National Food Institute, Technical University of Denmark, 2800 Kgs Lyngby, Denmark.
Department of Biotechnology and Biomedicine, Technical University of Denmark, 2800 Kgs Lyngby, Denmark.
FEMS Microbiol Lett. 2022 Feb 12;368(21-24). doi: 10.1093/femsle/fnac006.
There is increasing interest in gluten-degrading enzymes for use during food and drink processing. The industrially available enzymes usually work best at low to ambient temperatures. However, food manufacturing is often conducted at higher temperatures. Therefore, thermostable gluten-degrading enzymes are of great interest. We have identified a new thermostable gluten-degrading proline-specific prolyl endoprotease from the archaea Thermococcus kodakarensis. We then cloned and expressed it in Escherichia coli. The prolyl endoprotease was found to have a size of 70.1 kDa. The synthetic dipeptide Z-Gly-Pro-p-nitroanilide was used to characterize the prolyl endoprotease and it had maximum activity at pH 7 and 77°C. The Vmax, Km and kcat values of the purified prolyl endoprotease were calculated to be 3.14 mM/s, 1.10 mM and 54 s-1, respectively. When the immunogenic gluten peptides PQPQLPYPQPQLPY (α-gliadin) and SQQQFPQPQQPFPQQP (γ-hordein) were used as substrates, the prolyl endoprotease was able to degrade these. Furthermore, gluten in wort was reduced when the prolyl endoprotease was used during mashing of barley malt. The discoveries open up new food processing possibilities and further the understanding of proline-specific protease diversity.
人们对用于食品和饮料加工的降解谷蛋白的酶越来越感兴趣。工业上可用的酶通常在低温到环境温度下效果最佳。然而，食品制造通常在较高的温度下进行。因此，耐热的谷蛋白降解酶非常有吸引力。我们从古细菌 Thermococcus kodakarensis 中鉴定出一种新的耐热谷蛋白降解脯氨酸特异性脯氨酰内肽酶。然后，我们在大肠杆菌中克隆并表达了它。该脯氨酰内肽酶的大小为 70.1 kDa。合成二肽 Z-Gly-Pro-p-硝基苯胺用于表征脯氨酰内肽酶，其在 pH 7 和 77°C 时具有最大活性。纯化的脯氨酰内肽酶的 Vmax、Km 和 kcat 值分别计算为 3.14 mM/s、1.10 mM 和 54 s-1。当免疫原性谷蛋白肽 PQPQLPYPQPQLPY（α-麦醇溶蛋白）和 SQQQFPQPQQPFPQQP（γ-麦谷蛋白）用作底物时，该脯氨酰内肽酶能够降解这些底物。此外，当在大麦麦芽糖化过程中使用脯氨酰内肽酶时，麦汁中的谷蛋白减少。这些发现为新的食品加工可能性开辟了道路，并进一步了解了脯氨酸特异性蛋白酶的多样性。
