Patil Umesh, Baloch Khurshid Ahmed, Nile Shivraj Hariram, Kim Jun Tae, Benjakul Soottawat
International Center of Excellence in Seafood Science and Innovation, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai 90110, Songkhla, Thailand.
Division of Food and Nutritional Biotechnology, National Agri-Food Biotechnology Institute (NABI), Sector 81, Sahibzada Ajit Singh Nagar 140306, Punjab, India.
Foods. 2023 Aug 2;12(15):2937. doi: 10.3390/foods12152937.
The study aimed to purify trypsin from the pyloric caeca of Asian seabass (), and investigate its proteolytic capability toward acid-soluble collagen (ASC) in comparison with commercial porcine trypsin (CPT). Trypsin was purified from pyloric caeca, a leftover from the evisceration process, via ammonium sulphate (40-60% saturation) precipitation, and a soybean trypsin inhibitor (SBTI)-Sepharose 4B column. A 18.5-fold purification and a yield of 15.2% were obtained. SDS-PAGE analysis confirmed a single band of trypsin with a molecular weight of 23.5 kDa. Purified trypsin also showed the single band in native-PAGE. The optimal pH and temperature of trypsin for BAPNA (the specific substrate for amidase) hydrolysis were 8.5 and 60 °C, respectively. The trypsin was stable within the pH range of 7.0-9.5 and temperature range of 25-55 °C. Protease inhibition study confirmed that the purified enzyme was trypsin. The purified trypsin had a Michaelis-Menten constant (K) and catalytic constant (k) of 0.078 mM and 5.4 s, respectively, when BAPNA was used. For the hydrolysis of TAME (the specific substrate for esterase), the K and K were 0.09 mM and 4.8 s, respectively. Partially purified seabass trypsin (PPST) had a slightly lower hydrolysis capacity toward ASC than CPT, as evidenced by the lower degree of hydrolysis and protein degradation when the former was used. Both the α-chain and β-chain became more degraded as the hydrolysis time increased. Based on MALDI-TOP, peptides with MW of 2992-2970 Da were dominant in the hydrolysates. Therefore, seabass trypsin could be used in the production of hydrolyzed collagen. It could have economic importance to the market, by replacing some commercial proteases, which have religious constraints.
该研究旨在从尖吻鲈的幽门盲囊中纯化胰蛋白酶，并与市售猪胰蛋白酶（CPT）相比，研究其对酸溶性胶原蛋白（ASC）的蛋白水解能力。通过硫酸铵（40 - 60%饱和度）沉淀和大豆胰蛋白酶抑制剂（SBTI）-琼脂糖4B柱，从去内脏过程中的剩余物幽门盲囊中纯化胰蛋白酶。获得了18.5倍的纯化倍数和15.2%的产率。SDS-PAGE分析证实胰蛋白酶为一条分子量为23.5 kDa的单带。纯化的胰蛋白酶在非变性聚丙烯酰胺凝胶电泳中也显示为单带。胰蛋白酶对BAPNA（酰胺酶的特异性底物）水解的最适pH和温度分别为8.5和60℃。该胰蛋白酶在pH值7.0 - 9.5和温度25 - 55℃范围内稳定。蛋白酶抑制研究证实纯化的酶为胰蛋白酶。当使用BAPNA时，纯化的胰蛋白酶的米氏常数（K）和催化常数（k）分别为0.078 mM和5.4 s⁻¹。对于TAME（酯酶的特异性底物）的水解，Kₘ和kₐₜ分别为0.09 mM和4.8 s⁻¹。部分纯化的鲈鱼胰蛋白酶（PPST）对ASC的水解能力略低于CPT，这可通过使用前者时较低的水解程度和蛋白质降解来证明。随着水解时间的增加，α链和β链都降解得更多。基于基质辅助激光解吸电离飞行时间质谱（MALDI-TOP），水解产物中分子量为2992 - 2970 Da的肽占主导。因此，鲈鱼胰蛋白酶可用于水解胶原蛋白的生产。通过替代一些存在宗教限制的商业蛋白酶，它可能对市场具有经济重要性。
