Biosynthesis method of multi-effect cosmetic short peptide and long peptide connected in series

Abstract

The invention discloses a biosynthesis method of multi-effect cosmetic oligopeptides and long peptides connected in series, and relates to the technical field of biosynthesis, the method comprises the following specific steps: oligopeptide selection: collecting information of known oligopeptides with skin care efficacy, and screening out a target oligopeptide sequence. A plurality of selected typical short peptides with different skin care effects are connected in series to form medium peptides, then a plurality of different medium peptides are obtained by disorganizing the sequence and connecting the medium peptides in series for many times, and finally the medium peptides are connected in series to form the long peptide with the molecular weight of 50-60kDa and no repeatability, so that the long peptide sequence is theoretically easier to express, and compared with the prior art, the expression quantity is increased by several times; the molecular weight of the long peptide can be conveniently detected by PAGE (Polyacrylamide Gel Electrophoresis), the long peptide can be conveniently purified by a membrane filtration method, dozens of short peptides can be synthesized at one time, and the requirement of consumers on multiple effects of cosmetics is met.

Description

Technical Field

[0001] The present invention relates to the technical field of biosynthesis, and in particular to a biosynthesis method for connecting a short peptide of a multi-functional cosmetic with a long peptide in series. Background Art

[0002] In today's booming beauty and personal care industry, cosmetics, as an important product to meet consumers' pursuit of skin health and beauty, have always been the focus of industry attention in terms of research and development and innovation. With the continuous advancement of biotechnology, the application of bioactive ingredients in the cosmetics field is becoming more and more extensive. Short peptides with specific biological activities have become a hot spot in the research and development of cosmetic raw materials due to their small molecular weight, clear structure, easy penetration of the skin barrier and ability to regulate specific cell signaling pathways.

[0003] In terms of short peptide synthesis technology, chemical synthesis and biosynthesis are the two main routes. Although chemical synthesis can precisely control the reaction process, it has many disadvantages. The reaction conditions of chemical synthesis are often harsh and have high requirements for reaction equipment and environment. This not only increases production costs but also makes large-scale production face many difficulties. At the same time, the chemical synthesis process may use toxic or explosive solvents and produce a large number of by-products. These substances not only affect the purity of the product, but also may cause potential irritation and harm to the skin, and pose a major safety hazard. Biosynthesis technology is based on the metabolic mechanism of the organism itself, with mild reaction conditions. It is usually carried out in an environment close to physiological conditions, which greatly reduces the requirements for equipment and energy consumption, making production costs lower. Moreover, the biosynthesis process is highly specific, does not require toxic or explosive solvents, and has few by-products. It can effectively avoid the potential risks brought by toxic or explosive solvents and by-products in chemical synthesis, and the short peptide products produced are of higher purity and better safety.

[0004] At present, although there are technical attempts to synthesize short peptides using biological genetic engineering methods, that is, first forming a mid-peptide by tandem repeating the amino acid sequence of the short peptide, and then obtaining the short peptide by enzymatic cleavage, this traditional technology has many obvious disadvantages. First, the high tandem repetition of the amino acid sequence causes great difficulties in the gene synthesis and gene expression of the mid-peptide, which seriously affects the synthesis efficiency of the short peptide. In addition, the molecular weight range of the mid-peptide is 5-15 kDa, which is not very suitable for biological genetic engineering expression, further limiting the synthesis scale of the short peptide and making it difficult to detect the expression using PAGE gel electrophoresis. In addition, in the subsequent separation and purification stage, when the mid-peptide is separated by membrane filtration, it is difficult to separate and purify due to its close molecular weight to some small molecules originally in the organism, and it is difficult to separate it from small molecule odorous substances, making it difficult to effectively reduce the product odor and affecting product quality. Moreover, during the gene synthesis process, the highly tandem repeated sequence is very prone to erroneous recombination, resulting in synthesis failure or the production of erroneous products. In addition, the design length of the mid-peptide is limited, and the number of oligopeptides contained is small, so only 1-2 short peptides can be synthesized at a time, with a single function, which cannot meet the market demand for multi-functional cosmetics. Summary of the Invention

[0005] The purpose of the present invention is to make up for the deficiencies of the prior art and provide a biosynthesis method for a multi-functional cosmetic short peptide connected in series with a long peptide. The method can first connect multiple different short peptides in series to form a mid-peptide through a unique connection method, and then perform multiple rounds of scrambling and connection operations on the short peptides in the mid-peptide to obtain multiple different mid-peptides. These different mid-peptides are then connected in series again to form long peptides, thereby eliminating the repeatability of the long peptides after connection, and strictly controlling the molecular weight of the long peptide to be 50-60kDa. In the biosynthesis step, the polypeptide sequence corresponding to the long peptide is converted into a nucleotide sequence, and the nucleotide sequence is obtained by gene synthesis technology and then inserted into an expression vector, which is then transformed into an expression strain for expression, and the expressed long peptide is detected by PAGE gel electrophoresis, and then used The membrane filtration method separates and purifies long peptides, and finally the expressed long peptides are enzymatically cleaved into the original short peptides using trypsin. The long peptides eliminated by the present invention are easier to express, and the expression amount is increased several times. The molecular weight range of 50-60kDa is more consistent with the expression of biological genetic engineering methods. Sequence error recombination is not easy to occur during gene synthesis, which reduces the difficulty of synthesis. The molecular weight range of 50-60kDa is suitable for PAGE gel electrophoresis detection. In addition, in the subsequent separation and purification stage, when the membrane filtration method is used to separate the long peptides, since the molecular weight of the long peptides is very different from the molecular weight of some small molecules originally in the organism, it is easy to separate and purify, and it is easy to separate from small molecule odorous substances, so that the product odor can be effectively reduced and the product quality can be improved. Due to the elimination of sequence repetitiveness, the long peptide sequence can be designed to be longer than the traditional method, containing more oligopeptides, achieving more skin care effects, and dozens of short peptides can be synthesized at a time. The synthesis efficiency is high and the efficacy is diverse. At the same time, trypsin is used for enzymatic cleavage, and the cost is low, providing a high-efficiency, low-cost, and multi-functional short peptide synthesis solution for the field of cosmetic raw material synthesis technology.

[0006] In order to solve the above technical problems, the present invention provides the following technical solution: a biosynthesis method of a multifunctional cosmetic short peptide connected in series with a long peptide, the method comprising the following specific steps:

[0007] Short peptide selection: Collect information on short peptides known to have skin care effects and screen out target short peptide sequences;

[0008] Mid-peptide synthesis: The selected short peptides are concatenated into mid-peptides, and then the order is disrupted and concatenated multiple times, and then re-concatenated into multiple different mid-peptides;

[0009] Long peptide synthesis: multiple different medium peptides are concatenated into long peptides to eliminate repetitiveness and control the molecular weight to 50-60kDa;

[0010] Biosynthesis preparation: convert the long peptide amino acid sequence into nucleotide sequence according to the genetic codon table and strain codon preference, synthesize the DNA fragment, construct the recombinant expression vector and verify it;

[0011] Transformation and expression: Prepare Escherichia coli or Pichia pastoris competent cells, transform the recombinant expression vector into them, screen positive clones and induce or non-induced expression to express the long peptide.

[0012] Electrophoresis: PAGE gel electrophoresis was used to detect the expressed long peptides.

[0013] Separation and purification: Use membrane filtration to separate long peptides and remove some small molecules and odorous substances originally present in the organism.

[0014] Acquisition of short peptides: long peptides were cleaved with trypsin, separated and purified by HPLC technology, and then detected and identified.

[0015] Furthermore, in the short peptide selection step, information on short peptides known to have skin care effects is collected, and the short peptides include: YR, VK, CK, GHK, GHR, HFR, GPR, GPK, AHK, NCR, AQK, GQPR, ALAK, HLLR, MHIR, YIGSR, LPPSR, LQDGVR and YLPCPVTSK.

[0016] Furthermore, in the mid-peptide synthesis step, short peptides are connected in series to form a mid-peptide, and then the order is shuffled and then connected in series to form different mid-peptides, and these short peptides are connected in series to form multiple different mid-peptides after multiple rounds of shuffling, including but not limited to the following sequences:

[0017] GHRYRVKCKGHKHFRGPRGPKAHKNCRAQKGQPRALAKHLLRMHIRYIGSRLPPSRLQDGVRYLPCPVTSK;

[0018] CKMHIRGQPRLQDGVRYRGPKYIGSRGHRNCRYLPCPVTSKVKHFRHLLRGPRALAKAQKLPPSRHKAHK;

[0019] GQPRCKMHIRYLPCPVTSKYRGPKLPPSRAQKGHKHLLRGPRLQDGVRGHRALAKNCRVKAHKYIGSRHFR;

[0020] YIGSRGPRHLLRCKNCRYLPPCPVTSKYRAHKLQDGVRGHRALAKVKGQPRLPPSRHFRMHIRGPKAQKGHK;

[0021] GHKNCRYIGSRGPKHLLRYLPCPVTSKVKLPPSRHFRGQPRCKGHRYRALAKAHKLQDGVRGPRAQKMHIR;

[0022] ALAKVKYIGSRGHRAHKLQDGVRGHKAQKYRMHIRYLPCPVTSKGPRGQPRCKNCRLPPSRHFRHLLRGPK;

[0023] NCRMHIRYRGPKGHKLQDGVRHFRALAKCKLPPSRAQKYLPCPVTSKGPRGQPRVKHLLRAHKGHRYIGSR.

[0024] Furthermore, in the long peptide synthesis step, a plurality of different mid-peptides are connected to form a long peptide with repetitive sequences eliminated, and the long peptide includes but is not limited to the following sequence: GHRYRVKCKGHKHFRGPRGPKAHKNCRAQKGQPRALAKHLLRMHIRYIGSRLPPSRLQDGVRYLPCPVTSKCKMHIRGQPRLQDGVRYRGPKYIGSRGHRNCRYLPCPVTSKVKHFRHLLRGPRALAKAQKLPPSRGHKAHKGQPRCKMHIRYLPCPVTSKYRGPKLPPSRAQKGHKHLLRGPRLQDGVRGHRALAKNCRVKAHKYIGSRHFRYIGSRGPRH LLRCKNCRYLPPCPVTSKYRAHKLQDGVRGHRALAKVKGQPRLPPSRHFRMHIRGPKAQKGHKGHKNCRYIGSRGPKHLLRYLPCPVTSKVKLPPSRHFRGQPRCKGHRYRALAKAHKLQDGVRGPRAQKMHIRALAKV KYIGSRGHRAHKLQDGVRGHKAQKYRMHIRYLPCPVTSKGPRGQPRCKNCRLPPSRHFRHLLRGPKNCRMHIRYRGPKGHKLQDGVRHFRALAKCKLPPSRAQKYLPCPVTSKGPRGQPRVKHLLRAHKGHRYIGSR.

[0025] Furthermore, in the long peptide synthesis step, the number and length of the intermediate peptides are controlled during the synthesis process so that the molecular weight of the finally synthesized long peptide is in the range of 50-60 kDa.

[0026] Furthermore, in the biosynthesis preparation step, the amino acid sequence of the long peptide is reverse-translated into a nucleotide sequence using a software tool based on the genetic codon table. During the translation process, the nucleotide sequence is optimized according to the codon preference of the selected expression strain so that the codon adaptation index target value reaches 0.8 or above. The optimized nucleotide sequence is synthesized into the corresponding DNA fragment by chemical synthesis, and the pPICZ series vector for Pichia pastoris expression or the pET series vector for Escherichia coli expression is selected. The expression vector and the synthesized DNA fragment are double-digested with the corresponding restriction endonuclease, using 5-10 units of enzyme per microgram of DNA, and the reaction time is 1-3 hours. The DNA fragment after enzyme digestion is ligated with the vector using DNA ligase, and the molar ratio of DNA fragment to vector is 3:1-10:1. The amount of T4 DNA ligase used is 1-3 units of enzyme per microgram of DNA. The reaction temperature is 16°C and the reaction time is 12-16 hours. The recombinant expression vector is constructed, and the recombinant vector containing the insert is preliminarily screened by PCR amplification. The recombinant vector is then sequenced and compared with the original design sequence to confirm the successful construction of the vector.

[0027] Furthermore, in the transformation and expression steps, competent Escherichia coli or Pichia pastoris cells are prepared. Specifically, for Escherichia coli, Escherichia coli cells in the logarithmic growth phase are suspended in a 0.1M ice-cold calcium chloride solution and ice-bathed for 30-60 minutes to render the cells competent. During heat shock transformation, the recombinant vector is mixed with the competent cells and then ice-bathed. They are quickly placed at 42°C for a heat shock of 45-90 seconds, then cooled in an ice bath for 2-5 minutes, and then cultured with culture medium. For Pichia pastoris, Pichia pastoris cells in the logarithmic growth phase are washed and resuspended in an ice-cold sorbitol solution to prepare competent cells. After mixing the recombinant vector with the competent Pichia pastoris cells, they are subjected to electric pulse treatment under specific voltage and capacitance conditions, and then cultured with culture medium.

[0028] 13. Furthermore, in the transformation and expression steps, after screening for positive clones, expression is induced or non-induced. Specifically, the transformed cells are plated on a plate containing corresponding antibiotics, including ampicillin and bleomycin, and positive clones containing the recombinant vector are screened. Single colonies are picked for PCR identification or sequencing verification to determine positive clones, and the positive clones are inoculated into liquid culture medium. For the Escherichia coli expression system, IPTG inducer is added to induce recombinant protein expression. The IPTG concentration is 0.1-1 mM, the induction temperature is 16-37°C, and the induction time is 4-24 hours. For the Pichia pastoris pPIC series expression system, methanol medium is replaced to induce expression. The methanol concentration is initially 0.5%-1% during induction and adjusted to 1%-2% based on subsequent growth conditions. The induction temperature is 28-30°C and the induction time is 48-96 hours. For the Pichia pastoris pGAP series expression system, no induction is required.

[0029] Furthermore, in the short peptide acquisition step, after the induction expression is completed, the expressed long peptide is detected by PAGE gel electrophoresis.

[0030] Furthermore, membrane filtration is used to separate long peptides and remove some small molecules and odorous substances originally present in the organism.

[0031] Trypsin is added to the long peptide in a ratio of trypsin to long peptide of 1:50-1:200. The enzymatic cleavage reaction is carried out at 37°C and pH 7.5-8.5 for a reaction time of 4-24 hours. Samples are taken regularly during the reaction and the progress of the enzymatic cleavage is analyzed by HPLC. After the enzymatic cleavage reaction is completed, the enzymatic cleavage product is separated and purified by HPLC. The HPLC parameters are set as follows: the mobile phase adopts a water-acetonitrile system, the acetonitrile gradient is adjusted according to the properties of the short peptide, ranging from 5% to 60%, the flow rate is 0.5-2mL / min, the detection wavelength is 214nm or 280nm, the molecular weight of the short peptide is determined by mass spectrometry to determine its accuracy, the amino acid composition of the short peptide is determined by amino acid analysis, and the biological activity of the short peptide is evaluated through activity detection experiments to ensure that the obtained short peptide meets the expected skin care efficacy requirements.

[0032] Compared with the existing technology, this biosynthesis method of multi-functional cosmetic short peptides connected in series with long peptides has the following beneficial effects:

[0033] 1. The present invention uses a unique polypeptide concatenation strategy to first concatenate a variety of typical short peptides with different skin care effects to form a medium peptide, then shuffle the order and concatenate multiple times to obtain multiple different medium peptides, and finally concatenate these medium peptides into long peptides with a molecular weight of 50-60kDa and no repetitiveness. This design makes long peptide sequences theoretically easier to express, and the expression amount is increased several times compared to the existing technology. At the same time, dozens of short peptides can be synthesized at one time, meeting consumers' demand for multiple functions of cosmetics. In addition, when enzymatically cleaving long peptides to obtain short peptides, the use of low-cost trypsin effectively reduces the synthesis cost of short peptides, thereby improving the economy and competitiveness of this technology in the field of cosmetic raw material synthesis.

[0034] 2. The present invention eliminates repetitiveness through long peptide sequences, greatly reduces the possibility of sequence error recombination, significantly reduces the difficulty of gene synthesis, improves the accuracy and success rate of gene synthesis, and makes it easy to detect the expressed long peptides by PAGE gel electrophoresis. At the same time, after the long peptide sequence is eliminated from repetitiveness, it can contain more oligopeptides, thereby achieving more skin care effects and broadening the application range of cosmetic raw materials. During the long peptide synthesis process, the molecular weight is precisely controlled within the appropriate range of 50-60kDa, making it more suitable for expression by biological genetic engineering methods. Moreover, in subsequent separation and purification steps, due to the large molecular weight of the long peptide sequence, it is easy to use membrane filtration to separate the long peptide, remove some small molecules and odorous substances originally in the organism, reduce the risk of impurity mixing, and be more conducive to obtaining high-purity, low-odor short peptide products, thereby ensuring the quality and stability of the final cosmetic raw materials.

[0035] Other advantages, objects and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art based on an examination of the following or may be learned from the practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0037] Figure 1 This is a flow chart of the biosynthesis method of a multi-functional cosmetic short peptide connected in series with a long peptide;

[0038] Figure 2 The present invention is a flow chart of a biosynthetic method of a multifunctional cosmetic short peptide connected in series with a long peptide. DETAILED DESCRIPTION

[0039] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0040] Example 1

[0041] 19 typical short peptide sequences used in cosmetics, such as YR, VK, and CK, were screened out from literature and professional books.

[0042] At room temperature, short peptides are first concatenated in a certain order to form a mid-peptide. For example, the first concatenation obtains the GHRYRVKCKGHKHFRGPRGPKAHKNCRAQKGQPRALAKHLLRMHIR YIGSRLPPSRLQDGVRYLPCPVTSK sequence. The order is shuffled using a computer program and concatenated again, such as obtaining the CKMHIRGQPRLQDGVRYRGPKYIGSRGHRNCRYLPCPVTSKVKHFRHLLRGPRALAKAQKLPPSRGHKAHK sequence. This process is repeated many times to obtain multiple different mid-peptides.

[0043] Connect multiple different peptides in series, such as GHRYRVKCKGHKHFRGPRGPKAHKNCRAQKGQPRALAKHLLRMHIR YIGSRLPPSRLQDGVRYLPCPVTSK;

[0044] CKMHIRGQPRLQDGVRYRGPKYIGSRGHRNCRYLPCPVTSKVKHFRHLLRGPRALAKAQKLPPSRHKAHK;

[0045] GQPRCKMHIRYLPCPVTSKYRGPKLPPSRAQKGHKHLLRGPRLQDGVRGHRALAKNCRVKAHKYIGSRHFR;

[0046] YIGSRGPRHLLRCKNCRYLPPCPVTSKYRAHKLQDGVRGHRALAKVKGQPRLPPSRHFRMHIRGPKAQKGHK;

[0047] GHKNCRYIGSRGPKHLLRYLPCPVTSKVKLPPSRHFRGQPRCKGHRYRALAKAHKLQDGVRGPRAQKMHIR;

[0048] ALAKVKYIGSRGHRAHKLQDGVRGHKAQKYRMHIRYLPCPVTSKGPRGQPRCKNCRLPPSRHFRHLLRGPK;

[0049] NCRMHIRYRGPKGHKLQDGVRHFRALAKCKLPPSRAQKYLPCPVTSKGPRGQPRVKHLLRAHKGHRYIGSR

[0050] The same mid-peptides are connected in series to form long peptides with repeated sequences eliminated. The molecular weight is regulated by controlling the number and length of mid-peptides, so that the molecular weight of the long peptide reaches 50-60kDa.

[0051] According to the genetic codon table, the amino acid sequence of the long peptide was reverse translated into a nucleotide sequence. According to the codon preference of Escherichia coli, the nucleotide sequence was optimized using software to achieve a codon adaptation index (CAI) of 0.85. The optimized nucleotide sequence was synthesized, and restriction endonuclease recognition sites were added to both ends. The sequence was then subjected to double enzyme digestion and ligation with the pET-28a vector. 8 units of restriction endonuclease were used per microgram of DNA, and the reaction was carried out at 37°C for 2 hours. The molar ratio of DNA fragment to vector was set to 5:1. 2 units of T4 DNA ligase were used per microgram of DNA, and the reaction was carried out at 16°C for 14 hours. The recombinant expression vector was constructed, and the successful construction of the vector was verified by PCR screening and sequencing.

[0052] Escherichia coli BL21 competent cells were prepared by the calcium chloride method. The Escherichia coli cells were suspended in 0.1M ice-cold calcium chloride solution and ice-bathed for 45 minutes. The recombinant expression vector was transformed into the cells. The cells were heat-shocked at 42°C for 60 seconds and then ice-bathed for 3 minutes. Positive clones were screened on plates containing ampicillin, and single colonies were picked and inoculated into liquid culture medium and cultured to the logarithmic growth phase. IPTG was added to induce long peptide expression. The IPTG concentration was set to 0.5mM, the induction temperature was 20°C, and the induction time was 12 hours.

[0053] After the induction expression was completed, the bacteria were collected by centrifugation at 4°C and 6000 rpm for 12 minutes, and the long peptide was released by ultrasonic disruption. The expressed long peptide was detected by PAGE gel electrophoresis.

[0054] Membrane filtration is used to separate long peptides and remove some small molecules and odorous substances originally present in the organism.

[0055] Trypsin was added for enzymatic cleavage reaction, the mass ratio of trypsin to long peptide was set to 1:100, and the reaction was carried out at 37°C for 16 hours. The enzymatic cleavage product was separated and purified by high performance liquid chromatography, the mobile phase acetonitrile gradient was set to 10%-50%, the flow rate was 1.5 mL / min, and the detection wavelength was 214 nm. Mass spectrometry, amino acid analysis and other methods were used to detect the molecular weight and amino acid composition of the short peptide, and its skin care activity was evaluated through activity detection experiments.

[0056] The expression level of the long peptides without repetitiveness was significantly improved in E. coli, increasing by approximately three times compared to the expression level of traditional tandem peptides. This significantly increased the yield of short peptides obtained subsequently, meeting the needs of large-scale production. During the construction of the recombinant expression vector, since the long peptide sequence eliminated repetitiveness, no sequence errors or recombination occurred during gene synthesis, and operations such as enzyme cleavage and ligation were smooth, improving the success rate and efficiency of the experiment. The molecular weight range of 50-60kDa is suitable for PAGE gel electrophoresis detection. In addition, in the subsequent separation and purification stage, when the long peptides are separated by membrane filtration, the molecular weight of the long peptides is significantly different from that of some small molecules originally present in the organism, making them easy to separate and purify, and easy to separate from small odorous substances, which can effectively reduce product odor and improve product quality. The designed long peptides are of appropriate length and contain multiple oligopeptides. After enzyme cleavage, dozens of different short peptides can be obtained at one time. After testing, these short peptides have good activity and have multiple skin care effects such as anti-wrinkle, whitening, anti-inflammatory, and promoting collagen regeneration, expanding the functional diversity of cosmetic raw materials.

[0057] Example 2

[0058] 19 typical short peptides with skin care effects such as YR, VK, and CK were selected from academic literature and professional materials.

[0059] At room temperature, short peptides are first connected in a certain order to form medium peptides, like the first connection.

[0060] GQPRCKMHIRYLPPCPVTSKYRGPKLPPSRAQKGHKHLLRGPRLQDGVRGHRALAKNCRVKAHKYIGSRHFR

[0061] The sequence is then shuffled and concatenated using a computer program, for example, to obtain YIGSRGPRHLLRCKNCRYLPCPVTSKYRAHKLQDGVRGHRALAKVKG QPRLPPSRHFRMHIRGPKAQKGHK

[0062] The process was repeated many times to obtain multiple different peptides.

[0063] Concatenate multiple different mid-peptides into long peptides, such as GHRYRVKCKGHKHFRGPRGPKAHKNCRAQKGQPRALAKHLLRMHIR YIGSRLPPSRLQDGVRYLPCPVTSK;

[0064] CKMHIRGQPRLQDGVRYRGPKYIGSRGHRNCRYLPCPVTSKVKHFRHLLRGPRALAKAQKLPPSRHKAHK;

[0065] GQPRCKMHIRYLPCPVTSKYRGPKLPPSRAQKGHKHLLRGPRLQDGVRGHRALAKNCRVKAHKYIGSRHFR;

[0066] YIGSRGPRHLLRCKNCRYLPPCPVTSKYRAHKLQDGVRGHRALAKVKGQPRLPPSRHFRMHIRGPKAQKGHK;

[0067] GHKNCRYIGSRGPKHLLRYLPCPVTSKVKLPPSRHFRGQPRCKGHRYRALAKAHKLQDGVRGPRAQKMHIR;

[0068] ALAKVKYIGSRGHRAHKLQDGVRGHKAQKYRMHIRYLPCPVTSKGPRGQPRCKNCRLPPSRHFRHLLRGPK;

[0069] NCRMHIRYRGPKGHKLQDGVRHFRALAKCKLPPSRAQKYLPCPVTSKGPRGQPRVKHLLRAHKGHRYIGSR

[0070] The middle peptides are connected together, and the molecular weight is regulated by controlling the number and length of the middle peptides, so that the molecular weight of the long peptide is between 50-60kDa.

[0071] According to the genetic codon table, the amino acid sequence of the long peptide was reverse translated into a nucleotide sequence. According to the codon preference of Pichia pastoris, the nucleotide sequence was optimized using software to achieve a codon adaptation index (CAI) of 0.82. The optimized nucleotide sequence was synthesized, and restriction endonuclease recognition sites were added to both ends. The sequence was then subjected to double enzyme digestion and ligation reactions with the pET-28a vector. Six units of restriction endonuclease were used per microgram of DNA, and the reaction was carried out at 37°C for 2.5 hours. The molar ratio of DNA fragment to vector was set to 4:1, 1.5 units of T4 DNA ligase were used per microgram of DNA, and the reaction was carried out at 16°C for 15 hours. The recombinant expression vector was constructed, and the successful construction of the vector was verified by PCR screening and sequencing.

[0072] Pichia pastoris GS115 competent cells were prepared using electroporation. Pichia pastoris cells in the logarithmic growth phase were washed and resuspended in ice-cold sorbitol. The recombinant expression vector was then transformed into the competent cells. Electric pulses were then applied under specific voltage and capacitance conditions. The transformed cells were plated on ampicillin-containing plates to screen for positive clones. Single colonies were picked and inoculated into liquid culture medium and cultured until the logarithmic growth phase. For the Pichia pastoris pPIC expression system, methanol was added to induce long peptide expression. The initial methanol concentration was 0.6%, the induction temperature was 29°C, and the induction time was 60 hours. No induction was required for the Pichia pastoris pGAP expression system.

[0073] After induction, the cells were collected by centrifugation at 7000 rpm for 10 minutes at 4°C, and the long peptides were released by high-pressure homogenization. The long peptides were detected by PAGE gel electrophoresis.

[0074] The membrane filtration method is used to separate long peptides, remove some small molecules and odorous substances originally in the organism, and improve product quality.

[0075] Trypsin was added for enzymatic cleavage reaction with a mass ratio of trypsin to long peptide of 1:80. The reaction was carried out at 37°C for 12 hours. The enzymatic cleavage product was separated and purified by high performance liquid chromatography with an acetonitrile gradient of 8%-55% for the mobile phase at a flow rate of 1.2 mL / min and a detection wavelength of 280 nm. The molecular weight and amino acid composition of the short peptide were detected by mass spectrometry and amino acid analysis, and its skin care activity was evaluated through activity detection experiments.

[0076] The expression level of long peptides in Pichia pastoris was significantly improved, about 2.5 times higher than that of traditional methods, which provides a guarantee for the subsequent large-scale acquisition of short peptides. Due to the elimination of long peptide sequence repetitiveness, the gene synthesis and vector construction process was smooth, no obvious errors occurred, and the success rate of the experiment was improved. The molecular weight range of 50-60kDa is suitable for PAGE gel electrophoresis detection. In addition, in the subsequent separation and purification stage, when the long peptides are separated by membrane filtration, since the molecular weight of the long peptides is very different from that of some small molecules originally in the organism, it is easy to separate and purify, and it is easy to separate from small molecule odorous substances, so that the product odor can be effectively reduced and the product quality can be improved. The short peptides obtained by enzymatic cleavage are rich in variety and have various skin care effects, such as anti-wrinkle, moisturizing, anti-oxidation, etc., which meet the diversified needs of cosmetics.

[0077] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A biosynthetic method for a multifunctional cosmetic short peptide connected in series with a long peptide, characterized in that: The method comprises the following specific steps: Short peptide selection: Collect information on short peptides known to have skin care effects and screen out target short peptide sequences; Mid-peptide synthesis: multiple selected short peptides are concatenated into mid-peptides, and after multiple concatenations in a disrupted order, they are re-concatenated into different mid-peptides; Long peptide synthesis: Multiple different mid-peptides are connected in series to form a long peptide without internal repetitive sequences, with a controlled molecular weight of 50-60kDa; Biosynthesis preparation: convert the long peptide amino acid sequence into nucleotide sequence according to the genetic codon table and strain codon preference, synthesize the DNA fragment, construct the recombinant expression vector and verify it; Transformation and expression: Prepare E. coli or Pichia pastoris competent cells, transform them with the recombinant expression vector, screen positive clones, and then induce or non-induced expression; Long peptide detection: PAGE gel electrophoresis was used to detect the molecular weight of the long peptide, which was consistent with the theoretical value; Long peptide purification: long peptides are purified by membrane filtration; Acquisition of short peptides: The purified long peptide is digested with trypsin to obtain the desired short peptide; the short peptide is separated and purified by HPLC technology and then detected and identified.

2. The biosynthesis method of a multifunctional cosmetic short peptide connected in series with a long peptide according to claim 1, characterized in that: In the short peptide selection step, information on short peptides known to have skin care effects is collected, and the short peptides include: YR, VK, CK, GHK, GHR, HFR, GPR, GPK, AHK, NCR, AQK, GQPR, ALAK, HLLR, MHIR, YIGSR, LPPSR, LQDGVR and YLPCPVTSK.

3. The biosynthesis method of a multifunctional cosmetic short peptide connected in series with a long peptide according to claim 1, characterized in that: In the long peptide synthesis step, a plurality of different short peptides are first connected to form a medium peptide.

4. The biosynthesis method of a multifunctional cosmetic short peptide connected in series with a long peptide according to claim 1, characterized in that: In the mid-peptide synthesis step, the mid-peptide formed by connecting multiple different short peptides for the first time is disrupted in order multiple times and then connected in series to form multiple different mid-peptides.

5. The biosynthesis method of a multifunctional cosmetic short peptide connected in series with a long peptide according to claim 1, characterized in that: In the long peptide synthesis step, a plurality of different mid-peptides are connected to form a long peptide that eliminates repetitiveness.

6. The biosynthesis method of a multifunctional cosmetic short peptide connected in series with a long peptide according to claim 1, characterized in that: In the long peptide synthesis step, the number and length of the mid-peptides are controlled during the synthesis process, and the molecular weight of the long peptide is calculated using online software so that the molecular weight of the finally synthesized long peptide is in the range of 50-60 kDa.

7. The biosynthesis method of a multifunctional cosmetic short peptide connected in series with a long peptide according to claim 1, characterized in that: In the biosynthesis preparation step, the amino acid sequence of the long peptide is reverse-translated into a nucleotide sequence using a software tool according to a genetic codon table. During the translation process, the nucleotide sequence is optimized according to the codon preference of the selected expression strain so that the codon adaptation index target value reaches 0.8 or above. The optimized nucleotide sequence is synthesized into a corresponding DNA fragment by chemical synthesis. A pPICZ series vector for Pichia pastoris expression or a pET series vector for Escherichia coli expression is selected. The expression vector and the synthesized DNA fragment are double-digested with corresponding restriction endonucleases, using 5-10 units of enzyme per microgram of DNA and a reaction time of 1-3 hours. The digested DNA fragment is ligated to the vector using DNA ligase, with a molar ratio of DNA fragment to vector of 3:1-10:1, and an amount of T4 DNA ligase of 1-3 units per microgram of DNA. The reaction temperature is 16° C. and the reaction time is 12-16 hours. A recombinant expression vector is constructed, and a recombinant vector containing an insert is preliminarily screened by PCR amplification. The recombinant vector is then sequenced and compared with the original design sequence to confirm that the vector construction is successful.

8. The biosynthesis method of a multifunctional cosmetic short peptide connected in series with a long peptide according to claim 1, characterized in that: In the transformation and expression steps, Escherichia coli or Pichia pastoris competent cells are prepared. Specifically, for Escherichia coli, Escherichia coli cells in the logarithmic growth phase are suspended in a 0.1M ice-cold calcium chloride solution and ice-bathed for 30-60 minutes to render the cells competent. During heat shock transformation, the recombinant vector is mixed with the competent cells and then ice-bathed. They are quickly subjected to a heat shock at 42°C for 45-90 seconds, then cooled in an ice bath for 2-5 minutes, and then cultured with a culture medium. For Pichia pastoris, Pichia pastoris cells in the logarithmic growth phase are washed and resuspended in an ice-cold sorbitol solution to prepare competent cells. After mixing the recombinant vector with the competent Pichia pastoris cells, they are subjected to electric pulse treatment under specific voltage and capacitance conditions, and then cultured with a culture medium.

9. The biosynthesis method of a multifunctional cosmetic short peptide connected in series with a long peptide according to claim 1, characterized in that: In the transformation and expression steps, positive clones are screened and then expression is induced or non-induced. Specifically, the transformed cells are spread on a plate containing corresponding antibiotics, including ampicillin and bleomycin, and positive clones containing the recombinant vector are screened. Single colonies are picked for PCR identification or sequencing verification to determine positive clones, and the positive clones are inoculated into liquid culture medium. For the Escherichia coli expression system, IPTG inducer is added to induce recombinant protein expression, the IPTG concentration is 0.1-1mM, the induction temperature is 16-37°C, and the induction time is 4-24 hours. For the Pichia pastoris pPIC series expression system, methanol culture medium is replaced to induce expression, the methanol concentration is 0.5%-1% at the initial induction stage, and is adjusted to 1%-2% according to subsequent growth conditions, the induction temperature is 28-30°C, and the induction time is 48-96 hours. No induction is required for the Pichia pastoris pGAP series expression system.

10. The biosynthesis method of a multifunctional cosmetic short peptide connected in series with a long peptide according to claim 1, characterized in that: The molecular weight of the long peptide detected by PAGE gel electrophoresis was consistent with the theoretical value.

11. The biosynthesis method of a multifunctional cosmetic short peptide connected in series with a long peptide according to claim 1, characterized in that: Purification of long peptides by membrane filtration.

12. The biosynthesis method of a multifunctional cosmetic short peptide connected in series with a long peptide according to claim 1, characterized in that: In the short peptide acquisition step, after purifying the long peptide, trypsin is added to the long peptide, and the ratio of trypsin to long peptide is 1:50-1:

200. The enzymatic cleavage reaction is carried out at 37°C and pH 7.5-8.

5. The reaction time is 4-24 hours. Samples are taken at regular intervals during the reaction, and the progress of the enzymatic cleavage is analyzed by HPLC. After the enzymatic cleavage reaction is completed, the enzymatic cleavage product is separated and purified by HPLC. The HPLC parameters are set as follows: the mobile phase adopts a water-acetonitrile system, the acetonitrile gradient is adjusted according to the properties of the short peptide, ranging from 5% to 60%, the flow rate is 0.5-2mL / min, the detection wavelength is 214nm or 280nm, the molecular weight of the short peptide is determined by mass spectrometry to determine its accuracy, the amino acid composition of the short peptide is determined by amino acid analysis, and the biological activity of the short peptide is evaluated by activity detection experiments to ensure that the obtained short peptide meets the expected skin care efficacy requirements.

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