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Thermophilic Geobacillus kaustophilus HTA426 genome possesses a monoacylglycerol lipase (MAGL) gene. MAGLs can synthesize emulsifiers for use in the food and pharmaceutical industries from fatty acids and glycerol. They can also be used to analyze monoacylglycerol (MAG) levels in serum and food. The MAGL gene from strain HTA426 was artificially synthesized and heterologously expressed in Escherichia coli BL21(DE3). The recombinant His-tag fused MAGL (GkMAGL) was purified using a Ni2+-affinity column. The purified enzyme showed a temperature optimum at 65 °C and was stable up to 75 °C after 30 min incubation. In addition, the enzyme exhibited a pH optimum of 7.5 and was stable from pH 5.0 to 11.0. The enzyme hydrolyzed monoacylglycerols and showed the highest activity toward 1-monolauroylglycerol. The enzyme was stable in the presence of various organic solvents and detergents. The addition of Triton X-100 significantly increased GkMAGL activity. The thermal stability of the enzyme was higher than that of thermostable MAGL from Geobacillus sp. 12AMOR1 (12AMOR1_MAGL). Circular dichroism spectral analysis showed that the conformational stability of the GkMAGL was higher than that of 12AMOR1_MAGL at higher temperatures. These results indicate that the GkMAGL has useful features that can be used for various biotechnological applications. [ABSTRACT FROM AUTHOR]

Cholesterol oxidases (COXases) have a diverse array of applications including analysis of blood cholesterol levels, synthesis of steroids, and utilization as an insecticidal protein. The COXase gene from Janthinobacterium agaricidamnosum was cloned and expressed in Escherichia coli. The purified COXase showed an optimal temperature of 60 °C and maintained about 96 and 72% of its initial activity after 30 min at 60 and 70 °C, respectively. In addition, the purified COXase exhibited a pH optimum at 7.0 and high pH stability over the broad pH range of 3.0–12.0. The pH stability of the COXase at pH 12.0 was higher than that of highly stable COXase from Chromobacterium sp. DS-1. The COXase oxidized cholesterol and β-cholestanol at higher rates than other 3β-hydroxysteroids. The Km, Vmax, and kcat values for cholesterol were 156 μM, 13.7 μmol/min/mg protein, and 14.4 s−1, respectively. These results showed that this enzyme could be very useful in the clinical determination of cholesterol in serum and the production of steroidal compounds. This is the first report to characterize a COXase from the genus Janthinobacterium. [ABSTRACT FROM AUTHOR]

The extracellular cholesterol oxidase of Pseudomonas sp. strain ST-200 was purified to investigate the role of this activity in ST-200 strain-mediated cholesterol oxidation in an organic solvent. The oxidase effectively oxidized cholesterol in the organic solvents but was not as effective in oxidizing cholesterol in the assay solution. It is speculated that oxidase had a mediating effect in the early stages of the cholesterol conversion reactions in the ST-200 culture.

Pseudomonas sp. strain ST-200 dehydrogenates and oxygenates cholesterol in a two-phase water-organic solvent system to reduce cholesterol content in the system. The oxygenation induces the formation of two major compounds, (6)beta-hydroxycholest-4-en-3-one and cholest-4-ene-3,6-dione, and three minor compounds. The ST-200 strain uses a ketone or hydroxyl group to oxidize the C-3 and C-6 cholesterol positions.

Gene expression profiles were collected from Escherichia coli strains before and after exposure to organic solvents, and the six genes that showed higher gene expression were selected. Among these genes, glpC encoding the anaerobic glycerol-3-phosphate dehydrogenase subunitC remarkably increased the organic solvent tolerance.