Lysinibacillus sphaericus for producing lipase and application thereof
By using Lysinibacillus capsiciYin-202501 for fermentation to produce lipase, combined with specific culture media and purification methods, the problem of insufficient bacterial lipase activity was solved, achieving the preparation of lipase with high activity and strong stability, suitable for various industrial applications.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-05
- Publication Date
- 2026-04-14
AI Technical Summary
Existing bacterial lipases have low enzyme activity, insufficient thermal stability, and inadequate pH tolerance, which limits their industrial application.
Lipase was produced by fermentation using Lysinibacillus capsiciYin-202501. High-activity lipase solution was prepared by purifying the lipase using LB liquid medium and olive oil or olive oil emulsion as an inducer, combined with precipitation of metal ions and ammonium sulfate.
A high-activity lipase solution was obtained, with an enzyme activity of up to 3 U/mL. It has excellent thermal stability and pH adaptability, is suitable for a variety of organic solvents, and is suitable for industrial applications.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial enzymology, and in particular to a lysine-producing Bacillus and its applications. Background Technology
[0002] Lipases (EC 3.1.1.3) are key enzymes that catalyze the hydrolysis and synthesis of triglycerides, and are widely used in food processing, detergents, biodiesel production, pharmaceuticals, and waste treatment. With the development of green biomanufacturing technologies, the demand for novel, efficient, stable, and adaptable lipases is increasing. However, the industrial application of natural lipases still faces many challenges, including low enzyme activity, poor thermal stability, insufficient substrate specificity, and high production costs. Therefore, exploring novel lipase-producing microbial resources and optimizing their expression systems has become a current research hotspot in the field of biocatalysis.
[0003] Currently, the main strains for industrial lipase production include fungi (such as Aspergillus niger and Aspergillus oryzae) and bacteria (such as Bacillus subtilis and Pseudomonas). Although these strains have been commercially applied, their inherent limitations restrict further development: fungal lipases have long culture cycles, are susceptible to contamination, and have high downstream separation and purification costs; bacterial lipases have limited enzyme production levels (<50 U / mL) and insufficient thermostability or pH tolerance. Lysine-containing Bacillus (… Lysinibacillus (Bacillus) is a group of Gram-positive, thermostable, spore-forming bacteria widely distributed in soil, compost, and extreme environments. Previous studies have shown that some strains of this genus have the potential to secrete industrial enzymes such as proteases and amylases, but the lipase production capacity of the same strains has not been systematically studied.
[0004] Bacteria remain the primary source of lipase production. Screening for high-lipase-producing strains and optimizing enzyme production conditions play a crucial role in improving lipase production efficiency and promoting large-scale lipase production, thus meeting the growing demand for high-activity lipases in technological processes. Summary of the Invention
[0005] The purpose of this invention is to address the problem of low enzyme activity in the current production of lipase using bacterial fermentation, and to provide a high-lipase-producing bacterium—Bacillus lysine-producing, and a method for preparing high-activity lipase using it.
[0006] This invention proposes a lysine-containing Bacillus, specifically a lysine-containing Bacillus. Lysinibacillus capsici Yin-202501, with accession number CCTCC No. M2025574.
[0007] This invention proposes the application of any of the above-mentioned lysine-containing Bacillus species in the production of lipase.
[0008] Furthermore, the method for preparing lipase using Bacillus lysine includes the following steps:
[0009] The lysine-containing Bacillus strain was inoculated into a liquid culture medium for activation. The activated strain was then inoculated into a fermentation culture medium for fermentation. After centrifugation, the supernatant obtained from the fermentation broth was the crude lipase solution.
[0010] Furthermore, the liquid culture medium is LB liquid culture medium, wherein the LB liquid culture medium is composed of the following components: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride.
[0011] Furthermore, the fermentation medium includes LB liquid medium.
[0012] Furthermore, the fermentation medium also includes at least one of an inducer or a metal ion.
[0013] Furthermore, the volume fraction of the inducer in the fermentation medium is 1%-5%;
[0014] The inducing agent includes at least one of olive oil or olive oil emulsion.
[0015] Furthermore, the concentration of metal ions in the fermentation medium is 1-5 g / L;
[0016] The metal ions include Cu 2+ Fe 2+ Ca 2+ NH4 2+ or Mg 2+ At least one of them.
[0017] Furthermore, the fermentation temperature is 30-45℃;
[0018] The fermentation time is 0.5-3 days;
[0019] After activation, the strain was inoculated into the fermentation medium at a volume ratio of 1%-5%.
[0020] Furthermore, it also includes adjusting the saturation of the obtained supernatant with ammonium sulfate, collecting the protein precipitate by centrifugation, and reconstituted to obtain the purified enzyme solution; wherein ammonium sulfate is added to a saturation of 10-100%.
[0021] This invention has the following advantages:
[0022] The *Bacillus lysinicus* Yin-202501 proposed in this invention possesses a high lipase-producing capacity. The method for preparing high-activity lipase solution using *Bacillus lysinicus* Yin-202501 is simple to operate, has mild culture conditions, good reproducibility, and yields high extracellular lipase activity, with the highest lipase activity reaching 3 U / mL, significantly higher than that of other common bacterial lipases. The obtained lipase exhibits extremely strong thermal stability and adaptability, as well as strong pH adaptability, and can be well used in a variety of organic solvents, demonstrating good application value and providing a foundation for the industrial application of lipases.
[0023] The Lysine Bacillus proposed in this invention is named Lysinibacillus capsici Yin-202501. This bacterium was deposited at the China Center for Type Culture Collection (CCTCC) on March 24, 2025, with accession number CCTCC No. M2025574, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Attached Figure Description
[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0025] Figure 1 The precipitation zone phenomenon is observed in 14 lipase-producing strains screened using lipase screening medium in Example 1 of this invention.
[0026] Figure 2 The standard curve for p-nitrophenol provided in Example 1 of this invention.
[0027] Figure 3 Provided for Embodiment 2 of the present invention Lysinibacillus capsici Phylogenetic tree of Yin-202501.
[0028] Figure 4 Provided for Embodiment 3 of the present invention Lysinibacillus capsici Determination of the optimal growth temperature for Yin-202501.
[0029] Figure 5 Provided for Embodiment 3 of the present invention Lysinibacillus capsici Growth curve of Yin-202501.
[0030] Figure 6 Provided for Embodiment 4 of the present invention Lysinibacillus capsici Enzyme production curve of Yin-202501.
[0031] Figure 7 The inducing agent provided in Example 5 of the present invention Lysinibacillus capsici Effects of Yin-20250 on enzyme production capacity.
[0032] Figure 8 Examples of different metals provided in Embodiment 6 of the present invention Lysinibacillus capsici Effects of Yin-20250 on enzyme production capacity.
[0033] Figure 9 The effect of ammonium sulfate with different saturations on lipase precipitation is shown in Example 7 of this invention.
[0034] Figure 10 This is a diagram of the purified enzyme solution product provided in Example 7 of the present invention.
[0035] Figure 11 The effect of temperature on enzyme activity is provided in Example 8 of the present invention.
[0036] Figure 12 The effect of temperature on the thermal stability of enzymes is provided in Example 8 of the present invention.
[0037] Figure 13 The effect of pH on enzyme stability is provided in Example 9 of the present invention.
[0038] Figure 14 The effect of different organic solvents on enzyme stability provided in Example 10 of the present invention.
[0039] Figure 15 The lyophilized lipase powder provided in Example 12 of this invention.
[0040] Figure 16 The effect of metal ions on lipase activity provided in Example 11 of the present invention. Detailed Implementation
[0041] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other.
[0042] On one hand, embodiments of the present invention provide a lysine-containing Bacillus, specifically a... Lysinibacillus capsici Yin-202501, with accession number CCTCC No. M2025574.
[0043] The Lysinibacillus proposed in this embodiment of the invention is named Lysinibacillus capsiciYin-202501 was deposited on March 24, 2025, at the China Center for Type Culture Collection (CCTCC), with accession number M2025574, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. This *Bacillus lysine* can efficiently produce extracellular lipase, enabling the application of *Bacillus lysine* in the preparation of high-activity lipase solutions.
[0044] In one embodiment of the present invention, the Lysinibacillus capsici The 16S rRNA sequence of Yin-202501 is shown in the sequence listing SEQ ID NO:1.
[0045] On the other hand, one embodiment of the present invention also proposes the application of any of the above-mentioned lysine-containing Bacillus in the production of lipase.
[0046] In one embodiment of the present invention, a method for preparing lipase using Bacillus lysine includes the following steps:
[0047] The lysine-containing Bacillus strain was inoculated into a liquid culture medium for activation. The activated strain was then inoculated into a fermentation culture medium for fermentation. After centrifugation, the supernatant obtained from the fermentation broth was the crude lipase solution.
[0048] In one embodiment of the present invention, the fermentation temperature is 30-45℃. Specifically, it can be 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, etc. Preferably, the fermentation temperature is 37℃.
[0049] In one embodiment of the present invention, the fermentation time is 0.5-3 days. Specifically, the fermentation time can be 0.5 days, 1.0 days, 1.5 days, 2.0 days, 2.5 days, 3.0 days, etc. Preferably, the fermentation time is 2 days.
[0050] In one embodiment of the present invention, fermentation is carried out at 200 rpm.
[0051] In one embodiment of the present invention, the activated strain is inoculated into the fermentation medium at an inoculation amount of 1%-5% by volume. Specifically, the activated strain is inoculated into the fermentation medium at an inoculation amount of 1%, 2%, 3%, 4%, or 5% by volume.
[0052] In one embodiment of the present invention, the liquid culture medium is LB (Luria Bertani) liquid culture medium. The LB liquid culture medium is composed of the following components: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride.
[0053] In one embodiment of the present invention, the fermentation medium comprises the above-described LB liquid medium. Preferably, the fermentation medium further comprises at least one of an inducer and metal ions.
[0054] More preferably, the inducer has a volume fraction of 1%-5% in the fermentation medium. Preferably, the inducer has a volume fraction of 1% in the liquid medium. Specifically, the inducer includes at least one of olive oil and olive oil emulsion.
[0055] More preferably, the concentration of metal ions in the fermentation medium is 1-5 g / L. Preferably, the concentration of metal ions in the fermentation medium is 1 g / L. Specifically, the metal ions include Cu. 2+ Fe 2+ Ca 2+ NH4 2+ or Mg 2+ At least one of them.
[0056] In a preferred embodiment of the present invention, the fermentation medium comprises the following components: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 10-50 ml / L olive oil emulsion, and 1-5 g / L ferrous sulfate. The olive oil emulsion has a mass concentration of 20%.
[0057] In one embodiment of the present invention, the centrifugation conditions are 8000-10000 r / min for 5-10 min. Centrifugation removes insoluble precipitates.
[0058] In one embodiment of the present invention, the method further includes adjusting the saturation of the obtained supernatant with ammonium sulfate, collecting the protein precipitate by centrifugation, and reconstituted to obtain a purified enzyme solution.
[0059] In a preferred embodiment of the present invention, ammonium sulfate is added until the saturation is 10-100%.
[0060] In one embodiment of the present invention, the purified enzyme solution can be adapted to a temperature range of 1~100℃. Specifically, it can be 4℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 100℃, etc. Preferably, the purified enzyme solution exhibits the best enzyme activity stability at 30℃.
[0061] In one embodiment of the present invention, the purified enzyme solution can be adapted to a pH range of 3-9; preferably, the purified enzyme solution can be adapted to a pH of 7.
[0062] In one embodiment of the present invention, the organic solvent suitable for the purified enzyme solution includes methanol, ethanol, isopropanol, acetone, acetonitrile, etc.
[0063] The present invention will now be described in detail with reference to the accompanying drawings.
[0064] Example 1: Screening of lipase-producing strains and determination of their enzyme activity
[0065] Soil samples were collected in Yichun, Jiangxi Province. 4 g of soil sample was weighed and immediately placed in 36 mL of sterile water containing glass beads. The mixture was shaken at 160 rpm for 30 min to prepare a soil leachate with a concentration of 10%. -1 Dilute it to 10. -3 10 -4 10 -5 Inoculate 150 μL onto beef extract peptone medium (3 plates each), spread evenly using a spreader, and incubate at 30°C for 3 days. Pick a single colony and transfer it to beef extract peptone liquid medium, incubate at 30°C for 3 days. Inoculate 150 μL onto LB solid medium, spread evenly using a spreader, and incubate at 30°C for 3 days. Pick a single colony and transfer it to LB liquid medium, incubate for 3 days. Repeat the above steps, inoculating the culture into slant tubes prepared with LB medium and storing the culture at 4°C.
[0066] The initially screened strains were activated onto LB solid medium and then added to lipase screening and selection medium (LB solid medium + Tween 80 + CaCl2). The medium was incubated at 30 °C. Based on the fact that lipase can decompose Tween 80 and that the product can react with CaCl2, the lipase was selected. 2+ Ion binding formed a white precipitate ring, and 14 lipase-producing strains were preliminarily screened (see...). Figure 1 ).
[0067] The enzyme-producing strains obtained from the initial screening were inoculated onto LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) for activation. After activation, the inoculum was added to fermentation medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) at a volume ratio of 1%. The temperature was set at 30℃, the rotation speed was set at 160 r / min, and the culture was carried out on a shaker for 32 h. A certain amount of fermentation broth was taken and centrifuged at 8000 r / min for 2 min. The supernatant was then used for enzyme activity determination.
[0068] Lipase activity was determined using the p-nitrophenol method, with p-nitrophenol palmitate (PMP) as the ester. p Using lipase as a substrate, lipase hydrolyzes the substrate to produce colored p-nitrophenol (-NPP). p Lipase activity can be determined by measuring its absorbance at a wavelength of 405 nm (-NP, molecular weight 139.11). The specific procedure is as follows:
[0069] (1) Construction of the standard curve of lipase activity
[0070] Weigh out 0.1391 g p-NP was dissolved in 50 mL of Tris-HCl buffer (0.05 mol / L, pH 8.0) to prepare a 20 mM stock solution. 10 mL of this stock solution was then diluted to 100 mL with Tris-HCl buffer to obtain the 2 mM working solution. The dosages of various reagents are shown in the table below. The reaction conditions for preparing the standard curve were consistent with those used to determine the enzyme activity of the samples in the experiment. The absorbance of each concentration was measured at 405 nm. Based on the results, a standard curve for p-nitrophenol was plotted (see...). Figure 2 ), and calculate the absorbance value and p -Formulas related to NP content:
[0071] Y = ax + b (Y: p -NP concentration; x: absorbance value; a, b: reaction coefficients);
[0072] Table 1 p Determination of the standard curve using the NPP method
[0073]
[0074] (2) The enzyme activity of the fermentation broth was determined using... p -NPP as substrate method: 2.1 mL Tris-HCl buffer (0.05 mol / L, pH 8.0), 200 μL p -NPP (7.5 mmol / L, methanol) and 100 μL of appropriately diluted enzyme sample were added to 5 mL EP tubes. The reaction mixture was stirred in a 37 °C water bath for 10 min. After the reaction was completed, the tubes were placed in an ice-water bath, and 100 μL of ZnSO4 (0.1 mol / L) was added to terminate the reaction. The mixture was filtered through a 0.45 μm aqueous filter membrane and then measured at 405 nm.
[0075] according to p -NP standard curve, from which the formula for calculating lipase activity is derived:
[0076] The enzyme activity of the sample (U / μmol) = (Ab) / a;
[0077] In the formula, A is the absorbance of the sample at 405 nm, and a and b are... p - The coefficients in the NP standard curve formula.
[0078] The enzyme activities of the 14 strains are as follows:
[0079] Table 2 Enzyme activities of 14 enzyme-producing strains
[0080]
[0081] Example 2 Identification of lipase-producing strains
[0082] Based on enzyme activity, strain No. 3, producing high-activity lipase, was selected from 14 lipase-producing strains and sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rRNA sequence determination. Further identification of the bacterial strain was performed using next-generation sequencing. BLAST analysis of the gene sequence against the NCBI database was conducted to construct a phylogenetic tree. Figure 3 The results showed that strain Yin-202501 shared 99.05% sequence homology with Lysinibacillus_capsici_PB300 GCA_003367505.1, exceeding 95%, thus identifying it as a species. Second-generation sequencing confirmed that the bacterium was a Lysinibacillus capsici, and it was named... Lysinibacillus capsici Yin-202501. This bacterium was deposited at the China Center for Type Culture Collection (CCTCC) on March 24, 2025, with accession number CCTCC No. M2025574, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.
[0083] Lysinibacillus capsici The 16S rRNA sequence of strain Yin-202501 is shown in SEQ ID NO:1 in the sequence listing, as follows:
[0084] tccggaatta ttgggcgtaa agcgcgcgca ggcggtcctt taagtctgat gtgaaagccc
[0085] acggctcaac cgtggagggt cattggaaac tgggggactt gagtgcagaa gaggaaagtg
[0086] gaattccaag tgtagcggtg aaatgcgtag agatttggag gaacaccagt ggcgaaggcg
[0087] actttctggt ctgtaactga cgctgaggcg cgaaagcgtg gggagcaaac aggattagat
[0088] accctggtag tccacgccgt aaacgatgag tgctaagtgt tagggggttt ccgcccctta
[0089] gtgctgcagc taacgcatta agcactccgc ctggggagta cggtcgcaag actgaaactc
[0090] aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc
[0091] gaagaacctt accaggtctt gacatcccgt tgaccactgt agagatatag tttccccttc
[0092] gggggcaacg gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt
[0093] taagtcccgc aacgagcgca acccttgatc ttagttgcca tcatttagtt gggcactcta
[0094] aggtgactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca tcatgcccct
[0095] Example 3 Lysinibacillus capsici Optimal growth temperature and growth curve of Yin-202501
[0096] 1. Determination of the optimal growth temperature for lipase-producing strains
[0097] The activated strain was inoculated into liquid culture medium and cultured in a shaker at different temperatures (30℃, 37℃, and 45℃) and a rotation speed of 200 r / min for 24 h. The OD value at 420 nm was measured to determine the optimal growth temperature of the strain. Figure 4 ).
[0098] Temperature Lysinibacillus capsici The growth effects of Yin-202501 are as follows: Figure 4 As shown, the optimal temperature is 37℃, and it exhibits high activity in the range of 30-45℃.
[0099] 2. Determination of growth curves of lipase-producing strains
[0100] The activated strain was inoculated into liquid culture medium, the temperature was set to the optimum temperature of 37℃, the rotation speed was set to 200 r / min, and the culture was carried out on a shaker for 48 h. Samples were taken every 4 h in a clean bench to measure the OD value at 600 nm and a growth curve was plotted. Figure 5 ).
[0101] Example 4 Determination of enzyme production curve
[0102] The strain was activated by inoculating it into LB liquid medium at a 1% (v / v) inoculation rate into the fermentation medium. The temperature was set at 37℃, the rotation speed at 200 r / min, and the culture was carried out on a shaker for 48 h. Every 4 h, the fermentation broth was collected in a clean bench to measure enzyme activity and plot the enzyme production curve. Figure 6 ).
[0103] Example 5: Inducer on Lysinibacillus capsici Effect of Yin-202501 on enzyme production capacity
[0104] enzyme-producing strains Lysinibacillus capsici Yin-202501 was inoculated onto LB liquid medium for activation;
[0105] The activated strain was inoculated into LB liquid medium, with a blank control group without the addition of an inducer. An inducer with a volume fraction of 1% was added, namely olive oil and olive oil emulsion (mass concentration of 20%). The temperature was set at 37℃, the rotation speed was set at 200 r / min, and the culture was carried out on a shaker for 48 h. A certain amount of fermentation broth was taken and centrifuged at 8000 r / min for 2 min at 4℃. The supernatant was used for enzyme activity determination.
[0106] Inducer Lysinibacillus capsici The results of the effect of Yin-202501 on enzyme production capacity are as follows: Figure 7 As shown, olive oil helps lipase-producing strains produce lipase, and homogenizing the oil into an emulsion can better promote its enzyme activity.
[0107] Example 6 Metal ion pairs Lysinibacillus capsici Effect of Yin-202501 on enzyme production capacity
[0108] enzyme-producing strains Lysinibacillus capsici Yin-202501 was inoculated onto LB liquid medium for activation;
[0109] The activated strain was inoculated into LB liquid medium, with olive oil emulsion added as an inducer. Five metal ions (Cu, Cu, and O2) were weighed separately. 2+ Fe 2+ Ca 2+ NH4 2+ Mg 2+ Add 1 g / L to the fermentation medium, with no metal ions added as the blank group. The temperature was set at 37℃, the rotation speed at 200 r / min, and the culture was carried out on a shaker for 48 h. A certain amount of fermentation broth was taken and centrifuged at 8000 r / min for 2 min at 4℃. The supernatant was then used for enzyme activity determination.
[0110] Metal ion pairs Lysinibacillus capsiciThe results of the effect of Yin-202501 on enzyme production capacity are as follows: Figure 8 As shown, at a concentration of 1 g / L, Fe 2+ It has an activating effect on lipase activity.
[0111] It should be noted that Examples 1, 4, 5, and 6 are all different batches of experiments. Because each batch of experimental strains undergoes activation, revival, and fermentation culture for enzyme production, there are errors between different batches. Therefore, only the effect of the same batch of bacteria recorded in the same example has comparative value, and the experiments of different batches recorded in different examples do not have comparative value.
[0112] Example 7 Ammonium sulfate precipitation and dialysis concentration
[0113] The supernatant obtained by centrifuging the bacterial culture was used to obtain the crude enzyme solution (prepared according to the crude enzyme solution obtained in Example 1). Ammonium sulfate powder was added to saturation (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%), and the solution was allowed to stand at 4°C for 2 hours. After freezing and centrifugation, the supernatant was collected to determine the enzyme activity. A graph was plotted between enzyme activity and ammonium sulfate saturation. Figure 9 ).
[0114] The activity curve of Bacillus lysine lipase in the supernatant of ammonium sulfate-precipitated strains showed that the enzyme activity was high when the saturation was 10%~40%, and the enzyme activity decreased rapidly when the saturation increased to 50%. When the saturation reached 100%, the enzyme activity was less than 1U, indicating that most of the lipase had precipitated at this saturation level.
[0115] The fermentation broth was precipitated based on the determined ammonium sulfate saturation. The precipitate was discarded, and the broth was reconstituted with an equal volume of phosphate buffer. The precipitate was then transferred to an 8000-12000 mL dialysis bag and dialyzed at 4°C for 12 hours, with the dialysis buffer changed every 2 hours. A small sample of the enzyme solution was tested with saturated calcium chloride solution. If no precipitate formed, the dialysis was considered successful. The purified enzyme solution is shown below. Figure 10 As shown.
[0116] Example 8: Effect of temperature on enzyme activity and thermal stability of enzymes
[0117] An appropriate amount of purified enzyme solution (prepared by adding ammonium sulfate powder to 100% saturation according to Example 7) was subjected to enzyme reaction at 4℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 100℃, respectively. The reaction was terminated immediately after 15 min, and the enzyme activity was measured. At the same time, it was incubated at 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 1 h, and the activity was measured every 15 min to investigate the thermostability of lipase.
[0118] Enzyme activity assay results Figure 11 This indicates that lipase exhibits its strongest lipase activity at 30℃, reaches 80% at 40℃, and retains 38% relative activity even at 4℃. It retains approximately 25% relative activity between 60 and 80℃, until complete inactivation at 100℃. The crude purified enzyme solution was incubated at various temperatures for the same duration. Figure 12 The residual enzyme activity was measured. The strain exhibited the best enzyme activity stability at 30℃, and strong thermal stability. Enzyme activity showed an increasing trend after 1 hour at 40-60℃, possibly because 30℃ is its kinetic optimum temperature, resulting in the highest enzyme activity. At temperatures between 40-60℃, the lipase underwent violent structural motion, disrupting its precise catalytic conformation and leading to a disordered state. Driven by thermal energy, the lipase adapted to the environment with a more stable but different conformation. These results indicate that the lipase produced by *Bacillus fusiformis* possesses extremely strong thermal stability and adaptability.
[0119] Example 9: Effect of pH on enzyme activity and pH stability of enzymes
[0120] Enzyme solution (purified crude enzyme solution obtained according to Example 7) was added to buffer solutions with pH values of 3, 4, 5, 6, 7, 8, and 9, respectively. After being placed at the optimal enzyme reaction temperature for 60 min, the enzyme activity was measured to study the effect of pH value on the enzyme.
[0121] Lipases exhibit the strongest enzymatic activity under neutral conditions. Figure 13 It can maintain 80% relative enzyme activity in acidic conditions 3-6, indicating that it has strong acid resistance, and it can also maintain close to 80% in alkaline environments 8-9, indicating that this lipase has extremely strong pH adaptability.
[0122] Example 10 Effect of organic solvents on enzyme activity
[0123] Adding a certain concentration of organic solvent can play an important role in enzymatic organic synthesis applications. A 50% concentration system was prepared using methanol, ethanol, isopropanol, acetone, and acetonitrile. The purified enzyme solution (the crude enzyme solution purified according to Example 7) was then mixed with this system, and the enzyme activity was measured.
[0124] All five selected organic solvents promoted enzyme activity. Figure 14 This indicates that the lipase can be well applied in the organic chemical industry.
[0125] Example 11 Effect of metal ions on enzyme activity
[0126] Cu 2+ Ba 2+ Fe 2+ Cd 2+ Mg + Ca 2+Na + Mn 2+ The metal ions were added to the enzyme reaction to achieve a final concentration of 1 mmol / L. Using a control group without added metal ions, the effects of different metal ions on lipase activity were investigated. The results are shown below. Figure 16 .Depend on Figure 16 It can be obtained that Cu 2+ and Na + Significantly promotes enzyme activity, of which Na + The enzymatic activity of [a specific enzyme] is the most significant. This demonstrates that metal ions play an important role in enzymatic organic synthesis.
[0127] Example 12 Lyophilized Extraction and Enzyme Activity Determination of Lipase
[0128] The purified lipase solution (with ammonium sulfate powder added to 100% saturation according to Example 7) was lyophilized to remove excess water to obtain lipase powder. Figure 15 Weigh 20 mg of lipase solid powder, dissolve 20 mg of lyophilized lipase in 100 μL of ultrapure water to prepare 100 μL of enzyme solution, and perform the remaining calculations as in Example 1. The enzyme activity was measured to be 72.729 U.
[0129] This embodiment is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A lysine-containing Bacillus, specifically a... Lysinibacillus capsici Yin-202501, with accession number CCTCC No. M2025574.
2. The use of the lysine-containing Bacillus as described in claim 1 in the production of lipase.
3. The application according to claim 2, characterized in that, A method for preparing lipase using Bacillus lysinensis includes the following steps: The lysine-containing Bacillus strain was inoculated into a liquid culture medium for activation. The activated strain was then inoculated into a fermentation culture medium for fermentation. After centrifugation, the supernatant obtained from the fermentation broth was the crude lipase solution.
4. The application according to claim 3, characterized in that, The liquid culture medium is LB liquid culture medium, which is composed of the following components: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride.
5. The application according to claim 4, characterized in that, The fermentation medium includes LB liquid medium.
6. The application according to claim 5, characterized in that, The fermentation medium also includes at least one of an inducer or a metal ion; The metal ion is Fe. 2+ .
7. The application according to claim 6, characterized in that, The inducer has a volume fraction of 1%-5% in the fermentation medium; The inducing agent includes at least one of olive oil or olive oil emulsion.
8. The application according to claim 6, characterized in that, The concentration of metal ions in the fermentation medium is 1-5 g / L.
9. The application according to claim 3, characterized in that, The fermentation temperature is 30-45℃; The fermentation time is 0.5-3 days; After activation, the strain was inoculated into the fermentation medium at a volume ratio of 1%-5%.
10. The application according to claim 3, characterized in that, It also includes adjusting the saturation of the obtained supernatant with ammonium sulfate, collecting the protein precipitate by centrifugation, redissolving, and obtaining a purified enzyme solution; wherein ammonium sulfate is added to a saturation of 10-100%.
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