A halophilic extracellular protease from a halophilic archaebacterium strain 172 P1

Abstract

An unidentified halophilic archaebacterium strain 172 P1 produced three extracellular proteases in media containing 15-27% salts. One component, F-II, was purified to homogeneity. It is a serine protease that can be inhibited by phenylmethylsulfonyl fluoride and chymostatin. A high concentration of NaCl was required for its stability; in the presence of 25% NaCl, only 4% of the activity was lost by incubating at 60 degrees C for 30 min, while complete inactivation occurred in the presence of 5% NaCl. F-II is a thermophilic and halophilic protease. High activity was obtained at 75-80 degrees C when F-II was assayed in the presence of 25% NaCl. The optimal concentration of NaCl required was 10-14% when assayed at 70 degrees C with azocasein as substrate, though a halophilic characteristic was not distinct at lower temperatures. Hydrolyses of the synthetic substrates succinyl-alanyl-alanyl-prolyl-phenylalanyl-4-methylcoumaryl-7-amide or succinyl-alanyl-alanyl-alanyl-p-nitroanilide at 26 degrees C were maximal at 25 and 30% NaCl, respectively. F-II was most stable at pH 6-7, and its optimal pH was 10.7. Its molecular weight was estimated as 44,000-46,000 by sodium dodecyl sulfate--polyacrylamide gel electrophoresis and by gel filtration--high-pressure liquid chromatography. The sequence of the 35 N-terminal amino acid residues was determined and compared with that of other serine proteases.

Molecular cloning and sequencing of the gene for a halophilic alkaline serine protease (halolysin) from an unidentified halophilic archaea strain (172P1) and expression of the gene in Haloferax volcanii

“…Though a few articles have dealt with the purification and characterization of proteases from Halobacterium salinarium (14,18) and H. halobium (12), detailed mechanisms to explain the halophilicity of these enzymes have not been elucidated because of their extreme instability even in the presence of 3 to 4 M NaCl (12). In a previous article, Kamekura and Seno reported the purification and characterization of a halophilic alkaline serine protease (F-II) of a halophilic archaebacterium (strain 172P1) and also determined the sequence of the first 35 N-terminal amino acids (13). F-II is thus the first serine protease from archaebacteria (alternatively called archaea, according to a new proposal by Woese et al [29]) whose amino acid sequence has been partially determined.…”

“…The protease halolysin was purified, and a powdered phenylmethane sulfonyl-halolysin (PMS-halolysin) was obtained as described in a previous article (13). The PMS-halolysin was digested completely with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-trypsin, and resultant oligopeptides were separated by highpressure liquid chromatography (HPLC) with a column of VYDAC 218TP54.…”

“…volcanii WFD11 was transformed with plasmid pMDS13 by the method described previously (9,10). Transformants were plated on 1.5% agar plates of transformation medium containing 18% seawater, 15% sucrose, 0.8% Bacto skim milk (13), and 0.3 pug of novobiocin per ml and incubated at 37°C. Transformants expressing the halolysin gene hly were detected as colonies surrounded by an area of clearing in the milk agar (i.e., a clear halo).…”

Extreme Environments: Goldmine of Industrially Valuable Alkali-stable Proteases

“…A few proteases from extreme halophiles have been reported from archaeal halophiles [171,175,176], but some from moderately halophilic and halotolerant bacteria have also been purified and studied [27,172,177,178]. As moderate halophiles are adapted to grow over a wide salt range (optimal growth at 3-15% NaCl), they represent an interesting group of organisms with great industrial potential [177].…”

Cloning, expression, and nucleotide sequence of the alpha-amylase gene from the haloalkaliphilic archaeon Natronococcus sp. strain Ah-36

“…Only a few extracellular enzymes have been purified from strains in the domain Archaea (2,8,10,11). Among them, halolysin is the only enzyme up to now with a determined primary structure (9).…”