Enzyme for hydrolyzing collagen and application thereof

Abstract

The application relates to the technical field of collagen, in particular to an enzyme for hydrolyzing collagen and application. The enzyme has an amino acid sequence with at least 90% sequence identity with SEQ ID NO: 3. The enzyme can hydrolyze collagen under the condition of 80 DEG C enzymolysis, has high enzyme activity, and has hydrolysis activity on insoluble collagen.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of collagen, in particular to an enzyme for hydrolyzing collagen and application thereof. BACKGROUND

[0002] Collagen is the most abundant structural protein in animals. Low molecular weight collagen peptides can be obtained by hydrolyzing collagen using acid-base hydrolysis, enzymatic hydrolysis, microbial fermentation, etc. Compared with macromolecular collagen, collagen peptides are more easily absorbed by the intestinal tract and have various physiological activities, such as antioxidant, antitumor, antibacterial, immune regulation, and moisturizing. Enzymatic hydrolysis of collagen is the most common method for large-scale production of collagen peptides at present. It refers to specific hydrolysis of collagen by a single protease or a combination of multiple proteases, and the process is controllable and the yield of peptides is high. Collagen has a unique stable cross-linked triple helix structure, which is mainly hydrolyzed by some collagenases from mammals or bacteria, such as matrix metalloproteinases, serine proteases, and metalloproteinases. SUMMARY

[0003] Thermally denatured collagen is more easily hydrolyzed under high-temperature enzymatic hydrolysis conditions, so the hydrolysis efficiency of collagen can be improved and the risk of microbial contamination during the enzymatic hydrolysis process can be reduced. The present application provides an enzyme for hydrolyzing collagen, which can hydrolyze collagen under 80℃ enzymatic hydrolysis conditions, and has high enzyme activity and hydrolysis activity on insoluble collagen.

[0004] To this end, the embodiments of the present application disclose at least the following technical solutions:

[0005] In a first aspect, the embodiments disclose an enzyme having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 3.

[0006] In a second aspect, the embodiments disclose a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 4.

[0007] In a third aspect, the embodiments disclose an expression vector comprising a nucleotide sequence as shown in SEQ ID NO: 4.

[0008] In a fourth aspect, the embodiments disclose an expression strain, which is a chassis cell carrying the expression vector of the third aspect.

[0009] In a fifth aspect, the embodiments disclose a method for preparing the enzyme of the first aspect. The method comprises: obtaining the expression strain of the fourth aspect; fermenting the expression strain; and harvesting the enzyme from the fermentation broth.

[0010] In a sixth aspect, the embodiments disclose use of the enzyme of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, or the expression strain of the fourth aspect in the preparation of a collagen peptide. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 Relative enzyme activity curves of the enzymes BTC23 and BTC324 provided by the embodiments for casein enzymolysis at different enzymolysis temperatures. DETAILED DESCRIPTION

[0012] In order to make the purposes, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not used to limit the present application. The reagents not specifically described in the present application are conventional reagents and can be obtained from commercial channels; the methods not specifically described are conventional experimental methods and can be known from the prior art.

[0013] The term "identity" (%) is used herein to refer to a comparison between polynucleotides or polypeptides, and is determined by comparing two optimally aligned sequences in a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window can comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage is calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue or a nucleic acid base or amino acid residue that will align with the identical nucleic acid base or amino acid residue, occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. Those skilled in the art understand that there are many established algorithms that can be used to align two sequences. Optimal alignment of sequences for comparison can be conducted by any suitable method, including, but not limited to, the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482

[1981] ), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443

[1970] ), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444

[1988] ), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or by visual inspection, as is known in the art. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include, but are not limited to, the BLAST and BLAST 2.0 algorithms, which are described by Altschul et al. (see, respectively, Altschul et al., J. Mol. Biol., 215:403-410

[1990] ; and Altschul et al., Nucl. Acids Res., 3389-3402

[1977] ). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued score threshold T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (see, Altschul et al., supra).These initial neighborhood word hits act as seeds to initiate the search to find longer HSPs containing them. Then the word hits are extended in each direction along each sequence until an incrementally scored alignment that is better than the previous one is obtained. For nucleotide sequences, the scores of the individual residue pair matches are counted using the parameters M (for a match between residues; always > 0) and N (for a mismatch between residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

[1989] ). An example of a sequence comparison with % sequence identity can be determined using the BESTFIT or GAP programs in the GCG Wisconsin Software Package (Accelrys, Madison WI), using the provided default parameters.

[0014] The embodiments provide an enzyme capable of hydrolyzing collagen, which has good degradation activity on bovine bone type I insoluble collagen under high-temperature hydrolysis conditions without the need for an expansion process. Moreover, the enzyme can hydrolyze collagen under 80°C enzymatic hydrolysis conditions, has high enzyme activity, and also has hydrolysis activity on insoluble collagen.

[0015] In some embodiments, the enzyme has an amino acid sequence as set forth in SEQ ID NO: 3.

[0016] In one aspect, the embodiments disclose an enzyme having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 3.

[0017] In some embodiments, the enzyme has an amino acid sequence with at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to SEQ ID NO: 3.

[0018] In some embodiments, the enzyme has at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% identity compared to SEQ ID NO: 3.

[0019] In another aspect, embodiments disclose a nucleic acid molecule, the nucleotide sequence of which is set forth in SEQ ID NO: 4. In some embodiments, the nucleic acid molecule can be an isolated free DNA, RNA, or chimera of both, and is capable of being transcribed, translated, and expressed by an organism (e.g., a microorganism) to synthesize a protein molecule (e.g., the enzyme described above).

[0020] In another aspect, embodiments disclose an expression vector comprising the nucleotide sequence set forth in SEQ ID NO: 4.

[0021] In some embodiments, the expression vector further comprises a base plasmid, and the base plasmid is pBT101.

[0022] In another aspect, embodiments disclose an expression strain, which is a chassis cell carrying the expression vector of the third aspect.

[0023] In some embodiments, the chassis cell is selected from Bacillus subtilis, Bacillus licheniformis, or Bacillus amyloliquefaciens. In some embodiments, the Bacillus subtilis is Bacillus subtilis SCK6.

[0024] In another aspect, embodiments disclose a method for preparing the enzyme provided in the above embodiments. The method comprises: obtaining the expression strain provided in the above embodiments; fermenting the expression strain; and harvesting the enzyme from the fermentation broth.

[0025] In another aspect, embodiments disclose the use of the enzyme described in the above embodiments, the nucleic acid described in the above embodiments, the expression vector described in the above embodiments, or the expression strain described in the above embodiments in the preparation of a collagen peptide.

[0026] The present application will be described in more detail below with reference to specific embodiments, but the present application is not limited to the embodiments.

[0027] Objective sequence and expression vector

[0028] The embodiment provides a preparation process of the basic plasmid pTB101, including: PCR amplification of a sequence fragment containing an E. coli replicon and an ampicillin resistance gene, as shown in SEQ ID NO: 5; PCR amplification of a sequence fragment containing a Bacillus replicon, as shown in SEQ ID NO: 6; chemical synthesis of a sequence fragment containing a tetracycline resistance gene, a promoter, a signal peptide and a terminator, as shown in SEQ ID NO: 7; seamless cloning linkage of the three DNA fragments; transformation of the linking product into E. coli DH5a; screening of positive colonies; extraction of the culture of the positive colonies and sequence determination to obtain the basic plasmid pTB101.

[0029] The embodiment provides the DNA as shown in SEQ ID NO: 4 as a target sequence. The target sequence can be obtained by PCR amplification and chemical synthesis, and the embodiment is not limited.

[0030] The embodiment further provides an expression vector pBT103 carrying the nucleotide sequence as shown in SEQ ID NO: 4. The preparation process of the expression vector includes: connecting the DNA as shown in SEQ ID NO: 4 with a basic plasmid fragment (pTB101); transforming the linking product into E. coli DH5a; screening of positive colonies; extraction of the culture of the positive colonies and sequence determination to obtain the expression vector pBT103.

[0031] As a comparison, the comparative example further provides an expression vector pBT102 carrying the nucleotide sequence as shown in SEQ ID NO: 2. The preparation process of the expression vector includes: connecting the DNA as shown in SEQ ID NO: 2 with a basic plasmid fragment (pTB101); transforming the linking product into E. coli DH5a; screening of positive colonies; extraction of the culture of the positive colonies and sequence determination to obtain the expression vector pBT102.

[0032] Preparation of expression strain

[0033] The embodiment further provides an expression strain carrying the expression vector. The preparation method of the expression strain includes: transforming the expression vector provided in the embodiment into Bacillus subtilis SCK6 (BNCC359202, Beina Bio); screening of positive colonies from the transformants, which are the expression strain.

[0034] The expression vector is transformed into Bacillus subtilis SCK6, including:

[0035] The strain Bacillus subtilis SCK6 is activated by using an LB solid plate, and is cultured at 37 DEG C for 12 hours. 2-3 single colonies are selected into 10 mL fresh LB liquid medium, and are cultured at 37 DEG C, 220 r / min for 5-6 hours, so that the bacterial density OD600 about 0.8-1.0. A xylose solution with a final concentration of 1.5% was added, and the induction culture was continued for 2h to obtain the competent cells of the strain SCK6. 200μL of the competent cells were mixed with 10μL of the plasmid pBT103, and the mixture was incubated for 1h, and then spread on a skim milk plate containing tetracycline with a final concentration of 10μg / mL, and incubated at 37℃ for 12h.

[0036] In which, the positive colonies were screened from the transformants, including:

[0037] The transformant with the largest proteolytic circle on the transformation plate was picked, and streaked on a skim milk plate containing tetracycline with a final concentration of 10μg / mL to obtain a single colony with good hydrolysis activity, i.e., the expression strain B. subtilis KH103 of the protease BTC324. As a control, the expression plasmid provided in the comparative example was transformed into B. subtilis SCK6 to obtain the expression strain B. subtilis KH102 of the enzyme BTC23.

[0038] Preparation of enzyme for hydrolyzing collagen

[0039] The embodiments also provide a method for preparing a collagen-hydrolyzing enzyme. The method comprises: obtaining the expression strain provided in the above embodiments; fermenting the expression strain; and harvesting the enzyme from the fermentation broth.

[0040] In some embodiments, 3-5 single colonies of the expression strains KH102 and KH103 were inoculated into 20mL of LB solution containing 10μg / mL of tetracycline, and incubated at 37℃, 220r / min for 6-7h, and the cell density OD 600 about 1.0-1.2. 6mL of the seed liquid was transferred into 300mL of fermentation medium (1% yeast powder, 2% tryptone, 0.2% sodium chloride, 2% glucose, 1% potassium phosphate dibasic), and incubated at 37℃, 220r / min for 60h. The fermentation broth was centrifuged at 8000g for 10min, and the supernatant was collected to obtain an enzyme liquid containing the target protease. The enzyme prepared by using the expression strain KH103 provided in the embodiments is BTC324, and the enzyme prepared by using the expression strain KH102 provided in the embodiments is BTC23.

[0041] Enzymolysis temperature

[0042] According to GB / T 23527-2009 Proteinase Preparations, the casein hydrolysis activity of the proteases BTC23 and BTC324 at 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃ and 80℃ was determined at pH 9.0, respectively. For example, Figure 1As shown, the optimum enzymolysis temperature of enzyme BTC23 is 55°C, and the optimum enzymolysis temperature of enzyme BTC324 is increased to 70°C, which indicates that enzyme BTC324 is more heat-resistant and more suitable for enzymolysis of bovine bone insoluble collagen under high temperature conditions.

[0043] Enzymolysis of bovine bone type I insoluble collagen

[0044] 1. Collagen activity

[0045] The activities of BTC23 and BTC324 on bovine bone type I insoluble collagen were determined by the indantrione coloration method (Nagano et al. 2000). The enzyme activity unit was defined as the amount of 1 μmol of leucine required for the degradation of 1 mg of insoluble collagen per hour per mg of protein at a specific temperature.

[0046] Enzyme activity determination under 55°C conditions: 800 μL of 50 mmol / L Tris-HCl (pH 9.0), 4 mmol / L CaCl2, 10 mg of bovine bone type I insoluble collagen (ABM-5162, Aibright Biotech Co., Ltd.) substrate, and shaking and mixing, 55°C water bath for 30 min. 200 μL of BTC324 enzyme solution diluted to 1 mg of protein with 50 mmol / L Tris-HCl (pH 9.0) was added, and the enzyme was hydrolyzed at 55°C for 2 h, and 1 mL of 0.1 mol / L acetic acid was added to terminate the reaction. Centrifugation at 10,000 g for 10 min gave an enzyme hydrolysate aqueous solution. 40 μL of the enzyme hydrolysate aqueous solution was taken, 200 μL of an indantrione-sodium citrate mixture was mixed, and then boiled for 20 min. After cooling to room temperature, 1 mL of 50% n-propanol solution was added, mixed, and 200 μL was taken to an enzyme-labeled plate, and the absorbance value was determined at 600 nm using an enzyme-labeled instrument. The enzyme activity of wild-type BTC23 was determined synchronously. To eliminate the background effect of the solution in the detection system, a blank control group was set. The blank control group was 200 μL of boiled and inactivated diluted enzyme solution reacted with 10 mg of bovine bone insoluble collagen, and the rest was the same as the experimental group. Different concentrations of 40 μL of leucine solution were reacted with the indantrione-sodium citrate mixture, and the absorbance value at 600 nm was determined to prepare a standard curve. The enzyme activity determination methods under 60°C, 65°C, 70°C, 75°C, and 80°C conditions were referred to 55°C.

[0047] Table 1

[0048] Temperature (°C) Enzyme activity of BTC23 (U / mg) Enzyme activity of BTC324 (U / mg) 55 678.3 702.2 60 642.7 787.3 65 492.5 955.7 70 343.4 1135.1 75 228.2 902.4 80 75.1 633.6

[0049] As shown in Table 1, as the enzymatic temperature increased from 55℃ to 80℃, the enzyme activities of enzymes BTC23 and BTC324 both showed a trend of first increasing and then decreasing to different degrees, but the optimal enzymatic temperature of enzyme BTC23 was 55℃, and the optimal enzymatic temperature of enzyme BTC324 was 70℃. And the enzyme activity at the respective optimal enzymatic temperature, enzyme BTC324 was significantly higher than enzyme BTC23, which showed that enzyme BTC324 was more suitable for high-temperature enzymatic hydrolysis of bovine bone type I insoluble collagen.

[0050] 2. Preparation of bovine bone type I insoluble collagen enzymatic hydrolysate

[0051] The examples also use BTC23 and BTC324 enzymes to hydrolyze bovine bone type I insoluble collagen, and obtain BTC23 enzymatic hydrolysate and BTC324 enzymatic hydrolysate.

[0052] Specifically, 10 mg of bovine bone type I insoluble collagen was dissolved in 2 mL of 50 mmol / L Tris-HCl (pH 9.0) solution at 70℃, and 0.8‰ (W / W) of enzyme BTC324 or BTC23 was added, and the reaction was oscillated for 2 h, 3 h and 4 h. The temperature was raised to 90-100℃, and the enzyme was inactivated for 10 min. Centrifugation at 10000g for 10 min to obtain BTC23 enzymatic hydrolysate and BTC324 enzymatic hydrolysate.

[0053] The ratio of the content of amino nitrogen in the BTC23 enzymatic hydrolysate and the content of total nitrogen in the substrate was determined, and the degree of hydrolysis (DH) of the substrate protein was calculated (Lu Chen et al. 2024), and the enzymatic performance of mutant BTC324 on bovine bone insoluble collagen was evaluated. According to GB 5009.5-2016 "National Food Safety Standard Determination of Protein in Food", the total nitrogen content in bovine bone insoluble collagen was determined by Kjeldahl nitrogen determination method. The content of amino nitrogen in the enzymatic hydrolysate was determined by formaldehyde automatic potentiometric titration method, and the results are shown in Table 2.

[0054] Table 2

[0055] Enzymolysis time (h) DH of BTC23 (%) DH of BTC324 (%) 2 63.1 82.9 3 65.6 84.5 4 66.2 86.2

[0056] As shown in Table 2, at an enzymatic temperature of 70℃, after 2h of enzymolysis, the degree of hydrolysis of the substrate by enzyme BTC324 reached 82.9%, which was 131.4% of enzyme BTC23. After 3h of enzymolysis, the degree of hydrolysis of enzyme BTC324 increased to 128.9% of enzyme BTC23.

[0057] Therefore, it is shown that enzyme BTC324 has excellent heat resistance and can enzymolysis bovine bone insoluble collagen at high enzymolysis temperature, which has significant economic value for downstream applications.

[0058] 3. Preparation of bovine bone collagen peptide

[0059] A bovine bone collagen solution was prepared with a substrate concentration of 6-8% (W / V) and a pH of 9. At a temperature of 70°C, 0.8‰ (W / W) of BTC324 enzyme solution was added to the collagen, and the reaction was stirred for 2 hours. The pH was adjusted to 9.0 using NaOH solution. The enzyme solution was cooled to 50°C, and the pH was adjusted to 6.5. Then, 0.2‰ (W / W) of neutral protease (50000 U / mg) was added to the collagen, and the reaction was continued for 1 hour. The enzyme solution was heated to 90-100°C for 20 minutes to inactivate the enzyme and terminate the enzymolysis. A crude bovine bone collagen peptide solution was obtained. Activated carbon was added at a ratio of 1-1.5% (w / v) and stirred for 60 minutes for adsorption, decolorization, and debittering. The activated carbon was removed by ceramic membrane and ultrafiltration to obtain a clear bovine bone collagen peptide solution. The proportion of different molecular weight peptide segments in the enzyme solution was determined according to GB 31645-2018, and the results are shown in Table 3.

[0060] Table 3

[0061] Molecular weight (Da) Proportion of peptide segment (%) Number average molecular weight Weight average molecular weight ＞3000 4.2 3362 3421 3000～2000 6.1 2354 2471 2000～1000 7.5 1426 1603 1000～500 46.7 680 713 ＜500 35.5 245 263

[0062] The above description is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any changes or replacements within the technical scope disclosed in the present application can be easily thought of by those skilled in the art, and should be covered within the protection scope of the present application.

Claims

1. An enzyme having the amino acid sequence shown in SEQ ID NO:

3.

2. A nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO:

4.

3. An expression vector comprising a nucleotide sequence as shown in SEQ ID NO:

4.

4. An expression strain comprising a chassis cell carrying the expression vector of claim 3.

5. The expression strain according to claim 4, wherein the chassis cells are selected from Bacillus subtilis, Bacillus licheniformis, or Bacillus amyloliquefaciens.

6. A method for preparing the enzyme as described in claim 1, comprising: Obtain the expression strain as described in any one of claims 4 to 5; The expressed strain was fermented; Enzymes are harvested from the fermentation broth.

7. Use of the enzyme of claim 1, the nucleic acid molecule of claim 2, the expression vector of claim 3, or the expression strain of any one of claims 4 to 5 in the preparation of collagen peptides.

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