Dataset Information

High-level synthesis and secretion of laccase, a metalloenzyme biocatalyst, by the halophilic archaeon Haloferax volcanii.

ABSTRACT: Haloarchaea and their enzymes have extremophilic properties desirable for use as platform organisms and biocatalysts in the bioindustry. These GRAS (generally regarded as safe) designated microbes thrive in hypersaline environments and use a salt-in strategy to maintain osmotic homeostasis. This unusual strategy has resulted in the evolution of most of the intracellular and extracellular enzymes of haloarchaea to be active and stable not only in high salt (2-5M) but also in low salt (0.2M). This salt tolerance is correlated with a resilience to low water activity, thus, rendering the haloarchaeal enzymes active and stable in organic solvent and temperatures of 50-60°C used in the enzymatic biodelignification and saccharification of lignocellulosic materials. High-level secretion of haloarchaeal enzymes to the extracellular milieu is useful for many applications, including enzymes that deconstruct biomass to allow for lignin depolymerization and simultaneous fermentation of sugars released from hemicellulose and cellulose fractions of lignocellulosics. Here we detail strategies and methods useful for high-level secretion of a laccase, HvLccA, that mediates oxidation of various phenolics by engineering a recombinant strain of the haloarchaeon Haloferax volcanii.

SUBMITTER: Kasirajan L

PROVIDER: S-EPMC8867731 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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High-level synthesis and secretion of laccase, a metalloenzyme biocatalyst, by the halophilic archaeon Haloferax volcanii.

Kasirajan Lakshmi L Adams Zachary Z Couto-Rodriguez Ricardo L RL Gal Daniel D Jia Huiyong H Mondragon Paula P Wassel Paul C PC Yu David D Uthandi Sivakumar S Maupin-Furlow Julie A JA

Methods in enzymology 20210727

Haloarchaea and their enzymes have extremophilic properties desirable for use as platform organisms and biocatalysts in the bioindustry. These GRAS (generally regarded as safe) designated microbes thrive in hypersaline environments and use a salt-in strategy to maintain osmotic homeostasis. This unusual strategy has resulted in the evolution of most of the intracellular and extracellular enzymes of haloarchaea to be active and stable not only in high salt (2-5M) but also in low salt (0.2M). This ...[more]

PMID: 34752290

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Project description:A 20S proteasome, composed of alpha(1) and beta subunits arranged in a barrel-shaped structure of four stacked rings, was purified from a halophilic archaeon Haloferax volcanii. The predominant peptide-hydrolyzing activity of the 600-kDa alpha(1)beta-proteasome on synthetic substrates was cleavage carboxyl to hydrophobic residues (chymotrypsin-like [CL] activity) and was optimal at 2 M NaCl, pH 7.7 to 9.5, and 75 degrees C. The alpha(1)beta-proteasome also hydrolyzed insulin B-chain protein. Removal of NaCl inactivated the CL activity of the alpha(1)beta-proteasome and dissociated the complex into monomers. Rapid equilibration of the monomers into buffer containing 2 M NaCl facilitated their reassociation into fully active alpha(1)beta-proteasomes of 600 kDa. However, long-term incubation of the halophilic proteasome in the absence of salt resulted in hydrolysis and irreversible inactivation of the enzyme. Thus, the isolated proteasome has unusual salt requirements which distinguish it from any proteasome which has been described. Comparison of the beta-subunit protein sequence with the sequence deduced from the gene revealed that a 49-residue propeptide is removed to expose a highly conserved N-terminal threonine which is proposed to serve as the catalytic nucleophile and primary proton acceptor during peptide bond hydrolysis. Consistent with this mechanism, the known proteasome inhibitors carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132) and N-acetyl-leucinyl-leucinyl-norleucinal (calpain inhibitor I) were found to inhibit the CL activity of the H. volcanii proteasome (K(i) = 0.2 and 8 microM, respectively). In addition to the genes encoding the alpha(1) and beta subunits, a gene encoding a second alpha-type proteasome protein (alpha(2)) was identified. All three genes coding for the proteasome subunits were mapped in the chromosome and found to be unlinked. Modification of the methods used to purify the alpha(1)beta-proteasome resulted in the copurification of the alpha(2) protein with the alpha(1) and beta subunits in nonstoichometric ratios as cylindrical particles of four stacked rings of 600 kDa with CL activity rates similar to the alpha(1)beta-proteasome, suggesting that at least two separate 20S proteasomes are synthesized. This study is the first description of a prokaryote which produces two separate 20S proteasomes and suggests that there may be distinct physiological roles for the two different alpha subunits in this halophilic archaeon.

Dataset Information

High-level synthesis and secretion of laccase, a metalloenzyme biocatalyst, by the halophilic archaeon Haloferax volcanii.

Publications

High-level synthesis and secretion of laccase, a metalloenzyme biocatalyst, by the halophilic archaeon Haloferax volcanii.

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