Aneta Białkowska

Antarctic marine bacterium Pseudoalteromonas sp. 22b as a source of cold-adapted β-galactosidase

The marine, psychrotolerant, rod-shaped and Gram-negative bacterium 22b (the best of 41 beta-galactosidase producers out of 107 Antarctic strains subjected to screening), classified as Pseudoalteromonas sp. based on 16S rRNA gene sequence, isolated from the alimentary tract of Antarctic krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted beta-galactosidase, which efficiently hydrolyzes lactose at 0-20 degrees C, as indicated by its specific activity of 21-67 U mg(-1) of protein (11-35% of maximum activity) in this temperature range, as well as k(cat) of 157 s(-1), and k(cat)/K(m) of 47.5 mM(-1) s(-1) at 20 degrees C. The maximum enzyme synthesis (lactose as a sufficient inducer) was observed at 6 degrees C, thus below the optimum growth temperature of the bacterium (15 degrees C). The enzyme extracted from cells was purified to homogeneity (25% recovery) by using the fast, three-step procedure, including affinity chromatography on PABTG-Sepharose. The enzyme is a tetramer composed of roughly 115 kDa subunits. It is maximally active at 40 degrees C (190 U mg(-1) of protein) and pH 6.0-8.0. PNPG is its preferred substrate (50% higher activity than against ONPG). The Pseudoalteromonas sp. 22b beta-galactosidase is activated by thiol compounds (70% rise in activity in the presence of 10 mM dithiotreitol), some metal ions (K(+), Na(+), Mn(2+)-40% increase, Mg(2+)-15% enhancement), and markedly inactivated by pCMB and heavy metal ions, particularly Cu(2+). Noteworthy, Ca(2+) ions do not affect the enzyme activity, and the homogeneous protein is stable at 4 degrees C for at least 30 days without any stabilizers.

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 Euphasiasuperba Dana, and classified as Pseudoalteromonas sp. strain 643A. A genomic DNA library of strain 643A was introduced into Escherichiacoli 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°C. The optimal temperature for EstA was 35°C. EstA was stable between pH 9 and 11.5. The enzyme showed activity for esters of short- to medium-chain (C4 and C10) 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. Mg2+, Co2+ and Cu2+ led to the reduction of enzymatic activity and the enzyme was slightly activated (∼30%) by Ca2+ ions.

Purification, characterisation and expression in Saccharomyces cerevisiae of LipG7 an enantioselective, cold-adapted lipase from the Antarctic filamentous fungus Geomyces sp. P7 with unusual thermostability characteristics.

A lipase, LipG7, has been purified from the Antarctic filamentous fungus Geomyces sp. P7 which was found to be cold-adapted and able to retain/regain its activity after heat denaturation. The LipG7 exhibits 100% residual activity following 1h incubation at 100°C whilst simultaneously showing kinetic adaptations to cold temperatures. LipG7 was also found to have industrial potential as an enantioselective biocatalyst as it is able to effectively catalyse the enantioselective transesterification of a secondary alcohol. The LipG7 coding sequence has been identified and cloned using 454 pyrosequencing of the transcriptome and inverse PCR. The LipG7 protein has been heterologously expressed in Saccharomyces cerevisiae BJ5465 and shown to exhibit the same characteristics as the native protein.

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.

Extremophilic Proteases: Developments of Their Special Functions, Potential Resources and Biotechnological Applications

Currently, microbiological proteases, along with lipases, are the most significant enzymes for biotechnology. They are produced in great quantity and since they are qualitatively diversified, they can be successfully applied in various branches of industry, including medicine. For several decades, the number as well as the significance of studies on extremophilic proteases have been growing. Extremophilic proteases are isolated from extremophiles which, given their unique kinetic and structural adaptations, can be used at low and high temperatures and in extreme environments (alkaline, acidic, saline). These enzymes have already enriched the range of commercial proteases and the studies on their properties in relation to their structural features stimulated a rational engineering of conventional proteases, aimed at enhancing their ability to adapt to specific conditions. In the chapter below, we characterized selected representatives of this most significant, in terms of economy, group of extremophilic proteases and discussed possible directions for their application in biotechnology.

Application of byproducts from food processing for production of 2,3-butanediol using Bacillus amyloliquefaciens TUL 308

ABSTRACT A nonpathogenic bacterial strain Bacillus amyloliquefaciens TUL 308 synthesized minor 2,3-butanediol (2,3-BD) amounts from glucose, fructose, sucrose, and glycerol, and efficiently produced the diol from molasses and hydrolysates of food processing residues. Batch fermentations yielded 16.53, 10.72, and 5 g/L 2,3-BD from enzymatic hydrolysates of apple pomace, dried sugar beet pulp, and potato pulp (at initial concentrations equivalent to 45, 20, and 30 g/L glucose, respectively), and 25.3 g/L 2,3-BD from molasses (at its initial concentration equivalent to 60 g/L saccharose). Fed-batch fermentations in the molasses-based medium with four feedings with either glucose or sucrose (in doses increasing their concentration by 25 g/L) resulted in around twice higher maximum 2,3-BD concentration (of about 60 and 50 g/L, respectively). The GRAS Bacillus strain is an efficient 2,3-BD producer from food industry byproducts.

Miscellaneous Cold-Active Yeast Enzymes of Industrial Importance

Cold-adapted organisms, thriving permanently at near-zero temperatures, synthesize cold-active enzymes to sustain their cell cycle. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. In this chapter, we describe the main properties of enzymes from cold-adapted yeasts and describe some of their potential biotechnological applications. The useful applications of these enzymes are widespread to a large number of industries like textile industry, food and dairy industry, brewing and wine industry, laundry, etc. Cold-active hydrolytic enzymes like lipases (reviewed in Chap.16), proteases, cellulases, and amylases can be used as an active agent in detergents applied for cold washing. Other potential applications of psychrophilic enzymes, apart from these, are in processes such as the hydrolysis of lactose in milk using β-galactosidases, extraction and clearing fruit juices using pectinases, meat tenderization or taste improvement of refrigerated meat using proteases, betterment of bakery products using glycosidases (e.g., amylases, xylanases).

Genetic and biochemical characterization of yeasts isolated from Antarctic soil samples

The Polish Arctowski Station is situated in the maritime Antarctic on the western shore of Admiralty Bay and encompasses terrestrial habitats which are not permanently covered by ice, in contrast to more than 90% of the island’s surface area. Over the past several decades, studies exploring the soils of those habitats have revealed a considerable diversity of bacteria, filamentous fungi, and, to a lesser extent, yeasts; however, characterization of this complex microbiome, especially at the molecular level, is still far from satisfactory. The isolates were assigned to their respective genera and species based on genetic analysis of the D1/D2 and ITS1-5.8S-ITS2 regions of rDNA. In the studied soil samples, the most abundant microorganisms belonged to the genera Cryptococcus, Rhodotorula, and Debaryomyces. Physiological and biochemical analysis of Cryptococcus gilvescens (pro tempore Goffeauzyma gilvescens) and Rhodotorula mucilaginosa showed only a limited level of intraspecies diversity. Cellular DNA content and karyotypes were determined using flow cytometry and pulsed-field gel electrophoresis for several selected strains. For the first time, genome size and electrophoretic karyotypes were investigated in C. gilvescens (pro tem G. gilvescens), Cryptococcus saitoi (pro tem Naganishia globosa), Cryptococcus gastricus (pro tem Goffeauzyma gastrica), and Cryptococcus albidus (pro tem Naganishia albida). In addition, plate tests showed Antarctic yeasts to be a potential source of biotechnologically important enzymes. This study in biodiversity, presenting physiological and molecular characterization of psychrotolerant yeast strains isolated from the soils of western Admiralty Bay, contributes to a better understanding of the microbial ecology of this unique ecosystem.

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.