Correction to: Molecular, biochemical and kinetic analysis of a novel, thermostable lipase (LipSm) from Stenotrophomonas maltophilia Psi­1, the first member of a new bacterial lipase family (XIX).

[This corrects the article DOI: 10.1186/s40709-018-0074-6.].

Molecular, biochemical and kinetic analysis of a novel, thermostable lipase (LipSm) from Stenotrophomonas maltophilia Psi-1, the first member of a new bacterial lipase family (XVIII).

BACKGROUND: Microbial lipases catalyze a broad spectrum of reactions and are enzymes of considerable biotechnological interest. The focus of this study was the isolation of new lipase genes, intending to discover novel lipases whose products bear interesting biochemical and structural features and may have a potential to act as valuable biocatalysts in industrial applications. RESULTS: A novel lipase gene (lipSm), from a new environmental Stenotrophomonas maltophilia strain, Psi-1, originating from a sludge sample from Psittaleia (Greece), was cloned and sequenced. lipSm was further overexpressed in E. coli BL21(DE3) and the overproduced enzyme LipSm was purified and analyzed in respect to its biochemical and kinetic properties. In silico analysis of LipSm revealed that it is taxonomically related to several uncharacterized lipases from different genera, which constitute a unique clade, markedly different from all other previously described bacterial lipase families. All members of this clade displayed identical, conserved consensus sequence motifs, i.e. the catalytic triad (S, D, H), and an unusual, amongst bacterial lipases, Y-type oxyanion hole. 3D-modeling revealed the presence of a lid domain structure, which allows LipSm to act on small ester substrates without interfacial activation. In addition, the high percentage of alanine residues along with the occurrence of the AXXXA motif nine times in LipSm suggest that it is a thermostable lipase, a feature verified experimentally, since LipSm was still active after heating at 70 °C for 30 min. CONCLUSIONS: The phylogenetic analysis of LipSm suggests the establishment of a new bacterial lipase family (XVIII) with LipSm being its first characterized member. Furthermore, LipSm is alkaliphilic, thermostable and lacks the requirement for interfacial activation, when small substrates are used. These properties make LipSm a potential advantageous biocatalyst in industry and biotechnology.

Novel FRET-substrates of Rhizomucor pusillus rennin: Activity and mechanistic studies.

The development of sensitive, easy and reliable methods for the determination of Rhizomucor pusillus rennin (MPR) activity, in free and in immobilized form, along with the elucidation of the mechanism of action, represent challenges for the widespread use of the enzyme in industrial cheese production. These could be accomplished by using highly specific and sensitive substrates, as well as direct assay methods. We designed and synthesized novel substrates based on Fluorescence Resonance Energy Transfer (FRET) for the MPR by employing computational simulation techniques and peptide synthesis in liquid phase. Three FRET-substrates (Abz-GFY-pNA, Abz-SFY-pNA and Abz-GFI-pNA) were found active, while the Abz-GFY-pNA showed the highest reliability, sensitivity and specificity among them. Subsequently, a novel mechanism of MPR action was elucidated, with the development of novel methods for assaying activity in free and immobilized form, which both may contribute in the wider use of rennin in cheese production and other biotechnological applications.

Scale-up for esters production from straw whiskers for biofuel applications.

Delignified wheat straw was fermented by a mixed bacterial anaerobic culture obtained from a UASB reactor to produce organic acids (OAs). Kissiris was used as immobilization carrier in a 2-compartment 82L bioreactor filled with 17L of fermentation broth for the first 7 fermentation batches and up to 40L for the subsequent batches. The amount of straw used was 30g/L and the temperature was set at 37°C for all experiments. The total OAs reached concentrations up to 17.53g/L and the produced ethanol ranged from 0.3 to 1mL/L. The main OAs produced was acetic acid (6-8g/L) and butyric acid (3-8g/L). The OAs were recovered from the fermentation broth by a downstream process using 1-butanol, which was the solvent with the best recovery yields and also served as the esterification alcohol. The enzymatic esterification of OAs resulted to 90% yield.

Sustainable production of a new generation biofuel by lipase-catalyzed esterification of fatty acids from liquid industrial waste biomass.

In this work we suggest a methodology comprising the design and use of cost-effective, sustainable, and environmentally friendly process for biofuel production compatible with the market demands. A new generation biofuel is produced using fatty acids, which were generated from acidogenesis of industrial wastes of bioethanol distilleries, and esterified with selected alcohols by immobilized Candida antarctica Lipase-B. Suitable reactors with significant parameters and conditions were studied through experimental design, and novel esterification processes were suggested; among others, the continuous removal of the produced water was provided. Finally, economically sustainable biofuel production was achieved providing high ester yield (<97%) along with augmented concentration (3.35M) in the reaction mixtures at relatively short esterification times, whereas the immobilized lipase maintained over 90% of its initial esterifying ability after reused for ten cycles.

Kinetic and computational analysis of the reversible inhibition of porcine pancreatic elastase: a structural and mechanistic approach.

Structural and mechanistic insights were revealed for the reversible inhibition of Porcine Pancreatic Elastase (PPE); the kinetics of uninhibited and inhibited hydrolysis of substrate Suc-AAA-pNA was analyzed thoroughly. Additionally, the interactions between PPE and its inhibitor were studied by computational techniques. The uninhibited hydrolysis of Suc-AAA-pNA by PPE proceeds through a virtual transition state, involving an inferior physical and another dominating chemical step, where two stabilized reactant states precede the predominant acyl-enzyme. Different kinds of bonding with the PPE-backbone residues, including those of the catalytic triad, were found during the MD simulation of 5 ns, as key interactions favoring a higher stabilization of the best ranked complex PPE-CF3C(O)-KA-NHPh-p-CF3. The proton inventories of the inhibited hydrolysis of Suc-AAA-pNA by PPE, were ruled out the existence of any virtual transition state and thus they argue for a different mode of catalysis involving a structurally disturbed PPE molecule. Thereafter, a novel inhibition mechanism was suggested.

Economic evaluation of technology for a new generation biofuel production using wastes.

An economic evaluation of an integrated technology for industrial scale new generation biofuel production using whey, vinasse, and lignocellulosic biomass as raw materials is reported. Anaerobic packed-bed bioreactors were used for organic acids production using initially synthetic media and then wastes. Butyric, lactic and acetic acid were predominately produced from vinasse, whey, and cellulose, respectively. Mass balance was calculated for a 16,000L daily production capacity. Liquid-liquid extraction was applied for recovery of the organic acids using butanol-1 as an effective extraction solvent which serves also as the alcohol for the subsequent enzyme-catalyzed esterification. The investment needed for the installation of the factory was estimated to about 1.7million€ with depreciation excepted at about 3months. For cellulosics, the installation investment was estimated to be about 7-fold higher with depreciation at about 1.5years. The proposed technology is an alternative trend in biofuel production.