated for 1 h at 20°C and pH 8 in different concentrations of different surfactants (including ionic and nonionic surfactants) as shown in Table 1. The residual activity of marine bacteria lipase MNL on C10 substrate hydrolysis was measured. The reaction system and enzyme activity assay method were the same as in Example 3.

[0068] The effects of different surfactants on the activity of Marine Bacterium lipase MNL were expressed as relative enzyme activity, with the enzyme activity measured in the experimental group without any surfactant being taken as 100%. All experiments were repeated three times. The results are shown in Table 1.

[0069] Table 1

[0070]

[0071] Table 1 shows that Marine Bacillus lipase MNL has good tolerance to ionic surfactants, and its enzyme activity is enhanced when exposed to ionic surfactants. It also has good stability under the action of other detergent additives such as anti-redeposition agents and bleaching enhancers. Some surfactants also have an activating effect on Marine Bacillus lipase MNL, such as MES and STPP, which can increase the enzyme activity to 231% and 126% of the original enzyme activity at 1 mM and 10 mM, respectively.

[0072] Example 6: Evaluation of the detergency effect of Marine Bacillus lipase MNL on JB-04 edible oil-stained cloth.

[0073] Includes the following steps:

[0074] (1) Measure the whiteness value F1 of the soiled cloth test piece (JB-04) under a calibrated whiteness meter (JJG 512) to obtain the whiteness value F1 before washing;

[0075] (2) Add 500ml of tap water, 500ml of tap water + commercial detergent (laundry detergent pods, Ou Ni Ai brand) (0.4%), 500ml of tap water + marine bacillus lipase MNL (4mg), 500ml of tap water + commercial detergent (0.2%) + marine bacillus lipase MNL (2mg) and 500ml of tap water + commercial detergent (0.2%) + Novozymes lipase Lipoclean (2mg) to five beakers respectively. Completely immerse the test piece from (1) in the mixture, control the temperature at 30℃±2℃, start the stirrer and stir at 150r / min for 30 minutes.

[0076] (3) Place the sample piece processed in (2) into an oven to dry at a temperature of 37°C;

[0077] (4) Measure the whiteness of the sample treated in (3) using a whiteness meter to obtain the whiteness value F2 after measurement, and calculate the stain removal value.

[0078] (5) Perform acid-base titration on the remaining waste liquid from washing in (2). Add 2 ml of 0.1% phenolphthalein solution to the waste liquid, and then add 0.05 M sodium hydroxide solution dropwise until the solution turns light red. Record the volume of sodium hydroxide added and calculate the content of free fatty acids produced.

[0079] (6) Calculate the staining value according to the staining value calculation method mentioned in GB / T 13174-2021; the amount of sodium hydroxide consumed by acid-base titration can be regarded as the content of free fatty acids produced during washing.

[0080] The results are shown in Table 2.

[0081] Table 2

[0082]

[0083] This embodiment uses JB-04 edible oil-stained cloth as specified in the national standard for experiments, and uses commercially available detergent and Novozymes lipase Lipoclean as controls. Table 2 shows that the cleaning value of the blank group using only tap water was 4.275, while the cleaning value of Marine Bacillus lipase MNL alone was 7.93, indicating its good oil-removing ability. The cleaning value of commercially available detergent alone was 8.45, while the cleaning value of Marine Bacillus lipase MNL combined with commercially available detergent was 9.18, a significant improvement, proving that the combined use of the two is more effective at removing oil stains than the combination of Novozymes lipase Lipoclean and commercially available detergent (8.98).

[0084] The washed wastewater was subjected to acid-base titration to determine the content of free fatty acids produced by lipase hydrolysis of oils. The fatty acid content of the blank group using only tap water was 28 μM, the fatty acid content of the group using only commercial detergent was 152 μM, the fatty acid content of the group using only Marine Bacillus lipase MNL was 99.17 μM, and the fatty acid content of the group using Marine Bacillus lipase MNL in combination with commercial detergent increased to 176.83 μM, which is slightly lower than the fatty acid content of the group using Novozymes lipase Lipoclean in combination with commercial detergent (204.83 μM).

[0085] The results of this embodiment show that Marine Bacillus lipase MNL has a good ability to remove oil stains. Adding Marine Bacillus lipase MNL to commercial detergents further enhances their ability to remove oil stains, and the effect is comparable to adding Novozymes' commercial lipase Lipoclean to commercial detergents.

[0086] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0087] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of a marine bacillus lipase MNL in oil removal, characterized in that, The amino acid sequence of the marine bacillus lipase MNL is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the marine bacillus lipase MNL is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The marine bacillus lipase MNL is expressed by a recombinant expression vector containing the coding gene of marine bacillus lipase MNL, the nucleotide sequence of which is shown in SEQ ID NO.

1.

4. The application of a marine bacillus lipase MNL in the preparation of detergents, characterized in that, The amino acid sequence of the marine bacillus lipase MNL is shown in SEQ ID NO.

2.

5. The application according to claim 4, characterized in that, The nucleotide sequence of the gene encoding the marine bacillus lipase MNL is shown in SEQ ID NO.

1.

6. The application according to claim 4, characterized in that, The marine bacillus lipase MNL is expressed by a recombinant expression vector containing the coding gene of marine bacillus lipase MNL, the nucleotide sequence of which is shown in SEQ ID NO.

1.

7. The application according to any one of claims 4 to 6, characterized in that, The cleaning products mentioned are detergents.

8. The application of engineered bacteria in the removal of oil stains, characterized in that, The engineered bacteria were transformed with a recombinant expression vector, which is an expression vector containing the coding gene for Marinebacterium lipase MNL, and the nucleotide sequence of the coding gene for Marinebacterium lipase MNL is shown in SEQ ID NO.

1.

9. A method for removing oil stains, characterized in that, The method includes the following steps: using Marinebacterium lipase MNL in a detergent, the amino acid sequence of which is shown in SEQ ID NO.2.

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