Molecular Analysis of a Gene Encoding a Cell-Bound Esterase from Streptomyces chrysomallus

ABSTRACT A gene (estA) encoding a 42-kDa cell-bound esterase, EstA, was found to be located 75 bp upstream of the cyclophilin A gene (cypA) of Streptomyces chrysomallus. Western blot analysis revealed the presence of EstA (42 kDa) in cell extracts of S. chrysomallus X2 and Streptomyces lividans. EstA specifically hydrolyzes short-chainp-nitrophenyl esters. EstA formation starts at the end of growth phase, and its activity level remains constant throughout stationary phase. Expression of estA from the melanin (mel) promoter in plasmid pIJ702 led to a substantial increase of total esterase activity in streptomycetes.

Esterase activity and associated insecticide resistance in the damson-hop aphid,Phorodon humuli(Schrank) (Hemiptera: Aphididae)

Phorodon humuli(Schr.) was obtained from both wild and cultivated hop plants, cultured, and bioassayed using seven different foliar spray insecticides, and the results were assesssed by probit analysis to determine LC50s and LC95s. The wild aphids and their progeny were highly susceptible to the insecticides, whereas those from cultivated hops were generally resistant to them. The level of resistance was related to the source of the original aphid population and the insecticide used. Clones of insecticide-resistant and susceptible aphids were maintained under identical conditions, and individual adult apterae were assayed for total esterase and esterase isoenzyme activities. Total esterase activity was appreciably higher in resistant aphids than in susceptible ones. Resistant aphids were associated with high esterase isoenzyme activity of complex bands II–V, while in susceptible aphids these bands were virtually missing. An insecticide-resistant clone ofP. humuliwas selected for low esterase activity and a susceptible clone was selected for high esterase activity, over eight generations. Selection pressure was then reversed for a further eight generations. The resistant clone lost its esterase activity by generation six and it remained low thereafter, whereas that of the control stock remained high. By contrast, the esterase activity of the susceptible clone remained unaltered by selection. The resistant clone also lost its resistance to both methomyl and methidathion.

FRACTIONATION OF MOUSE LIVER MICROSOMAL ESTERASES

Mouse liver microsomal esterases were fractionated on DEAE-cellulose after the solution of these enzymes in a solution containing 0.1 M glycyl glycine buffer, pH 7.0, and 5 × 10−4 M Lubrol W, a nonionic detergent. Elution of the enzymes from the DEAE-cellulose was accomplished by using NaCl in the glycyl glycine – Lubrol W solution. Microsomes isolated from sucrose and glycerol homogenates have three esterase peaks in common and two peaks not in common. The glycerol supernatant fraction from whole liver homogenate, which contains about 50% of the total esterase activity, and the sucrose supernatant fraction, which contains about 6% of the total esterase activity, have four common esterase peaks, one of which is different from both the microsomes isolated from glycerol and sucrose liver homogenates. Incorporation of Lubrol W in the solution that was employed for the elution of the esterases from the DEAE-cellulose was necessary to prevent extensive loss or inactivation of these enzymes. The nature of the changes induced by glycerol on liver esterases is briefly discussed.