Fusion of Glutamate Dehydrogenase and Formate Dehydrogenase Yields a Bifunctional Efficient Biocatalyst for the Continuous Removal of Ammonia

A novel fusion protein has been rationally designed, combining the hexameric glutamate dehydrogenase from Clostridium symbiosum with the dimeric formate dehydrogenase from Candida boidinii. The former enzyme consumes ammonia for the reductive amination of α-ketoglutarate using NADH, while the latter biocatalyst regenerates continuously the cofactor. This enzymes fusion opens new perspectives for the detection and the removal of ammonia. The bifunctional biocatalyst has been successfully created, expressed, and then characterized. The two fused protein domains retained identical properties and catalytic activity of the individual enzymes. Additionally, the immobilization on a methacrylate resin optimized the assembly providing a reusable and stable biocatalyst. This is an example of immobilization of a fusion protein, so that efficiency and sustainability of the process are enhanced. The immobilized biocatalyst could be recycled 10 times retaining still half of the initial activity. Such preparation outperforms the co-immobilized wild-type enzymes in the conversion of 300 mM of ammonia, which could be carried out also in continuous mode.

Lactiplantibacillus plantarum Used as Single, Multiple, and Mixed Starter Combined with Candida boidinii for Table Olive Fermentations: Chemical, Textural, and Sensorial Characterization of Final Products

In this study, four different kinds of table olive fermentations belonging to Olea europaea L. Itrana cultivar were evaluated: A, spontaneous fermentation; B, fermentation with a single inoculum (Lactiplantibacillus plantarum B1); C, fermentation with multiple inoculum (L. plantarum B1 + L. plantarum B51 + L. plantarum B124, 1:1:1); and D, fermentation with mixed (bacterium + yeast) inoculum (L. plantarum B1 + Candida boidinii). This research focuses on the correlation between the different mixes of inoculations and their effect under the chemical, sensorial, and textural profiles in the final products (olives) for potential applications on table olive fermentation. During the fermentation, some specific parameters were monitored: chemical characterization of oil fraction (pigments, tocopherols, fatty acids, alkyl esters, and sterol composition), Texture Profile Analysis (TPA), determination of olive color, and sensory evaluation of the final products. The use of LAB starters (single and multiple inocula) compared to spontaneous process revealed a greater performance in preventing the spoilage process and in developing favorable physico-chemical conditions during the fermentation. In fact, the highest values of fatty acid alkyl esters were reached in spontaneous fermentation (~480 mg/kg in jar A). The presence of C. boidinii as inoculum in jar D was involved in table olive softening: the fermented olives showed the lowest values of the parameters related to consistence of fruit as hardness (~2300 g) and gumminess (~990 g) and high value of fatty acid methyl esters (~110 mg/kg).

Removal of Phenols in Table Olive Processing Wastewater by Using a Mixed Inoculum of Candida boidinii and Bacillus pumilus: Effects of Inoculation Dynamics, Temperature, pH, and Effluent Age on the Abatement Efficiency

The main goal of this paper was to assess the ability of a combination of Candida boidinii and Bacillus pumilus to remove phenol in table olive processing water, as a function of some variables, like temperature, pH, a dilution of waste and the order of inoculation of the two microorganisms. At this purpose C. boidinii and B. pumilus were sequentially inoculated in two types of table olive processing water (fresh wastewater, FTOPW and wastewater stored for 3 months-aged wastewater, ATOPW). pH (6 and 9), temperature (10 and 35 °C) and dilution ratio (0, 1:1) were combined through a 2k fractional design. Data were modeled using two different approaches: Multifactorial Analysis of Variance (MANOVA) and multiple regression. A higher removal yield was achieved by inoculating B. pumilus prior to the yeast (192 vs. 127 mg/L); moreover, an increased efficiency was gained at 35 °C (mean removal of 200 mg/L). The use of two statistic approach suggested a different weight of variables; temperature was a global variable, that is a factor able to affect the yield of the process in all conditions. On the other hand, an alkaline pH could increase the removal of phenol at 10 °C (25–43%).

Bioelectricity Production from Blueberry Waste

Global warming and the increase in organic waste from agro-industries create a major problem for the environment. In this sense, microbial fuel cells (MFC) have great potential for the generation of bioelectricity by using organic waste as fuel. This research produced low-cost MFC by using zinc and copper electrodes and taking blueberry waste as fuel. A peak current and voltage of 1.130 ± 0.018 mA and 1.127 ± 0.096 V, respectively, were generated. The pH levels were acid, with peak conductivity values of 233. 94 ± 0.345 mS/cm and the degrees Brix were descending from the first day. The maximum power density was 3.155 ± 0.24 W/cm2 at 374.4 mA/cm2 current density, and Cándida boidinii was identified by means of molecular biology and bioinformatics techniques. This research gives a new way to generate electricity with this type of waste, generating added value for the companies in this area and helping to reduce global warming.

Recombinant Enzymatic Redox Systems for Preparation of Aroma Compounds by Biotransformation

The aim of this study was to develop immobilized enzyme systems that reduce carbonyl compounds to their corresponding alcohols. The demand for natural aromas and food additives has been constantly growing in recent years. However, it can no longer be met by extraction and isolation from natural materials. One way to increase the availability of natural aromas is to prepare them by the enzymatic transformation of suitable precursors. Recombinant enzymes are currently being used for this purpose. We investigated trans-2-hexenal bioreduction by recombinant Saccharomyces cerevisiae alcohol dehydrogenase (ScADH1) with simultaneous NADH regeneration by recombinant Candida boidinii formate dehydrogenase (FDH). In a laboratory bioreactor with two immobilized enzymes, 88% of the trans-2-hexenal was transformed to trans-2-hexenol. The initial substrate concentration was 3.7 mM. The aldehyde destabilized ScADH1 by eluting Zn2+ ions from the enzyme. A fed-batch operation was used and the trans-2-hexenal concentration was maintained at a low level to limit the negative effect of Zn2+ ion elution from the immobilized ScADH1. Another immobilized two-enzyme system was used to reduce acetophenone to (S)-1-phenylethanol. To this end, the recombinant alcohol dehydrogenase (RrADH) from Rhodococcus ruber was used. This biocatalytic system converted 61% of the acetophenone to (S)-1-phenylethanol. The initial substrate concentration was 8.3 mM. All enzymes were immobilized by poly-His tag to Ni2+, which formed strong but reversible bonds that enabled carrier reuse after the loss of enzyme activity.

Comparison of Enzymatic and Acidic Fractionation of Corn Fiber for Glucose-rich Hydrolysate and Bioethanol Production by Candida boidinii

Corn fiber is a by-product of the corn wet milling process and a promising raw material to produce bioethanol in a bio-refinery process. In this study, enzymatic and acidic fractionations of corn fiber were compared with particular attention to produce glucose-rich hydrolyzates. The acidic fractionation contained two, sequential, sulphuric acid-catalyzed, hydrolysis steps based on our previous study. In the enzymatic fractionation process, corn fiber was pre-treated by soaking in aqueous ammonia (18.5 % (w/w) dry matter, 15 % (w/w) ammonia solution, 24 hours) and then hydrolyzed by using Hemicellulase (NS 22002) enzyme cocktail. The cellulose part of the solid residues obtained after the acidic and enzymatic fractionation processes was enzymatically hydrolyzed by using Cellic Ctec2 and Novozymes 188 (12.5 % (w/w) dry matter, 50 °C, 72 hours). Cellulose hydrolysis after the acidic and enzymatic fractionation resulted in a supernatant containing 64 g/L and 25 g/L glucose, respectively. Therefore, ethanol fermentation experiments were performed in Separated Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF) configurations after the acidic fractionation of corn fiber. SHF configuration was found to be more advantageous regarding the achievable ethanol yield. Although the fermentation with Candida boidinii NCAIM Y.01308 was accomplished within longer time (43 hours) compared to Saccharomyces cerevisiae (5 hours), the achieved ethanol yields were similar (79%) during the SHF process. It was concluded that acidic fractionation is more efficient to produce glucose-rich hydrolyzate from corn fiber compared to enzymatic fractionation, and Candida boidinii is suitable for ethanol fermentation on the glucose-rich hydrolyzate.

Effect of confinement of horse heart cytochrome c and formate dehydrogenase from Candida boidinii on mesoporous carbons on their catalytic activity

This study reports the immobilization of two biocatalysts (e.g., cytochrome c—Cyt c—and the non-metalloenzyme formate dehydrogenase from Candida boidinii–cbFDH) on a series of mesoporous carbons with controlled pore sizes. The catalytic activity of the nanoconfined proteins was correlated with the pore size distribution of the carbon materials used as supports. The electrochemical behaviour of nanoconfined Cyt c showed direct electron transfer electroactivity in pore sizes matching tightly the protein dimension. The pseudo-peroxidase activity towards H2O2 reduction was enhanced at pH 4.0, due to the protein conformational changes. For cbFDH, the reduction of CO2 towards formic acid was evaluated for the nanoconfined protein, in the presence of nicotinamide adenine dinucleotide (NADH). The carbons displayed different cbFDH uptake capacity, governed by the dimensions of the main mesopore cavities and their accessibility through narrow pore necks. The catalytic activity of nanoconfined cbFDH was largely improved, compared to its performance in free solution. Regardless of the carbon support used, the production of formic acid was higher upon immobilization with lower nominal cbFDH:NADH ratios.

Probing the Role of the Conserved Arg174 in Formate Dehydrogenase by Chemical Modification and Site-Directed Mutagenesis

The reactive adenosine derivative, adenosine 5′-O-[S-(4-hydroxy-2,3-dioxobutyl)]-thiophosphate (AMPS-HDB), contains a dicarbonyl group linked to the purine nucleotide at a position equivalent to the pyrophosphate region of NAD+. AMPS-HDB was used as a chemical label towards Candida boidinii formate dehydrogenase (CbFDH). AMPS-HDB reacts covalently with CbFDH, leading to complete inactivation of the enzyme activity. The inactivation kinetics of CbFDH fit the Kitz and Wilson model for time-dependent, irreversible inhibition (KD = 0.66 ± 0.15 mM, first order maximum rate constant k3 = 0.198 ± 0.06 min−1). NAD+ and NADH protects CbFDH from inactivation by AMPS-HDB, showing the specificity of the reaction. Molecular modelling studies revealed Arg174 as a candidate residue able to be modified by the dicarbonyl group of AMPS-HDB. Arg174 is a strictly conserved residue among FDHs and is located at the Rossmann fold, the common mononucleotide-binding motif of dehydrogenases. Arg174 was replaced by Asn, using site-directed mutagenesis. The mutant enzyme CbFDHArg174Asn was showed to be resistant to inactivation by AMPS-HDB, confirming that the guanidinium group of Arg174 is the target for AMPS-HDB. The CbFDHArg174Asn mutant enzyme exhibited substantial reduced affinity for NAD+ and lower thermostability. The results of the study underline the pivotal and multifunctional role of Arg174 in catalysis, coenzyme binding and structural stability of CbFDH.

Carbonic anhydrase/ formate dehydrogenase bienzymatic system for CO2 capture, utilization and storage

In order to establish carbon capture, utilization, and storage (CCUS) technology, we focused on the system consisting of two different biocatalysts (formate dehydrogenase from Candida boidinii; CbFDH and carbonic anhydrase...