Neumann S, Kula M-R
Institute of Enzyme Technology, Heinrich-Heine University Düsseldorf, 52426, Jülich, Germany.
Appl Microbiol Biotechnol. 2002 May;58(6):772-80. doi: 10.1007/s00253-002-0943-6. Epub 2002 Mar 8.
The peptide amidase (Pam) from the gram-negative bacterium Stenotrophomonas maltophilia catalyzes predominantly the hydrolysis of the C-terminal amide bond in peptide amides. Its gene ( pam) was isolated by Southern hybridization using a DNA probe derived from the known N-terminal amino acid sequence. Pam is a member of the amidase signature family and was identified as a periplasmic protein by an N-terminal signal peptide found in the gene. The processed protein consists of 503 amino acids with a molecular mass of 53.5 kDa. The recombinant enzyme with a C-terminal His(6) tag has a monomeric structure and its isoelectric point is 6.3. The dipeptide amide L-Ala- L-Phe-NH(2) is hydrolyzed in the absence of cofactors to L-Ala- L-Phe-OH and ammonia with V(max)=194 U/mg and K(m) <0.5 mM. The natural function of Pam remains unclear. Chymostatin ( K(i)<0.3 microM) and Pefabloc SC ( K(i) not determined) were identified as inhibitors. When the gene was expressed in Escherichia coli on a 12-l scale, the specific activity in the crude extract was 60 U/mg, compared to 0.24 U/mg in S. maltophilia. In the expression system, Pam made up about 31% of the total soluble cell protein. From 75 g wet cells, 2.1 g of >95% pure enzyme was obtained, which corresponds to a total activity of 416,000 units.
嗜麦芽窄食单胞菌这种革兰氏阴性菌中的肽酰胺酶（Pam）主要催化肽酰胺中C末端酰胺键的水解。利用从已知N末端氨基酸序列衍生而来的DNA探针，通过Southern杂交分离出了其基因（pam）。Pam是酰胺酶特征家族的成员，通过该基因中发现的N末端信号肽被鉴定为周质蛋白。加工后的蛋白质由503个氨基酸组成，分子量为53.5 kDa。带有C末端His(6)标签的重组酶具有单体结构，其等电点为6.3。在没有辅因子的情况下，二肽酰胺L-Ala-L-Phe-NH₂被水解为L-Ala-L-Phe-OH和氨，V(max)=194 U/mg，K(m)<0.5 mM。Pam的天然功能仍不清楚。抑肽酶（K(i)<0.3 microM）和苯甲磺酰氟（Pefabloc SC，K(i)未测定）被鉴定为抑制剂。当该基因在12升规模的大肠杆菌中表达时，粗提物中的比活性为60 U/mg，而嗜麦芽窄食单胞菌中的为0.24 U/mg。在该表达系统中，Pam约占总可溶性细胞蛋白的31%。从75克湿细胞中获得了2.1克纯度>95%的酶，其总活性为416,000单位。
