The Bacteroidetes Aequorivita sp. and Kaistella jeonii Produce Promiscuous Esterases With PET-Hydrolyzing Activity

Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 μl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the IsPETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as IsPETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation.

Highly Stable, Cold-Active Aldehyde Dehydrogenase from the Marine Antarctic Flavobacterium sp. PL002

Stable aldehyde dehydrogenases (ALDH) from extremophilic microorganisms constitute efficient catalysts in biotechnologies. In search of active ALDHs at low temperatures and of these enzymes from cold-adapted microorganisms, we cloned and characterized a novel recombinant ALDH from the psychrotrophic Flavobacterium PL002 isolated from Antarctic seawater. The recombinant enzyme (F-ALDH) from this cold-adapted strain was obtained by cloning and expressing of the PL002 aldH gene (1506 bp) in Escherichia coli BL21(DE3). Phylogeny and structural analyses showed a high amino acid sequence identity (89%) with Flavobacterium frigidimaris ALDH and conservation of all active site residues. The purified F-ALDH by affinity chromatography was homotetrameric, preserving 80% activity at 4 °C for 18 days. F-ALDH used both NAD+ and NADP+ and a broad range of aliphatic and aromatic substrates, showing cofactor-dependent compensatory KM and kcat values and the highest catalytic efficiency (0.50 µM−1 s−1) for isovaleraldehyde. The enzyme was active in the 4–60 °C-temperature interval, with an optimal pH of 9.5, and a preference for NAD+-dependent reactions. Arrhenius plots of both NAD(P)+-dependent reactions indicated conformational changes occurring at 30 °C, with four(five)-fold lower activation energy at high temperatures. The high thermal stability and substrate-specific catalytic efficiency of this novel cold-active ALDH favoring aliphatic catalysis provided a promising catalyst for biotechnological and biosensing applications.

Structure and in silico simulations of a cold-active esterase reveals its prime cold-adaptation mechanism

Here we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical α/β hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7's closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one substrate access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are important for of EstN7's cold adaptation rather than changes in dynamics.

Cold-Active Lipase-Based Biocatalysts for Silymarin Valorization through Biocatalytic Acylation of Silybin

Extremophilic biocatalysts represent an enhanced solution in various industrial applications. Integrating enzymes with high catalytic potential at low temperatures into production schemes such as cold-pressed silymarin processing not only brings value to the silymarin recovery from biomass residues, but also improves its solubility properties for biocatalytic modification. Therefore, a cold-active lipase-mediated biocatalytic system has been developed for silybin acylation with methyl fatty acid esters based on the extracellular protein fractions produced by the psychrophilic bacterial strain Psychrobacter SC65A.3 isolated from Scarisoara Ice Cave (Romania). The extracellular production of the lipase fraction was enhanced by 1% olive-oil-enriched culture media. Through multiple immobilization approaches of the cold-active putative lipases (using carbodiimide, aldehyde-hydrazine, or glutaraldehyde coupling), bio-composites (S1–5) with similar or even higher catalytic activity under cold-active conditions (25 °C) have been synthesized by covalent attachment to nano-/micro-sized magnetic or polymeric resin beads. Characterization methods (e.g., FTIR DRIFT, SEM, enzyme activity) strengthen the biocatalysts’ settlement and potential. Thus, the developed immobilized biocatalysts exhibited between 80 and 128% recovery of the catalytic activity for protein loading in the range 90–99% and this led to an immobilization yield up to 89%. The biocatalytic acylation performance reached a maximum of 67% silybin conversion with methyl decanoate acylating agent and nano-support immobilized lipase biocatalyst.

Optimization of exogenous carbohydrases supplemented in broiler diets using in vitro simulated gastrointestinal digestion and response surface methodology

The present study aimed to explore the optimal zymogram of combination of 6 carbohydrases (glucoamylase, pullulanase, maltase, thermostable α-amylase, medium temperature α-amylase, and cold-active α-amylase) supplemented in corn-soybean based diet of broilers aged 1 to 3 wk for the maximum starch digestibility, by using in vitro simulated gastrointestinal digestion and response surface method. The third generation of simulated monogastric animal digestion system was used for in vitro digestion experiment. By using single factor completely random design, the optimal supplement levels of single carbohydras were determined by the reducing sugar release amount and improved dry matter digestibility, which were the parameters representing the starch digestibility of the diet. Additionally, Box-Behnken response surface method was used to predict the optimal combination of 6 carbohydrases. The results showed that the optimistic zymogram of 6 carbohydrases in corn-soybean based diet for broilers aged 1 to 3 wk were 297.39 U/g glucoamylase, 549.72 U/g pullulanase, 3.01 U/g maltase, 1,455.73 U/g thermostable α-amylase, 278.64 U/g medium temperature α-amylase, and 1,985.97 U/g cold-active α-amylase, and the associated reduced sugar release amount and improved dry matter digestibility were 215.98 mg/g, and 6.23%, respectively. Furthermore, we conducted in vitro digestion experiments with diets supplemented with the predicted optimistic zymogram and found that the experimental reduced sugar release amount and improved dry matter digestibility were 219.26 mg/g and 6.31% respectively, whose errors to the predicted optimistic reducing sugar release amount and the improved dry matter digestibility were 1.05% and 1.02%. To sum up, the predicted optimal zymogram of 6 carbohydrases in the present study were capable to improve the starch digestibility in diet for broilers aged 1 to 3 wk, which were represented by increased reduced sugar release amount and improved dry matter digestibility.

Cold-Active Shewanella glacialimarina TZS-4T nov. Features a Temperature-Dependent Fatty Acid Profile and Putative Sialic Acid Metabolism

Species of genus Shewanella are among the most frequently identified psychrotrophic bacteria. Here, we have studied the cellular properties, growth dynamics, and stress conditions of cold-active Shewanella strain #4, which was previously isolated from Baltic Sea ice. The cells are rod-shaped of ~2μm in length and 0.5μm in diameter, and they grow between 0 and 25°C, with an optimum at 15°C. The bacterium grows at a wide range of conditions, including 0.5–5.5% w/v NaCl (optimum 0.5–2% w/v NaCl), pH 5.5–10 (optimum pH 7.0), and up to 1mM hydrogen peroxide. In keeping with its adaptation to cold habitats, some polyunsaturated fatty acids, such as stearidonic acid (18:4n-3), eicosatetraenoic acid (20:4n-3), and eicosapentaenoic acid (20:5n-3), are produced at a higher level at low temperature. The genome is 4,456kb in size and has a GC content of 41.12%. Uniquely, strain #4 possesses genes for sialic acid metabolism and utilizes N-acetyl neuraminic acid as a carbon source. Interestingly, it also encodes for cytochrome c3 genes, which are known to facilitate environmental adaptation, including elevated temperatures and exposure to UV radiation. Phylogenetic analysis based on a consensus sequence of the seven 16S rRNA genes indicated that strain #4 belongs to genus Shewanella, closely associated with Shewanella aestuarii with a ~97% similarity, but with a low DNA–DNA hybridization (DDH) level of ~21%. However, average nucleotide identity (ANI) analysis defines strain #4 as a separate Shewanella species (ANI score=76). Further phylogenetic analysis based on the 92 most conserved genes places Shewanella strain #4 into a distinct phylogenetic clade with other cold-active marine Shewanella species. Considering the phylogenetic, phenotypic, and molecular characterization, we conclude that Shewanella strain #4 is a novel species and name it Shewanella glacialimarina sp. nov. TZS-4T, where glacialimarina means sea ice. Consequently, S. glacialimarina TZS-4T constitutes a promising model for studying transcriptional and translational regulation of cold-active metabolism.

Exogenous Production of Cold-Active Cellulase From Psychrophilic Actinobacteria With Increased Cellulose Hydrolysis Efficiency

Actinobacteria form the largest phylum consisting of diverse, ecologically unique and biologically active members. The actinobacteria are omnipresent and occur in various habitats such as cold environment, aquatic, desert and terrestrial ecosystems. Though the studies are available on actinobacteria at various habitats very few reports are available on cold tolerant/loving actinobacteria in the Southern Ocean part of the Antarctic Ocean. In this context, the present work was designed to isolate and characterize the actinobacteria in the Polar Front region of the Southern Ocean waters and species of Nocardiopsis and Streptomyces were identified. Among those, the psychrophilic actinobacterium, Nocardiopsis dassonvillei PSY13 was found to have good cellulolytic activity and it was further studied for the production and characterization of cold-active cellulase enzyme. The latter was found to have a specific activity of 6.36 U/mg and a molar mass of 48 kDa with a 22.9-fold purification and 5% recovery at an optimum pH of 7.5 and a temperature of 10 ºC. Given the importance of psychrophilic actinobacteria N. dassonvillei PSY13 can be further exploited for its benefits, meaning that the Southern Ocean harbours biotechnologically important microorganisms that can be further explored for versatile biotechnological and industrial applications.