Jørgensen Casper M, Madsen Søren M, Vrang Astrid, Hansen Ole C, Johnsen Mads G
Bioneer A/S, Kogle Alle 2, 2970 Hørsholm, Denmark.
Protein Expr Purif. 2013 Dec;92(2):148-55. doi: 10.1016/j.pep.2013.09.009. Epub 2013 Sep 29.
Thermitase (EC 3.4.21.66) is a thermostable endo-protease with the ability to convert various food relevant substrates into low-molecular weight peptides. A thermitase produced by Laceyella sacchari strain DSM43353 was found to have a mature amino acid sequence nearly identical to that of the original thermitase isolated from Thermoactinomyces vulgaris. The DSM43353 thermitase gene sequence contains a pro-peptide including parts of an I9 inhibitor motif. Expression of the thermitase gene in the Lactococcus lactis P170 expression system allowed secretion of stable thermitase in an auto-induced fermentation setup at 30°C. Thermitase accumulated in the culture supernatant during batch fermentations and was easily activated at 50°C or by prolonged dialysis. The activation step resulted in an almost complete degradation of endogenous L. lactis host proteins present in the supernatant. Mature activated product was stable at 50°C and functional at pH values between pH 6 and pH 11, suggesting that substrate hydrolysis can be performed over a broad range of pH values. The L. lactis based P170 expression system is a simple and safe system for obtaining food compatible thermitase in the range of 100 mg/L.
嗜热菌蛋白酶（EC 3.4.21.66）是一种耐热的内切蛋白酶，能够将各种与食品相关的底物转化为低分子量肽。人们发现，嗜糖莱西氏菌DSM43353菌株产生的一种嗜热菌蛋白酶，其成熟氨基酸序列与从普通嗜热放线菌中分离出的原始嗜热菌蛋白酶几乎相同。DSM43353嗜热菌蛋白酶基因序列包含一个前肽，其中包括部分I9抑制剂基序。在乳酸乳球菌P170表达系统中表达嗜热菌蛋白酶基因，可在30℃的自动诱导发酵设置中分泌稳定的嗜热菌蛋白酶。在分批发酵过程中，嗜热菌蛋白酶积聚在培养上清液中，并在50℃或通过长时间透析容易被激活。激活步骤导致上清液中存在的内源性乳酸乳球菌宿主蛋白几乎完全降解。成熟的激活产物在50℃下稳定，在pH值介于6和11之间时具有活性，这表明底物水解可以在很宽的pH值范围内进行。基于乳酸乳球菌的P170表达系统是一种简单且安全的系统，可用于获得浓度在100 mg/L范围内的食品相容性嗜热菌蛋白酶。
