In-situ screening and identification method of high-activity rice bran endogenous lipase and application of in-situ screening and identification method

Abstract

The invention relates to the technical field of food biochemistry and lipase detection, in particular to an in-situ screening and identification method of high-activity rice bran endogenous lipase and application of the in-situ screening and identification method. Comprising the following steps: carrying out electrophoretic separation on rice bran protein, incubating separated gel in a solution containing a 4-methylumbelliferone fatty acid ester substrate, and carrying out in-situ identification on a lipase strip in a protein mixture by detecting a fluorescence signal. According to the method, the cross-modal luminescence characteristic of 4-methylumbelliferone in a nucleic acid imaging environment is utilized, and the LC-MS / MS technical means is combined, so that the sequence information of enzyme is retrieved and determined through a bioinformatics database, and the identity attribution of representative lipase in rice bran is defined. The method has the characteristics of rapidness and sensitivity, and lays a foundation for explaining a mechanism of rice bran rancidity caused by rice bran lipase and excavating high-activity lipase.

Description

Technical Field

[0001] This invention relates to the fields of food biochemistry and lipase detection technology, and in particular to an in-situ screening and identification method for highly active rice bran endogenous lipase and its application. Background Technology

[0002] Rice bran is a high-quality and inexpensive raw material in the food processing industry. As a major byproduct of rice processing, it contains 64% of the nutritional essence of rice and is hailed as a "gift from nature." Rice bran contains about 20% fat and is a common raw material for rice bran oil production. Rice bran oil is rich in unsaturated fatty acids and hundreds of bioactive substances, making it a high-quality edible oil. Defatted rice bran, obtained after oil extraction, contains 15-20% protein. This rice bran protein has low allergenicity and a reasonable amino acid composition. At the same time, defatted rice bran retains dietary fiber, B vitamins, vitamin E, and trace elements, making it suitable for the development of meal replacement powders and other products. However, the problem of rice bran rancidity restricts the utilization of this resource. Freshly ground rice bran experiences a significant increase in free fatty acid (FFA) content within hours, producing a musty smell; after a month of storage, the free fatty acid content can reach 70%. When the free fatty acid content in rice bran oil exceeds 5%, it is unsuitable for refining; when it exceeds 10%, it is unsuitable for consumption. Therefore, effectively deactivating the lipase in rice bran is the key to delaying its rancidity.

[0003] A thorough understanding of the enzymatic characteristics of endogenous lipases in rice bran and their mediated triglyceride hydrolysis mechanisms is crucial for mitigating rice bran rancidity and achieving high-value utilization of rice bran. However, current research on rice bran lipases is still in its early stages. The specific types of representative lipases in rice bran and the mechanisms by which they efficiently catalyze rice bran oil production remain unclear. This is mainly due to the challenges in the precise identification and functional characterization of rice bran lipases at this stage, specifically in the following two aspects: First, traditional methods for extracting natural proteins face the dual challenges of yield and purity. Traditional methods such as ammonium sulfate fractionation or acid-base precipitation to directly extract proteins from rice bran, followed by multi-stage chromatographic separation and purification of the target enzyme using ion exchange, molecular sieving, and hydrophobic chromatography, are often limited by cumbersome preparation processes, low enzyme activity recovery rates, and interference from other proteins. Due to the complex matrix of rice bran and its low lipase abundance, traditional methods struggle to obtain high-purity protein samples that meet the requirements for enzymatic kinetic characterization and structural analysis. Rajeshwara et al. directly isolated rice bran lipase from rice bran, but analysis revealed that the enzyme had lost most of its activity. Yu et al. isolated two highly similar lipases from rice bran, but failed to obtain pure enzymes due to the difficulty in separation; furthermore, Yu et al. found that approximately 25% of the lipases in rice bran exist in a bound state, making effective extraction difficult using conventional methods.

[0004] Second, modern molecular biology strategies have limitations in tissue-specific localization. While searching and screening rice (Oryza sativa) lipase genes in databases such as NCBI for heterologous recombination expression can overcome the bottlenecks of "yield" and "purity" in natural protein extraction, there is significant uncertainty regarding their biological classification. Given the large number of lipase family members in the rice genome (such as the TGL and GDSL families), and the high spatial heterogeneity of subtypes in tissue distribution (such as roots, stems, leaves, endosperm, and cortex), blindly using predicted whole-genome sequences of rice to select lipase genes and perform recombination expression cannot confirm the true expression level of the recombinant enzyme in rice bran tissue and its representative role in the rancidity process.

[0005] In summary, establishing a comprehensive strategy that integrates tissue spatial localization, activity-guided discovery, and efficient preparation to accurately identify and purify key lipases in the rice bran rancidity process is a problem that urgently needs to be solved. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a method for rapidly identifying lipases from rice bran protein mixtures. This method utilizes the cross-modal luminescence properties of 4-methylumbelliferone in a nucleic acid imaging environment, combined with LC-MS / MS technology, to determine the enzyme's sequence information through bioinformatics database searches, thus identifying representative lipases in rice bran. This method is rapid and sensitive, laying the foundation for elucidating the mechanism by which rice bran lipases cause rice bran rancidity and for the discovery of highly active lipases.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an in-situ screening and identification method for highly active rice bran endogenous lipase, comprising the following steps: separating rice bran proteins by electrophoresis, incubating the separated gel in a solution containing 4-methylumbelliferone fatty acid ester substrate, and identifying lipase bands in the protein mixture in situ by detecting fluorescence signals.

[0008] Preferably, the electrophoresis is SDS-PAGE or non-denaturing PAGE; the incubation time is 5~20 min.

[0009] Preferably, it further includes the following steps: S1: Rice bran protein was extracted from defatted rice bran using ammonium sulfate precipitation with different saturations; S2: Separate rice bran proteins by electrophoresis; S3: Prepare a substrate solution containing 4-methylumbelliferone fatty acid ester; S4: Incubate the electrophoresis gel with the substrate solution in the dark; S5: Use a gel imaging system or ultraviolet analyzer to capture fluorescence signals and identify lipase bands; S6: Extract the fluorescent bands for protein recovery and LC-MS / MS mass spectrometry identification; S7: Determine the enzyme gene sequence based on the mass spectrometry results, and perform recombinant expression and enzyme activity verification.

[0010] Preferably, the saturation of ammonium sulfate in step S1 is 40%~80%.

[0011] Preferably, the substrate solution in step S3 is 4-methylumbelliferyl ketone butyrate, heptanoate, laurate, or oleate; the concentration of the substrate solution is 0.2~0.5 mM; the solvent of the substrate solution is a buffer solution with pH 7.0~8.5; and the substrate solution further includes DMSO or an emulsifier.

[0012] Preferably, in step S4: for SDS-PAGE gels, refolding, elution, and equilibration are required before incubation with the substrate; for non-denaturing PAGE gels, equilibration is performed directly before incubation with the substrate.

[0013] Preferably, the LC-MS / MS mass spectrometry identification in step S6 includes trypsin digestion, peptide sequencing, and bioinformatics database retrieval.

[0014] This invention provides a rice bran endogenous lipase identified by the method described above.

[0015] Preferably, the amino acid sequence of the rice bran endogenous lipase is shown in SEQ ID NO:1: MASSAAPFHMVQIQRDDTTFDAYVVGKENAPGIVVLQEWWGVDYEIKNHAVHISQIGEGYRALIPDLYRGKVALDVAEAQHLMEGLDWPGAVKDIQASVKWLKANGSPKVGVTGYCMGGALS IASGVSVPEVDAVVAFYGTPPSELADASKAQAPIQAHFGELDSFVGFADVTAAKSLEEKLKSSGVPHEVHIYPGCSHAFMNTSPEAVKRRKEMGLTDENQAAIDLAWSRFSTWMGRFLGSA.

[0016] This invention provides the application of the rice bran endogenous lipase in oil hydrolysis, food quality control, or biocatalyst preparation.

[0017] Compared with the prior art, the present invention has the following beneficial effects: Economic efficiency and accessibility: This invention constructs a cross-modal imaging scheme that can detect the fluorescence signal generated by the hydrolysis of 4-methylumbelliferone fatty acid esters using conventional nucleic acid gel imaging systems (such as EB mode), achieving highly sensitive visual analysis of lipase activity, significantly reducing the equipment dependence threshold, and improving the scalability of this technology in laboratory and production environments.

[0018] Highly efficient and precisely localized: This invention optimizes elution, renaturation, and substrate incubation conditions, shortening the identification cycle to 5-20 minutes. This enables rapid and accurate anchoring of lipase activity bands from complex rice bran protein systems. Compared to traditional colorimetric methods, this method offers higher screening efficiency and localization accuracy, and is suitable for in-situ screening of lipases from various sources, including animal and plant tissues and microorganisms.

[0019] The technology is highly scalable and supports multidimensional enzymatic characterization: This invention is not only applicable to the rapid screening of lipases, but also possesses the capability for multidimensional enzymatic property analysis. By combining parallel electrophoresis with in-situ colorimetric techniques for substrates of different chain lengths, the substrate selectivity and catalytic efficiency of the enzyme can be directly evaluated. Simultaneously, Native-PAGE allows for parallel comparison of multiple lipases in the same reaction system, avoiding errors caused by differences in substrate dispersibility in traditional liquid-phase reactions, thus providing a reliable platform for screening highly efficient catalysts.

[0020] A novel, highly active rice bran lipase has been discovered, possessing both scientific research value and application potential: A novel rice bran lipase (UniProt ID: A0A8J8YJR4) was successfully identified using this method. K cat / K m Reaching 3.75×10 5 M -1 min -1 Furthermore, its sequence similarity to known rice lipases is less than 30%, indicating that it is a novel plant lipase with a unique evolutionary background. This enzyme exhibits significantly superior catalytic activity towards rice bran oil compared to commercially available lipases, providing a key molecular target for elucidating the mechanism of rice bran rancidity. It also shows promising application prospects in oil processing, biodiesel production, and other fields. Attached Figure Description

[0021] Figure 1 The images show rice bran protein extracted using ammonium sulfate at saturations of 40%, 50%, 60%, and 70% in this invention, after gradient Native-PAGE gel separation, incubated with 4-methylumbelliferyl ketobutyrate (4-MUB), and imaged in nucleic acid gel mode on a gel imaging system (left image); and the imaged protein gel mode after Coomassie brilliant blue staining (right image). Porcine pancreatic lipase and Candida lipase were used as positive controls in the experiment.

[0022] Figure 2 The invention demonstrates the imaging results of rice bran protein extracted with 40% ammonium sulfate, separated by 12% SDS-PAGE gel, incubated with 4-methylumbelliferyl ketone butyrate (4-MUB), and then incubated with 4-methylumbelliferyl ketone butyrate in the nucleic acid gel mode of a gel imaging system; and the imaging results of protein gel in the protein gel mode after Coomassie brilliant blue staining, wherein the fluorescent bands of the protein are indicated by red arrows.

[0023] Figure 3 The images show the results of rice bran protein extracted by precipitation with ammonium sulfate at 60% and 70% saturation, before and after heating, separated by 10% SDS-PAGE gel, and incubated with 4-methylumbelliferyl ketone heptanoate (4-MUH) for 5 min, as well as the imaging results in nucleic acid gel mode of a gel imaging system (left image); and the imaging results in protein gel mode of a gel imaging system after Coomassie brilliant blue staining (right image).

[0024] Figure 4 The images show the results of porcine pancreatic lipase and Candida lipase after separation by Native-PAGE gel, incubation with 4-methylumbelliferone lauryl ester (4-MUL) for 15 min, and imaging in nucleic acid gel mode of a gel imaging system (left image); and the results of imaging in protein gel mode of a gel imaging system after Coomassie brilliant blue staining (right image).

[0025] Figure 5 The images show the results of rice bran protein, Candida lipase, and porcine pancreatic lipase extracted by salting out with ammonium sulfate at saturations of 40%, 50%, 60%, and 70% in this invention, after separation by Native-PAGE gel, incubation with 4-methylumbelliferyl oleate (4-MUO) for 20 min, and imaging in nucleic acid gel mode of a gel imaging system (left image); and the results of imaging in protein gel mode of a gel imaging system after Coomassie brilliant blue staining (right image).

[0026] Figure 6 This invention demonstrates Figure 1 LC-MS / MS mass spectrometry identification results of the target band protein (Mascot search) and sequence coverage map of the target band protein.

[0027] Figure 7The results of the bioinformatics analysis of the target protein identified in this invention are presented. (A) A phylogenetic tree of the target protein constructed based on maximum likelihood, showing the evolutionary relationship between the target protein (Uniprot ID: A0A8J8YJR4) and various plant-derived lipases; the sequence marked in red is the target protein, with Diutina rugosa (P2026.1) as the outgroup for root identification. (B) Comparison of amino acid sequences of the target protein and homologous plant lipases. The target protein and rice ( ) were analyzed using ClustalW software. Oryza sativa ), soybeans Glycine soja ) and wheat ( Triticum aestivum Sequence alignment of lipases from species such as ( ) was performed.

[0028] Figure 8 The isolation, purification, and activity verification of the target enzyme in this invention are shown. (A) SDS-PAGE gel image of the target enzyme after affinity chromatography purification. (B) The catalytic activity of the target enzyme was analyzed by titration using rice bran oil and palm oil as substrates, respectively, with porcine pancreatin and Candida lipase as controls.

[0029] Figure 9 The catalytic kinetics of the target enzyme A0A8J8YJR4 in this invention are presented. (A) Michaelis-Menten kinetics analysis of the substrate 4-methylumbelliferyl ketone butyrate by the target enzyme A0A8J8YJR4, porcine pancreatic lipase, and Candida albicans lipase. (B) Left: Imaging results of enzyme A0A8J8YJR4, Candida albicans lipase, and porcine pancreatic lipase after separation by Native-PAGE gradient gel, followed by incubation with 4-methylumbelliferyl ketone butyrate (4-MUB) for 5 min in nucleic acid gel imaging mode; Right: Imaging results of enzyme A0A8J8YJR4 after Coomassie brilliant blue staining in protein gel imaging mode, with rice bran protein prepared by alkali dissolution and acid precipitation as a negative control. (C) The catalytic kinetic parameters of the three enzymes were obtained by nonlinear regression fitting, among which the catalytic kinetic parameters of enzyme A0A8J8YJR4 were... K cat / K m The value reached 3.75×10 5 M -1 min -1 .

[0030] Figure 10 A flowchart of the technical solution of this invention is shown. Detailed Implementation

[0031] In one embodiment of the present invention, such as Figure 1As shown, the method for in-situ identification of lipase in rice bran protein mixture using Native-PAGE is as follows: First, the protein mixture in defatted rice bran was obtained by salting out with ammonium sulfate at saturations of 40%, 50%, 60%, and 70%. Next, the obtained rice bran proteins were separated by electrophoresis using gradient Native-PAGE gels. After electrophoresis, the gel was immersed in a CaCl2 equilibration buffer solution. The equilibrated gel was then incubated with 4-methylumbelliferyl ketobutyrate (4-MUB) for 5 min, and then photographed using the nucleic acid gel imaging mode of a gel imaging system. The lipase bands showed fluorescence. Next, the gel was immersed in Coomassie Brilliant Blue staining solution for staining. After destaining, the protein gel imaging mode of the gel imaging system was used to observe the distribution of protein bands. Finally, the fluorescent target bands were cut out using a UV analyzer with gel cutting function. Subsequent processing included protein recovery and processing from the target bands, LC-MS / MS mass spectrometry identification, protein sequence analysis, and identification analysis. The buffer used for protein loading should be a native-pAGE non-reducing loading buffer, and heating should be avoided. A commercial lipase was used as a positive control in the experiment. The standard protein used was a common pre-stained protein standard. Since it has no reference value in native-pAGE gel, the molecular weight of the standard was not labeled.

[0032] In another embodiment of the invention, such as Figure 2 As shown, the method for in-situ identification of lipases in rice bran protein mixtures using SDS-PAGE gel is as follows: First, rice bran protein obtained by precipitation with 40% ammonium sulfate saturation is subjected to SDS-PAGE electrophoresis; after electrophoresis, the gel is immersed in a buffer containing detergent for refolding to remove SDS and allow the protein in the gel to refold; then, the gel is immersed in the above-mentioned buffer without detergent to wash away excess detergent and avoid non-specific fluorescence in subsequent experiments; next, the gel is placed in an equilibration buffer containing CaCl2 for equilibration; the equilibrated gel is then co-incubated with 4-methylumbelliferyl ketone butyrate (4-MUB) for 5-10 minutes. The gel was then photographed using the nucleic acid gel imaging mode of a gel imaging system, where the lipase band showed fluorescence. Next, the gel was immersed in Coomassie Brilliant Blue staining solution for staining. After destaining, the gel was photographed using the protein gel imaging mode of the gel imaging system to observe the distribution of protein bands. Finally, the fluorescent target band was cut out using a UV analyzer with gel cutting function. Subsequent processing included protein recovery from the band, mass spectrometry identification, protein sequence analysis, and identification analysis. It should be noted that because proteins in denatured SDS-PAGE gels are difficult to fully renature, the fluorescence of the target protein band will be weaker than that obtained by separating the target protein using a native-PAGE gel under the same conditions.

[0033] In another embodiment of the present invention, in the above-mentioned "method for in-situ identification of lipases in rice bran protein mixtures using SDS-PAGE gel", the substrate used is 4-methylumbelliferyl ketoheptanate (4-MUH), a medium- and long-chain fatty acid, and the heated rice bran protein is used as a negative control. The results are as follows: Figure 3 As shown.

[0034] In another embodiment of the present invention, in the above-mentioned "method for in-situ identification of lipases in rice bran protein mixtures using Native-PAGE gel", the substrate used is the long-chain fatty acid 4-methylumbelliferyl laurate (4-MUL), and the results are as follows: Figure 4 As shown.

[0035] In another embodiment of the present invention, in the above-mentioned "method for in-situ identification of lipases in rice bran protein mixtures using Native-PAGE gel", the substrate used is long-chain fatty acid 4-methylumbelliferyl oleate (4-MUO), and the results are as follows: Figure 5 As shown.

[0036] In another embodiment of the present invention, this study utilized a 40% saturated ammonium sulfate fractionation technique to pre-enrich the target protein from rice bran, and combined this with LC-MS / MS proteometry to successfully identify and locate a novel lipase. Figure 6 After in-depth analysis of its sequence characteristics using bioinformatics methods, a heterologous expression system was constructed, and high-purity recombinant target enzymes were obtained through affinity chromatography. Figure 7 and Figure 8 In the functional characterization stage, this study used rice bran oil and palm oil as natural substrates and quantitatively analyzed the catalytic activity of the enzyme using acid-base titration. Simultaneously, using the fluorescent substrate 4-methylumbelliferyl ketone butyrate (4-MUB) as a simulated substrate, the Michaelis-Menten kinetic parameters of the recombinant enzyme were systematically determined. Figure 9 A).

[0037] In another embodiment of the present invention, a method for comparing enzyme activity based on native-PAGE electrophoresis was established: by diluting the target lipase, Candida albicans lipase, and porcine pancreatic lipase to equal mass concentrations, electrophoresis on the same plate, and then incubating them in situ with 4-MUB substrate followed by fluorescence imaging analysis. This method not only visually presents the catalytic activity of enzymes in situ on the gel, but also provides a precise evaluation method for efficiently comparing the catalytic efficiency of enzymes from different sources on the same substrate under completely identical reaction microenvironments. Figure 9 B).

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1 - Method for in-situ screening and identification of lipases in rice bran protein using Native-PAGE

[0040] This embodiment provides an in situ identification method for lipase in rice bran protein mixtures using Native-PAGE electrophoresis. The parameters are optimized, and the steps are as follows: extract rice bran protein; perform Native-PAGE electrophoresis on the obtained rice bran protein; prepare 4-MUB substrate solution; pre-equilibrate the electrophoresis gel in a specific buffer solution; incubate the equilibrated gel with the substrate solution; and finally place the incubated gel in an imaging system for detection and imaging.

[0041] (1) Extraction of rice bran protein by ammonium sulfate fractionation precipitation method

[0042] Weigh 120g of rice bran (from Jinbasu Rice Industry Co., Ltd., Yuanyang County, Henan Province), wash with 360 mL of n-hexane and stir for 30 min, repeating 3 times to achieve defatting. Place the rice bran in a fume hood and air dry until the n-hexane completely evaporates. Then, mix the defatted rice bran with 10 mM Tris-HCl buffer (pH 7.5) containing 1.0 mM ethylenediaminetetraacetic acid (EDTA), stir at 4°C for 12 hours, and then centrifuge at 13000 rpm for 10 min at 4°C.

[0043] The supernatant extracted in the above steps was treated using a saturated ammonium sulfate precipitation fractionation method (40%, 50%, 60%, and 70%, w / v). After continuous stirring at room temperature for 30 minutes, it was allowed to stand at 4°C for 1 hour. The precipitates from each fraction were collected by centrifugation at 10,000 rpm for 10 minutes at 4°C. Ammonium sulfate was added slowly in steps, i.e., small amounts of solid ammonium sulfate were added to the supernatant multiple times. The fractionated precipitates were resuspended in 20 mM Tris-HCl (pH 7.5) + 1 mM EDTA solution, and salts were removed using a dialysis bag with 20 mM Tris-HCl (pH 7.5) + 1 mM EDTA solution. Dialysis was performed at 4°C for 24 hours, and BaCl2 detection was used to ensure complete removal of ammonium sulfate. The rice bran protein sample was freeze-dried and stored at -20°C.

[0044] (2) Native-PAGE electrophoresis to separate rice bran protein

[0045] Given the limited effectiveness of native-pAGE in separating high molecular weight or protein complexes with significant molecular weight differences, a 4%-20% gradient gel electrophoresis was used to optimize the separation of rice bran protein samples. Five mg of rice bran protein samples obtained from ammonium sulfate fractionation at different saturations were added to 500 μL of non-denaturing, non-reducing protein loading buffer. After thorough mixing, the samples were centrifuged at 10,000 r / min for 10 min at 4°C, and the supernatant was collected. 10 μL of sample was loaded into each well, and electrophoresis was performed at a constant voltage of 90 V until the bromophenol blue indicator migrated to the bottom of the gel.

[0046] (3) Prepare 4-MUB substrate working solution

[0047] A 100 mM 4-MUB stock solution was prepared using DMSO. The 4-MUB stock solution was diluted to 10 mM with DMSO. 1 mL of the 10 mM 4-MUB substrate was dissolved in 49 mL of equilibration buffer (50 mM Tris-HCl solution containing 1 mM CaCl2, pH 8.0) to achieve a final 4-MUB concentration of 0.2 mM in the substrate solution, with the DMSO content not exceeding 2%.

[0048] (4) Gel imaging was performed after co-incubating the PAGE gel with the substrate solution.

[0049] After electrophoresis, the protein gel was placed in equilibration buffer (50 mM Tris-HCl solution containing 1 mM CaCl2, pH 8.0) and gently shaken on a shaker at room temperature for 15 min to pre-equilibrate, adjusting the gel environment to the optimal pH for enzyme reaction. Subsequently, the equilibrated gel was transferred to the substrate working solution and incubated at room temperature for 5 min before imaging detection. Specific detection modes of the gel imaging system (such as nucleic acid imaging EB mode) can be used for signal capture and imaging, or observation can be performed using a UV analyzer. The lipase activity and location can be determined based on the fluorescence intensity of the bands. Within a certain time range, the color intensity of the target band in the gel will increase with prolonged incubation time, but the fluorescence band will begin to decay after incubation time exceeding 40 min.

[0050] (5) Recovery and processing of proteins in the target band

[0051] The excised gel strips containing the target lipase were cut into small gel pieces and placed in sterile 1.5 mL centrifuge tubes. Decolorization was performed using a decolorizing solution (50 mM ammonium bicarbonate and acetonitrile mixed in a 1:1 volume ratio) until the gel pieces became transparent. Reductive alkylation was then performed: 10 mM DTT was added and reduced at 56°C for 1 h, followed by alkylation at room temperature in the dark for 1 h. The gel particles were dehydrated with acetonitrile, and after drying, PNGase F was added and enzymatically hydrolyzed at 37°C for 6 h to remove sugar chains, followed by washing with 25 mM ammonium bicarbonate. After further dehydration with acetonitrile, trypsin was added and in situ hydrolyzed at 37°C for 16 h. The gel particles were then subjected to a gradient extraction using 25 mM ammonium bicarbonate, 0.1% FA acetonitrile solution, and pure acetonitrile. The extracts were combined, vacuum evaporated, and the peptides were reconstituted with 0.1% formic acid aqueous solution for later use.

[0052] (6) LC-MS / MS mass spectrometry identification of the target protein

[0053] The reconstituted peptides were identified using a Nano LC-MS / MS system. The peptides were dissolved in mobile phase A (an aqueous solution containing 0.1% formic acid) and loaded onto an Acclaim PepMap C18 reversed-phase guard column (75 μm × 2 cm) using a Dionex Ultimate 3000 LC system. Gradient separation was then performed on a reversed-phase C18 analytical column (75 μm × 10 cm). The elution gradient was set as follows: 0–6 min, 5%–8% B; 6–40 min, 8%–30% B; 40–45 min, 30%–60% B; 45–48 min, 60%–80% B; 48–56 min, maintaining 80% B (mobile phase B was an acetonitrile solution containing 0.1% formic acid). The flow rate was maintained at 400 nL / min.

[0054] The eluted products were directly fed into a Q Exactive mass spectrometer (Thermo Fisher Scientific) using positive ion mode and data-dependent acquisition (DDA). Full scan (Full MS) ranged from 350 to 2000 m / z with a resolution of 70,000; secondary mass spectrometry (MS / MS) resolution was 17,500. The MS / MS scan trigger threshold was 1E+5, and the separation window was 2 Da. High-collision energy dissociation (HCD) mode was used for peptide fragmentation, with the normalized collision energy (NCE) set to 28%.

[0055] (7) Target protein sequence analysis and identification analysis

[0056] The peptide sequences obtained by mass spectrometry sequencing were submitted to the Mascot search engine for searching in relevant non-redundant protein databases of NCBI. Based on the identified full-length protein sequences, sequence homology comparison was performed using the BLAST algorithm, and combined with phylogenetic tree analysis and domain prediction, the family affiliation of the target protein was finally confirmed, and it was determined whether it belonged to a novel lipase.

[0057] The experimental results of Embodiment 1 of the present invention are as follows: Figure 1 and Figure 6-9 As shown. First, the rice bran protein components obtained by ammonium sulfate gradient precipitation at different saturations were preliminarily separated using Native-PAGE, and in-situ color development was performed using 4-methylumbelliferyl ketone butyrate (4-MUB) as a fluorescent substrate. The results showed that clear and significantly bright fluorescent bands were observed in the rice bran protein components precipitated at 40% saturation. Figure 1 Subsequently, by excising and recovering the protein from the target fluorescent band, the amino acid sequence information of the target protein was successfully identified using LC-MS / MS technology. Figure 6 ).

[0058] Bioinformatics analysis was used to predict the functional domains and perform sequence homology alignment on the identified polypeptide sequences. The results confirmed that the protein belongs to the lipase family, and phylogenetic analysis showed that it has an independent evolutionary background, classifying it as a novel rice bran lipase. Figure 7 To verify its biological function, this invention achieved soluble expression of the enzyme in *E. coli* using heterologous expression technology, obtaining a recombinant target enzyme with a purity higher than 90%. Its hydrolytic activity against rice bran oil was determined using acid-base titration, and the results showed that the catalytic activity of the target enzyme was significantly superior to that of commercially available lipases of the same type. Figure 8 Further quantitative characterization of the target enzyme's reaction kinetics was performed, specifically the ratio of its measured catalytic constant to the Michaelis constant. k cat / K m The value reached 3.75 × 10 5 M -1 min -1 This kinetic indicator confirms that the target enzyme has a higher conversion efficiency for the substrate than existing commercially available lipases, demonstrating extremely high industrial catalytic potential. Figure 9 ).

[0059] Example 2 - In-situ screening of lipases in rice bran protein using SDS-PAGE

[0060] This embodiment provides a method for in-situ identification of lipases in rice bran protein mixtures using refolded SDS-PAGE. Compared with native-PAGE, the SDS-PAGE system offers higher resolution, resulting in better separation of rice bran protein mixtures and clearer, sharper protein bands, which facilitates precise targeting of lipase bands. Although the denaturing agent (SDS) and reducing agent (DTT) used during electrophoresis may cause some conformational changes in the proteins, the enzymatic activity can be effectively restored while maintaining high-resolution separation through a specific refolding process. Therefore, this method can serve as an important supplement and evolution to native-PAGE, significantly improving the screening efficiency of lipases in complex protein components.

[0061] This implementation case optimizes the experimental parameters involved, and the specific steps are as follows: extract rice bran protein; separate the obtained rice bran protein by SDS-PAGE electrophoresis; prepare 4-MUB substrate solution; incubate the electrophoresis gel in a refolding buffer containing nonionic detergent to competitively replace SDS to promote the spatial conformation recovery of the protein in the gel, thereby achieving in-situ refolding of the protein; then wash the gel multiple times in the same system buffer without detergent to completely remove residual detergent components; pre-equilibrate the eluted gel in a specific equilibration buffer; incubate the equilibrated gel with the substrate solution; finally, place the incubated gel in an imaging system for detection and imaging.

[0062] In this implementation case, the extraction of rice bran protein, the preparation of the 4-MUB substrate solution, the equilibration after gel elution, and the imaging detection are consistent with those in the aforementioned implementation case 1, and will not be repeated here. Only the remaining steps will be explained: (1) SDS-PAGE electrophoresis to separate rice bran protein Take 5 mg of rice bran protein samples obtained from fractionation precipitation with ammonium sulfate at different saturations, add 500 μL of protein loading buffer, mix thoroughly, and centrifuge at 10000 r / min for 10 min at 4°C. Collect the supernatant. Use a 12% SDS-PAGE gel, loading 10 μL per well, and perform electrophoresis at a constant voltage of 90 V until the bromophenol blue indicator migrates to the bottom of the gel. The gel concentration can be selected in the range of 8-12% for different samples, and needs to be optimized based on the protein molecular weight.

[0063] (2) In situ renaturation of proteins in SDS-PAGE gel

[0064] After electrophoresis, the gel was placed in 50 mM Tris-HCl (pH 8.0) buffer containing 2.5% Triton-X 100 and washed twice, 30 min each time. During the procedure, it should be ensured that the washing buffer completely covers the gel to promote the restoration of the protein spatial conformation in the gel by competitively displacing SDS.

[0065] (3) SDS-PAGE gel elution

[0066] The gel was thoroughly eluted in 50 mM Tris-HCl (pH 8.0) buffer, repeated three times for 15 min each time. During the procedure, it is crucial to ensure the eluent completely covers the gel to thoroughly remove any residual nonionic detergent, Triton X-100. This step aims to prevent excess detergent molecules carrying the generated fluorescent product from migrating nonspecifically within the gel matrix during subsequent incubation, thus effectively avoiding background fluorescence interference within the lanes and ensuring clear band boundaries. Incomplete elution can result in residual detergent forming tiny hydrophobic environments that bind 4-methylumbelliferone molecules, causing fluorescence signal diffusion and interfering with the accurate localization of the lipase band.

[0067] The experimental results of Embodiment 2 of the present invention are as follows: Figure 2 As shown. The rice bran protein fraction obtained by 40% ammonium sulfate precipitation was separated by SDS-PAGE, and in-situ fluorescence development was performed using 4-methylumbelliferyl ketobutyrate (4-MUB) as a substrate. The results showed that two clearly defined fluorescent bands were observed in the 40% saturation fraction; compared with Example 1. Figure 1 In comparison, the protein band resolution in SDS-PAGE in this embodiment is significantly higher than that in Native-PAGE, and the band morphology is sharper, which helps to accurately locate the target lipase.

[0068] Meanwhile, due to the introduction of strong denaturing agents (SDS) and reducing agents (DTT) into the SDS-PAGE system, the native spatial conformation of the target protein is disrupted to some extent, resulting in a decrease in enzyme activity compared to the undenatured state. Therefore, compared to the color development results of Native-PAGE, the fluorescence intensity within the SDS-PAGE gel shows a weakening trend.

[0069] Example 3 - In situ identification of rice bran lipases with activity against medium-chain fatty acid substrates using SDS-PAGE

[0070] This embodiment provides a method for in-situ identification of lipases with catalytic activity against medium- and long-chain fatty acid substrates in rice bran protein mixtures using refolded SDS-PAGE technology. In the aforementioned embodiment, the universality of this system was verified using 4-methylumbelliferyl ketone butyrate (4-MUB) as a substrate; however, for lipid processing and metabolism studies, enzymes with catalytic activity against medium- and long-chain fatty acid esters typically exhibit more typical biochemical characteristics of true lipases.

[0071] This embodiment leverages the potential interfacial catalytic properties of lipases, which exhibit catalytic activity against medium- and long-chain fatty acids, and optimizes relevant experimental parameters using 4-methylumbelliferyl ketone heptanoate (4-MUH) as a substrate. The specific operational procedure is consistent with that in Example 2 above. The steps are as follows: (1) Preparation of 4-methylumbelliferyl ketoheptanate (4-MUH) substrate working solution First, a 100 mM 4-MUH stock solution was prepared using DMSO. The 4-MUH stock solution was diluted to 10 mM with DMSO. 1 mL of the 10 mM 4-MUH substrate was dissolved in 49 mL of equilibration buffer (50 mM Tris-HCl solution containing 1 mM CaCl2 and 0.3% sodium deoxycholate, pH 8.0) to achieve a final 4-MUH concentration of 0.2 mM in the substrate solution, with the DMSO content not exceeding 2%. The substrate solution preparation process must be carried out under light-protected conditions to avoid substrate decomposition. The 0.3% sodium deoxycholate can also be replaced with 0.1–0.3% of a surfactant (such as Triton X-100).

[0072] (2) Gel imaging was performed after co-incubating the protein electrophoresis gel with the substrate solution.

[0073] The eluted protein gel was placed in equilibration buffer (50 mM Tris-HCl solution containing 1 mM CaCl2, pH 8.0) and gently shaken on a shaker at room temperature for 15 min to pre-equilibrate, adjusting the gel environment to the optimal pH for enzyme reaction. Subsequently, the equilibrated gel was transferred to the substrate working solution and incubated at room temperature. After 5 min of incubation, imaging detection could be performed.

[0074] The experimental results of Example 3 of the present invention are as follows: Figure 3 As shown, rice bran protein groups obtained by precipitation with 60% and 70% saturated ammonium sulfate were subjected to thermal denaturation treatment at 98℃ for 5 min, respectively. Subsequently, the protein samples before and after heating were separated by SDS-PAGE electrophoresis, and in-situ colorimetric detection was carried out using 4-methylumbelliferyl ketoheptanate (4-MUH) as a fluorescent substrate.

[0075] Experimental results showed that in the fraction obtained by salting out at 60% saturation, a clearly defined fluorescent band was observed in the molecular weight range of 33 kDa to 43 kDa; while in the fraction obtained by salting out at 70% saturation, only a distinct diffuse fluorescent signal was observed at the top of the separating gel. Notably, no fluorescent signal was observed in any of the control group samples after thermal denaturation treatment. This inactivation phenomenon confirms that the captured fluorescent band is heat-sensitive, consistent with the typical characteristics of protein enzyme catalysts, thus ruling out interference from non-enzymatic chemical reactions.

[0076] The results of Example 3 fully validate the feasibility of the present invention in screening specific lipases from complex protein components using medium-chain fatty acid ester substrates.

[0077] Example 4 - In-situ identification of rice bran lipases with activity against medium- and long-chain fatty acid substrates using native-PAGE

[0078] This embodiment provides a method for in-situ identification of lipases with long-chain fatty acid substrate catalytic activity in rice bran protein mixtures based on native-PAGE technology. Previous implementations have used short-chain (4-MUB) and medium-to-long-chain fatty acid esters (4-MUH) as substrates, verifying the feasibility of this identification method. However, due to the poor solubility and large molecular weight of long-chain fatty acid substrates, and the fact that lipases with catalytic activity against long-chain fatty acids typically contain a "cap" domain and exhibit interfacial activation characteristics, in-situ activity detection after PAGE gel separation presents a challenge. To address these issues, this embodiment maintains the basic procedure consistent with Example 1 above, but optimizes the experimental conditions based on the interfacial catalytic characteristics of lipases and the large molecular weight of long-chain substrates (4-MUL and 4-MUO). The steps are as follows: (1) Native-PAGE electrophoresis to separate rice bran protein Compared to the short-chain substrate 4-MUB, the long-chain substrates 4-MUL and 4-MUO have significantly increased molecular weights. Since excessively high concentrations of the Native-PAGE gel can result in excessively small gel pores, hindering substrate entry, a low concentration (e.g., 8%) gel is more suitable. Five mg of rice bran protein samples obtained from ammonium sulfate fractionation at different saturations were taken and added to 500 μL of non-denaturing, non-reducing protein loading buffer. After thorough mixing, the samples were centrifuged at 10,000 r / min for 10 min at 4°C, and the supernatant was collected. 10 μL of sample was loaded into each well, and electrophoresis was performed at a constant voltage of 90 V until the bromophenol blue indicator migrated to the bottom of the gel.

[0079] (2) Preparation of substrate working solutions of 4-methylumbelliferone laurate (4-MUL) and oleate (4-MUO)

[0080] First, prepare a 100 mM stock solution of 4-MUL or 4-MUO using DMSO. When using, dilute the stock solution to 10 mM with DMSO. Take 1 mL of the 10 mM 4-MUH substrate and dissolve it in 49 mL of equilibration buffer (50 mM Tris-HCl solution containing 1 mM CaCl2 and 1% gum arabic powder, pH 8.0) to achieve a final substrate concentration of 0.2 mM, with the DMSO content not exceeding 2%.

[0081] (3) Gel imaging was performed after co-incubating the PAGE gel with the substrate solution.

[0082] After electrophoresis, the Native-PAGE gel was placed in buffer (50 mM Tris-HCl solution containing 1 mM CaCl2 and 0.3% sodium deoxycholate, pH 8.0) and gently incubated on a shaker at room temperature for 45 min to create an interfacial activation environment for the proteins in the gel. Subsequently, the incubated gel was placed in equilibration buffer (50 mM Tris-HCl solution containing 1 mM CaCl2, pH 8.0) and gently equilibrated twice at room temperature, 15 min each time. The equilibrated gel was then transferred to 4-MUL or 4-MUO substrate working solution and incubated at room temperature. Imaging and detection were performed after 10 min of incubation.

[0083] The experimental results of embodiment 4 of the present invention are as follows: Figure 4 and Figure 5 As shown, commercial lipase and rice bran protein components were separated by non-denaturing electrophoresis using Native-PAGE, and in situ colorimetric detection was performed using 4-methylumbelliferone laurate (4-MUH) and 4-methylumbelliferone oleate (4-MUO) as fluorescent substrates, respectively. Figure 4 The results showed that after incubation with 4-MUH for 10 min, obvious fluorescent bands were observed in the lane containing Candida lipase, indicating that this method can effectively identify lipases that are active against medium and long chain substrates. Figure 5 Further results showed that after incubation with the long-chain substrate 4-MUO for 10-15 min, significant fluorescence signals were observed in the rice bran fraction obtained by salting out at 70% saturation and in the lanes of porcine pancreatic lipase.

[0084] The results of Example 4 fully demonstrate that the in-situ colorimetric identification system constructed in this invention is not only applicable to short-chain and medium-chain substrates, but can also effectively screen specific lipases with catalytic activity on long-chain fatty acid esters, reflecting the broad applicability of this method.

[0085] Example 5 - Activity Analysis of the Identified Rice Bran Lipase

[0086] This embodiment experimentally verifies the activity of rice bran lipase obtained by following the above steps and combining it with bioinformatics methods. The relevant experimental steps are as follows: Detection of the catalytic activity of the target enzyme A0A8J8YJR4 against natural oils: The substrate was prepared into a 5% (v / v) emulsion using 50 mM pH 7.5 buffer containing 2% (w / v) gum arabic powder. All substrate solutions were prepared fresh for use and ultrasonically emulsified immediately before use (500 W, 3 s sonication, 3 s interval, total 20 min). 2 mL of the substrate solution was mixed with 0.5 mL of appropriately diluted enzyme solution and reacted at 37°C and pH 7.5 with shaking for 10 min. Immediately, 20 mL of 50% ethanol was added to terminate the reaction, followed by the addition of 100 µL of phenolphthalein indicator. The solution was titrated with standardized 20 mM NaOH solution until it turned red and did not fade after 30 s of shaking. The volume of NaOH consumed was recorded. The specific activity unit of the enzyme is defined as the amount of enzyme required to generate 1 μmol of fatty acid within 1 min under the above reaction conditions.

[0087] Kinetic Analysis of Target Enzyme A0A8J8YJR4: This invention quantitatively evaluated the catalytic performance of the target enzyme A0A8J8YJR4 through enzyme-catalyzed reaction kinetic experiments, using porcine pancreatin and Candida albicans enzyme as controls. First, a series of 4-methylumbelliferone standard solutions were prepared with DMSO, and a standard curve of fluorescence intensity versus product yield was established in the same buffer system as the enzyme-catalyzed reaction. Kinetic measurements were performed on a 96-well black plate, with 100 μL of the target enzyme solution and 100 μL of 4-MUB substrate buffer at different concentration gradients added to each well (to ensure a final substrate concentration in the range of 0–750 μM). A blank control group was also included, with an equal volume of PBS buffer used instead of the enzyme solution. A multifunctional fluorescent microplate reader was used to dynamically monitor the changes in fluorescence intensity in each well under excitation wavelength of 365 nm and emission wavelength of 445 nm. The signal slope at the initial stage of the reaction was converted into initial velocity (V0) using the standard curve. Finally, the Michaelis-Menten equation was fitted using nonlinear regression to calculate the Michaelis constant of the target enzyme. K m and catalytic turnover ( K cat ).

[0088] Comparative analysis of the activity of the target enzyme A0A8J8YJR4 with commercially available enzymes: Commercial Candida lipase, porcine pancreatic lipase (both purchased from Shanghai Yuanye Biotechnology Co., Ltd.) and the target enzyme identified in this invention were diluted to the same protein concentration, and an appropriate volume of non-denaturing and non-reducing loading buffer was added. Then, Native-PAGE electrophoresis and fluorescence detection were carried out according to the steps described in Example 1.

[0089] The experimental results of embodiment 5 of the present invention are as follows: Figure 8 (B) and Figure 9 As shown. Enzymatic activity evaluation results showed that the target lipase had a specific activity of 216 U / mg for the hydrolysis of rice bran oil, significantly superior to the commercially available lipase used in the comparison. This quantitative analysis confirmed from a biochemical experimental perspective that the identified protein is a highly active endogenous lipase (…). Figure 8 B).

[0090] Further analysis using enzyme reaction kinetics revealed that the catalytic constant of the target enzyme A0A8J8YJR4 ( k cat / K m The value reached 3.75 × 10 5 M -1 min -1 Its catalytic efficiency is significantly higher than that of commercially available lipases ( Figure 9 (A and 9C). In particular, the in-situ identification system constructed using this invention enables direct evaluation of the catalytic efficiency of lipases from different sources in Native-PAGE gels. For example... Figure 9 As shown in Figure C, under consistent substrate incubation conditions, the fluorescence signal intensity of the band region containing the target enzyme was significantly higher than that of the two commercially available control enzymes. This visualization result is consistent with the conclusions of the aforementioned kinetic quantitative analysis. This not only provides intuitive evidence from a physical visual perspective of the superior catalytic potential of the target enzyme A0A8J8YJR4 of this invention, but also fully demonstrates that the in-situ identification method for lipases described in this invention possesses excellent technical scalability and can meet the complex research needs ranging from high-throughput screening to multi-dimensional enzymatic characterization.

[0091] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for in-situ screening and identification of highly active rice bran endogenous lipases, characterized in that, Includes the following steps: Rice bran proteins were separated by electrophoresis, and the separated gels were incubated in a solution containing 4-methylumbelliferone fatty acid ester substrate. Lipase bands in the protein mixture were identified in situ by detecting fluorescence signals.

2. The method according to claim 1, characterized in that, The electrophoresis is SDS-PAGE or non-denaturing PAGE; the incubation time is 5~20 min.

3. The method according to claim 1, characterized in that, Further steps include: S1: Rice bran protein was extracted from defatted rice bran using ammonium sulfate precipitation with different saturations; S2: Separate rice bran proteins by electrophoresis; S3: Prepare a substrate solution containing 4-methylumbelliferone fatty acid ester; S4: Incubate the electrophoresis gel with the substrate solution in the dark; S5: Use a gel imaging system or ultraviolet analyzer to capture fluorescence signals and identify lipase bands; S6: Extract the fluorescent bands for protein recovery and LC-MS / MS mass spectrometry identification; S7: Determine the enzyme gene sequence based on the mass spectrometry results, and perform recombinant expression and enzyme activity verification.

4. The method according to claim 3, characterized in that, In step S1, the saturation of ammonium sulfate is 40%~80%.

5. The method according to claim 3, characterized in that, The substrate solution in step S3 is 4-methylumbelliferyl ketone butyrate, heptanoate, laurate, or oleate; the concentration of the substrate solution is 0.2~0.5 mM; the solvent of the substrate solution is a buffer solution with pH 7.0~8.5; and the substrate solution also includes DMSO or an emulsifier.

6. The method according to claim 3, characterized in that, In step S4: For SDS-PAGE gels, refolding, elution, and equilibration are required before incubation with the substrate; for non-denaturing PAGE gels, equilibration is performed directly before incubation with the substrate.

7. The method according to claim 3, characterized in that, The LC-MS / MS mass spectrometry identification in step S6 includes trypsin digestion, peptide sequencing, and bioinformatics database retrieval.

8. A rice bran endogenous lipase identified by the method described in any one of claims 1 to 7.

9. The rice bran endogenous lipase according to claim 8, characterized in that, The amino acid sequence of the rice bran endogenous lipase is shown in SEQ ID NO:

1.

10. The application of the rice bran endogenous lipase according to claim 8 or 9 in oil hydrolysis, food quality control or preparation of biocatalysts.

Images