Proteolytic Enzymes from Extremely Halophilic Bacteria

Abstract

S U M M A R YHalobacterium strains produce a truly extracellular proteinase which degrades gelatine and casein. It has a pH optimum of about 8 and depends upon divalent cations and a high concentration of NaCl or KC1 for activity and stability. Proteolytic enzymes were also found in cell homogenates obtained by ultra sonic treatment. A caseinolytic enzyme, probably different from the extracellular one, is associated with particles which sediment upon ultracentrifugation. A soluble peptidase of lower molecular weight is also present in the extract. Both enzymes are dependent upon divalent cations and a high concentration of NaCl or KCI for activity. In contrast to other halophilic enzymes, the proteolytic enzymes of Halobacterium sdharium are more active in the presence of NaCl than KCl at equimolar concentrations. I N T R O D U C T I O NBacteria of the genus Halobacterium are widely distributed in brines of high salt concentration. They are thus conspicuous in solar evaporation ponds of salt works, where they often become the dominating type of organisms when the salt concentration rises above 25 %. The extremely halophilic bacteria are best cultivated in complex media containing peptones and yeast extract to which at least 15 % (wlv) NaCl has been added. Best growth is obtained in media containing about 25 % salt, and when bacteria are transferred to solutions with a salt concentration of less than about 10 % they lyse rapidly.The unusual properties of halophilic bacteria make them interesting objects of study for both microbiologists and biochemists, and Larsen (1967) summarized the result of a large number of investigations on them. Special methods are often necessary in studies of their metabolism and conventional procedures are unsuitable for the purification and assay of ' halophilic ' enzymes.The extreme halophiles do not generally grow well on carbohydrates but they have a well-developed enzyme apparatus for metabolizing amino acids. In order to utilize the proteins of dead organisms in salt brines the bacteria must form proteolytic enzymes. Gibbons (1957) found that 45 out of 49 tested strains were gelatinolytic, and most of these strains also degraded casein. However, a closer study of the properties of the proteolytic enzymes of such strains has apparently not been made. It is known that the bacteria have an intracellular concentration of NaCl + KCI, which is approximately the same as the NaCl concentration of the growth medium. The halophilic bacteria's enzymes are thus active at salt concentrations which inhibit or even denature many enzymes of non-halophilic organisms. This must be reflected in marked differences in the composition and properties of the protein molecules. Ingram (1947) has

Evidence for Covalent Attachment of Diphytanylglyceryl Phosphate to the Cell-surface Glycoprotein of Halobacterium halobium

“…In the course of the current experiments, we became aware of the occasional appearance of radioactive protein bands with molecular masses Ͻ35-45 kDa. Halobacteria are known to produce extracellular protease (13), and Mescher et al (14) have already reported that isolated halobacterial cell-surface glycoprotein preparations contain a protease activity. Therefore, we tested the action of endogenous protease during protein preparation because we have used incubation conditions (37°C, Ͼ10 min) that reduce the viscosity of the cell lysis solution by DNase I treatment.…”

Transfer of Natrialba asiatica B1T to Natrialba taiwanensis sp. nov. and description of Natrialba aegyptiaca sp. nov., a novel extremely halophilic, aerobic, non-pigmented member of the Archaea from Egypt that produces extracellular poly(glutamic acid).

“…Several authors have established that biochemical tests depend on the culture media utilized rather than on the substrate under test (Gibbons, 1957 ;Norberg & v. Hofsten, 1969 ;Sehgal & Gibbons, 1960 ;Tomlinson & Hochstein, 1972). In order to obtain comparable results, we carried out the biochemical tests for the three strains, strain 40 T , Natrialba asiatica 172P1 T and Natrialba asiatica B1T, in parallel on the same media under the same conditions.…”

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

“…Their extraordinary ability to grow in hypertonic solution (above 300 g of NaCl per liter) and their potential ability to hydrolyze proteins (7) are the main reasons for the reddening of salted fish. 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).…”