Heterologous biosynthesis and characterization of a glycocin from a thermophilic bacterium.

The genome of the thermophilic bacterium, Aeribacillus pallidus 8, encodes the bacteriocin pallidocin. It belongs to the small class of glycocins and is posttranslationally modified, containing an S-linked glucose on a specific Cys residue. In this study, the pallidocin biosynthetic machinery is cloned and expressed in Escherichia coli to achieve its full biosynthesis and modification. It targets other thermophilic bacteria with potent activity, demonstrated by a low minimum inhibitory concentration (MIC) value. Moreover, the characterized biosynthetic machinery is employed to produce two other glycopeptides Hyp1 and Hyp2. Pallidocin and Hyp1 exhibit antibacterial activity against closely related thermophilic bacteria and some Bacillus sp. strains. Thus, heterologous expression of a glycocin biosynthetic gene cluster including an S-glycosyltransferase provides a good tool for production of hypothetical glycocins encoded by various bacterial genomes and allows rapid in vivo screening.

Characterization and application of keratinolytic paptidases from Bacillus spp.

Solid keratin-rich waste management is one of essential research area in nowadays. Conventional chemical and high thermal keratin waste decomposition methods are fully explored and not enough effective for future biotechnology perspectives. However, traditional keratin-rich waste decomposition methods could be replaced by environmentally-friendly and economical microbial keratin waste biodegradation methods without energy wastage and essential amino acids and nutrition elements loss. In this study BPKer and BAKer keratinolytic peptidases from Bacillus sp. AD-W and Bacillus sp. AD-AA3 strains, respectively, were successfully produced, purified and biochemically characterized. Physical and chemical characterization of native BPKer and BAKer suggested that new keratinolytic peptidases are powerful biocatalysts for efficient keratin waste biodegradation and can replace conventional insufficient non-biological hydrolysis processes without energy, important amino acids and nutritional elements loss. High value bio-active hydrolysis products - peptides obtained from keratin waste biodegradation by BPKer and BAKer are suitable for industrial applications in white and green biotechnology.

Construction of a novel lipolytic fusion biocatalyst GDEst-lip for industrial application.

The gene encoding esterase (GDEst-95) from Geobacillus sp. 95 was cloned and sequenced. The resulting open reading frame of 1497 nucleotides encoded a protein with calculated molecular weight of 54.7 kDa, which was classified as a carboxylesterase with an identity of 93-97% to carboxylesterases from Geobacillus bacteria. This esterase can be grouped into family VII of bacterial lipolytic enzymes, was active at broad pH (7-12) and temperature (5-85 °C) range and displayed maximum activity toward short acyl chain p-nitrophenyl (p-NP) esters. Together with GD-95 lipase from Geobacillus sp. strain 95, GDEst-95 esterase was used for construction of fused chimeric biocatalyst GDEst-lip. GDEst-lip esterase/lipase possessed high lipolytic activity (600 U/mg), a broad pH range of 6-12, thermoactivity (5-85 °C), thermostability and resistance to various organic solvents or detergents. For these features GDEst-lip biocatalyst has high potential for applications in various industrial areas. In this work the effect of additional homodomains on monomeric GDEst-95 esterase and GD-95 lipase activity, thermostability, substrate specificity and catalytic properties was also investigated. Altogether, this article shows that domain fusing strategies can modulate the activity and physicochemical characteristics of target enzymes for industrial applications.

Combining prebiotics with probiotic bacteria can enhance bacterial growth and secretion of bacteriocins.

There is a growing interest in supporting human health by using prebiotics, such as oligosaccharides, and beneficial bacteria, also called probiotics. Combining these two components we can develop synbiotics. In order to create successful combination of synbiotic it is very important to evaluate the influence of prebiotic oligosaccharides to probiotic bacteria and their behavior, such as growth and secretion of health related biomolecules, including bacteriocins. In this study seven type strains of probiotic bacteria (five Lactobacillus sp. and two Lactococcus sp.) and two Lactobacillus sp. strains, isolated from probiotic yoghurt, were cultivated with various commercially available and extracted oligosaccharides (OS). The aim of this study was to evaluate the influence of these OS on type and isolated bacterial strains growth and antibacterial activity. Obtained results suggest that combination of certain OS with probiotic strains may considerably improve their growth and/or antibacterial activity. We also determined the antibacterial activity spectrum of investigated strains with combination of OS against common food borne pathogens. Results of this work show that prebiotic OS can be useful for modulating probiotic bacteria growth, antibacterial activity and even specificity of this activity.

Detection of Asp371, Phe375, and Tyr376 Influence on GD-95-10 Lipase Using Alanine Scanning Mutagenesis.

GD-95-10 and GD-95-20 lipases are modified GD-95 lipase variants, which lack 10 and 20 C-terminal amino acids, respectively. Previous analysis showed that GD-95-10 lipase has higher activity than GD-95 lipase, while GD-95-20 lipase almost completely loses its activity. Analysis in silico suggested three conservative amino acids at region between 369 and 378 amino acids which can be relevant to the activity of GD-95-10 lipase. These amino acids have direct contacts with residues involved in substrate binding, stabilization of the serine loop or form oxyanion hole. In this work, the role of Asp371, Phe375, and Tyr376 on activity, functionality, and structure of GD-95-10 lipase was analyzed by Ala scanning mutagenesis. We showed that even a single mutation can impact the main structure and activity of Geobacillus lipases. Our experiments provide new knowledge about lipases from thermophilic Geobacillus bacteria and are important for protein engineering and synthetic biology. These enzymes and their engineering can be basis for future biocatalysts applied in production of biofuel or other industrial esters.

Genetic engineering of Geobacillus spp.

Members of the genus Geobacillus are thermophiles that are of great biotechnological importance, since they are sources of many thermostable enzymes. Because of their metabolic versatility, geobacilli can be used as whole-cell catalysts in processes such as bioconversion and bioremediation. The effective employment of Geobacillus spp. requires the development of reliable methods for genetic engineering of these bacteria. Currently, genetic manipulation tools and protocols are under rapid development. However, there are several convenient cloning vectors, some of which replicate autonomously, while others are suitable for the genetic modification of chromosomal genes. Gene expression systems are also intensively studied. Combining these tools together with proper techniques for DNA transfer, some Geobacillus strains were shown to be valuable producers of recombinant proteins and industrially important biochemicals, such as ethanol or isobutanol. This review encompasses the progress made in the genetic engineering of Geobacillus spp. and surveys the vectors and transformation methods that are available for this genus.

Keratinous waste decomposition and peptide production by keratinase from Geobacillus stearothermophilus AD-11.

A keratinolytic proteinase was cloned from thermophilic bacterium Geobacillus stearothermophilus AD-11 and was expressed in Escherichia coli BL21(DE3). Recombinant keratinolytic proteinase (RecGEOker) with an estimated molecular weight of 57 kDa was purified and keratinase activity was measured. RecGEOker showed optimal activity at pH 9 and 60 °C. Recombinant keratinolytic proteinase showed the highest substrate specificity toward keratin from wool > collagen > sodium caseinate > gelatin > and BSA in descending order. RecGEOker is applicable for efficient keratin waste biodegradation and can replace conventional non-biological hydrolysis processes. High-value small peptides obtained from enzymatic biodegradation by RecGEOker are suitable for industrial application in white and/or green biotechnology for use as major additives in various products.

Development of synbiotics with inulin, palatinose, alpha-cyclodextrin and probiotic bacteria.

Success in creating a synbiotic depends on compatibility between the chosen components--prebiotic and probiotic. In this work the interactions between Lactobacillus sp. strains isolated from yogurts and type strains of Lactobacillus sp. and Lactococcus sp., and the dependence of their growth and antibacterial activity on three oligosaccharides (OS)--palatinose, inulin and alpha-cyclodextrin were investigated. All isolated lactobacilli produce antibacterial compounds, which possibly are the bacteriocins of Lactobacillus casei ATCC334 strain. Results of growth analysis with different OS revealed that part of lactobacilli isolated from yogurts can effectively ferment inulin and may be used for the development of synbiotics. Palatinose and Lactobacillus acidophilus could be used as symbiotics with effective antibacterial activity. One of the types of Lactococcus sp. strains can assimilate palatinose and alpha-cyclodextrin, so they both can be used as components of synbiotics with the investigated lactococci. Results of this analysis suggest that the investigated isolated and type strains of Lactobacillus sp. and Lactoccocus sp. can be useful as probiotics in the development of synbiotics. Together with prebiotics--palatinose, inulin and alpha-cyclodextrin, the synbiotics, which could regulate not only the growth of beneficial bacteria in the gastrointestinal tract, but also their antibacterial activity, can be created.

Keratinolytic proteinase from Bacillus thuringiensis AD-12.

A new isolated strain noted to produce a novel detergent-stable serine keratinolytic proteinase and identified as Bacillus thuringiensis AD-12. Native keratinolytic proteinase from B. thuringiensis (BtKER) was purified and characterized. The purified BtKER enzyme is a monomer with a molecular mass of 39kDa. Biochemical characterization assays revealed that the BtKER attained optimal activity at pH 7 and 30°C. Residual activity after 1h incubation at 50°C was higher than 80%. The enzyme was activated and stabilized by Mn(2+) and Li(+) metal ions but inactivated by organic solvents. Purified BtKER showed the highest substrate specificity toward keratin from wool>sodium caseinate>collagen>BSA>gelatin in descending order. BtKER is the first reported keratinolytic proteinase from B. thuringiensis and obtained results suggested that new characterized enzyme can be a powerful biocatalyst in peptide production associated to hydrolysis of keratinous and/or keratin-like waste.

Ras/PKA signal transduction pathway participates in the regulation of Saccharomyces cerevisiae cell apoptosis in an acidic environment.

The acidification of the medium is observed during yeast cell growth. This process contributes to the emission of organic acids, mainly acetic acid. Acetic acid is known as the inducer of apoptosis in the yeast Saccharomyces cerevisiae. In this study, we showed that hydrochloric acid can also induce apoptosis in yeast cells, and the apoptotic phenotype triggered by treating yeast cells with hydrochloric acid is modulated by the Ras/PKA pathway. The Ras/PKA pathway is highly conserved between all eukaryotic organisms, as well as cell processes that are related to apoptosis and aging. In this research, we demonstrated that the activation of the Ras/PKA pathway by insertion of Ras2(Val19) allele or deletion of PDE2 gene increases cell death, displaying the markers of apoptosis in an acidic environment. Downregulation of the pathway by deletion of RAS2, RAS1, PDE1, and insertion of the Ha-ras gene increases the cell viability and diminishes cell death with the apoptotic phenotypes. The deletion of PDE1 gene and double deletion of both phosphodiesterase genes prevent the induction of apoptosis in the cells. Modulations in the Ras/PKA pathway affect cell viability and apoptosis during natural gradual acidification of the medium as well as in acid stress conditions.

Influence of N- and/or C-terminal regions on activity, expression, characteristics and structure of lipase from Geobacillus sp. 95.

GD-95 lipase from Geobacillus sp. strain 95 and its modified variants lacking N-terminal signal peptide and/or 10 or 20 C-terminal amino acids were successfully cloned, expressed and purified. To our knowledge, GD-95 lipase precursor (Pre-GD-95) is the first Geobacillus lipase possessing more than 80% lipolytic activity at 5 °C. It has maximum activity at 55 °C and displays a broad pH activity range. GD-95 lipase was shown to hydrolyze p-NP dodecanoate, tricaprylin and canola oil better than other analyzed substrates. Structural and sequence alignments of bacterial lipases and GD-95 lipase revealed that the C-terminus forms an α helix, which is a conserved structure in lipases from Pseudomonas, Clostridium or Staphylococcus bacteria. This work demonstrates that 10 and 20 C-terminal amino acids of GD-95 lipase significantly affect stability and other physicochemical properties of this enzyme, which has never been reported before and can help create lipases with more specific properties for industrial application. GD-95 lipase and its modified variants GD-95-10 can be successfully applied to biofuel production, in leather and pulp industries, for the production of cosmetics or perfumes. These lipases are potential biocatalysts in processes, which require extreme conditions: low or high temperature, strongly acidic or alkaline environment and various organic solvents.

Characterization of two novel plasmids from Geobacillus sp. 610 and 1121 strains.

We describe two cryptic low molecular weight plasmids, pGTD7 (3279bp) and pGTG5 (1540bp), isolated from Geobacillus sp. 610 and 1121 strains, respectively. Homology analysis of the replication protein (Rep) sequences and detection of ssDNA indicate that both of them replicate via rolling circle mechanism. As revealed by sequence similarities of dso region and Rep protein, plasmid pGTD7 belongs to pC194/pUB110 plasmid family. The replicon of pGTD7 was proved to be functional in another Geobacillus host. For this purpose, a construct pUCK7, containing a replicon of the analyzed plasmid, was created and transferred to G. stearothermophilus NUB3621R strain by electroporation. Plasmid pGTG5, based on Rep protein sequence similarity, was found to be related mostly to some poorly characterized bacterial plasmids. Rep proteins encoded by these plasmids contain conservative motifs that are most similar to those of Microviridae phages. This feature suggests that pGTG5, together with other plasmids containing the same motifs, could constitute a new family of bacterial plasmids. To date, pGTG5 is the smallest plasmid identified in bacteria belonging to the genus Geobacillus. The two plasmids described in this study can be used for the construction of new vectors suitable for biotechnologically important bacteria of the genus Geobacillus.

Complete nucleotide sequence of pGS18, a 62.8-kb plasmid from Geobacillus stearothermophilus strain 18.

The complete nucleotide sequence (62.8 kb) of pGS18, the largest sequenced plasmid to date from the species Geobacillus stearothermophilus, was determined. Computational analysis of sequence data revealed 65 putative open reading frames (ORFs); 38 were carried on one strand and 27 were carried on the other. These ORFs comprised 84.1% of the pGS18 sequence. Twenty-five ORFs (38.4%) were assigned to putative functions; four ORFs (6.2%) were annotated as pseudogenes. The amino acid sequences obtained from 29 ORFs (44.6%) had the highest similarity to hypothetical proteins of the other microorganisms, and seven (10.8%) had no significant similarity to any genes present in the current open databases. Plasmid replication region, strongly resembling that of the theta-type replicon, and genes encoding three different plasmid maintenance systems were identified, and a putative discontinuous transfer region was localized. In addition, we also found several mobile genetic elements and genes, responsible for DNA repair, distributed along the whole sequence of pGS18. The alignment of pGS18 with two other large indigenous plasmids of the genus Geobacillus highlighted the presence of well-conserved segments and has provided a framework that can be exploited to formulate hypotheses concerning the molecular evolution of these three plasmids.