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Proteins with JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) domains are widespread among all domains of life, yet poorly understood. Here we report the purification and characterization of an archaeal JAMM/MPN+ domain protein (HvJAMM1) from Haloferax volcanii that cleaves ubiquitin-like small archaeal modifier proteins (SAMP1/2) from protein conjugates. HvJAMM1 cleaved SAMP1/2 conjugates generated in H. volcanii as well as isopeptide-and linear-linked SAMP1-MoaE in purified form. Cleavage of linear linked SAMP1-MoaE was dependent on the presence of the SAMP domain and the C-terminal VSGG motif of this domain. While HvJAMM1 was inhibited by size exclusion chromatography and metal chelators, its activity could be restored by addition of excess ZnCl2. HvJAMM1 residues (Glu31, His88, His90, Ser98 and Asp101) that were conserved with the JAMM/MPN+ active-site motif were required for enzyme activity. Together, these results provide the first example of a JAMM/MPN+ zinc metalloprotease that independently catalyzes the cleavage of ubiquitin-like (isopeptide and linear) bonds from target proteins. In archaea, HvJAMM1 likely regulates sampylation and the pools of ‘free’ SAMP available for protein modification. HvJAMM1-type proteins are thought to release the SAMPs from proteins modified post-translationally as well as those synthesized as domain fusions.

Laccases couple the oxidation of phenolic compounds to the reduction of molecular oxygen and thus span a wide variety of applications. While laccases of eukaryotes and bacteria are well characterized, these enzymes have not been described in archaea. Here, we report the purification and characterization of a laccase (LccA) from the halophilic archaeon Haloferax volcanii. LccA was secreted at high levels into the culture supernatant of a recombinant H. volcanii strain, with peak activity (170 ؎ 10 mU ⅐ ml ؊1 ) at stationary phase (72 to 80 h). LccA was purified 13-fold to an overall yield of 72% and a specific activity of 29.4 U ⅐ mg ؊1 with an absorbance spectrum typical of blue multicopper oxidases. The mature LccA was processed to expose an N-terminal Ala after the removal of 31 amino acid residues and was glycosylated to 6.9% carbohydrate content. Purified LccA oxidized a variety of organic substrates, including bilirubin, syringaldazine (SGZ), 2,2,-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and dimethoxyphenol (DMP), with DMP oxidation requiring the addition of CuSO 4 . Optimal oxidation of ABTS and SGZ was at 45°C and pH 6 and pH 8.4, respectively. The apparent K m values for SGZ, bilirubin, and ABTS were 35, 236, and 670 M, with corresponding k cat values of 22, 29, and 10 s ؊1 , respectively. The purified LccA was tolerant of high salt, mixed organosolvents, and high temperatures, with a half-life of inactivation at 50°C of 31.5 h.Multicopper oxidases (MCOs) are a family of enzymes that include laccases (p-diphenol: dioxygen oxidoreductases; EC 1.10.3.2), ascorbate oxidases (EC 1.10.3.3), ferroxidases (EC 1.16.3.1), bilirubin oxidases (EC 1.3.3.5), and other enzyme subfamilies (27,65). MCOs couple the oxidation of organic and/or inorganic substrates to the four-electron reduction of molecular oxygen to water. These enzymes often have four Cu atoms classified into type 1 (T1), type 2 (T2), and type 3 (T3) centers, in which a mononuclear T1 center on the surface of the enzyme provides long-range intramolecular one-electron transfer from electron-donating substrates to an internal trinuclear T2-T3 center formed by a T2 Cu coordinated with a T3 Cu pair. The T2-T3 cluster subsequently reduces dioxygen to water.Enzymes of the laccase subfamily oxidize a broad range of compounds, including phenols, polyphenols, aromatic amines, and nonphenolic substrates, by one-electron transfer to molecular oxygen and thus have a wide variety of applications from biofuels to human health. The best-known application is the use of a laccase from the lacquer tree Rhus vernicifera in paint and adhesives for more than 6,000 years in East Asia (29). Laccases have also been used in the delignification of pulp, bleaching of textiles and carcinogenic dyes, detoxification of water and soils, removal of phenolics from wines, improving adhesive properties of lignocellulosic products, determination of bilirubin levels in serum, and transformation of antibiotics and steroids (60). In addition, laccases have demonstrated pote...

Background: Cellulose, the most versatile biomolecule on earth, is available in large quantities from plants. However, cellulose in plants is accompanied by other polymers like hemicellulose, lignin, and pectin. On the other hand, pure cellulose can be produced by some microorganisms, with the most active producer being Acetobacter xylinum. A. senengalensis is a gram-negative, obligate aerobic, motile coccus, isolated from Mango fruits in Senegal, capable of utilizing a variety of sugars and produce cellulose. Besides, the production is also influenced by other culture conditions. Previously, we isolated and identified A. senengalensis MA1, and characterized the bacterial cellulose (BC) produced. Results: The maximum cellulose production by A. senengalensis MA1 was pre-optimized for different parameters like carbon, nitrogen, precursor, polymer additive, pH, temperature, inoculum concentration, and incubation time. Further, the pre-optimized parameters were pooled, and the best combination was analyzed by using Central Composite Design (CCD) of Response Surface Methodology (RSM). Maximum BC production was achieved with glycerol, yeast extract, and PEG 6000 as the best carbon and nitrogen sources, and polymer additive, respectively, at 4.5 pH and an incubation temperature of 33.5°C. Around 20% of inoculum concentration gave a high yield after 30 days of inoculation. The interactions between culture conditions optimized by CCD included alterations in the composition of the HS medium with 50 mL L − 1 of glycerol, 7.50 g L − 1 of yeast extract at pH 6.0 by incubating at a temperature of 33.5°C along with 7.76 g L − 1 of PEG 6000. This gave a BC yield of wet weight as 469.83 g L − 1. Conclusion: The optimized conditions of growth medium resulted in enhanced production of bacterial cellulose by A. senegalensis MA1, which is around 20 times higher than that produced using an unoptimized HS medium. Further, the cellulose produced can be used in food and pharmaceuticals, for producing high-quality paper, wound dressing material, and nanocomposite films for food packaging.

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