Lipase mutant and application thereof in preparation of defatted hippocampus powder

Abstract

This invention discloses a lipase mutant and its application in the preparation of defatted dried seahorse. The lipase mutant is derived by mutation of the wild-type transaminase with the amino acid sequence shown in SEQ ID NO.1. This invention significantly enhances enzyme activity, making it suitable for the defatting process of dried seahorse. After enzymatic hydrolysis, the dried seahorse retains its intact shape and color. The application of this lipase mutant is expected to achieve a greener, more efficient, and standardized degreasing process for seahorse, improving the quality stability and market competitiveness of seahorse products, and yielding significant economic and social benefits.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a lipase mutant and its application in the preparation of defatted dried hippocampus. Background Technology

[0002] Seahorses, as a precious marine biological resource, are rich in protein, amino acids, fatty acids, and various bioactive substances, possessing multiple physiological functions such as anti-aging, immune enhancement, and anti-tumor effects. With increasing health awareness and the rapid development of the traditional Chinese medicine industry, the market demand for seahorses and their products in the health supplement, functional food, and pharmaceutical sectors continues to grow.

[0003] The preparation of dried seahorses is the main form through which they enter the market and are used clinically. Traditional processing methods for dried seahorses mainly include washing and drying; however, these simply processed products often contain a high fat content. To address the problem of excessive fat content in dried seahorses, traditional industries mainly use methods such as high-temperature cooking, hot water extraction, and mechanical pressing. While these methods are simple to operate, they suffer from high energy consumption, incomplete degreasing, and a tendency to cause protein denaturation and loss of active ingredients.

[0004] In recent years, with the development of biotechnology, enzyme engineering technology has shown great application potential in food processing and biomanufacturing. Enzymatic defatting, as a green and efficient processing technology, has significant advantages over traditional methods: the reaction conditions are mild, generally carried out at neutral pH and suitable temperature, which can maximize the preservation of the natural quality and bioactivity of the raw materials; enzyme catalysis has high specificity, acting only on fat molecules without affecting other components such as proteins and polysaccharides; the reaction process does not require the use of toxic or harmful chemical reagents, significantly improving product safety and environmental friendliness. However, applying enzymatic defatting technology to the processing of dried seahorses still faces many challenges. The dense structure of seahorses, with its fat components tightly bound to proteins and connective tissue, makes it difficult for lipases to fully contact the substrate and exert their catalytic effect; conventional low-performance lipases have low decomposition efficiency for this type of fat, requiring the synergistic action of other enzymes such as proteases to improve the decomposition effect, thus increasing process costs. Seahorse fat contains a high proportion of unsaturated fatty acids, which are easily oxidized during processing, requiring a faster and more thorough defatting process. Furthermore, as a high-value raw material, seahorse requires processing to preserve its complete shape and color as much as possible, which places higher demands on degreasing technology. Currently, commercially available lipase products generally suffer from insufficient enzyme activity, poor stability, and low efficiency in seahorse degreasing applications, resulting in long degreasing times, unsatisfactory effects, and localized collapse of the seahorse's shape, making it difficult to meet the needs of industrial production.

[0005] Therefore, developing a high-performance lipase mutant optimized for hippocampal defatting processes has significant economic value and practical application implications. Summary of the Invention

[0006] The purpose of this invention is to provide a lipase mutant and its application in the preparation of defatted dried seahorse. Its enzyme activity is significantly improved, making it suitable for the degreasing process of dried seahorse. After enzymatic hydrolysis, it can maintain the complete shape and color of dried seahorse, solving the problems of low efficiency, poor quality, and insufficient safety in traditional degreasing methods, and promoting the technological upgrading and sustainable development of the seahorse processing industry.

[0007] The technical solution adopted by this invention to solve its technical problem is: A lipase mutant, derived by mutation of a wild-type transaminase with the amino acid sequence shown in SEQ ID NO.1, wherein the mutation site is selected from one or more combinations of the following schemes: (1) Valine V at position 40 is mutated to isoleucine I; (2) Threonine T at position 46 is mutated to lysine K; (3) Serine S at position 53 is mutated to leucine L; (4) Leucine L at position 60 is mutated to glycine G; (5) Glutamine Q at position 71 is mutated to histidine H; (6) Asparagine N at position 104 is mutated to serine S; (7) Lysine K at position 149 is mutated to valine V; (8) Threonine T at position 163 is mutated to asparagine. Amide N, (9) Valine V at position 174 is mutated to glutamine Q, (10) Threonine T at position 189 is mutated to cysteine ​​C, (11) Proline P at position 203 is mutated to tyrosine Y, (12) Aspartic acid D at position 225 is mutated to alanine A, (13) Isoleucine I at position 247 is mutated to glutamine Q, (14) Leucine L at position 253 is mutated to tryptophan W, (15) Glycine G at position 262 is mutated to histidine H.

[0008] This invention uses site-directed mutagenesis to obtain mutants with significantly enhanced enzyme activity, which can be better applied to the preparation of dried hippocampus.

[0009] Wild-type transaminase is derived from Moesziomyces antarcticus.

[0010] The most preferred option is a combination of four mutation sites: glutamine Q at position 71 is mutated to histidine H, asparagine N at position 104 is mutated to serine S, threonine T at position 189 is mutated to cysteine ​​C, and leucine L at position 253 is mutated to tryptophan W.

[0011] A polynucleotide sequence encoding the lipase mutant described above.

[0012] A recombinant vector comprising the aforementioned polynucleotide sequence.

[0013] A host cell comprising the recombinant vector. The host cell is preferably *Escherichia coli*.

[0014] The application of the lipase mutant as an enzyme catalyst in the preparation of defatted dried hippocampus.

[0015] A method for preparing defatted dried seahorse involves enzymatically hydrolyzing the dried seahorse using the aforementioned lipase mutant, followed by enzyme inactivation, filtration, washing, and drying.

[0016] The conditions for enzymatic hydrolysis are: pH 7.5-8.5, temperature 40-60℃, and hydrolysis time 2-6 hours. The preferred enzyme dosage is 30-40 U / g hippocampus.

[0017] The beneficial effects of this invention are: 1. Compared with the parental lipase, the lipase mutant provided by this invention has significantly improved enzyme activity, up to about 10 times, and has a superior performance in decomposing fat, shortening the defatting process time, improving production efficiency, and better meeting the needs of the dried seahorse preparation process.

[0018] 2. The application of this lipase mutant is expected to achieve greening, efficiency and standardization of the seahorse defatting process, improve the quality stability and market competitiveness of seahorse products, and have significant economic and social benefits. Attached Figure Description

[0019] Figure 1 This is an image of dried seahorse after the parental lipase has been defatted. Figure 2 This is an image of the hippocampus after defatting treatment of the Clip-22 mutant. Detailed Implementation

[0020] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0021] The *E. coli* host TOP10, BL21(DE3), expression vector pET-30a(+), plasmid extraction kit, and DNA gel recovery kit used in this invention were all purchased from Beijing Qingke Biotechnology Co., Ltd. The restriction endonucleases, high-fidelity polymerases, and DNA ligases used were all purchased from Nanjing Novizan Biotechnology Co., Ltd. Other reagents, raw materials, and equipment, unless otherwise specified, were commercially available. In the following specific examples, unless otherwise specified, all experimental operations can be performed according to conventional methods and conditions in the art or the manufacturer's recommended instructions.

[0022] The culture medium formulation used in this invention is as follows: LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl; LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 g / L agar.

[0023] Seahorse crude fat extraction Accurately weigh dried seahorse powder and place it in a round-bottom flask. Add petroleum ether at a solid-liquid ratio of 1g:10mL, and extract twice by reflux, 1 hour each time. Combine all extracts. Remove the organic solvent from the combined extract by vacuum distillation until constant weight is achieved to obtain crude seahorse fat. Transfer the obtained crude fat to a desiccator for drying and storage, as a substrate for subsequent enzymatic hydrolysis.

[0024] Lipase screening enzyme activity detection method Lipase hydrolysis of substrates to produce free fatty acids leads to a decrease in the pH of the reaction system. Enzyme activity is compared based on the amount of alkali consumed per unit time to maintain a constant pH in the reaction system. Using extracted crude seahorse fat as a substrate, 50 mg of substrate was added to 5 mL of enzyme solution, and the reaction was carried out at pH 8.0 and 40℃ for 2 h. After the reaction was completed, the enzymatic reaction was terminated by boiling water bath, phenolphthalein was added, and the relative enzyme activity was compared by titration with 0.05M sodium hydroxide standard solution.

[0025] Methods for detecting the enzyme activity of lipase mutants Add 5 mL of olive oil emulsion substrate to 5 mL of enzyme solution and react for 15 min at pH 8.0 and 60℃. After the reaction, add an equal volume of 95% ethanol to terminate the reaction, then add phenolphthalein and titrate with sodium hydroxide standard solution. Compare enzyme activities based on the volume of sodium hydroxide consumed. Calculate the relative enzyme activities of different mutant lipases, taking the parental lipase activity as 100%.

[0026] Olive oil emulsion: Mix olive oil and 4% polyvinyl alcohol solution at a volume ratio of 1:3, homogenize using a high-pressure homogenizer, and obtain olive oil emulsion for use after complete homogenization.

[0027] Example 1 Screening and Obtaining of Parental Lipase Engineered Bacteria Wild-type lipases from Moesziomyces antarcticus (GenBank ID: BBC47797.1), Limtongozyma cylindracea (GenBank: CAA46806.1), Rhizopusoryzae (GenBank: AAF32408.1), and Pseudomonas aeruginosa (GenBank: BAF92628.1) were synthesized by Beijing Qingke Biotechnology Co., Ltd. by codon optimization of the gene sequence encoding the amino acid. To facilitate subsequent molecular biology operations, an NdeI restriction site was added to the 5' end of the gene fragment, and a KpnI restriction site was added to the 3' end.

[0028] The synthesized gene and vector pET-30a(+) were double-digested with restriction endonucleases NdeI and KpnI, respectively. The fragments were then recovered using a DNA gel extraction kit, ligated with T4 DNA ligase, and transformed into *E. coli* TOP10. The fragments were plated on LB agar plates containing Kan antibody (50 mg / L) for screening to identify positive clones. These positive clones were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing to obtain recombinant lipase expression vectors from different sources.

[0029] The successfully constructed vector plasmid was transformed into Escherichia coli BL21(DE3) by heat shock. The bacterial culture was then plated on LB agar plates containing Kan resistance (50 mg / L) to obtain positive engineered bacteria capable of expressing lipase.

[0030] Preparation of lipase expression from different sources Lipase expression strains from different sources were inoculated at a 1% inoculum into 10 mL of LB liquid medium containing Kan resistance (50 mg / L) and cultured overnight at 180 rpm and 37°C in a shaker. The overnight culture was then transferred at a 1% inoculum into 100 mL of LB liquid medium containing Kan resistance (50 mg / L) and cultured at 200 rpm and 37°C in a shaker until OD (digestion / extraction) was achieved. 600 When the protein content reaches approximately 0.6-0.8, IPTG at a final concentration of 0.1 mM is added to induce protein expression. The mixture is then cultured at 180 rpm and 24°C for 16 h to obtain the fermentation broth. The fermentation broth is then centrifuged at 4°C and 8000 rpm to obtain the fermented cells, which are ready for use.

[0031] Weigh the fermentation cells and dissolve them in 50 mM Tris-HCl buffer (pH 8.0) to a concentration of 50 mg / mL. Place the cells in an ice-water mixture for ultrasonic disruption, centrifuge, and retain the supernatant enzyme solution for relative enzyme activity detection to screen for dominant lipases.

[0032] Table 1 Dominant wild-type lipases .

[0033] The dominant lipase Clip, selected from Table 1, was used as the parental lipase for mutation screening. Its amino acid sequence is shown in SEQ ID NO.1: MKLLSLTGVAGVLATCVAATPLVKRLPSGSDPAFSQPKSVLDAGLTCQGASPSSVSKPILLVPGTGTTGPQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQVNTEYMVNAITTLYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRLMAFAPDYKGTVLAGPLDALA VSAPSVWQQTTGSALTTALRNSGGLTQIVPTTNLYSATDEIVQPQVSNSPLDSSYLFNGKNVQAVCGPLFVIDHAGSLTSQFSYVVGRSALRSTTGQARSADYGITDCNPLPANDLTPEQKVAAAALLAPAAAAIVAGPKQNCEPDLMPYARPFAVGKRTCSGIVTP(SEQ ID NO.1).

[0034] Example 2 Construction of lipase single-point mutant library Using the parental lipase recombinant expression vector constructed in Example 1 as a template, a single-point mutant library was constructed using site-directed mutagenesis. The primer design is shown in Table 2. Table 2 Primer Table

[0035] PCR was performed based on the original parental lipase sequence. The PCR reaction system and reaction conditions are shown in Tables 3 and 4 below. Table 3 PCR amplification system ; Table 4 PCR Amplification Conditions .

[0036] After PCR, the PCR products were digested with the restriction endonuclease DpnI at 37°C for 1 hour, and then transformed into E. coli host cells BL21(DE3). Positive clones with successful mutations were screened using LB plates containing Kan resistance. The DpnI digestion system is shown in the table below: Table 5 Enzyme digestion system .

[0037] Example 3 Parental and mutant lipase expression preparation The parental lipase and single-point mutant expression strain were inoculated at a rate of 1% into 10 mL of LB liquid medium containing Kan resistance (50 mg / L) and cultured overnight at 180 rpm and 37°C in a shaker. The overnight culture was then transferred at a rate of 1% to 100 mL of LB liquid medium containing Kan resistance (50 mg / L) and cultured at 200 rpm and 37°C in a shaker until OD (outcome limit) was reached. 600 When the protein content reaches approximately 0.6-0.8, IPTG at a final concentration of 0.1 mM is added to induce protein expression. The mixture is then cultured at 180 rpm and 24°C for 16 h to obtain the fermentation broth. The fermentation broth is then centrifuged at 4°C and 8000 rpm to obtain the fermented cells, which are ready for use.

[0038] Weigh the fermentation cells and dissolve them in 50 mM Tris-HCl buffer (pH 8.0) to a concentration of 50 mg / mL. Place the cells in an ice-water mixture for ultrasonic disruption, centrifuge, and retain the supernatant enzyme solution for relative enzyme activity detection to screen for dominant mutants.

[0039] Single-point mutants with enzyme activity higher than the parental strain were sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing, and the dominant mutant strains were obtained, as shown in Table 6: Table 6 Dominant Single-Point Mutants .

[0040] Example 4 Construction of lipase multi-point mutant library Using the dominant mutant gene sequence screened in Example 3 as a template, iterative mutations were continued. Primer design was the same as in Table 2, PCR reaction and transformation procedures were the same as in Example 2, and enzyme solution preparation and enzyme activity detection methods were the same as in Example 3. The enzyme activity detection screening results are shown in the table below: Table 7 Advantageous multi-point mutants .

[0041] Starting with the parental lipase strain, after multiple rounds of mutant library construction, screening, and subculturing, the lipase activity of the mutant strains was detected and screened using standard enzyme activity detection methods. Finally, a superior tetramutant strain with stable genetic traits and significantly enhanced enzyme activity was obtained, named Clip-22 (Q71H / N104S / T189C / L253W). Compared with the original parental lipase strain, this mutant strain showed approximately 10-fold increased lipase activity under the same culture conditions and enzyme activity detection conditions, and its enzymatic reaction stability remained good, demonstrating significant potential for industrial application.

[0042] Example 5 This embodiment compares the defatting effects of using parental lipase and multi-point dominant mutants in the defatting process of dry hippocampus.

[0043] Hippocampus defatting process: Fresh seahorses were washed and pre-dried in a 60℃ oven until the moisture content was ≤15%, obtaining semi-dried seahorse samples. 10 g of the semi-dried seahorse sample was weighed and added to 100 mL of Tris-HCl buffer (50 mM, pH 8.0). Parental lipase solution and other multi-site dominant mutant lipase solutions (final enzyme activity approximately 30 U / g seahorse) were added separately. The total volume was brought up with Tris-HCl buffer to ensure consistency across all experimental groups. The mixture was incubated at 55℃ and 150 rpm for 4 h for enzymatic digestion. After the reaction, the mixture was centrifuged at 3000 rpm for 10 min, the precipitate was collected, washed three times with deionized water, and freeze-dried to constant weight to obtain the defatted dried seahorse product.

[0044] Index testing: Fat content was determined and defatting rate was calculated using Soxhlet extraction method. For specific methods, please refer to the national standard GB5009.6—2016.

[0045] Table 8 below compares the defatting performance of the parental lipase and the multi-point dominant mutant during the defatting process of hippocampal dry matter: Table 8 Comparison of defatting performance between parents and multi-point advantage mutants .

[0046] Example 6 This embodiment compares the effects of using parental lipase and the tetram mutant (Clip-22, i.e., Q71H / N104S / T189C / L253W) in the process of defatting dried hippocampus.

[0047] Degreasing process of seahorses: Fresh seahorses were washed and pre-dried in a 60℃ oven until the moisture content was ≤15% to obtain semi-dried seahorse samples. 10 g of the semi-dried seahorse sample was weighed and 100 mL of Tris-HCl buffer was added. The following solutions were added separately: Experimental Group 1: 30 mL of parental lipase solution (final enzyme activity approximately 30 U / g seahorse) and Experimental Group 2: 3 mL of Clip-22 lipase solution (final enzyme activity approximately 30 U / g seahorse). The total volume was adjusted with Tris-HCl buffer to ensure consistency between the experimental groups. The mixture was placed at 55℃ and 150 rpm for 4 h for enzymatic hydrolysis. After the reaction, the mixture was centrifuged at 3000 rpm for 10 min, the precipitate was collected, washed three times with deionized water, and freeze-dried to constant weight to obtain the defatted dried seahorse product.

[0048] Index testing: Fat content was determined and defatting rate was calculated using Soxhlet extraction. Specific methods are detailed in national standard GB5009.6—2016.

[0049] Sensory evaluation is conducted by three researchers who score the odor and texture (1-5 points, with 5 being the best). For specific evaluation methods, please refer to GB / T 37062-2018 "Guidelines for Sensory Evaluation of Aquatic Products".

[0050] Table 8 below compares the performance of the parental lipase and the Clip-22 mutant in the process of defatting hippocampus: Table 9. Performance Comparison between Parental Line and Clip-22 Mutant .

[0051] The dried seahorses treated with parental lipase had poorer morphology. Figure 1 ), while the hippocampal stem treated with the Clip-22 mutant had a better morphology ( Figure 2 The Clip-22 lipase mutant exhibits higher catalytic efficiency, better product protection, and superior sensory quality in the dry degreasing process of hippocampus. It can replace the traditional degreasing process and has significant industrial application value.

[0052] Example 7 This example is an industrial-scale application of the lipase mutant Clip-22 in the defatting of dry hippocampus, compared with traditional hot water extraction and defatting with commercially available lipase.

[0053] Traditional hot water extraction method for degreasing: Take 80kg of fresh seahorses, wash them with running water, drain the surface water, and blanch them in 95-100℃ slightly boiling pure water for 15-25 seconds to degrease them. During this time, gently stir 1-2 times to ensure even heating. When the body surface tightens slightly, the body color turns slightly lighter and oil droplets are separated, immediately take them out and quickly put them into sterile water at room temperature to cool to room temperature for 3-5 minutes. Gently wipe the body surface oil, mucus and foam with a sterile soft cloth to remove them, and drain them again. Then put them into a 60℃ vacuum dryer to dry until the moisture content is ≤10%.

[0054] Seahorse enzymatic defatting process: 80 kg of fresh seahorses were washed with running water and drained. They were pretreated in a 65℃ hot air drying oven for 4 hours to reduce the moisture content to 18-20%. The pretreated seahorses were then placed in an enzymatic hydrolysis tank, and 400 L of 50 mM Tris-HCl buffer (pH 8.0) was added. 800 mL of Clip-22 lipase mutant enzyme solution (enzyme activity approximately 300 U / mL, final enzyme activity approximately 30 U / g seahorse) and 24 g of commercial lipase (enzyme activity approximately 100,000 U / g, final enzyme activity approximately 30 U / g seahorse) were added. The reaction conditions were controlled at 55℃, stirring speed at 60 rpm, and reaction time at 5 hours. During the process, samples were taken every hour to detect pH and free fatty acid release; the pH was fine-tuned with 1M NaOH if necessary. After the reaction, the temperature was raised to 85℃ and held for 10 minutes to inactivate the enzyme. The solid and liquid were separated using a plate and frame filter press, and the solid was washed twice with 200 L of pure water. Then dry in a vacuum dryer at 60°C until the moisture content is ≤10%.

[0055] The comparison results are shown in Table 9 below.

[0056] Table 10 Comparison of Clip-22 mutant application in hippocampal defatting with conventional methods .

[0057] After three consecutive batches, the defatting rates of the Clip-22 mutant were 81.6%, 82.1%, and 80.9%, respectively, with RSD < 1.5%.

[0058] After three cycles of enzyme solution recycling, the enzyme activity retention rate was >85%. The product's moisture and fat content met the standards for dried seahorse products in the Chinese Pharmacopoeia. In this industrial-scale test, compared to traditional hot water extraction and degreasing with commercial lipases, the Clip-22 lipase mutant applied to the degreasing process of dried seahorse demonstrated good stability, high degreasing efficiency, and excellent product quality. This process has significant advantages in degreasing rate, time cost, product yield, and sensory quality, and has the potential for large-scale production application.

[0059] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications may be made without departing from the technical solutions described in the claims.

Claims

1. A lipase mutant, characterized in that, The transaminase with the amino acid sequence shown in SEQ ID NO.1 was mutated from a wild-type transaminase, and the mutation site was selected from one or more combinations of the following schemes: (1) Valine V at position 40 is mutated to isoleucine I; (2) Threonine T at position 46 is mutated to lysine K; (3) Serine S at position 53 is mutated to leucine L; (4) Leucine L at position 60 is mutated to glycine G; (5) Glutamine Q at position 71 is mutated to histidine H; (6) Asparagine N at position 104 is mutated to serine S; (7) Lysine K at position 149 is mutated to valine V; (8) Threonine T at position 163 is mutated to asparagine. Amide N, (9) Valine V at position 174 is mutated to glutamine Q, (10) Threonine T at position 189 is mutated to cysteine ​​C, (11) Proline P at position 203 is mutated to tyrosine Y, (12) Aspartic acid D at position 225 is mutated to alanine A, (13) Isoleucine I at position 247 is mutated to glutamine Q, (14) Leucine L at position 253 is mutated to tryptophan W, (15) Glycine G at position 262 is mutated to histidine H.

2. The lipase mutant according to claim 1, characterized in that, Wild-type transaminase is derived from Ustilago maydis in Antarctica.

3. A lipase mutant according to claim 1, characterized in that, The mutation sites are a combination of four mutations: glutamine Q at position 71 is mutated to histidine H, asparagine N at position 104 is mutated to serine S, threonine T at position 189 is mutated to cysteine ​​C, and leucine L at position 253 is mutated to tryptophan W.

4. A polynucleotide sequence, characterized in that, It encodes the lipase mutant of claim 1.

5. A recombinant vector, characterized in that, It contains the polynucleotide sequence as described in claim 3.

6. A host cell, characterized in that, It comprises the recombinant vector as described in claim 4.

7. The application of the lipase mutant as described in claim 1 as an enzyme catalyst in the preparation of defatted dried hippocampus.

8. A method for preparing defatted dried seahorse, characterized in that, The dried hippocampus was subjected to enzymatic hydrolysis using the lipase mutant as described in claim 1, followed by enzyme inactivation, filtration, washing, and drying.

9. The preparation method according to claim 8, characterized in that, The conditions for enzymatic hydrolysis are: pH 7.5-8.5, temperature 40-60℃, and hydrolysis time 2-6 hours.

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