Method for preparing collagenase and method for preparing collagen tripeptide using same
A collagen tripeptide and collagenase technology, which is applied in the preparation method of peptide, biochemical equipment and method, tripeptide, etc., can solve the problems of difficulty in obtaining high content of tripeptide and low content of tripeptide.
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[0028] The method for preparing collagenase of the present invention comprises: the first step: centrifuging and separating the Bacillus subtilis strain; the second step: concentrating the centrifuged supernatant; and the third step: purifying by ion exchange chromatography.
[0029] And, the method for preparing collagen tripeptide by using collagenase in different embodiments of the present invention includes: the first step (S10): mixing the pretreated fish scales and water in a weight ratio of 2:8; the second step (S20) : the mixture was heat-treated at 90°C for 5 hours; the 3rd step (S30): adding the collagenase prepared according to claim 1 and degrading it at 35°C for 12 hours; the 4th step (S40): from The 3rd step removes impurities from the ingredients; the 5th step (S50): purify the ingredients according to ion exchange chromatography; the 6th step (S60): concentrate the purified ingredients; the 7th step (S70): utilize Activated charcoal purification of the concentr...
Embodiment 1
[0031] Screening for new bacterial strains with collagen degrading activity
[0032] As a generally recognized as safe (GRAS) strain, Bacillus subtilis is known to be harmless to humans, and it is widely used in food manufacturing because it can produce various proteolytic enzymes, lipid-decomposing enzymes, and sugar-inverting enzymes and feed addition. And, it is also reported that a part of Bacillus subtilis has collagen hydrolysis activity (Nagano H et al., Biosci Biotehnol Biochem, 2000, 64 (1): 181-3; Tran LH et al., J Food Sci, 2002, 67(3):1184-7). In the present invention, a collagen-degrading enzyme derived from a novel Bacillus subtilis was isolated, and its use in a collagen-degrading process was attempted.
[0033] After collecting soil samples in Jinju, South Gyeongsang Province, suspend 30g of soil in 270ml of sterile PBS buffer, and leave it at room temperature for about 30 minutes to remove coarse soil particles and impurities through sedimentation. 10ml of...
Embodiment 2
[0039] Isolation of New Collagenase (BP)
[0040] Collagenase (BP) derived from Bacillus was isolated and purified. The bacterial strain was cultured in 1000ml mTB (2.4% yeast extract, 1.2% tryptone, 1% glycerol, 2.31% KH 2 PO 4 , 12.54%K 2 HPO 4 ) at 30° C. and 180 rpm for 24 hours, and then centrifuged at 6500 rpm for 15 minutes. The resulting supernatant was carefully transferred to a new tube and concentrated 5-fold using a 30 kDa filter. The concentrated supernatant was purified by ion exchange chromatography using an AKTA prime unit.
[0041] The purification conditions used were: buffer A (50 mM Tris-HCl (pH 7.5)) was used as a binding buffer, and buffer B (50 mM Tris-HCl (pH 7.5), 0.5 M NaCl) was used for gradient elution. The flow rate was 5ml / min. Purification was carried out using cation-exchange chromatography and anion-exchange chromatography, and as the ion resins, the cation resin——SP Sepharose resin (GE Healthcare, USA, New Jersey) and the anion resin——...
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