Application of LIP in preparation of fat hydrolysis preparation

Abstract

The invention discloses application of LIP in preparation of a fat hydrolysis preparation. Specifically, the invention also discloses a fat hydrolysis preparation and a preparation method thereof. The fat hydrolysis preparation provided by the invention contains novel extracellular low-temperature alkaline lipase rLip, and has certain application potential in the industrial fields of low-temperature alkaline environments such as food processing, detergents, low-temperature environment restoration and the like.

Description

Technical Field

[0001] The invention relates to the field of biotechnology and application of LIP in preparing fat hydrolysis preparations. Background Art

[0002] Lipase (Triacylglycerol esterhydrolases, EC 3.1.1.3), unlike most hydrolases, has a heterogeneous catalytic reaction system. It can not only catalyze the hydrolysis of various forms of triacylglycerol esters into glycerol, water and free fatty acids at the oil-water interface, but also promote a variety of relatively complex chemical reactions such as transesterification, esterification, alcoholysis and acidolysis in micro-aqueous or non-aqueous phases. Lipase has a very rich source and a wide variety of species. It is widely present in animals, plants and microorganisms. It has the advantages of diverse catalytic activity and high stability. Therefore, it has important uses and broad application prospects in traditional and new industrial fields, such as medicine, food, biodiesel and detergent industries.

[0003] Low-temperature lipase refers to a type of lipase that has catalytic activity under low temperature conditions (0-40℃) and has an optimal enzyme activity temperature of 30℃. Unlike lipases produced by other animals and plants, most low-temperature lipases are produced by microorganisms under low temperature stress to adapt to extreme environments. This type of microorganism mainly includes Aeromonas, Pseudomonas, Bacillus, Candida, etc. Compared with lipases from thermophilic microorganisms, low-temperature lipases have evolved unique structures and physicochemical characteristics with high flexibility in order to adapt to low-temperature environments, such as increasing hydrophilic arginine to enhance organic solvent tolerance and increasing the flexibility of polypeptide chains to modify substrates to make them easier to adapt to low-temperature environments. In addition, low-temperature lipases have a wider pH adaptation range, prefer C3-C10 chain length esters, high catalytic activity, and genetic stability. Therefore, low-temperature lipases have great application potential and economic value in reducing energy costs in the fields of low-temperature cleaning, food processing and preservation, wastewater treatment, and fine chemical catalysis. Summary of the invention

[0004] The present invention uses the Deinococcus sp. RM strain isolated and screened in this laboratory as the object, clones and expresses its highly expressed extracellular lipase to study its enzymatic properties, aiming to provide a theoretical basis and technical guidance for the further industrial application of lipase.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] Application of LIP in the preparation of fat hydrolysis preparations.

[0007] Preferably, the LIP is the LIP gene or the protein expressed by the LIP gene.

[0008] Preferably, the protein expressed by the LIP gene is rLIP lipase obtained by exogenous expression using yeast as an engineered bacterium; the fat hydrolyzing agent is used to hydrolyze fats including: at least one of p-nitrophenyl caprylate, p-nitrophenol caproate, p-nitrophenyl laurate, p-nitrophenyl palmitate, and p-nitrophenyl stearate.

[0009] The invention also provides a fat hydrolysis preparation, which contains lip protein or rLIP lipase.

[0010] Preferably, the fat hydrolysis preparation is characterized by further comprising a soluble magnesium salt.

[0011] Preferably, the soluble magnesium salt is magnesium chloride.

[0012] Preferably, an alcohol organic solvent is also included.

[0013] Preferably, the alcohol organic solvent is at least one of ethanol, isopropanol, methanol and tert-butanol.

[0014] Preferably, Aprotinin is also included.

[0015] Preferably, the method for preparing the fat hydrolyzed preparation comprises the following steps:

[0016] (1) Using LIP gene as target gene, recombinant yeast was constructed; the yeast was induced to express and crude enzyme solution was obtained;

[0017] (2) The crude enzyme solution is dialyzed, chromatographed, and concentrated to obtain the recombinant rLIP enzyme after vernalization, that is, the fat hydrolysis preparation is prepared.

[0018] The delivery system of the present invention may have, but is not limited to, the following beneficial effects:

[0019] The present invention sequences the genome and transcriptome of the Deinococcus sp. RM strain, selects the lipase gene with high expression, recombines it into the Pichiapastoris GS115 strain for induced expression, separates and purifies the recombinant lipase, and measures the enzymatic properties. The results show that the RM strain has a total of 4132 genes, 3931 genes encode amino acid sequences, of which there are 6 lipase genes and only 1 extracellular lipase. The Lip gene was successfully expressed in the P. pastoris GS115 strain to obtain the recombinant lipase rLip, whose optimal substrates are p-nitrophenyl laurate and p-nitrophenyl palmitate, the optimal reaction temperature is 30°C, and it has higher catalytic activity and stability below 30°C, the optimal pH is 8.0, and the lipase activity can be maintained at more than 50% at pH 7.0-9.0. Mg 2+ It has a certain promoting effect on enzyme activity. Therefore, rLip secreted by Deinococcus sp. RM strain is a new type of extracellular low-temperature alkaline lipase, which has certain application potential in industrial fields of low-temperature alkaline environment such as food processing, detergents, and low-temperature environmental repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to clearly demonstrate the specific implementation methods of the present invention and certain detection technologies used in the experiments, the implementation methods and the technologies used will be described below, mainly in the form of drawings.

[0021] Figure 1 3D structure diagram of LIP homology modeling; columns represent α-helices; stripes represent β-folds; lines represent curls; blue circles represent Zn atoms; red and yellow represent the formed cap domain; the CPK model represents the catalytic active residues D314, H354, S109, and the Zn binding residues D55, H75, H81 and D225.

[0022] Figure 2 Hydrolysis circle diagram produced by recombinant Pichia pastoris engineered strains on screening medium.

[0023] Figure 3 The substrate specificity of rLIP lipase is: C2, p-nitrophenyl acetate; C4, p-nitrophenyl butyrate; C6, p-nitrophenyl hexanoate; C8, p-nitrophenyl octanoate; C10, p-nitrophenyl decanoate; C12, p-nitrophenyl laurate; C16, p-nitrophenyl palmitate; C18, p-nitrophenyl palmitate.

[0024] Figure 4 Effect of temperature on rLIP lipase activity (A) and stability (B).

[0025] Figure 5Effect of pH on rLIP lipase activity (A) and stability (B).

[0026] Figure 6 Effects of metal ions on rLIP lipase activity.

[0027] Figure 7 The effects of different inhibitors on rLIP lipase activity.

[0028] Specific implementation methods

[0029] The specific implementation methods of the present invention are explained with the aid of examples. Except that the technology used for detection does not impose any form of limitation on the present invention, some schemes in the described embodiments belong to part of the embodiments of the invention. The embodiments obtained by ordinary technical operators in this field without creative results all belong to the protection scope of the present invention.

[0030] Example 1 Materials and Methods

[0031] 1.1 Experimental materials and main reagents

[0032] Escherichia coli DH5α strain, Deinococcus sp. RM strain, pICH plasmid expression vector, and Pichia pastoris GS115 strain were provided by the Microbiology Laboratory of our research group; PCR enzyme and pEASY-Blunt cloning plasmid were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; the chicken feather fermentation medium formula for RM strain: 4.0% chopped chicken feather powder, 0.15% K2HPO4, 0.0025% CaCl2, 0.0025% MgSO4·7H2O, 0.0015% FeSO4 and 100 mL distilled water.

[0033] 1.2 Whole genome and transcriptome sequencing of strains

[0034] The RM strain was inoculated in LB liquid medium, cultured at 28°C and 160rpm, and then centrifuged to collect the bacteria. The bacterial genome was extracted using the acid-washed glass bead method and sequenced using the Illumina Hiseq 2500 second-generation sequencing platform (Shanghai Bioengineering Co., Ltd.). The strain was cultured in chicken feather fermentation medium, and the bacterial precipitate was collected by centrifugation. The transcriptome was sequenced using the Illumina platform (Shanghai Painuosen Biotechnology Co., Ltd.). 1.3 Analysis and screening of lipase gene expression

[0035] The genome sequence obtained by sequencing was used as the reference sequence of the transcriptome, and the sequence alignment analysis was performed using Bowtie2 software. Then, the Read Count value on each gene was counted using HTseq-count software as the original expression of the gene. In order to measure the relative expression between different genes, the FPKM value was further used for comparison (the gene with FPKM value>1 indicates expression, and the larger the value, the higher the relative expression). The lipase gene was analyzed and screened using the screening index of gene annotation as lipase and FPKM value>1.

[0036] 1.4 Cloning of LIP lipase gene, construction and screening of engineered bacteria

[0037] According to the lip gene sequence (GenBank: QYCQ01000277.1), the specific sequence is shown in SEQ ID NO: 1; the expression primers synthesized by Sangon Biotech (Shanghai) Co., Ltd. are specifically shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the underlines are Bam HI and Xho I restriction sites, respectively. The amplified LIP gene does not contain the coding 1-33 signal peptide sequence. The genomic DNA of the RM strain was extracted as a template, and PCR amplification was performed using primers P1 and P2. The PCR product was connected to the pEASY-Blunt cloning vector and sequenced and confirmed; the lip gene PCR product was double-digested with Bam HI and Xho I, and connected to the expression vector pICH to construct the recombinant expression vector pICH / lip. The recombinant expression vector was linearized with Sal I, and electroporated into the P. pastoris GS115 strain using the Gene Pulser Xcell electroporation system (Bio-Rad, USA), spread on MD plates and cultured for 2-4 days. A single colony was picked and inoculated into a modified lipase screening medium (10.0% tributyrin emulsion, 0.67% YNB, 1.0% methanol, 1.5% agar) and cultured for 2-4 days. The production of a transparent hydrolysis circle by the recombinant yeast was used as a screening marker, and the positive expression was screened for genomic PCR to confirm that the LIP gene was integrated into the yeast genome. At the same time, the expression empty vector pICH was electroporated into yeast, and the recombinant transformant obtained was used as an experimental control.

[0038] 1.5 Induction of expression of recombinant engineered bacteria in triangular flasks and determination of lipase activity

[0039] The recombinant engineered bacteria were inoculated into BMGY medium, cultured at 30°C and 160 rpm for 1 day, and then centrifuged at 8000 rpm for 1 min to collect the bacteria. 600The mixture was stirred at 28°C and 160 rpm for 5 days, and the supernatant was collected by centrifugation at 6000 rpm for 20 minutes to obtain the crude enzyme solution. The specific sequence of the LIP lipase is shown in SEQ ID NO: 2.

[0040] The method for determining the activity of the recombinant lipase is as follows: (1) Preparation of p-nitrobenzene laurate substrate solution: Weigh 3.0 mg p-nitrobenzene laurate and dissolve it in 1.0 mL isopropanol, then add 9.0 mL Tris-HCl buffer (20 mM, pH 8.0), mix thoroughly, and store in a brown bottle at 4.0°C. (2) Operation steps: ① Experimental treatment group: Take 75.0 μL of enzyme solution and add it to 375.0 μL of p-nitrobenzene laurate substrate solution and mix well. After reacting in a water bath at 30°C for 10 minutes, add 375.0 μL of 0.5 M trichloroacetic acid to the mixture to terminate the reaction, then keep it in a water bath at 30°C for 10 minutes, add 375.0 μL of 0.5 M Na2CO3 solution, and measure the absorbance at a wavelength of 410 nm. ②Blank control group: Add 375.0μL 0.5M trichloroacetic acid to 75.0μL enzyme solution to inactivate enzyme activity. Other operation steps are the same as those of the experimental treatment group. ③Preparation of standard curve: Weigh 27.89mg p-nitrophenol and dissolve it in 100mL Tris-HCl buffer (20mM, pH 8.0) to prepare a mother solution, then take 0.02, 0.04, 0.06, 0.08, 0.12 and 0.16mL of p-nitrophenol mother solution, dilute to 4.0mL with Tris-HCl buffer (20mM, pH 8.0), measure the absorbance at a wavelength of 410nm, and draw a standard curve. According to the standard curve, the activity of lipase is calculated according to the absorbance difference between the experimental treatment and the blank control group. ④ Lipase activity unit: At 30°C, the amount of enzyme required for lipase to catalyze the hydrolysis of p-nitrophenyl laurate to produce 1 μmol of p-nitrophenol per minute is defined as one lipase activity unit (U).

[0041] 1.6 Determination of enzymatic properties of rLIP

[0042] 1.6.1 Substrate specificity

[0043] Esters with different carbon chain lengths, including C2 (p-nitrophenyl acetate), C4 (p-nitrophenyl butyrate), C8 (p-nitrophenyl octanoate), C10 (p-nitrophenol decanoate), C12 (p-nitrophenyl laurate), C16 (p-nitrophenyl palmitate) and C18 (p-nitrophenyl stearate), were used as substrates. The lipase activity was determined by the method to determine the enzymatic effect of rLIP on different substrates. The relative enzyme activity of the most suitable substrate was set as 100%. 1.6.2 Effect of temperature on enzyme activity

[0044] (1) Using p-nitrophenyl laurate as substrate, the rLIP enzyme activity was measured at reaction temperatures of 4.0-45.0°C to determine the optimal reaction temperature, and the relative enzyme activity at the optimal temperature was set as 100%. (2) After the enzyme solution was incubated in a water bath at 4-45°C for 1h, 2h, 4h, 6h and 12h, the residual activity was measured to determine the thermal stability, and the relative enzyme activity without treatment was set as 100%.

[0045] 1.6.3 Effect of pH on enzyme activity

[0046] The enzyme solution was placed in different buffers (CH3COONa-CH3COOH (pH 5.0), NaH2PO4-Na2HPO4 (pH6.0-7.0), Tris-HCl (pH 8.0-9.0) and glycine / NaOH (pH10.0)) and dialyzed to the corresponding pH value, and then an appropriate amount of the enzyme solution was taken out to react with p-nitrophenyl laurate of the corresponding pH value as a substrate, and the enzyme activity was measured to determine the optimal reaction pH, and the relative enzyme activity at the optimal pH was set as 100%. After the enzyme solution was treated in the buffer for 12 hours, the residual activity was measured to determine the pH stability.

[0047] 1.6.4 Effects of metal ions, surfactants and inhibitors on enzyme activity

[0048] Add Cu to the enzyme solution 2+ Mg 2+ and Ni 2+After adding various metal ions (specifically CuSO4, MgSO4, NiSO4, MnCl2, KCl, NaCl, ZnSO4, FeSO4, Fe2(SO4)3 and CaCl2) to a final concentration of 5mM, and adding various inhibitors to a final concentration of 1.0mM EDTA, 5.0mM EDTA, 10.0mM DTT, 50.0mM DTT, 100.0mM DTT, 1.0mMPMSF, 3.0μM Pepstantin, 0.5mg / ml Leupeptin, 0.1μg / mL Aprotinin, the residual activity was measured after 30min at 4°C to determine the effect on enzyme activity. The enzyme solution without any reagent was used as a control, and the relative enzyme activity was set to 100%.

[0049] 1.6.5 Effect of organic solvents on enzyme activity

[0050] Ethanol, acetic acid, isopropanol, n-butanol, ethyl acetate, p-xylene, dichloromethane, tert-butanol, methanol and chloroform were added to the enzyme sample to make its volume fraction 10%, 30% and 50%, respectively. The enzyme sample added with an equal amount of 0.05M Tris-HCl buffer was set as the control group. After being kept in a 30℃ water bath for 2h, the lipase activity was measured to determine the effect of organic solvents on the enzyme activity. The enzyme activity of the control group was set as 100%.

[0051] 1.6.6 Data Analysis

[0052] All data in this experiment were obtained by three replicates, and GraphPad Prim 8 software was used to perform variance analysis, error value calculation, and enzymatic property result graphs on these data.

[0053] Example 2 Results and Analysis

[0054] 2.1 Genome sequencing analysis

[0055] The total length of the RM strain genome is 4013254bp, with a GC content of 67.81%; the N50 contig size is 21827bp, the longest contig is 79609bp, the average length is 12088.92bp, and it contains 332 contigs. The genome sequence has been submitted to GenBank (accession number no.QYCQ00000000.1). Analysis using the PGAP program in the GenBank database showed that the RM strain contains 4132 genes, of which 3931 genes encode amino acid sequences.

[0056] 2.2 Transcriptome analysis and screening of LIP genes

[0057] The expression of lipase genes in the RM strain is shown in Table 1. As shown in Table 1, the RM strain has 6 lipase genes (Gene IDs are D3W47_14600, D3W47_03800, D3W47_10280, D3W47_10265, D3W47_16670 and D3W47_20390) expressed, of which only the lipase gene LIP (Gene ID: D3W47_14600) has a signal peptide, the expression level FPKM is 152.91, and it is an extracellular lipase; therefore, this lipase gene was selected as the object of further study.

[0058] Table 1 Expression effects of the six lipase genes

[0059]

[0060] 2.3 LIP gene and its sequence analysis

[0061] Using the genome of RM strain as template, primers P1 and P2 were used to amplify the expected PCR product (1277 bp). The sequencing results of the PCR product were consistent with the genome sequence, indicating that the lip gene was successfully cloned.

[0062] The LIP gene encodes 453 amino acid residues. Using the Blastp program in the GenBank database to compare it, it can be seen that LIP is classified as extracellular lipase A, namely EstA (Conserved Protein Domain Family: COG1075); the sequence identity with other members of this lipase is low, and the highest sequence identity with the lipase of Pelosinusfermentans DSM 17108 strain (PDB code: 5ah0.1.A) is only 43.84%. Therefore, the 3D structure of Lip was modeled using 5ah0.1.A as a template (such as Figure 1 ).Depend on Figure 1It can be seen that LIP is composed of 6 α-helices, 1 310 helix, 17 β-folds and multiple random coils (the first amino acid residue K in the 3-D structure is defined as 1, and so on). Ser109, His354 and Asp314 residues are conserved catalytic triads in this family, among which Ser109 residue is located in the Gly-X-Ser-X-Gly conserved Motif structure in serine hydrolases. LIP is a monomeric enzyme belonging to the α / β hydrolase family. The center of the enzyme's spatial structure is composed of seven β-folds surrounded by α-helices. The two peptide chains N154-L231 and T259-A312 form a "cap"-like domain. When LIP is not hydrolyzed, the "cap" domain is in a closed conformation, covering the active site and catalytic triad residues, preventing the substrate from binding to the active site; when the substrate is hydrolyzed, the "cap" domain conformation becomes open, and the active site is exposed to bind to the substrate. The side chain groups of the conserved D55, H75, H81 and D225 residues of LIP are combined with a metal Zn atom, which contributes to the thermal stability of LIP. There is an interaction force between the residues near the metal Zn binding site and the long loop region in the "cap" structure, and the D225 residue is located in the "cap" domain; therefore, Zn has an important influence on the regulation of the opening of the "cap".

[0063] 2.4 Expression of rLIP in Pichia pastoris

[0064] The electrotransformed P. pastoris GS115 strain was spread on MD plates for culture, and a single colony was picked and transferred to a lipase screening medium plate. After 1-2 days of culture on the medium plate, curdling was observed around most transformants, and continued culture turned into a transparent hydrolysis circle. The transformant with the largest hydrolysis circle / colony diameter was selected for genomic PCR verification, and the LIP gene was successfully amplified, indicating that the LIP gene was successfully expressed in Pichia pastoris. The recombinant yeast strain was induced to express in a triangular flask. When induced for 120 hours, the enzyme activity of the fermentation broth was the highest, such as Figure 2 shown.

[0065] 2.5 Enzymatic properties of rLIP

[0066] 2.5.1 Substrate specificity

[0067] Depend on Figure 3 It can be seen that rLIP lipase (rLIP refers to recombinant lipase) specifically hydrolyzes medium- and long-chain p-nitrophenol esters, and its optimal substrates are C12 p-nitrophenyl laurate and C16 p-nitrophenyl palmitate, which is quite different from the substrate specificity of other low-temperature lipases.

[0068] 2.5.2 Effect of temperature on rLIP lipase activity and enzyme temperature stability

[0069] Effects of temperature on the activity and stability of rLIP lipase Figure 4 . The optimum reaction temperature of rLIP is 30°C. When the temperature is below 30°C, the enzyme can still maintain a high activity, about 80%, and has strong stability. When the temperature exceeds 35°C, its activity gradually decreases and its stability weakens. At 45°C, the enzyme still has a certain activity, about 40%, but after the enzyme sample is warmed for more than 1 hour, its residual enzyme activity drops sharply or even completely loses. According to the definition of Margsin et al., enzymes with an optimum reaction temperature below 40°C are low-temperature enzymes and have a certain degree of thermal stability. Therefore, rLIP lipase belongs to a low-temperature lipase.

[0070] 2.5.3 Effect of temperature on rLIP lipase activity and enzyme temperature stability

[0071] Effects of pH on the activity and stability of rLIP lipase Figure 5 The optimal reaction pH of rLIP is 8.0, and its stability is the strongest. It can still maintain a certain enzyme activity and stability at pH 6.0-8.0 or pH 8.0-9.0. When the pH is lower than 6.0 or higher than 9.0, the enzyme activity drops sharply and its stability is significantly reduced.

[0072] 2.5.4 Effects of metal ions on rLIP lipase activity

[0073] Effects of different metal ions on rLIP lipase activity Figure 6 .Cu 2+ 、Zn 2+ , Fe 3+ , Fe 2+ It has an inhibitory effect on rLIP activity. 2+ The rLIP enzyme activity decreased by 18.11%, Fe 2+ The rLIP enzyme activity decreased by 28.70%, Zn 2+ and Fe 3+ The inhibitory effect was the most significant, almost inhibiting the activity of rLIP. 2+ Ni has a certain promoting effect on rLIP activity. 2+ , Mn 2+ 、Na + , K + , Ca 2+ The impact is not significant.

[0074] 2.5.5 Effects of inhibitors on rLIP lipase activity

[0075] The effects of different inhibitors on rLIP lipase activity are shown in Figure 7When 5mM and 10mM EDTA were added, the activity of rLIP lipase decreased by 68.6% and 72.93%; when 10mM, 50mM, and 100mM DDT were added, the activity decreased by 13.53%, 15.4%, and 19.11% respectively; when 1mM / ml PMSF was added, the activity decreased by 5.85%; when 3μM / ml Pepstantin was added, the activity decreased by 15.53%; when 0.5mg / ml Leupeptin was added, the activity decreased by 5.88%; when 0.1μg / ml Aprotinin was added, the activity increased by 2.11%.

[0076] 2.5.6 Effect of organic solvents on rLIP lipase activity

[0077] rLIP lipase was treated in different volume fractions of 10%, 30% and 50% organic solvents and the enzyme activity was measured. As shown in Table 2, rLIP can still maintain a certain enzyme activity and good stability in 10% volume fraction of ethanol, isopropanol, methanol and tert-butanol, and the enzyme activity decreases significantly with the increase of volume fraction, but rLIP still has 61% enzyme activity in 30% methanol. n-Butanol, ethyl acetate, acetic acid, p-xylene, dichloromethane and chloroform have significant inhibitory effects on rLIP enzyme. In 50% organic solvents, all organic solvents show complete inhibition and the enzyme activity is completely lost.

[0078] Table 2 Effects of organic solvent on the activity of rLIP lipase

[0079]

[0080] Example 3 Discussion and Conclusion

[0081] 3.1 Discussion

[0082] Deinococcus belongs to the domain Bacteria, Deinococcus, Deinococcus-Thermus, Deinococci, Deinococcales, Deinococcaeae. In 1956, Anderson discovered the first strain of Deinococcus (Deinococcus Radiodurans) from canned meat after radiation sterilization. Deinococcus strains are a type of microorganisms that have evolved unique cell structures and physiological and biochemical characteristics over a long period of time to adapt to various extreme environments. Their cell morphology is generally spherical or short rod-shaped. Among them, spherical Deinococcus are mostly Gram-positive bacteria, and short rod-shaped Deinococcus are mostly Gram-negative bacteria. Deinococcus is a bacterium that has a strong resistance to radiation that causes cell lethal effects, so its growth tolerance temperature range is relatively large. Currently, this genus covers 20 valid published species.

[0083] At present, there are few studies on lipases expressed by Deinococcus, especially few reports on low-temperature lipases. In China, only Zhang Yage et al. screened and identified a new esterase EstDR4 from the extreme microorganism D. radiodurans. The enzyme is a cold-adapted alkaline esterase that exhibits maximum catalytic activity at 30°C and pH 8, and also has high catalytic activity at low temperatures of 0-10°C, and has the characteristics of preferring medium- and short-chain fatty acids. In this study, a highly expressed extracellular lipase gene lip was discovered through genome and transcriptome analysis of an unknown species in the genus Deinococcus sp. RM. Further homology analysis showed that the sequence identity of the extracellular lipase A to other members was low, and the highest sequence identity with the lipase of Pelosinusfermentans DSM 17108 strain (PDB code: 5ah0.1.A) was only 43.84%. Therefore, the enzyme has a unique structural feature similar to other low-temperature enzymes, namely a catalytic triad structure (Ser-Asp-His) centered on a serine (Ser) residue, and belongs to the α / β hydrolase family. The difference is that the nucleophilic serine residue of other lipases is located in a hairpin between a β-sheet and an α-helix in a highly conserved pentapeptide sequence (Gly-Xaa-Ser-Xaa-Gly), thus forming a characteristic "nucleophilic elbow", while the nucleophilic serine residue of Lip lipase is located in the Gly-X-Ser-X-Gly conserved motif structure. In summary, we believe that this enzyme is a new type of extracellular lipase with great application potential.

[0084] In order to study the physicochemical properties of the enzyme, we cloned and expressed the lip lipase gene through a prokaryotic system, and further expanded the induced expression through a eukaryotic expression system. Yeast, as a commonly used host bacteria in eukaryotic expression systems, not only retains the advantages of prokaryotes, but as a eukaryote it also has subcellular tissues that can undergo post-translational modifications. For example, Pan Xiaoxing heterologously expressed Geotrichum candidum lipase in Saccharomyces cerevisiae and Pichia pastoris, and the lipase activity of the recombinant Saccharomyces cerevisiae was 43.7U / g. In this study, we successfully expressed the lip lipase gene in the P. pastoris GS115 strain to obtain the recombinant lipase rLIP, whose enzyme activity in the fermentation broth reached 51.1U / mL. Further studies on the enzymatic properties of the modified enzyme revealed that the substrates of rLIP lipase prefer medium- and long-chain glycerolipids, and are more sensitive to C 10 ~C 18 Glycerolipids have a high catalytic efficiency, and their optimal substrate is C 12 p-Nitrophenyl laurate, C 16 Most of the reported low-temperature lipases prefer C3~C 10 The difference between the two indicates that rLIP, as a newly discovered lipase, can supplement and expand the application range of low-temperature lipases. Consistent with most low-temperature lipases, rLIP can maintain a high catalytic activity in a low-temperature environment, with an optimum temperature of 30°C. The difference is that rLIP can still maintain good activity and thermal stability after the temperature exceeds 30°C. Even after being warmed at 40°C for 12 hours and 45°C for 1 hour, it can still retain about 40% of residual enzyme activity. In addition, the tolerance of rLIP lipase to organic solvents is relatively general compared with other low-temperature lipases. For example, the JXJ-54 lipase reported by Yan Xiaoyun was significantly activated in the presence of 10% to 30% methanol, 10% acetonitrile and 30% ethanol, and has good tolerance to organic solvents such as isopropanol, ethanol, acetone, ethyl acetate, and chloroform. rLIP lipase can maintain good catalytic activity in 10% and 30% methanol, 10% ethanol, 10% isopropanol, and 10% tert-butanol, but the enzyme activity is significantly inhibited in n-butanol, acetic acid, ethyl acetate, p-xylene, dichloromethane, chloroform, and other volume fractions of methanol, ethanol, and isopropanol. The reason needs further study.

[0085] 3.2 Conclusion

[0086] (1) The genome of Deinococcus sp.RM strain is 4013254bp in length, with a GC content of 67.81%. It contains 4132 genes, 3931 of which encode proteins, of which 6 are lipase genes, and only one gene lip (GeneID: D3W47_14600) encodes extracellular lipase. Homology sequence analysis with other members of the extracellular lipase family, namely EstA (Conserved Protein Domain Family: COG1075), showed that the sequence identity of this gene with other members was low, with the highest similarity being only 43.84%.

[0087] (2) The LIP gene was heterologously expressed in Escherichia coli and Pichia pastoris expression systems, and the recombinant lipase rLIP was successfully obtained. The activity of the recombinant enzyme was as high as 51.1 U / mL when induced in a triangular flask. rLIP lipase has good catalytic activity for medium- and long-chain glycerides, and the optimal substrate is p-nitrophenyl laurate C 12 4-Nitrophenyl palmitate C 16 The optimum reaction temperature of the enzyme is 30°C. It can still maintain high activity and thermal stability at low temperatures (4-30°C). When the temperature is higher than 45°C, its activity and stability drop sharply. The optimum pH is 8.0. It can maintain more than 50% lipase activity at pH 7.0-9.0. Metal ions Cu 2+ 、Zn 2+ , Fe 3+ , Fe 2+ In the inhibition of its activity, Ni 2+ , Mn 2+ 、Na + , K + , Ca 2+ Mg 2 + It has little effect on enzyme activity, while Mg 2+ It has a promoting effect on it. EDTA above 5mM concentration can strongly inhibit rLIP enzyme activity, a certain dose of DTT, PMSF, Pepstantin, Leupeptin can inhibit rLIP enzyme activity, and Aprotinin inhibitor can promote its enzyme activity. Organic solvents will inhibit rLIP enzyme activity. Only in 10% and 30% methanol, 10% ethanol, 10% isopropanol, and 10% tert-butanol can rLIP maintain good catalytic activity. In summary, rLIP lipase is a new type of low-temperature alkaline lipase, which has certain application potential in industrial fields with low-temperature alkaline environments such as food processing, detergents, and low-temperature environmental remediation.

Claims

1. Application of LIP in the preparation of fat hydrolysis preparations.

2. The use according to claim 1, characterized in that: The LIP is the LIP gene or the protein expressed by the LIP gene.

3. The use according to claim 2, characterized in that: The protein expressed by the LIP gene is rLIP lipase obtained by exogenous expression using yeast as an engineering bacterium; the fat hydrolyzing agent is used to hydrolyze fats including: at least one of p-nitrophenyl octanoate, p-nitrophenol caproate, p-nitrophenyl laurate, p-nitrophenyl palmitate, and p-nitrophenyl stearate.

4. A fat hydrolysis preparation, characterized in that Contains lip protein or rLIP lipase.

5. The fat hydrolysis preparation according to claim 4, characterized in that: Also included are soluble magnesium salts.

6. The fat hydrolysis preparation according to claim 5, characterized in that: The soluble magnesium salt is magnesium chloride.

7. The fat hydrolysis preparation according to claim 6, characterized in that: Also included are alcohol organic solvents.

8. The fat hydrolysis preparation according to claim 7, characterized in that: The alcohol organic solvent is at least one of ethanol, isopropanol, methanol and tert-butanol.

9. The fat hydrolysis preparation according to claim 4, characterized in that: Also includes Aprotinin.

10. A method for preparing a fat hydrolyzed preparation according to any one of claims 4 to 9, characterized in that The steps include: (1) Using LIP gene as target gene, recombinant yeast was constructed; the yeast was induced to express and crude enzyme solution was obtained; (2) The crude enzyme solution is dialyzed, chromatographed, and concentrated to obtain the recombinant rLIP enzyme after vernalization, that is, the fat hydrolyzing preparation is prepared.

Images