Ultrasound-assisted dilute acid hydrolysis for production of essential oils, pectin and bacterial cellulose via a citrus processing waste biorefinery.

An orange peel waste biorefinery was developed employing a design of experiments approach to optimize the ultrasound-assisted dilute acid hydrolysis process applied for production of useful commodities. Central composite design-based response surface methodology was used to approximate the combined effects of process parameters in simultaneous production of essential oils, pectin and a sugar-rich hydrolyzate. Application of a desirability function determined the optimal conditions required for maximal production efficiency of essential oils, pectin and sugars as 5.75% solid loading, 1.21% acid concentration and 34.2 min duration. Maximum production yields of 0.12% w/w essential oils, 45% w/w pectin and 40% w/w sugars were achieved under optimized conditions in lab- and pilot-scale facilities. The hydrolyzate formed was applied in bacterial cellulose fermentations producing 5.82 g biopolymer per 100 g waste. Design of experiments was efficient for process analysis and optimization providing a systems platform for the study of biomass-based biorefineries.

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.

Plasmid transformation of Weissella paramesenteroides DX by electroporation.

The present investigation sought to provide a reliable and highly efficient electrotransformation method for the lactic acid bacterium Weissella paramesenteroides DX. Experiments were carried out with the shuttle vectors pVS44 (2910 bps), pTRKH3 (7766 bps) and its derivative pTRKH3-1 (4855 bps). Several parameters, including the concentration of transforming plasmid DNA, plasmid size, electric field strength, age of the culture, cell density, and the pretreatment of cells with dl-threonine, lysozyme, and combined treatment with lithium acetate and dithiothreitol, were investigated and proved to influence the efficiency of transformation. Electrocompetence was found to peak in the early stationary phase (OD600 1.2). Other optimized conditions included: the concentration of 10 µg/ml transforming DNA, the cell density of 10(10) cells/ml, a high-density electric field pulse of 2.5 kV, 25 µF and 200 Ω, pretreatment of cells with 40 mM dl-threonine and 2000 U/ml lysozyme, and yielded 3.5×10(4) transformants/µg DNA for pVS44 while 1.2×10(4) transformants/µg DNA for the large plasmid TRKH3. Compared to previously reported data, the obtained transformation efficiencies provided an 8.75-fold increase for pVS44 and ensured plasmid stability for 120 generations in non-selective medium.

Advances in lipase-catalyzed esterification reactions.

Lipase-catalyzed esterification reactions are among the most significant chemical and biochemical processes of industrial relevance. Lipases catalyze hydrolysis as well as esterification reactions. Enzyme-catalyzed esterification has acquired increasing attention in many applications, due to the significance of the derived products. More specifically, the lipase-catalyzed esterification reactions attracted research interest during the past decade, due to an increased use of organic esters in biotechnology and the chemical industry. Lipases, as hydrolyzing agents are active in environments, which contain a minimum of two distinct phases, where all reactants are partitioned between these phases, although their distribution is not fixed and changes as the reaction proceeds. The kinetics of the lipase-catalyzed reactions is governed by a number of factors. This article presents a thorough and descriptive evaluation of the applied trends and perspectives concerning the enzymatic esterification, mainly for biofuel production; an emphasis is given on essential factors, which affect the lipase-catalyzed esterification reaction. Moreover, the art of using bacterial and/or fungal strains for whole cell biocatalysis purposes, as well as carrying out catalysis by various forms of purified lipases from bacterial and fungal sources is also reviewed.

Purification, kinetic characterization and properties of a novel thermo-tolerant extracellular protease from Kluyveromyces marxianus IFO 0288 with potential biotechnological interest.

A novel extracellular hydrolase of ∼45kDa molecular mass was purified from Kluyveromyces marxianus IFO 0288 cultures and characterized as serine protease. The K(m)-value of protease (designated protease-KM-IFO-0288-A), which was found active in media containing elevated [NaCl] but lacking EDTAK(2), decreased with increasing [Ca(2+)]. The protease maintained considerable activity at the range of 10-60°C and pH 6.00-10.25, with optimum k(cat)/K(m)-value at 35.5°C and pH 7.75. It was strongly affected by specific irreversible inhibitors of serine proteases while was unaffected by inhibitors of cysteine proteases. Significant rate constants, activation energies, and proton inventories were estimated from the profiles of Michaelis-Menten parameters, versus pH, temperature and deuterium atom fraction, in the hydrolysis of Suc-AAPF-pNA showing that protease-KM-IFO-0288-A performs catalysis via a charge-relay system. The properties of protease-KM-IFO-0288-A suggest that K. marxianus represents a valuable source of extracellular protease of biotechnological interest which, given its GRAS status, could find several important applications.

Production of the antimicrobial protein weisselin A by Weissella paramesenteroides DX in batch fermentations: the type of carbohydrate used as the C-source in the substrate affects the association of production with growth.

The effect of the type of carbohydrate (glucose, fructose, sucrose or galactose) used as the carbon source in the substrate on weissellin A production by Weissella paramesenteroides DX was evaluated on a solid-state cultivation procedure and conventional batch fermentations. Solid-state cultivation was done on M17-based medium over 3- and 6-h incubation periods. Experimental data showed that glucose supports the highest production levels on a per cell basis. Stirred tank bioreactor fermentations carried out at 50 % dissolved oxygen tension revealed the superiority of glucose over the other carbohydrates. Glucose supports growth-associated production and increased production rates and productivities (1,120 AU/ml). Growth-associated production was maintained with fructose but with lower fermentation rates and productivities. Sucrose cannot support this type of production. Fermentations with sucrose were characterized of lower sugar uptake rates, and lower specific growth and production rates, with bacteriocin titres not exceeding 630 AU/ml, while product formation kinetics were of the intermediate type.

Experimental investigation and optimization of process variables affecting the production of extracellular lipase by Kluyveromyces marxianus IFO 0288.

In this study, the production and optimization of extracellular lipase from Kluyveromyces marxianus IFO 0288 was investigated by using optimized nutritional and cultural conditions in a yeast medium containing glucose as the carbon source in fully aerobic batch fermentation (150 rpm). The influence of four fermentation parameters (type of lipidic source, initial culture pH, temperature, and length of fermentation) on growth and lipase production was investigated and evaluated using the conventional "one variable at a time" approach and response surface methodology. An 18-fold increase in lipase production during 65 h of fermentation was obtained with optimized nutritional (0.5 % olive oil) and cultivation (pH 6.5, 35 °C) conditions by employing the conventional optimization method. By applying the response surface methodology technique the initial pH value of 6.4 and temperature of 32.5 °C were identified as optimal and led to further improvements (up to 18-fold) of extracellular lipase production. The results provide, for the first time, evidence that K. marxianus has the potential to be used as an efficient producer of extracellular lipase with prospective application in a variety of industrial and biotechnological areas.

Aspects on the catalysis of lipase from porcine pancreas (type VI-s) in aqueous media: development of ion-pairs

This article reports a first contribution for the elucidation of catalytic mechanism of Lipase from porcine pancreas, type VI-s (PPL), in hydrolyzing an ester substrate in aqueous media. The conclusions were based on the pH-profiles of Michaelis-Menten parameters k cat/Km, k cat and Km, as well as on the absolute temperature profile of k cat/Km, obtained during the hydrolysis of p-nitrophenyl laurate by PPL. It was found that (a) PPL performs catalysis by means of ion pairs formed either as Ser152-Ο-/His263-Im+H and/or Carbonyl-Ο-/His263-Im+H, (b) the parameter k cat/Km equals to k1 and thus ES is formed and destroyed in the course of a series of consecutive reactions governed by the dynamic constant K S = k2/k1, and (c) the hydrolysis of substrate is assisted by a hydrogen bond developed between deprotonated Asp176 and the positively charged imidazole of His263 across a pKa-value 3.85, necessary for efficient catalysis.

Purification, amino acid sequence and characterization of the class IIa bacteriocin weissellin A, produced by Weissella paramesenteroides DX.

Weissella paramesenteroides DX has been shown to produce a 4450-Da class IIa bacteriocin, weissellin A, composed of 43 amino acids with the sequence KNYGNGVYCNKHKCSVDWATFSANIANNSVAMAGLTGGNAGN. The bacteriocin shares 68% similarity with leucocin C from Leuconostoc mesenteroides. Computational analyses predict that the bacteriocin is a hydrophobic molecule with a beta-sheet type conformation. Weissellin A exhibited various levels of activity against all gram-positive bacteria tested, but was not active against Salmonella enterica Enteritidis. The antimicrobial activity was not associated with target-cell lysis. The bacteriocin retained activity after exposure to 121°C for 60 min or to -20°C for 6months, and to pH 2.0-10.0. It was not sensitive to trypsin, α-chymotrypsin, pepsin and papain, but was inactivated by proteinase K. At a dissolved oxygen concentration of 50%, weissellin A was produced with growth-associated kinetics. The properties of weissellin A make this bacteriocin a potentially suitable agent for food and feed preservation.

Necessary auxiliary background for efficient use of an existing computer program of non-parametric fitting of nonlinear equations

This work constituted a significant contribution for more efficient use of a valuable computer program of non-parametric fitting of nonlinear multiparametric equations to experimental data. However, prerequisite in this context was the transformation of nonlinear multiparametric equations into linear hyperplane forms before their incorporation within the computer program; this latter was decisive and a matter of proper programming practice. Herein, a series of widely used equations useful in different fields of chemical processes, in biochemistry and/or in biotechnology, along with their suitable transformations as well as the appropriate programming support are being reported.

Este trabalho constitui uma contribuição significativa para um uso mais eficiente de um programa de computador valioso do encaixe não-paramétrico de equações multiparamétricas não lineares aos dados experimentais. Entretanto, o pré-requisito neste contexto é a transformação de equações multiparamétricas não lineares em formulários lineares do hyperplano antes de sua incorporação dentro do programa de computador; este último é decisive e uma matéria da prática de programação apropriada. Nisto, nós relatamos uma série das equações extensamente usadas úteis em campos diferentes de processos químicos, em bioquímia e/ou em biotechnologia, junto com suas transformações apropriadas além a sustentação de programação apropriada.

Purification and kinetics of two novel thermophilic extracellular proteases from Lactobacillus helveticus, from kefir with possible biotechnological interest.

Two thermophilic extracellular proteases, designated Lmm-protease-Lh ( approximately 29 kDa) and Hmm-protease-Lh ( approximately 62 kDa), were purified from the Lactobacillus helveticus from kefir, and found active in media containing dithiothreitol; the activity of Lmm-protease-Lh was increased significantly in media containing also EDTAK(2). Both novel proteases maintained full activity at 60 degrees C after 1-h incubation at 10 degrees C as well as at 80 degrees C, showing optimum k(cat)/K(m) values at pH 7.00 and 60 degrees C. Only irreversible inhibitors specific for cysteine proteinases strongly inhibited the activity of both novel enzymes, while they remained unaffected by irreversible inhibitors specific for serine proteinases. Both enzymes hydrolyzed the substrate Suc-FR-pNA via Michaelis-Menten kinetics; conversely, the substrate Cbz-FR-pNA was hydrolyzed by Lmm-protease-Lh via Michaelis-Menten kinetics and by Hmm-protease-Lh via substrate inhibition kinetics. Valuable rate constants and activation energies were estimated from the temperature-(k(cat)/K(m)) profiles of both enzymes, and useful results were obtained from the effect of different metallic ions on their Michaelis-Menten parameters.

Proton inventories constitute reliable tools in investigating enzymatic mechanisms: application on a novel thermo-stable extracellular protease from a halo-alkalophilic Bacillus sp.

A novel protease designated protease-A-17N-1, was purified from the halo-alkalophilic Bacillus sp. 17N-1, and found active in media containing dithiothreitol and EDTAK(2). This enzyme maintained significant activity from pH 6.00 to 9.00, showed optimum k(cat)/K(m) value at pH 7.50 and 33 degrees C. It was observed that only specific inhibitors of cysteine proteinases inhibited its activity. The pH-(k(cat)/K(m)) profile of protease-A-17N-1 was described by three pK(a)s in the acid limb, and one in the alkaline limb. Both are more likely due t3o the protonic dissociation of an acidic residue, and the development and subsequent deprotonation of an ion-pair, respectively, in its catalytic site, characteristic for cysteine proteinases. Moreover, both the obtained estimates of rate constant k(1) and the ratio k(2)/k(-1) at 25 degrees C, from the temperature-(k(cat)/K(m)) profile of protease-A-17N-1, were found similar to those estimated from the proton inventories of the same parameter, verifying the reliability of the latter methodology. Besides, the bowed-downward proton inventories of k(cat)/K(m), as well as the large inverse SIE observed for this parameter, in combination with its dependence versus temperature, were showed unambiguously that k(cat)/K(m) = k(1). Such results suggest that the novel enzyme is more likely to be a cysteine proteinase functioning via a general acid-base mechanism.

The catalytic mode of cysteine proteinases of papain (C1) family.

The Proton Inventory (PI) method has been applied in the hydrolysis of synthetic substrates by papain, chymopapain and stem bromelain, comparing also their corresponding pH-(k(cat)/K(m)) profiles, and it was found: (a) k(cat)/K(m)=k(1), and thus K(S)=k(2)/k(1) is a dynamic equilibrium constant, (b) bowed-downward PI for k(cat)/K(m) exhibiting large inverse SIE, and (c) linear PI exhibiting large normal SIE for K(S), k(2) and k(3). A novel finding of this work is that the association of substrates onto all three studied cysteine proteinases proceeds via a stepwise pathway, in contrast to purely concerted pathways found previously for both acylation and deacylation. A hydrogen bond, which seems more likely to be developed across a pK(a)-value close to 4.00, connecting [see text] (papain/chymopapain or bromelain numbering), constitutes another novelty of this work.

The kinetics of dissolution of the carbonate minerals of phosphate ores using dilute acetic acid solutions: the case of pH range from 3.96 to 6.40.

In this manuscript we present, in detail, the mechanism of the selective dissolution of calcite from low-grade phosphate ores (Epirus area, Greece) by dilute acetic acid. It was found that mass transfer makes a significant contribution to the rate-determining step of the overall reaction of four separate pH regions discriminated within the pH range from 3.96 to 6.40. Moreover, two different reaction mechanisms were proposed and two different model equations best fitted the corresponding experimental data. By applying a particular treatment to the developed differential equations proposed by us earlier (see Ref. (2)), we estimated the rate constants of specific slow reactions referred to pH regions 3.96-4.95 and 4.95-5.50. However, for pH region 5.50-6.40, we cannot reach a similar conclusion so far.