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.

Native xylose-inducible promoter expands the genetic tools for the biomass-degrading, extremely thermophilic bacterium Caldicellulosiruptor bescii.

Regulated control of both homologous and heterologous gene expression is essential for precise genetic manipulation and metabolic engineering of target microorganisms. However, there are often no options available for inducible promoters when working with non-model microorganisms. These include extremely thermophilic, cellulolytic bacteria that are of interest for renewable lignocellulosic conversion to biofuels and chemicals. In fact, improvements to the genetic systems in these organisms often cease once transformation is achieved. This present study expands the tools available for genetically engineering Caldicellulosiruptor bescii, the most thermophilic cellulose-degrader known growing up to 90 °C on unpretreated plant biomass. A native xylose-inducible (P xi ) promoter was utilized to control the expression of the reporter gene (ldh) encoding lactate dehydrogenase. The P xi -ldh construct resulted in a both increased ldh expression (20-fold higher) and lactate dehydrogenase activity (32-fold higher) in the presence of xylose compared to when glucose was used as a substrate. Finally, lactate production during growth of the recombinant C. bescii strain was proportional to the initial xylose concentration, showing that tunable expression of genes is now possible using this xylose-inducible system. This study represents a major step in the use of C. bescii as a potential platform microorganism for biotechnological applications using renewable biomass.

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.

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.