Verhaert R M, Riemens A M, van der Laan J M, van Duin J, Quax W J
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
Appl Environ Microbiol. 1997 Sep;63(9):3412-8. doi: 10.1128/aem.63.9.3412-3418.1997.
Alcaligenes faecalis penicillin G acylase is more stable than the Escherichia coli enzyme. The activity of the A. faecalis enzyme was not affected by incubation at 50 degrees C for 20 min, whereas more than 50% of the E. coli enzyme was irreversibly inactivated by the same treatment. To study the molecular basis of this higher stability, the A. faecalis enzyme was isolated and its gene was cloned and sequenced. The gene encodes a polypeptide that is characteristic of periplasmic penicillin G acylase (signal peptide-alpha subunit-spacer-beta subunit). Purification, N-terminal amino acid analysis, and molecular mass determination of the penicillin G acylase showed that the alpha and beta subunits have molecular masses of 23.0 and 62.7 kDa, respectively. The length of the spacer is 37 amino acids. Amino acid sequence alignment demonstrated significant homology with the penicillin G acylase from E. coli A unique feature of the A. faecalis enzyme is the presence of two cysteines that form a disulfide bridge. The stability of the A. faecalis penicillin G acylase, but not that of the E. coli enzyme, which has no cysteines, was decreased by a reductant. Thus, the improved thermostability is attributed to the presence of the disulfide bridge.
粪产碱杆菌青霉素G酰化酶比大肠杆菌的酶更稳定。粪产碱杆菌酶的活性在50℃温育20分钟后不受影响，而同样处理会使超过50%的大肠杆菌酶不可逆地失活。为了研究这种更高稳定性的分子基础，分离了粪产碱杆菌酶并克隆和测序了其基因。该基因编码一种具有周质青霉素G酰化酶特征的多肽（信号肽-α亚基-间隔区-β亚基）。青霉素G酰化酶的纯化、N端氨基酸分析和分子量测定表明，α亚基和β亚基的分子量分别为23.0 kDa和62.7 kDa。间隔区长度为37个氨基酸。氨基酸序列比对显示与大肠杆菌青霉素G酰化酶有显著同源性。粪产碱杆菌酶的一个独特特征是存在形成二硫键的两个半胱氨酸。没有半胱氨酸的大肠杆菌酶的稳定性不受影响，而粪产碱杆菌青霉素G酰化酶的稳定性因还原剂而降低。因此，热稳定性的提高归因于二硫键的存在。
