CN-121975896-A - Method and control system for preparing functional peptide by cottonseed proteolysis

CN121975896ACN 121975896 ACN121975896 ACN 121975896ACN-121975896-A

Abstract

The invention relates to the technical field of functional peptide preparation, and discloses a method and a control system for preparing functional peptide by cottonseed proteolysis, wherein the method comprises the steps of constructing a molecular three-dimensional coordinate system and a dipole vector field, identifying a folding potential well region and a key stress tendinopeptide segment, and calculating ripple effect coefficients; defining a three-dimensional conformational coordinate system, screening out ideal cutting sites, formulating an enzymolysis strategy based on a scoring function, designing an enzymolysis coordination time sequence disc for the enzymolysis strategy, constructing capture catalytic microspheres, and combining a product separation mechanism to realize targeted harvesting of the target functional peptide. According to the invention, the enzymolysis optimization is improved from experience trial and error to a quantitative design level, the problem of low enzymolysis efficiency caused by conformational trapping is solved, and the yield and purity of the target functional peptide are obviously improved.

Inventors

LV SHAOYING
CUI ZHIYING
YU ZHONGLI
YUN HUI
WU HAITAO
WANG LUNXUE
LI TIANBI

Assignees

新疆希普生物科技股份有限公司
广东希普生物科技股份有限公司

Dates

Publication Date: 20260505
Application Date: 20260205

Claims (10)

1. A method for preparing a functional peptide by proteolysis of cotton seeds, comprising: Constructing a dipole vector field covering the whole protein molecule in the molecular three-dimensional coordinate system, identifying a folding potential well region and a key stress tendinopeptide segment based on the dipole vector field, and calculating ripple effect coefficients showing local conformational intensity based on the key stress tendinopeptide segment; Defining a three-dimensional conformational coordinate system according to the folding potential well region and the ripple effect coefficient, screening ideal cutting sites with optimal comprehensive unlocking efficiency for the protease in the three-dimensional conformational coordinate system, and formulating a scoring function for quantifying the comprehensive unlocking capacity of the protease based on the ideal cutting sites to obtain an enzymolysis strategy; And designing an enzymolysis coordination time sequence disc for programmed control of an enzymolysis strategy, constructing a functionally integrated capture catalytic microsphere according to the enzymolysis coordination time sequence disc, and executing a product separation mechanism on the capture catalytic microsphere based on the enzymolysis strategy to obtain the target functional peptide.
2. The method for preparing functional peptide by cottonseed proteolysis as claimed in claim 1, wherein the molecular three-dimensional coordinate system comprises: The three-dimensional structure data of the cottonseed protein molecule comprises a protein sequence, an atom type list and an atom coordinate set, wherein the protein sequence refers to a linear sequence formed by arranging the connection sequence of all arginine residues constituting the protein on a peptide chain, the atom type list refers to the chemical element type of each atom in the protein molecule, and the atom coordinate set refers to the three-dimensional coordinate set corresponding to each atom in the atom type list; traversing each atom in the atom type list, finding out the three-dimensional coordinate of each atom from an atom coordinate set, finding out the atomic weight corresponding to each atom from the chemical element periodic table, and combining the atomic weight with the three-dimensional coordinate value of the atom to calculate so as to obtain the molecular centroid position of the cottonseed protein molecule; And constructing a three-dimensional coordinate system by taking the molecular mass center position as an origin, namely a molecular three-dimensional coordinate system.
3. The method for preparing functional peptide by cottonseed proteolysis as claimed in claim 2, wherein the method for obtaining the key stress tendinopeptide fragment comprises the following steps: Dipole vector field comprising dipole vectors of all arginine residues; Setting a window with a fixed length, scanning protein sequences by a sliding window method, forming a candidate peptide segment to be analyzed by a sequence composed of arginine residues covered at each window position, and for each candidate peptide segment in the window, determining that the candidate peptide segment meets the first layer condition by calculating the cosine value of an included angle between dipole vectors of any two adjacent arginine residues on the candidate peptide segment, and when the cosine value of the included angle between all adjacent vectors in one peptide segment is greater than a preset consistency threshold; Checking the spatial position of the candidate peptide in a molecular three-dimensional coordinate system, traversing each arginine residue in the candidate peptide, calculating the Euclidean distance between the coordinate of the dipole vector of the arginine residue in the molecular three-dimensional coordinate system and the folded potential well region, and judging that the second layer condition is met if the Euclidean distance of at least one arginine residue in the candidate peptide is smaller than a preset contact distance threshold value; and simultaneously meeting the first layer condition and the second layer condition to obtain the candidate peptide chain, namely the key stress tendinopeptide segment.
4. The method for preparing the functional peptide by proteolysis of cotton seeds according to claim 3, wherein the method for constructing the three-dimensional conformational coordinate system comprises the following steps: Positioning all arginine residues embedded in the folded potential well region, calculating the accessible conformation space volume of the side chain of the arginine residues to be marked as the accessible conformation space volume before cutting by a Monte Carlo sampling algorithm for each arginine residue, virtually cutting a peptide bond on a key stress tendinopeptide segment, calculating ripple effect coefficients of the virtual cut peptide bond, and carrying out local conformation rearrangement of the simulated protein; Based on the protein conformation after local conformational rearrangement, performing a Monte Carlo sampling algorithm again, calculating the accessible conformational space volume of an arginine residue side chain, marking the accessible conformational space volume after cutting, and calculating the combination of the accessible conformational space volume before cutting and the accessible conformational space volume after cutting to obtain conformational entropy gain; Calculating an atomic coordinate set to obtain site accessibility, identifying all cleavable peptide bonds, namely potential cleavage sites, of protease on the whole cottonseed protein molecule in an obtained protein sequence, counting the total number of the potential cleavage sites, traversing the potential cleavage sites, identifying peptide bonds positioned in a key stress tendinopeptide segment, marking the peptide bonds as target cleavage site numbers, and dividing the target cleavage site numbers by the total number of the peptide bonds to obtain unlocking accuracy; The values on the X-axis of the three-dimensional conformational coordinate system are defined as conformational entropy gain, the values on the Y-axis are defined as site accessibility, and the values on the Z-axis are defined as unlocking precision.
5. The method for preparing functional peptide by proteolysis of cotton seeds according to claim 4, wherein the method for obtaining ideal cleavage site comprises: according to a specific production target, a conformational entropy gain threshold value, a site accessibility threshold value and an unlocking accuracy threshold value are set based on the conformational entropy gain, the site accessibility and the unlocking accuracy of a three-dimensional conformational coordinate system; the potential cleavage site is marked as an ideal cleavage site only if the conformational entropy gain, site accessibility, and unlocking precision corresponding to the coordinates of the potential cleavage site are greater than a conformational entropy gain threshold, site accessibility threshold, and unlocking precision threshold, respectively.
6. The method for preparing functional peptides by proteolysis of cottonseed of claim 5, wherein the enzymatic hydrolysis strategy comprises: screening ideal cleavage sites of each protease aiming at specific proteases, accumulating corresponding conformational entropy gains of the ideal cleavage sites of each protease in a three-dimensional conformational coordinate system to obtain a comprehensive score value, and taking the protease with the highest comprehensive score value as the optimal single enzyme; if no ideal cleavage site is generated by each protease, selecting a protease with the highest unlocking precision value as a first unlocking protease, generating a new protein conformation by the first unlocking protease according to a local conformation rearrangement method, recalculating the comprehensive score values of all proteases except the first unlocking protease, using the protease with the highest comprehensive score value of all proteases except the first unlocking protease as a second unlocking protease, and combining the first unlocking protease and the second unlocking protease as protease with matched cleavage capacity; and integrating the protease combination with the optimal single enzyme to obtain an enzymolysis strategy.
7. The method for preparing functional peptide by proteolysis of cotton seeds of claim 6, wherein the enzymolysis coordination time sequence disc comprises: Setting a total reaction time length, equally dividing the total reaction time length into N operation allocation sectors, and formulating a unique identifier for each operation allocation sector; If the enzymolysis strategy is the optimal single enzyme, the instruction added with the optimal single enzyme is distributed to a first operation distribution sector, and the subsequent operation distribution sectors are uniformly marked as continuous enzymolysis reaction until the total reaction time is over; If the enzymolysis strategy is protease combination, an instruction for adding a first type of unlocking protease is distributed to a first operation distribution sector, the number J of operation distribution sectors marked as standing waiting sectors is calculated, from a second operation distribution sector, all J continuous operation distribution sectors are marked as standing waiting sectors, namely all J continuous operation distribution sectors from the second operation distribution sector to the J+1th operation distribution sector are marked as standing waiting sectors, and an instruction for adding a second type of unlocking protease is distributed to the J+2th operation distribution sector, from the J+3rd operation distribution sector to the N continuous operation distribution sector, and the J continuous operation distribution sectors are marked as cooperative enzymolysis reaction until the total reaction duration is finished.
8. The method for preparing functional peptides by proteolysis of cottonseed of claim 7, wherein the capturing catalytic microspheres comprises: The capture catalytic microsphere comprises a microsphere core, target protease, a charge capture layer and a covalent connecting arm; Mixing and reacting the target protease with the microsphere core, wherein the target protease molecular surface substance reacts with the high-activity chemical groups on the microsphere core to form a stable covalent connecting arm; and carrying out chemical modification on the surface of the microsphere core to introduce a charge trapping layer, so that the surface of the microsphere core is provided with strong negative charges, and the trapping catalytic microsphere with trapping function and catalytic function is obtained.
9. The method for preparing functional peptides by proteolysis of cottonseed as claimed in claim 8 wherein the product separation mechanism comprises: the product separation mechanism comprises three stages, namely a structure unlocking stage, a target harvesting stage and a product separation and purification stage; A structure unlocking stage, namely adding cottonseed protein solution into a reaction kettle, injecting protease into the reaction kettle when the enzymolysis process enters a first operation distribution sector, and injecting the optimal single enzyme if the protease is the optimal single enzyme, wherein the reaction kettle enters a continuous enzymolysis reaction stage, and injecting the first unlocking protease in the protease combination if the protease combination is the protease; the target harvesting stage comprises determining a target harvesting delay time for the optimal single enzyme, adding capture catalytic microspheres into the reaction kettle after the target harvesting delay time passes after the continuous enzymolysis reaction starts, adding a second unlocking protease and simultaneously adding the capture catalytic microspheres into the reaction kettle when the enzymolysis process leaves the continuous standing waiting sector for protease combination; And in the product separation and purification stage, after the total reaction time is over, the capturing catalytic microsphere carrying the peptide fragment is separated from the reaction kettle, and eluent is added to the separated capturing catalytic microsphere to obtain the target functional peptide.
10. A control system for preparing a functional peptide by proteolysis of cotton seeds for realizing the method for preparing a functional peptide by proteolysis of cotton seeds according to any one of claims 1 to 9, characterized in that the system comprises: The target spot analysis module is used for acquiring the three-dimensional structure data of cottonseed protein molecules to construct a molecular three-dimensional coordinate system, constructing a dipole vector field covering the whole protein molecules in the molecular three-dimensional coordinate system, and identifying a folding potential well region and a key stress tendinopeptide segment based on the dipole vector field; The strategy generation module is used for defining a three-dimensional conformational coordinate system according to the folding potential well region and the ripple effect coefficient, screening ideal cutting sites with optimal comprehensive unlocking efficiency for the protease in the three-dimensional conformational coordinate system, and formulating a scoring function for quantifying the comprehensive unlocking capacity of the protease based on the ideal cutting sites to obtain an enzymolysis strategy; And the product harvesting module is used for designing an enzymolysis coordination time sequence disc for programmed control of an enzymolysis strategy, constructing a function-integrated capture catalytic microsphere according to the enzymolysis coordination time sequence disc, and executing a product separation mechanism on the capture catalytic microsphere based on the enzymolysis strategy to obtain the target functional peptide.

Description

Method and control system for preparing functional peptide by cottonseed proteolysis Technical Field The invention relates to the field of functional peptide preparation, in particular to a method and a control system for preparing functional peptide by cottonseed proteolysis. Background With the increasing market demand for arginine functional peptides, cottonseed proteins with high arginine content are attracting attention as a high quality raw material. However, the conventional enzymolysis methods, such as adjusting macroscopic process parameters such as enzyme dosage, reaction time or pH value, generally face yield bottlenecks, the actual yield of active peptide fragments is far lower than theoretical value, and the optimization effect is not yet stopped. The prior art has been largely relegated to the analysis of primary protein sequences or to the prediction of cleavage sites depending on the static, isolated three-dimensional structure of the protein. The conformational trapping effect caused by dynamic folding and reconstruction of the protein in the enzymolysis process is ignored, namely, the peptide segments rich in arginine are wrapped in the hydrophobic core, enzymes cannot be effectively contacted, and stress released in the enzymolysis process can induce new change of the protein conformation, and exposed sites are embedded again, so that the enzymolysis efficiency falls into a low valley of dynamic balance. Although the traditional molecular dynamics simulation can reveal part of mechanisms, huge calculated amount cannot be used for guiding the real-time and dynamic enzymolysis process control. Therefore, how to change from static structural analysis to dynamic mechanical prediction, macroscopic process parameter optimization is changed into accurate cutting of key peptide segments, so that the limitation of conformational trapping is broken through, and the method is a technical problem to be solved in the field. In the prior art, chinese patent application with the application publication number of CN120199335A discloses an enzymolysis extraction method of cottonseed protein peptide based on machine learning optimization. Setting key factors of enzymolysis, screening key factor variable values through a single factor experiment to form a set, adopting a full factor experimental design to generate experimental samples of different enzymolysis condition combinations, inputting the samples into a machine learning model to obtain response data, and searching for optimal enzymolysis conditions by combining a natural heuristic optimization algorithm. The method avoids blindness of the traditional trial-and-error method through data-driven parameter optimization, efficiently locates the optimal solution in a complex parameter space, and improves the extraction efficiency of cottonseed protein peptide to a certain extent. The Chinese patent with the publication number of CN102550794B discloses a method for extracting cottonseed protein from cottonseed meal, which comprises the steps of crushing the cottonseed meal, carrying out ultrasonic treatment at 25-35 kHz at 40-50 ℃ for 40-60 min, extracting active enzyme of cotton bollworms, carrying out enzymolysis at 55-60 ℃ for 1-2 h at pH 7.5-8.0, heating, inactivating enzyme, centrifuging, washing filter residues, merging supernatant liquid, removing impurities by a microfiltration membrane, concentrating by a nanofiltration membrane, and drying to obtain powdery cottonseed protein. The cost is reduced by replacing high-price commercial enzymes with the cotton bollworm active enzymes, and the ultrasonic cell disruption and membrane separation technology is combined, so that the cotton seed protein extraction rate and the functional activity of the product are improved. However, although the two prior arts have a certain value in terms of parameter optimization, cost control and extraction rate improvement, the problem of conformational trapping core pain points in the preparation of cottonseed protein arginine enrichment peptide segments cannot be solved. The Chinese patent with publication No. CN120199335A focuses only on static optimization of macroscopic technological parameters, does not consider conformational trapping effect caused by dynamic folding of protein in the enzymolysis process, cannot identify and break the problem that an arginine region is wrapped by a hydrophobic core, so that the yield of arginine branched peptide segments is difficult to break through a theoretical value bottleneck, and the Chinese patent with the publication No. CN102550794B is authorized to improve the extraction effect through ultrasonic assistance and a special enzyme source, but does not relate to the phenomenon of re-embedding of arginine sites caused by refolding of peptide segments in the enzymolysis process, does not establish a dynamic conformational prediction and real-time regulation mechanism, cannot cope with dynamic balance of the enzymolysis effici