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A CALB-like Cold-Active Lipolytic Enzyme from Pseudonocardia antarctica: Expression, Biochemical Characterization, and AlphaFold-Guided Dynamics

2026-01-14T07:42:06Z

Title: A CALB-like Cold-Active Lipolytic Enzyme from Pseudonocardia antarctica: Expression, Biochemical Characterization, and AlphaFold-Guided Dynamics Authors: Liu Lixiao; Do, Hackwon; Kim Jong-Oh; Lee, Jun Hyuck; Kim Hak Jun Abstract: Cold-active lipolytic enzymes enable low-temperature biocatalysis, but remain underexplored in Antarctic actinomycetes. Here, we report the discovery and first-step characterization of a CALB-like cold-active lipolytic enzyme (PanLip) from Pseudonocardia antarctica. Sequence and structure analyses revealed a canonical alpha/beta-hydrolase fold with a conserved Ser-Asp-His triad and short helical elements around the pocket reminiscent of CALB's alpha 5/alpha 10 lid. Mature PanLip was expressed primarily as inclusion bodies in E. coli; an N-terminally truncation (PanLip Delta N) improved solubility and PanLip Delta N was purified by Ni-NTA. Far-UV CD confirmed a folded alpha/beta architecture. PanLip Delta N favored short-chain substrates (p-NPA, kcat/KM = 2.4 x 105 M-1s-1) but also showed measurable hydrolytic activity toward natural triglycerides, consistently with a lipase-family esterase. The enzyme showed an activity optimum near 25 degrees C and pH 8.0. The enzyme tolerated low salt (maximal at 0.1 M NaCl), mild glycerol, and selected organic solvents (notably n-hexane), but was inhibited by high salt, Triton X-100, and SDS. AlphaFold predicted high local confidence for the catalytic core; DALI placed PanLip closest to fungal lipases (AFLB/CALB). Temperature-series MD and CABS-flex indicated enhanced surface breathing and flexible segments adjacent to the active site-including a region topologically matching CALB alpha 10-supporting a flexibility-assisted access mechanism at low temperature. Structure-based MSAs did not support a cold adaptation role for the reported VDLPGRS motif. Taken together, these findings position PanLip as a promising cold-active catalyst with CALB-like access control and potential for low-temperature biocatalysis.

Regulation of anti-CRISPR operons by structurally distinct families of Aca proteins

2026-02-10T04:21:03Z

Title: Regulation of anti-CRISPR operons by structurally distinct families of Aca proteins Authors: So Yeon Lee; Nils Birkholz; Lee, Jun Hyuck; Peter C. Fineran; Hyun Ho Park Abstract: CRISPR-Cas systems provide bacteria with adaptive immunity against bacteriophages and mobile genetic elements, driving an evolutionary arms race in which phages deploy anti-CRISPR (Acr) proteins. Acr proteins are often co-encoded in operons with anti-CRISPR-associated (Aca) proteins, which coordinate the regulation of acr gene expression. Here, we reveal the molecular basis of DNA binding that mediates transcriptional repression by two distinct Aca family members: Aca7 and Aca11. Crystal structures of Aca7 and Aca11 highlight conserved helix-turn-helix (HTH) motifs within α-helix bundles, providing a universal DNA-binding platform. Aca7 forms a symmetrical dimer to recognize a 19-bp inverted repeat (IR) within the acrIF11-aca7 operon. Strikingly, Aca11 binds 22-bp IRs in two distinct promoters, suggesting that Aca proteins can control multiple target operons. Mutagenesis and electrophoretic mobility shift assays (EMSAs) confirm that dimerization and sequence-specific IR recognition are essential for DNA binding. Despite mechanistic similarities, these and other Aca proteins exhibit notable differences. Structural comparisons across Aca families reveal that while monomer structures are generally similar with conserved HTH motifs, the structures of their dimeric functional units vary significantly. These structural differences might be essential for Aca proteins to bind to various promoters and regulate the expression of different Acr proteins.

Genomic and transcriptomic insights into benzoate and 4-hydroxybenzoate degradation in Arthrobacter sp. PAMC25564 isolated from cryoconite

2026-01-15T06:31:32Z

Title: Genomic and transcriptomic insights into benzoate and 4-hydroxybenzoate degradation in Arthrobacter sp. PAMC25564 isolated from cryoconite Authors: Ghimire Nisha; Kim Byeollee; Han So-Ra; Lee, Jun Hyuck; Oh Tae-Jin Abstract: Arthrobacter sp. has long been recognized for its metabolic versatility, particularly in the degradation of xenobiotic compounds. They have been isolated from diverse environments, including extreme environments. In this study, we conducted biotransformation, genomics and transcriptomics of Arthrobacter sp. PAMC25564 isolated from the cryoconite of Wurmkogel, Otztaler Alps, Austria, one of the extreme environments to unveil its aromatic compound-degrading potential. The strain possesses several genes associated with aromatic compound catabolism including at least one complete pathway to utilize two peripheral intermediates, 4-hydroxybenzoate (4-HB) and benzoate, which are mostly determined as wastewater contaminants. Arthrobacter sp. PAMC25564 tolerated and degraded benzoate and 4-HB up to 25 mM. Further, RNA-seq transcriptomics was done to investigate the benzoate and 4-HB degradation pathway in PAMC25564. The results indicate global gene regulation and pathway redundancy. Furthermore, growth of the strain in these aromatic compounds upregulates multiple genes associated with amino acid metabolism and phenylacetate degradation, which are associated with stress response. These findings contribute to a better understanding of aromatic compound degradation with a special focus on wastewater contaminants, benzoate and 4-HB degradation in Arthrobacter sp. PAMC25564.

Ice affinity purification system for recombinant proteins using a DUF3494 ice-binding protein

2025-08-22T04:47:57Z

Title: Ice affinity purification system for recombinant proteins using a DUF3494 ice-binding protein Authors: Hoang, Trang; Nguyen, Dieu Linh; Kim, Bo-Mi; Choi, Woong; Cho, Sung Mi; Kim, Han-Woo; Han, Se Jong; Kim, Kitae; Lee, Jun Hyuck; Do, Hackwon Abstract: Protein purification is essential for the isolation of specific proteins from mixtures. Conventional affinity tags have advanced recombinant protein purification. However, their reliance on costly resins and complex procedures often limits scalability and affordability. In this study, we identified three ice-binding domains (CoIBD1, CoIBD2, and CoIBD3) in Candidatus Cryosericum odellii SMC5 to evaluate their potential as protein purification tags. These domains exhibited hyperactive ice-binding properties, including high thermal hysteresis and ice recrystallization inhibition activities; additionally, they bound to multiple ice planes, enabling efficient attachment to ice surfaces. Through sequence and structural analyses, we engineered an enhanced variant that retained these ice-binding traits while achieving improved thermal and chemical stability: eCoIBD1. We then used eCoIBD1 as a fusion tag to develop the Ice Affinity Purification (IAP) system and evaluated its performance with GFP as a model protein. The IAP system achieved 87 % purity after two purification rounds, recovering 29 % of the initial protein from the crude extract. Consistent performance was observed in the presence of additives such as dithiothreitol and glycerol. The IAP system provides a cost-effective, environmentally friendly alternative to traditional methods by leveraging ice as a renewable binding medium, thereby eliminating the need for expensive resins or regeneration steps.