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首页> 外文期刊>Applied and Environmental Microbiology >Identification of Endopeptidase Genes from the Genomic Sequence of Lactobacillus helveticus CNRZ32 and the Role of These Genes in Hydrolysis of Model Bitter Peptides
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Identification of Endopeptidase Genes from the Genomic Sequence of Lactobacillus helveticus CNRZ32 and the Role of These Genes in Hydrolysis of Model Bitter Peptides

机译：从瑞士乳杆菌CNRZ32基因组序列鉴定内肽酶基因及其在模型苦味肽水解中的作用

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Genes encoding three putative endopeptidases were identified from a draft-quality genome sequence of Lactobacillus helveticus CNRZ32 and designated pepO3, pepF, and pepE2. The ability of cell extracts from Escherichia coli DH5α derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, β-casein (β-CN) (f193-209) and αS1-casein (αS1-CN) (f1-9), under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10°C) was examined. CNRZ32 PepO3 was determined to be a functional paralog of PepO2 and hydrolyzed both peptides, while PepE and PepF had unique specificities towards αS1-CN (f1-9) and β-CN (f193-209), respectively. CNRZ32 PepE2 and PepO did not hydrolyze either peptide under these conditions. To demonstrate the utility of these peptidases in cheese, PepE, PepO2, and PepO3 were expressed in Lactococcus lactis, a common cheese starter, using a high-copy vector pTRKH2 and under the control of the pepO3 promoter. Cell extracts of L. lactis derivatives expressing these peptidases were used to hydrolyze β-CN (f193-209) and αS1-CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides αS1-CN (f1-9), αS1-CN (f1-13), and αS1-CN (f1-16) were identified from Cheddar cheese serum and included in the defined peptide mix. Our results demonstrate that in all systems examined, PepO2 and PepO3 had the highest activity with β-CN (f193-209) and αS1-CN (f1-9). Cheese-derived peptides were observed to affect the activity of some of the enzymes examined, underscoring the importance of incorporating such peptides in model systems. These data indicate that L. helveticus CNRZ32 endopeptidases PepO2 and PepO3 are likely to play a key role in this strain's ability to reduce bitterness in cheese.
机译：从瑞士乳杆菌CNRZ32的草稿质量基因组序列中鉴定出编码三种推定的内肽酶的基因，并将其命名为pepO3，pepF和pepE2。表达CNRZ32内肽酶PepE，PepE2，PepF，PepO，PepO2和PepO3的大肠杆菌DH5α衍生物的细胞提取物水解模型苦味肽，β-酪蛋白（β-CN）（f193-209）和αS1-酪蛋白（在干酪成熟条件下（pH 5.1、4％NaCl和10°C）检查了αS1-CN）（f1-9）。 CNRZ32 PepO3被确定为PepO2的功能旁系同源物，并水解了这两种肽，而PepE和PepF分别对αS1-CN（f1-9）和β-CN（f193-209）具有独特的特异性。在这些条件下，CNRZ32 PepE2和PepO不会水解任何一种肽。为了证明这些肽酶在干酪中的实用性，使用高拷贝载体pTRKH2并在pepO3启动子的控制下，在常见的干酪发酵剂乳酸乳球菌中表达了PepE，PepO2和PepO3。表达这些肽酶的乳酸乳球菌衍生物的细胞提取物在单肽反应中，在确定的肽混合物中以及在单肽反应中，用于在干酪干酪化条件下水解β-CN（f193-209）和αS1-CN（f1-9）。切达干酪的血清。从切达干酪奶酪血清中鉴定出肽αS1-CN（f1-9），αS1-CN（f1-13）和αS1-CN（f1-16），并将其包括在确定的肽混合物中。我们的结果表明，在所有检查的系统中，PepO2和PepO3对β-CN（f193-209）和αS1-CN（f1-9）的活性最高。观察到干酪衍生的肽会影响某些酶的活性，从而强调了将这些肽掺入模型系统的重要性。这些数据表明，瑞士乳杆菌CNRZ32内肽酶PepO2和PepO3可能在该菌株减少奶酪苦味的能力中起关键作用。

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