胶原蛋白肽广泛应用于化妆品与保健品,主要依赖于物理和化学降解法制备。相比较,酶法降解制备胶原蛋白肽由于降解条件温和、分子质量可控等特点具有更大优势。该研究旨在挖掘并实现一种具有商业化潜在价值的胶原蛋白水解酶的高效表达制备,实现胶原蛋白肽的酶法制备。首先,通过密码子优化合成了蜡样芽孢杆菌来源的胶原蛋白水解酶ColVD021编码基因,通过N-端序列截短优化,实现了ColVD021-Δ30在大肠杆菌中的活性表达(8.96±0.21) U/mL。进一步通过优化添加金属离子Zn2+和Ca2+,ColVD021-Δ30的表达水平提高至(16.24±0.21) U/mL。在此基础上,确定了ColVD021-Δ30的最适pH为7.5,最适温度为37 ℃。酶解结果表明,重组ColVD021-Δ30对不同来源的胶原蛋白均具有高降解活力,胶原蛋白肽分子质量均低于1 000 Da。此外,农副产品鸡胸软骨的酶解结果表明,ColVD021-Δ30可以直接水解粉碎的新鲜鸡胸软骨生产胶原蛋白肽。该研究实现了蜡样芽孢杆菌胶原蛋白水解酶的高效表达,并建立了胶原蛋白肽的酶法制备工艺,为工业化酶法生产胶原蛋白肽提供了解决方案。
Collagen peptides are extensively applied in cosmetics and health products, primarily prepared through physical and chemical degradation methods.In comparison, enzymatic degradation for collagen peptide production offers significant advantages such as mild degradation conditions and controllable molecular weight.This study aimed to explore and achieve efficient expression and preparation of a collagenase with commercial potential, derived from Bacillus cereus, for enzymatic collagen peptide synthesis.Initially, the gene encoding collagenase ColVD021 from Bacillus cereus was synthesized with codon optimization.Through N-terminal sequence truncation optimization, the active expression of ColVD021-Δ30 was achieved in Escherichia coli (8.96±0.21) U/mL.Further enhancement of ColVD021-Δ30 expression levels to (16.24±0.21) U/mL was achieved by optimizing the addition of metal ions Zn2+ and Ca2+.The optimal pH and temperature for ColVD021-Δ30 were determined to be 7.5 and 37 ℃, respectively.Enzymatic hydrolysis results demonstrated that recombinant ColVD021-Δ30 exhibited high degradation activity on collagen from different sources, yielding collagen peptide molecules with molecular weights below 1 000 Da.Additionally, enzymatic digestion of agricultural by-product chicken breast cartilage indicated that ColVD021-Δ30 could directly hydrolyze crushed fresh chicken breast cartilage to produce collagen peptides.This study realized efficient expression of Bacillus cereus collagenase and established an enzymatic process for collagen peptide production, offering a solution for industrial-scale enzymatic synthesis of collagen peptides.
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