<Emphasis Type="Italic">Geobacillus zalihae</Emphasis> sp. nov., a thermophilic lipolytic bacterium isolated from palm oil mill effluent in Malaysia

Background  

Thermophilic Bacillus strains of phylogenetic Bacillus rRNA group 5 were described as a new genus Geobacillus. Their geographical distribution included oilfields, hay compost, hydrothermal vent or soils. The members from the genus Geobacillus have a growth temperatures ranging from 35 to 78°C and contained iso-branched saturated fatty acids (iso-15:0, iso-16:0 and iso-17:0) as the major fatty acids. The members of Geobacillus have similarity in their 16S rRNA gene sequences (96.5–99.2%). Thermophiles harboring intrinsically stable enzymes are suitable for industrial applications. The quest for intrinsically thermostable lipases from thermophiles is a prominent task due to the laborious processes via genetic modification.     

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.     

Analysis of Comparative Sequence and Genomic Data to Verify Phylogenetic Relationship and Explore a New Subfamily of Bacterial Lipases

Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca²⁺-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter P_lac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65°C and retained ≥ 97% activity after incubation at 50°C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents.     

Peculiarities and biotechnological potential of environmental adaptation by Geobacillus species

The genus Geobacillus comprises thermophilic bacilli capable of endospore formation. The members of this genus provide thermostable proteins and can be used in whole cell applications at elevated temperatures; therefore, these organisms are of biotechnological importance. While these applications have been described in previous reviews, the present paper highlights the environmental adaptations and genome diversifications of Geobacillus spp. and their applications in evolutionary-protein engineering. Despite their obligate thermophilic properties, Geobacillus spp. are widely distributed in nature. Because several isolates demonstrate remarkable properties for cell reproduction in their respective niches, they seem to exist not only as endospores but also as vegetative cells in diverse environments. This suggests their excellence in environmental adaptation via genome diversification; in fact, evidence suggests that Geobacillus spp. were derived from Bacillus spp. while diversifying their genomes via horizontal gene transfer. Moreover, when subjected to an environmental stressor, Geobacillus spp. diversify their genomes using inductive mutations and transposable elements to produce derivative cells that are adaptive to the stressor. Notably, inductive mutations in Geobacillus spp. occur more rapidly and frequently than the stress-induced mutagenesis observed in other microorganisms. Owing to this, Geobacillus spp. can efficiently generate mutant genes coding for thermostable enzyme variants from the thermolabile enzyme genes under appropriate selection pressures. This phenomenon provides a new approach to generate thermostable enzymes, termed as thermoadaptation-directed enzyme evolution, thereby expanding the biotechnological potentials of Geobacillus spp. In this review, we have discussed this approach using successful examples and major challenges yet to be addressed.

     

Bacillus and biopolymer: Prospects and challenges

The microbially derived polyhydroxyalkanoates biopolymers could impact the global climate scenario by replacing the conventional non-degradable, petrochemical-based polymer. The biogenesis, characterization and properties of PHAs by Bacillus species using renewable substrates have been elaborated by many for their wide applications. On the other hand Bacillus species are advantageous over other bacteria due to their abundance even in extreme ecological conditions, higher growth rates even on cheap substrates, higher PHAs production ability, and the ease of extracting the PHAs. Bacillus species possess hydrolytic enzymes that can be exploited for economical PHAs production. This review summarizes the recent trends in both non-growth and growth associated PHAs production by Bacillus species which may provide direction leading to future research towards this growing quest for biodegradable plastics, one more critical step ahead towards sustainable development.     

Recent discoveries and applications of Anoxybacillus

The Bacillaceae family members are a good source of bacteria for bioprocessing and biotransformation involving whole cells or enzymes. In contrast to Bacillus and Geobacillus, Anoxybacillus is a relatively new genus that was proposed in the year 2000. Because these bacteria are alkali-tolerant thermophiles, they are suitable for many industrial applications. More than a decade after the first report of Anoxybacillus, knowledge accumulated from fundamental and applied studies suggests that this genus can serve as a good alternative in many applications related to starch and lignocellulosic biomasses, environmental waste treatment, enzyme technology, and possibly bioenergy production. This current review provides the first summary of past and recent discoveries regarding the isolation of Anoxybacillus, its medium requirements, its proteins that have been characterized and cloned, bioremediation applications, metabolic studies, and genomic analysis. Comparisons to some other members of Bacillaceae and possible future applications of Anoxybacillus are also discussed.     

Engineering lipases for temperature adaptation: Structure function correlation

Bacillus lipases are industrially attractive enzymes due to their broad substrate specificity and optimum alkaline pH. However, narrow temperature range of action and low thermostability restrain their optimal use and thus, necessitate attention. Several laboratories are engaged in protein engineering of Bacillus lipases to generate variants with improved attributes for decades using techniques such as directed evolution or rational design. This review summarizes the effect of mutations on the conformational changes through in silico modeling and their manifestation with respect to various biochemical parameters. Various studies have been put together to develop a perspective on the molecular basis of biocatalysis of lipases holding industrial importance.     

Lipases from the genus Rhizopus: Characteristics,expression, protein engineering and application

Lipases are versatile catalysts that hydrolyze ester bonds of water-insoluble glycerides or carry out reversible reactions at the water/lipid interface. The remarkable characteristics of lipases from the genus Rhizopus are their high sn-1,3-positional specificity, enantioselectivity and activity in nonaqueous media, which make them one of the most desirable enzymes for many applications, including lipid modification and biodiesel and chiral organic compound synthesis. sn-1,3-Position-specific Rhizopus lipases are particularly useful for the production of structured triacylglycerols. Significant progress has been made regarding lipases from the genus Rhizopus, including gene sequencing, elucidation of the protein structure and catalytic function, heterologous expression and redesigning Rhizopus lipases for valuable properties, which is receiving increasing academic and industrial attention. In this review, we present a comprehensive overview of Rhizopus lipases, focusing on (a) the characteristics of Rhizopus lipases, (b) Rhizopus lipase genes and structural features, (c) strategies for heterologous expression of Rhizopus lipase genes in yeast system, (d) progress in protein engineering for the improvement of the properties of Rhizopus lipases, and (e) development of biotechnological applications.     

Abiotic and Microbiotic Factors Controlling Biofilm Formation by Thermophilic Sporeformers

One of the major concerns in the production of dairy concentrates is the risk of contamination by heat-resistant spores from thermophilic bacteria. In order to acquire more insight in the composition of microbial communities occurring in the dairy concentrate industry, a bar-coded 16S amplicon sequencing analysis was carried out on milk, final products, and fouling samples taken from dairy concentrate production lines. The analysis of these samples revealed the presence of DNA from a broad range of bacterial taxa, including a majority of mesophiles and a minority of (thermophilic) spore-forming bacteria. Enrichments of fouling samples at 55°C showed the accumulation of predominantly Brevibacillus and Bacillus, whereas enrichments at 65°C led to the accumulation of Anoxybacillus and Geobacillus species. Bacterial population analysis of biofilms grown using fouling samples as an inoculum indicated that both Anoxybacillus and Geobacillus preferentially form biofilms on surfaces at air-liquid interfaces rather than on submerged surfaces. Three of the most potent biofilm-forming strains isolated from the dairy factory industrial samples, including Geobacillus thermoglucosidans, Geobacillus stearothermophilus, and Anoxybacillus flavithermus, have been characterized in detail with respect to their growth conditions and spore resistance. Strikingly, Geobacillus thermoglucosidans, which forms the most thermostable spores of these three species, is not able to grow in dairy intermediates as a pure culture but appears to be dependent for growth on other spoilage organisms present, probably as a result of their proteolytic activity. These results underscore the importance of abiotic and microbiotic factors in niche colonization in dairy factories, where the presence of thermophilic sporeformers can affect the quality of end products.     

Analysis of Arabidopsis genes encoding putative class III lipases

Triacylglycerol lipases are class of enzymes which catalyze the hydrolysis of long-chain triglycerides. They are widely found in the plant kingdom, numerous genes putatively encoding triacylglycerol lipases are sequenced but only a few of them have been characterized. Here we systematically analyzed Arabidopsis gene sequences deposited in public databases, and identified 38 putative class III lipase proteins, all of which contain a highly conserved lipase_3 domain (Pfam ID: PF01764). These 38 genes are randomly distributed on all chromosomes, and their genomic sequences consist of variable numbers of introns from zero to 13. They can be divided into four groups based on homology of protein sequences, and their potential subcellular localization is predicted to cytosol, chloroplast, mitochondria or endoplasmic reticulum. Furthermore, ten typical genes are selected to investigate their expression patterns. Most of them show weak tissue- or organ-specificity expression pattern. Several of them significantly accumulates in some tissues or organs in addition to germinated seedlings. Some of them are specifically transcribed during seed germination while others are not detected during stages of normal growth which are probably induced by stresses. In conclusion, putative Arabidopsis class III lipases display polymorphism in their sequences, gene structures and expression patterns.     

Comparative analysis of the Geobacillus hemicellulose utilization locus reveals a highly variable target for improved hemicellulolysis

Background

Members of the thermophilic genus Geobacillus can grow at high temperatures and produce a battery of thermostable hemicellulose hydrolytic enzymes, making them ideal candidates for the bioconversion of biomass to value-added products. To date the molecular determinants for hemicellulose degradation and utilization have only been identified and partially characterized in one strain, namely Geobacillus stearothermophilus T-6, where they are clustered in a single genetic locus.

Results

Using the G. stearothermophilus T-6 hemicellulose utilization locus as genetic marker, orthologous hemicellulose utilization (HUS) loci were identified in the complete and partial genomes of 17/24 Geobacillus strains. These HUS loci are localized on a common genomic island. Comparative analyses of these loci revealed extensive variability among the Geobacillus hemicellulose utilization systems, with only seven out of 41–68 proteins encoded on these loci conserved among the HUS⁺ strains. This translates into extensive differences in the hydrolytic enzymes, transport systems and metabolic pathways employed by Geobacillus spp. to degrade and utilize hemicellulose polymers.

Conclusions

The genetic variability among the Geobacillus HUS loci implies that they have variable capacities to degrade hemicellulose polymers, or that they may degrade distinct polymers, as are found in different plant species and tissues. The data from this study can serve as a basis for the genetic engineering of a Geobacillus strain(s) with an improved capacity to degrade and utilize hemicellulose.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-836) contains supplementary material, which is available to authorized users.     

Screening and characterization of lipase producing bacteria isolated from the gut of a lepidopteran larvae,Samia ricini

Lipolytic enzymes are an important group of hydrolases that have found immense industrial application in biotechnology. In this study, the ability of gut bacteria isolated from the gut of the Eri silkworm, Samia ricini, to produce lipolytic enzymes was evaluated through qualitative and quantitative assays. The results of lipase screening showed that 28 isolates had lipolytic activity. The results of 16S ribosomal RNA sequencing indicated that the genus Bacillus comprised majority of the lipolytic bacterial isolates (71%) followed by Pseudomonas (15%); whilst Acinetobacter, Enterobacter and Enterococcus comprised 11%. Lipolytic activity was found in bacteria isolates identified from all the three gut compartments of S. ricini larvae with significant activity from isolates extracted from the foregut and midgut. The lipolytic index among the bacterial isolates ranged between 0.63 and 2.81 on Rhodamine B medium, and all isolates exhibited significant lipolytic activity with p-nitrophenyl butyrate (PNPB) with specific activity ranging from 0.52 to 0.82 μmol/min/mg. The effect of pH and temperature showed that lipase activity was optimum at 37 °C and pH 7–9. A phylogenetic relationship of lipase producing gut bacteria indicated high cluster stability for isolates from different stages (>50%) suggesting that the isolates persist across developmental stages of the host. The Eri silkworm is reared for its silk and the knowledge of its gut bacteria with the ability to produce lipases lies in the significance as far as boosting production of this insect via development of probiotics to enhance commercial Eri rearing. In addition, this insect may be a good resource for profiling novel lipolytic microbes for commercial production of lipases as lipases from microbial origin have assumed a great deal of importance as industrial enzymes due to their potential for use in biotechnology.     

New bacilli from shallow hydrothermal vents of Panarea Island (Italy) and their biotechnological potential

Aims: To characterize bacilli isolated from shallow hydrothermal vents of Panarea Island (Italy) and evaluate their biotechnological potential. Methods and Results: Fifteen isolates were characterized by culture and molecular methods. Eleven isolates were thermophilic, six isolates were alkalophilic and four of them were haloalkalophilic. After 16S rRNA gene sequencing, four strains, exhibiting sequence similarity below 95% with deposited strains, may represent novel species of bacilli. One strain was strictly related to Geobacillus subterraneus, but shared phenotypic characteristics for which it could be considered a new strain of this species. Four strains were affiliated with different Bacillus spp. Most isolates produced gelatinase, lipases and amylase, and some were mercury tolerant. Exopolysaccharides (EPS) production was tested adding different sugars (glucose, sucrose, trehalose, fructose, ribose, xylose and mannose, 1% w/v) as a carbon source in a minimal medium. The highest EPS yield (185 mg l^?1) was reached by strain 1A70 utilizing ribose as a carbon source. Conclusions: Novel strains of Geobacillus and indigenous ribotypes of Bacillus with biotechnological potential inhabit shallow vents of Panarea Island. Significance and Impact of the Study: New strains of thermophilic bacilli from Panarea are producers of useful biomolecules for industrial purposes as well as environmental and biotechnological applications.     

Phylogenetic diversity of thermophilic carbohydrate degrading bacilli from Bulgarian hot springs

Phylogenetic diversity of culturable bacteria from genus Bacillus and related genera, isolated from 18 Bulgarian hot springs was investigated in association with their functional diversity. Sixty-seven thermophilic and facultative thermophilic strains were isolated under aerobic conditions at 60°C. Sixty-six of them belonged to eight species in four genera from Bacillus group: Anoxybacillus, Geobacillus, Brevibacillus and Bacillus. Representatives of the genus Anoxybacillus predominated. Based on phylogenetic analysis (<97% sequence similarity) four strains belonged to groups representing potentially novel species. Producers of carbohydrases, degrading 12 from the tested 13 substrates were isolated. About half of the isolates degraded amylose by exo- or endo-mechanism of action of their enzymes. The isolates degrading hemicellulose carbohydrates like arabinan, arabinoxylan, β-glucan, galactan, galactomannan and xyloglucan were reached to. Some of the microorganisms were able to uptake microbial polysaccharides like curdlan and gellan and their enzymes were between first reported thermostable enzymes in their groups, like gellan lyase and curdlan lyase A relation between species affiliation and their functional activity was observed—all A. gonensis strains were producer of amylolytic enzymes, most of Brevibacillus ruber strains were able to grow in a minimal medium with xanthan.     

Application of rpoB sequence similarity analysis, REP-PCR and BOX-PCR for the differentiation of species within the genus Geobacillus

Aim:  To investigate the applicability of rpoB gene, which encodes the β subunit of RNA polymerase, to be used as an alternative to 16S rRNA for sequence similarity analysis in the thermophilic genus Geobacillus. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP‐ and BOX‐polymerase chain reaction) were also used. Methods and Results:  rpoB DNA (458 bp) were amplified from 21 Geobacillus‐ and Bacillus type strains, producing different BOX‐ and REP‐PCR profiles, in addition to 11 thermophilic isolates of Geobacillus and Bacillus species from a Santorini volcano habitat. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The results demonstrated between 90–100% (16S rRNA) and 74–100% (rpoB) similarity among examined bacteria. Conclusion:  BOX‐ and REP‐PCR can be applied for molecular typing within Geobacillus genus. rpoB sequence similarity analysis permits a more accurate discrimination of the species within the Geobacillus genus than the more commonly used 16S rRNA. Significance and Impact of the Study:  The obtained results suggested that rpoB sequence similarity analysis is a powerful tool for discrimination between species within the ecologically and industrially important strains of Geobacillus genus.     

Exploring the Mechanism Responsible for Cellulase Thermostability by Structure-Guided Recombination

Cellulases from Bacillus and Geobacillus bacteria are potentially useful in the biofuel and animal feed industries. One of the unique characteristics of these enzymes is that they are usually quite thermostable. We previously identified a cellulase, GsCelA, from thermophilic Geobacillus sp. 70PC53, which is much more thermostable than its Bacillus homolog, BsCel5A. Thus, these two cellulases provide a pair of structures ideal for investigating the mechanism regarding how these cellulases can retain activity at high temperature. In the present study, we applied the SCHEMA non-contiguous recombination algorithm as a novel tool, which assigns protein sequences into blocks for domain swapping in a way that lessens structural disruption, to generate a set of chimeric proteins derived from the recombination of GsCelA and BsCel5A. Analyzing the activity and thermostability of this designed library set, which requires only a limited number of chimeras by SCHEMA calculations, revealed that one of the blocks may contribute to the higher thermostability of GsCelA. When tested against swollen Avicel, the highly thermostable chimeric cellulase C10 containing this block showed significantly higher activity (22%-43%) and higher thermostability compared to the parental enzymes. With further structural determinations and mutagenesis analyses, a 3₁₀ helix was identified as being responsible for the improved thermostability of this block. Furthermore, in the presence of ionic calcium and crown ether (CR), the chimeric C10 was found to retain 40% residual activity even after heat treatment at 90°C. Combining crystal structure determinations and structure-guided SCHEMA recombination, we have determined the mechanism responsible for the high thermostability of GsCelA, and generated a novel recombinant enzyme with significantly higher activity.     

Isolation and characterization of a thermotolerant ene reductase from Geobacillus sp. 30 and its heterologous expression in Rhodococcus opacus

Rhodococcus opacus B-4 cells are adhesive to and even dispersible in water-immiscible hydrocarbons owing to their highly lipophilic nature. In this study, we focused on the high operational stability of thermophilic enzymes and applied them to a biocatalytic conversion in an organic reaction medium using R. opacus B-4 as a lipophilic capsule of enzymes to deliver them into the organic medium. A novel thermo- and organic-solvent-tolerant ene reductase, which can catalyze the enantioselective reduction of ketoisophorone to (6R)-levodione, was isolated from Geobacillus sp. 30, and the gene encoding the enzyme was heterologously expressed in R. opacus B-4. Another thermophilic enzyme which catalyzes NAD⁺-dependent dehydrogenation of cyclohexanol was identified from the gene-expression library of Thermus thermophilus and the gene was coexpressed in R. opacus B-4 for cofactor regeneration. While the recombinant cells were not viable in the mixture due to high reaction temperature, 634 mM of (6R)-levodione could be produced with an enantiopurity of 89.2 % ee by directly mixing the wet cells of the recombinant R. opacus with a mixture of ketoisophorone and cyclohexanol at 50 °C. The conversion rate observed with the heat-killed recombinant cells was considerably higher than that obtained with a cell-free enzyme solution, demonstrating that the accessibility between the substrates and enzymes could be improved by employing R. opacus cells as a lipophilic enzyme capsule. These results imply that a combination of thermophilic enzymes and lipophilic cells can be a promising approach for the biocatalytic production of water-insoluble chemicals.     

Sequence Analysis and Comparative Bioinformatics Study of Camelysin Gene (<Emphasis Type="Italic">calY</Emphasis>) Isolated from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Bacillus strains have been widely used for the production of fibrinolytic enzymes having role in the treatment of cardiovascular disorders. Purification and overproduction of such enzymes has increased their usage in medical fields including metalloproteinases with the ability to degrade extracellular matrix (ECM). Camelysin, a neutral metalloproteinase has been isolated from different species of bacteria like Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis with fibrinolytic, collagenolytic and actin degradation activity. This project successfully demonstrated the presence of 734-bp coding DNA sequence (CDS) encoding a 20.72331 kDa camelysin gene in local strain of Bacillus thuringiensis containing a signal peptide with cleavage site between residues 19 and 20. The sequence was submitted to GenBank (KT023597) and the sequence showed high homology with the camelysin protein of closely related Bacillus species. The alignment of related proteins through ClustalW displayed difference of four amino acids (“Q” replaced by “P” at position 169 and at position 182–184, “NQE” replaced by “HLK”) in the isolated protein. Comparison including structural and functional analysis of camelysin sequences isolated from different Bacillus species was carried out using different bioinformatics tools and software. The information would help in better understanding the properties of camelysin protein and its role in pathogenicity and clinical treatments.     

Assessing the Performance of Bacterial Cellulases: the Use of <Emphasis Type="Italic">Bacillus</Emphasis> and <Emphasis Type="Italic">Paenibacillus</Emphasis> Strains as Enzyme Sources for Lignocellulose Saccharification

Plant biomass offers a renewable and environmentally favorable source of sugars that can be converted to different chemicals, second-generation ethanol, and other liquid fuels. Cellulose makes up approximately 45 % of the dry weight of lignocellulosic biomass. Prior to the enzymatic hydrolysis of cellulose, lignin and hemicellulose must be structurally altered or removed, at least in part, by chemical and/or physical pretreatments. However, the high cost and low efficiency of the enzymatic hydrolysis prevent the process from being economically competitive. For this reason, it is necessary to find enzymes suitable for this type of process, with higher specific activities and greater efficiency. Members of the Bacillus and Paenibacillus genera have been traditionally used for the production of many enzymes for industrial applications. Cellulases produced by both genera have shown activity on soluble and crystalline cellulose and high thermostability and/or activity over a wide pH spectrum. In this review, the most recent information about the characterization of cellulolytic enzymes obtained from new strains of the Bacillus and Paenibacillus genera are reviewed. We focused on the variety of isoenzymes produced by these cellulolytic strains, their optimal production and reaction conditions, and their kinetic parameters and biotechnological potential.