Rational and random mutagenesis of GDEst-95 carboxylesterase: New functionality insights.

Lipolytic enzymes are important contributors in industrial processes from lipid hydrolysis to biofuel production or even polyester biodegradation. While these enzymes can be used in numerous applications, the genotype-phenotype space of certain promising enzymes is still poorly explored. This limits the effective application of such biocatalysts. In this work the genotype space of a 55 kDa carboxylesterase GDEst-95 from Geobacillus sp. 95 was explored using site-directed mutagenesis and directed evolution methods. In this study four site-directed mutants (Gly108Arg, Ala410Arg, Leu226Arg, Leu411Ala) were created based on previous analysis of GDEst-95 carboxylesterase. Error-prone PCR resulted three mutants: two of them with distal mutations: GDEst-RM1 (Arg75Gln), GDEst-RM2 (Gly20Ser Arg75Gln) and the third, GDEst-RM3, with a distal (Ser210Gly) and Tyr317Ala (amino acid position near to the active site) mutation. Mutants with Ala substitution displayed approximately twofold higher specific activity. Arg mutations lead a reduced specific activity, retaining 2.86 % (Gly108Arg), 10.95 % (Ala410Arg), and 44.23 % (Leu226Arg) of lipolytic activity. All three random mutants displayed increased specific activity as well as improved catalytic properties. This research provides the first deeper insights into the functionality of understudied Geobacillus spp. carboxylesterases with 55 kDa in size.

Engineered Geobacillus lipolytic enzymes - Attractive polyesterases that degrade polycaprolactones and simultaneously produce esters.

Plastic pollution is one of the biggest environmental problems plaguing the modern world. Polyester-based plastics contribute significantly to this ecological safety concern. In this study, lipolytic biocatalysts GD-95RM and GDEst-lip developed based on lipase/esterase produced by Geobacillus sp. 95 strain were applied for the degradation of polycaprolactone films (Mn 45.000 (PCL45000) and Mn 80.000 (PCL80000)). The degradation efficiency was significantly enhanced by the addition of short chain alcohols. Lipase GD-95RM (1 mg) can depolymerize 264.0 mg and 280.7 mg of PCL45000 and PCL80000, films respectively, in a 24 h period at 30 °C, while the fused enzyme GDEst-lip (1 mg) is capable of degrading 145.5 mg PCL45000 and 134.0 mg of PCL80000 films in 24 h. The addition of ethanol (25 %) improves the degradation efficiency ~2.5 fold in the case of GD-95RM. In the case of GDEst-lip, 50 % methanol was found to be the optimal alcohol solution and the degradation efficiency was increased by ~3.25 times. The addition of alcohols not only increased degradation speeds but also allowed for simultaneous synthesis of industrially valuable 6-hydroxyhexonic acid esters. The suggested system is an attractive approach for removing of plastic waste and supports the principles of bioeconomics.

Insights in MICP dynamics in urease-positive Staphylococcus sp. H6 and Sporosarcina pasteurii bacterium.

Microbially induced calcite precipitation (MICP) is an efficient and eco-friendly technique that has attracted significant interest for resolving various problems in the soil (erosion, improving structural integrity and water retention, etc.), remediation of heavy metals, production of self-healing concrete or restoration of different concrete structures. The success of most common MICP methods depends on microorganisms degrading urea which leads to the formation of CaCO3 crystals. While Sporosarcina pasteurii is a well-known microorganism for MICP, other soil abundant microorganisms, such as Staphylococcus bacteria have not been thoroughly studied for its efficiency in bioconsolidation though MICP is a very important proccess which can ensure soil quality and health. This study aimed to analyze MICP process at the surface level in Sporosarcina pasteurii and a newly screened Staphylococcus sp. H6 bacterium as well as show the possibility of this new microorganism to perform MICP. It was observed that Staphylococcus sp. H6 culture precipitated 157.35 ± 3.3 mM of Ca2+ ions from 200 mM, compared to 176 ± 4.8 mM precipitated by S. pasteurii. The bioconsolidation of sand particles was confirmed by Raman spectroscopy and XRD analysis, which indicated the formation of CaCO3 crystals for both Staphylococcus sp. H6 and S. pasteurii cells. The water-flow test suggested a significant reduction in water permeability in bioconsolidated sand samples for both Staphylococcus sp. H6 and S. pasteurii. Notably, this study provides the first evidence that CaCO3 precipitation occurs on the surface of Staphylococcus and S. pasteurii cells within the initial 15-30 min after exposure to the biocementation solution. Furthermore, Atomic force microscopy (AFM) indicated rapid changes in cell roughness, with bacterial cells becoming completely coated with CaCO3 crystals after 90 min incubation with a biocementation solution. To our knowledge, this is the first time where atomic force microscopy was used to visualize the dynamic of MICP on cell surface.

Study of individual domains' functionality in fused lipolytic biocatalysts based on Geobacillus lipases and esterases.

The prospects of industrial uses of microbial enzymes have increased greatly during the 21st century. Fused lipolytic enzymes (where one or both fused domains possess lipolytic activity) is a rapidly growing group of industrial biocatalysts. However, the most effective fusion strategy, catalytic behavior of each domain and influence of added linkers on physicochemical and kinetic characteristics of such biocatalysts has not been yet explored. In this study the functionality of individual domains in fused lipolytic enzymes, while using GDEst-lip, GDLip-lip and GDEst-est enzymes as a model system, is analyzed for the first time. Analysis of mutant GDEst-lip, GDLip-lip and GDEst-est variants, where one domain is inactive, showed that both domains retained their activity, although the reduction in specific activity of individual domains has been detected. Moreover, experimental data proposed that the N-terminal domain mostly influenced the thermostability, while the C-terminal domain was responsible for thermal activity. GDEst-lip variants fused by using rigid (EAAELAAE) and flexible (GGSELSGG) linkers indicated that a unique restriction site or a rigid linker is the most preferable fusion strategy to develop new chimeric biocatalysts with domains of Geobacillus lipolytic enzymes.

New engineered Geobacillus lipase GD-95RM for industry focusing on the cleaner production of fatty esters and household washing product formulations.

This study presents a new microbial lipolytic enzyme GD-95RM designed via random mutagenesis using previously characterized GD-95 lipase as a template. The improvement in activity of GD-95 lipase was caused by E100K, F154V and V174I mutations. Compared with GD-95 lipase, the GD-95RM lipase had 1.3-fold increased specific activity (2000 U/mg), demonstrated resistance to higher temperatures (75-85 °C), had fourfold increased Vmax towards p-NP dodecanoate and showed 2.5-fold lower KM for p-NP butyrate. It retained > 50% of its lipolytic activity when hydrolyzing short, medium and long acyl chain substrates at 30 °C and 55 °C reaction temperatures after 20 days' incubation with 25% of ethanol. GD-95RM also displayed long-term tolerance (40 d) to 5% NaCl, trisodium citrate, sodium perborate, urea, 0.1% boric acid, citric acid and Triton X-100. Moreover, oil hydrolysis and transesterification results revealed the capability of GD-95RM lipase to produce fatty acids or fatty acid esters through eco-friendly hydrolysis and transesterification reactions using a broad range of vegetable and fish oils, animal fat and different alcohols as substrates. GD-95RM lipase was successfully applied in synthesis reactions for ethyl oleate, octyl oleate and isoamyl oleate without giving to use additional reaction compounds or special reaction conditions.

Development of a new Geobacillus lipase variant GDlip43 via directed evolution leading to identification of new activity-regulating amino acids.

In this study three lipases GD-28, GD-95 and GD-66 (all 43 kDa in size), isolated from Geobacillus spp. were subjected to directed evolution experiments to yield a new synthetic lipolytic enzyme. This new lipase, obtained by DNA shuffling and epPCR, was named GDlip43 (also 43 kDa in size). It demonstrated increased thermoactivity, thermostability, an ability to hydrolyze short and long acyl chain p-NP esters and was activated by different organic solvents. Different activity of GDlip43 raised the hypothesis of new candidate amino acids which could be important for the activity of Geobacillus lipases. Based on the sequence alignment of parental and GDlip43 lipase, three candidate amino acids were selected. The importance of these amino acids, localized at positions 153, 154 and 247 (all of which are distant from the catalytic center of Geobacillus lipases) was investigated using site-directed mutagenesis. Directed evolution experiments also yielded another new lipase - GDlip30 (30 kDa in size). This low molecular mass derivative of GDlip43 had clearly detectable lipolytic activity (40 U/mg) and is the smallest currently known active Geobacillus lipase variant.

Usage of GD-95 and GD-66 lipases as fusion partners leading to improved chimeric enzyme LipGD95-GD66.

Lipases are used as biocatalysts in industrial processes mainly because of their stability at broad temperature and pH range, resistance to organic solvents and wide spectrum of substrates. The usage of several lipolytic domains, each with different activity and resistance profiles, enables both the flexibility and efficiency of industrial processes. In this study, GD-95 and GD-66 lipases produced by Geobacillus sp. 95 and Geobacillus sp. 66, respectively, were used as fusion partners to create a new fused lipolytic enzyme LipGD95-GD66. Chimeric LipGD95-GD66 lipase displayed tenfold increase in activity (200 U/mg) compared to parental GD-66 lipase, improved Vmax (10 µmol/min mg-1) and catalytic efficiency (2 ∗ 105 min-1 mM-1) for p-NP palmitate as a substrate and increased activity at 70-75 °C compared to both parental lipases. All three lipases also retained >50% of their lipolytic activity after incubation with methanol, n-hexane, ethanol and DMF for longer than three weeks, highlighting a great prospect for application in industrial processes. Moreover, transesterification results revealed the capability of parental GD-95 lipase to be the most promising biocatalyst for production of methyl and ethyl esters through eco-friendly transesterification using argan oil and ethanol/methanol as acceptors of acyl group.

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.