A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active ß-d-Galactosidase from Paracoccus sp. 32d.

Yeasts provide attractive host/vector systems for heterologous gene expression. The currently used yeast-based expression platforms include mesophilic and thermotolerant species. A eukaryotic expression system working at low temperatures could be particularly useful for the production of thermolabile proteins and proteins that tend to form insoluble aggregates. For this purpose, an expression system based on an Antarctic psychrotolerant yeast Debaryomyces macquariensis strain D50 that is capable of growing at temperatures ranging from 0 to 30 °C has been developed. The optimal physical culture conditions for D. macquariensis D50 in a fermenter are as follows: temperature 20 °C, pH 5.5, aeration rate of 1.5 vvm, and a stirring speed of 300 rpm. Four integrative plasmid vectors equipped with an expression cassette containing the constitutive GAP promoter and CYC1 transcriptional terminator from D. macquariensis D50 were constructed and used to clone and express a gene-encoding cold-active ß-d-galactosidase of Paracoccus sp. 32d. The yield was 1150 U/L of recombinant yeast culture. Recombinant D. macquariensis D50 strains were mitotically stable under both selective and non-selective conditions. The D. macquariensis D50 host/vector system has been successfully utilized for the synthesis of heterologous thermolabile protein, and it can be an alternative to other microbial expression systems.

Advanced Glycation End-Products (AGEs): Formation, Chemistry, Classification, Receptors, and Diseases Related to AGEs.

Advanced glycation end-products (AGEs) constitute a non-homogenous, chemically diverse group of compounds formed either exogeneously or endogeneously on the course of various pathways in the human body. In general, they are formed non-enzymatically by condensation between carbonyl groups of reducing sugars and free amine groups of nucleic acids, proteins, or lipids, followed by further rearrangements yielding stable, irreversible end-products. In the last decades, AGEs have aroused the interest of the scientific community due to the increasing evidence of their involvement in many pathophysiological processes and diseases, such as diabetes, cancer, cardiovascular, neurodegenerative diseases, and even infection with the SARS-CoV-2 virus. They are recognized by several cellular receptors and trigger many signaling pathways related to inflammation and oxidative stress. Despite many experimental research outcomes published recently, the complexity of their engagement in human physiology and pathophysiological states requires further elucidation. This review focuses on the receptors of AGEs, especially on the structural aspects of receptor-ligand interaction, and the diseases in which AGEs are involved. It also aims to present AGE classification in subgroups and to describe the basic processes leading to both exogeneous and endogeneous AGE formation.

Keratinases as Versatile Enzymatic Tools for Sustainable Development.

To reduce anthropological pressure on the environment, the implementation of novel technologies in present and future economies is needed for sustainable development. The food industry, with dairy and meat production in particular, has a significant environmental impact. Global poultry production is one of the fastest-growing meat producing sectors and is connected with the generation of burdensome streams of manure, offal and feather waste. In 2020, the EU alone produced around 3.2 million tonnes of poultry feather waste composed primarily of keratin, a protein biopolymer resistant to conventional proteolytic enzymes. If not managed properly, keratin waste can significantly affect ecosystems, contributing to environmental pollution, and pose a serious hazard to human and livestock health. In this article, the application of keratinolytic enzymes and microorganisms for promising novel keratin waste management methods with generation of new value-added products, such as bioactive peptides, vitamins, prion decontamination agents and biomaterials were reviewed.

Green Oxidation of Amines by a Novel Cold-Adapted Monoamine Oxidase MAO P3 from Psychrophilic Fungi Pseudogymnoascus sp. P3.

The use of monoamine oxidases (MAOs) in amine oxidation is a great example of how biocatalysis can be applied in the agricultural or pharmaceutical industry and manufacturing of fine chemicals to make a shift from traditional chemical synthesis towards more sustainable green chemistry. This article reports the screening of fourteen Antarctic fungi strains for MAO activity and the discovery of a novel psychrozyme MAOP3 isolated from the Pseudogymnoascus sp. P3. The activity of the native enzyme was 1350 ± 10.5 U/L towards a primary (n-butylamine) amine, and 1470 ± 10.6 U/L towards a secondary (6,6-dimethyl-3-azabicyclohexane) amine. MAO P3 has the potential for applications in biotransformations due to its wide substrate specificity (aliphatic and cyclic amines, pyrrolidine derivatives). The psychrozyme operates at an optimal temperature of 30 °C, retains 75% of activity at 20 °C, and is rather thermolabile, which is beneficial for a reduction in the overall costs of a bioprocess and offers a convenient way of heat inactivation. The reported biocatalyst is the first psychrophilic MAO; its unique biochemical properties, substrate specificity, and effectiveness predispose MAO P3 for use in environmentally friendly, low-emission biotransformations.

Novel Cold-Adapted Recombinant Laccase KbLcc1 from Kabatiella bupleuri G3 IBMiP as a Green Catalyst in Biotransformation.

Cold-adapted enzymes are useful tools in the organic syntheses conducted in mixed aqueous-organic or non-aqueous solvents due to their molecular flexibility that stabilizes the proteins in low water activity environments. A novel psychrophilic laccase gene from Kabatiella bupleuri, G3 IBMiP, was spliced by Overlap-Extension PCR (OE-PCR) and expressed in Pichia pastoris. Purified recombinant KbLcc1 laccase has an optimal temperature of 30 °C and pH of 3.5, 5.5, 6.0, and 7.0 in the reaction with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol, sinapic acid, and syringaldazine, respectively. Moreover, laccase KbLcc1 is highly thermolabile, as it loses 40% of activity after 30 min at 40 °C and is inactivated at 50 °C after the same period of incubation. The new enzyme remained active with 1 mM of Ni2+, Cu2+, Mn2+, and Zn2+ and with 2 mM of Co2+, Ca2+, and Mg2+, but Fe2+ greatly inhibited the laccase activity. Moreover, 1% ethanol had no impact on KbLcc1, although acetone and ethyl acetate decreased the laccase activity. The presence of hexane (40%, v/v) caused a 58% increase in activity. Laccase KbLcc1 could be applied in the decolorization of synthetic dyes and in the biotransformation of ferulic acid to vanillin. After 5 days of reaction at 20 °C, pH 3.5, with 1 mM ABTS as a mediator, the vanillin concentration was 21.9 mg/L and the molar yield of transformation reached 14.39%.

Screening of Novel Laccase Producers-Isolation and Characterization of Cold-Adapted Laccase from Kabatiella bupleuri G3 Capable of Synthetic Dye Decolorization.

Psychrophilic laccases catalyzing the bond formation in mild, environmentally friendly conditions are one of the biocatalysts at the focus of green chemistry. Screening of 41 cold-adapted strains of yeast and yeast-like fungi revealed a new laccase-producing strain, which was identified as Kabatiella bupleuri G3 IBMiP according to the morphological characteristics and analysis of sequences of the D1/D2 regions of 26S rDNA domain and the ITS1-5,8S-ITS2 region. The extracellular activity of laccase in reaction with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at the optimal pH 3.5 was 215 U/L after 15 days of growth in a medium with waste material and 126 U/L after 25 days of cultivation in a defined medium. Copper (II) ions (0.4 mM), Tween 80 (1.0 mM) and ascorbic acid (5.0 mM) increased the production of laccase. The optimum temperature for enzyme operation is in the range of 30-40 °C and retains over 60% of the maximum activity at 10 °C. New laccase shows high thermolability-half-life at 40 °C was only 60 min. Enzyme degradation of synthetic dyes was the highest for crystal violet, i.e., 48.6% after 1-h reaction with ABTS as a mediator. Outcomes of this study present the K. bupleuri laccase as a potential psychrozyme for environmental and industrial applications.

The psychrotrophic yeast Sporobolomyces roseus LOCK 1119 as a source of a highly active aspartic protease for the in vitro production of antioxidant peptides.

A psychrotrophic yeast strain producing a cold-adapted protease at low temperature was classified as Sporobolomyces roseus. In standard YPG medium, S. roseus LOCK 1119 synthesized an extracellular protease with an activity of approximately 560 U/L. Optimization of medium composition and process temperature considerably enhanced enzyme biosynthesis; an approximate 70% increase in activity (2060 U/L). The native enzyme was purified to homogeneity by cation exchange chromatography followed by a size exclusion step, resulting in a 103-fold increase in specific activity (660 U/mg) with 25% recovery. The enzyme displayed 10%-30% of its maximum activity at 0-25 °C, with the optimum temperature being 50°C. Protease G8 was strongly inactivated by pepstatin A, an aspartic protease inhibitor. The enzyme was used to hydrolyze four natural substrates, and their antioxidant activities were evaluated against 1,1-diphenyl-2-picrylhydrazyl. The highest antioxidant activity (69%) was recorded for beef casein.

Extremophilic proteases as novel and efficient tools in short peptide synthesis.

The objective of this review is to outline the crucial role that peptides play in various sectors, including medicine. Different ways of producing these compounds are discussed with an emphasis on the benefits offered by industrial enzyme biotechnology. This paper describes mechanisms of peptide bond formation using a range of proteases with different active site structures. Importantly, these enzymes may be further improved chemically and/or genetically to make them better suited for their various applications and process conditions. The focus is on extremophilic proteases, whose potential does not seem to have been fully appreciated to date. The structure of these proteins is somewhat different from that of the common commercially available enzymes, making them effective at high salinity and high or low temperatures, which are often favorable to peptide synthesis. Examples of such enzymes include halophilic, thermophilic, and psychrophilic proteases; this paper also mentions some promising catalytic proteins which require further study in this respect.

Strategies for efficient and economical 2,3-butanediol production: new trends in this field.

2,3-Butanediol (2,3-BD) is a promising bulk chemical with a potentially wide range of applications e.g., in the manufacture of printing inks, perfumes, synthetic rubber, fumigants, antifreeze agents, fuel additives, foodstuffs and pharmaceuticals. Its high heating value and ability to increase the octane number of fuels make 2,3-BD a promising drop-in fuel. It can also be converted to methyl-ethyl ketone (MEK), which is considered an effective liquid fuel additive. After combination with MEK and hydrogenation reaction, 2,3-BD can be converted to octane, which is used to produce high-quality aviation fuel. Currently 2,3-BD is mainly produced on an industrial scale by chemical methods. However, microbiological production of 2,3-BD offers a less expensive and more environmentally friendly alternative to traditional synthesis. This alcohol is generated from hexoses and pentoses mainly by bacterial strains of the genera Klebsiella, Bacillus, Serratia, and Enterobacter, which can convert waste products (such as glycerol and agricultural residues) and excess biomass (such as wood hydrolysates) to 2,3-BD. Recently, a significant improvement in microbial production has been achieved by the screening of efficient natural microbial strains, the application of alternative cost-effective substrates, and the genetic improvement of microbial producers. Furthermore, Klebsiella strains, which are regarded the most efficient natural 2,3-BD producers, have been subjected to genetic modifications aiming at the removal of pathogenic factors and the development of avirulent strains that could be used for the safe production of the diol. This review summarizes existing knowledge and experience concerning various strategies for efficient and economical microbial production of 2,3-BD.

Effects of genetic modifications and fermentation conditions on 2,3-butanediol production by alkaliphilic Bacillus subtilis.

Two recombinants of alkaliphilic Bacillus subtilis LOCK 1086, constructed via different strategies such as cloning the gene encoding bacterial hemoglobin from Vitreoscilla stercoraria (vhb) and overexpression of the gene encoding acetoin reductase/2,3-butanediol dehydrogenase (bdhA) from B. subtilis LOCK 1086, did not produce more 2,3-butanediol (2,3-BD) than the parental strain. In batch fermentations, this strain synthesized 9.46 g/L in 24 h and 12.80 g/L 2,3-BD in 46 h from sugar beet molasses and an apple pomace hydrolysate, respectively. 2,3-BD production by B. subtilis LOCK 1086 was significantly enhanced in fed-batch fermentations. The highest 2,3-BD concentration (75.73 g/L in 114 h, productivity of 0.66 g/L × h) was obtained in the sugar beet molasses-based medium with four feedings with glucose. In a medium based on the apple pomace hydrolysate with three feedings with sucrose, B. subtilis LOCK 1086 produced up to 51.53 g/L 2,3-BD (in 120 h, productivity of 0.43 g/L × h).

Application of enzymatic apple pomace hydrolysate to production of 2,3-butanediol by alkaliphilic Bacillus licheniformis NCIMB 8059.

2,3-Butanediol (2,3-BD) synthesis by a nonpathogenic bacterium Bacillus licheniformis NCIMB 8059 from enzymatic hydrolysate of depectinized apple pomace and its blend with glucose was studied. In shake flasks, the maximum diol concentration in fed-batch fermentations was 113 g/L (in 163 h, from the hydrolysate, feedings with glucose) while in batch processes it was around 27 g/L (in 32 h, from the hydrolysate and glucose blend). Fed-batch fermentations in the 0.75 and 30 L fermenters yielded 87.71 g/L 2,3-BD in 160 h, and 72.39 g/L 2,3-BD in 94 h, respectively (from the hydrolysate and glucose blend, feedings with glucose). The hydrolysate of apple pomace, which was for the first time used for microbial 2,3-BD production is not only a source of sugars but also essential minerals.

Extracellular secretion of Pseudoalteromonas sp. cold-adapted esterase in Escherichia coli in the presence of Pseudoalteromonas sp. components of ABC transport system.

Recently we described identification and characterization of GDSL esterase EstA from psychrotrophic bacterium Pseudoalteromonas sp. 643A. Attempts to obtain heterologous overexpression of this enzyme in Escherichia coli system were not satisfactory. The EstA protein was expressed as inclusion bodies, most of that were inactive after purification step, and the recovery of esterolytic activity was very low after refolding. Based on the sequence analysis we found that the esterase EstA gene is clustered with three genes encoding components of ABC transport system. These genes, designated abc1, abc2, and abc3 encode an ATP-binding protein (ABC1) and two permease proteins (ABC2 and ABC3). In present study, to obtain larger amounts of the active cold-adapted EstA esterase from Pseudoalteromonas sp. 643A, we designed a two-plasmid E. coli expression system where the gene encoding EstA enzyme was cloned into pET30b(+) expression vector and three genes encoding components of ABC transport system were cloned into pACYC-pBAD vector. It was shown that the created expression system was useful for extracellular production of active EstA enzyme which was purified from the culture medium. In the presence of all the three transporter proteins the secretion of EstA was at the highest level. When one or two of these components were missing, EstA secretion was also possible, but not so effective. It indicates that ABC2 and ABC3 proteins of Pseudoalteromonas sp. 643A could be replaced with their homologous proteins of E. coli.

A cold-adapted esterase from psychrotrophic Pseudoalteromas sp. strain 643A.

A psychrotrophic bacterium producing a cold-adapted esterase upon growth at low temperatures was isolated from the alimentary tract of Antarctic krill Euphasia superba Dana, and classified as Pseudoalteromonas sp. strain 643A. A genomic DNA library of strain 643A was introduced into Escherichia coli TOP10F', and screening on tributyrin-containing agar plates led to the isolation of esterase gene. The esterase gene (estA, 621 bp) encoded a protein (EstA) of 207 amino acid residues with molecular mass of 23,036 Da. Analysis of the amino acid sequence of EstA suggests that it is a member of the GDSL-lipolytic enzymes family. The purification and characterization of native EstA esterase were performed. The enzyme displayed 20-50% of maximum activity at 0-20 degrees C. The optimal temperature for EstA was 35 degrees C. EstA was stable between pH 9 and 11.5. The enzyme showed activity for esters of short- to medium-chain (C(4) and C(10)) fatty acids, and exhibited no activity for long-chain fatty acid esters like that of palmitate and stearate. EstA was strongly inhibited by phenylmethylsulfonyl fluoride, 2-mercaptoethanol, dithiothreitol and glutathione. Addition of selected divalent ions e.g. Mg(2+), Co(2+) and Cu(2+) led to the reduction of enzymatic activity and the enzyme was slightly activated ( approximately 30%) by Ca(2+) ions.