Enzymes and pathways in microbial production of 2,3-butanediol and 3-acetoin isomers.

2,3-Butanediol (BD) and acetoin (AC) are products of the non-oxidative metabolism of microorganisms, presenting industrial importance due to their wide range of applications and high market value. Their optical isomers have particular applications, justifying the efforts on the selective bioproduction. Each microorganism produces different isomer mixtures, as a consequence of having different butanediol dehydrogenase (BDH) enzymes. However, the whole scene of the isomer bioproduction, considering the several enzymes and conditions, has not been completely elucidated. Here we show the BDH classification as R, S or meso by bioinformatics analysis uncovering the details of the isomers production. The BDH was compared to diacetyl reductases (DAR) and the new enoyl reductases (ER). We observed that R-BDH is the most singular BDH, while meso and S-BDHs are similar and may be better distinguished through their stereo-selective triad. DAR and ER showed distinct stereo-triads from those described for BDHs, agreeing with kinetic data from the literature and our phylogenetic analysis. The ER family probably has meso-BDH like activity as already demonstrated for a single sequence from this group. These results are of great relevance, as they organize BD producing enzymes, to our known, never shown before in the literature. This review also brings attention to nontraditional enzymes/pathways that can be involved with BD/AC synthesis, as well as oxygen conditions that may lead to the differential production of their isomers. Together, this information can provide helpful orientation for future studies in the field of BD/AC biological production, thus contributing to achieve their production on an industrial scale.

Biocatalytic CO2 Absorption and Structural Studies of Carbonic Anhydrase under Industrially-Relevant Conditions.

The unprecedently high CO2 levels in the atmosphere evoke the urgent need for development of technologies for mitigation of its emissions. Among the alternatives, the biocatalytic route has been claimed as one of the most promising. In the present work, the carbonic anhydrase from bovine erythrocytes (BCA) was employed as a model enzyme for structural studies in an aqueous phase at alkaline pH, which is typical of large-scale absorption processes under operation. Circular dichroism (CD) analysis revealed a high enzymatic stability at pH 10 with a prominent decrease of the melting temperature above this value. The CO2 absorption capacity of the aqueous solutions were assessed by online monitoring of pressure decay in a stainless-steel cell, which indicated a better performance at pH 10 with a kinetic rate increase of up to 43%, as compared to non-biocatalytic conditions. Even low enzyme concentrations (0.2 mg g-1) proved to be sufficient to improve the overall CO2 capture process performance. The enzyme-enhanced approach of CO2 capture presents a high potential and should be further studied.

Characterization of esterase activity from an Acetomicrobium hydrogeniformans enzyme with high structural stability in extreme conditions.

The biotechnological and industrial uses of thermostable and organic solvent-tolerant enzymes are extensive and the investigation of such enzymes from microbiota present in oil reservoirs is a promising approach. Searching sequence databases for esterases from such microbiota, we have identified in silico a potentially secreted esterase from Acetomicrobium hydrogeniformans, named AhEst. The recombinant enzyme was produced in E. coli to be used in biochemical and biophysical characterization studies. AhEst presented hydrolytic activity on short-acyl-chain p-nitrophenyl ester substrates. AhEst activity was high and stable in temperatures up to 75 °C. Interestingly, high salt concentration induced a significant increase of catalytic activity. AhEst still retained ~ 50% of its activity in 30% concentration of several organic solvents. Synchrotron radiation circular dichroism and fluorescence spectroscopies confirmed that AhEst displays high structural stability in extreme conditions of temperature, salinity, and organic solvents. The enzyme is a good emulsifier agent and is able to partially reverse the wettability of an oil-wet carbonate substrate, making it of potential interest for use in enhanced oil recovery. All the traits observed in AhEst make it an interesting candidate for many industrial applications, such as those in which a significant hydrolytic activity at high temperatures is required.

Biotransformation of ethylene glycol to glycolic acid by Yarrowia lipolytica: A route for poly(ethylene terephthalate) (PET) upcycling.

Biological recycling of PET waste has been extensively investigated recently to tackle plastic waste pollution, and ethylene glycol (EG) is one of the main building blocks recovered from this process. Wild-type Yarrowia lipolytica IMUFRJ 50682 can be a biocatalyst to biodepolymerize PET. Herein, we report its ability to perform oxidative biotransformation of EG into glycolic acid (GA): a higher value-added chemical with varied industrial applications. We found that this yeast tolerates high EG concentrations (up to 2 M) based on maximum non-inhibitory concentration (MNIC) tests. Whole-cell biotransformation assays using resting yeast cells showed GA production uncoupled to cell growth metabolism, and 13 C nuclear magnetic resonance (NMR) analysis confirmed GA production. Moreover, higher agitation speed (450 vs. 350 rpm) resulted in a 1.12-fold GA production improvement (from 352 to 429.5 mM) during Y. lipolytica cultivation in bioreactors after 72 h. GA was constantly accumulated in the medium, suggesting that this yeast may also share an incomplete oxidation pathway (i.e., it is not metabolized to carbon dioxide) as seen in acetic acid bacterial group. Additional assays using higher chain-length diols (1,3-propanediol, 1,4-butanediol, and 1,6-hexanediol) revealed that C4 and C6 diols were more cytotoxic, suggesting that they underwent different pathways in the cells. We found that this yeast consumed extensively all these diols, however, 13 C NMR analysis from supernatant identified solely the presence of 4-hydroxybutanoic acid from 1,4-butanediol, along with GA from EG oxidation. Findings reported herein reveal a potential route for PET upcycling to a higher value-added product.

Chemical Recycling of PET Using Catalysts from Layered Double Hydroxides: Effect of Synthesis Method and Mg-Fe Biocompatible Metals.

The chemical recycling of poly(ethylene terephthalate) (PET) residues was performed via glycolysis with ethylene glycol (EG) over Mg-Fe and Mg-Al oxide catalysts derived from layered double hydroxides. Catalysts prepared using the high supersaturation method (h.s.c.) presented a higher surface area and larger particles, but this represented less PET conversion than those prepared by the low supersaturation method (l.s.c.). This difference was attributed to the smaller mass transfer limitations inside the (l.s.c.) catalysts. An artificial neural network model well fitted the PET conversion and bis(2-hydroxyethyl) terephthalate (BHET) yield. The influence of Fe in place of Al resulted in a higher PET conversion of the Mg-Fe-h.s.c. catalyst (~95.8%) than of Mg-Al-h.s.c. (~63%). Mg-Fe catalysts could be reused four to five times with final conversions of up to 97% with reaction conditions of EG: PET = 5:1 and catalyst: PET = 0.5%. These results confirm the Mg-Fe oxides as a biocompatible novel catalyst for the chemical recycling of PET residues to obtain non-toxic BHET for further polymerization, and use in food and beverage packaging.

Adding value to a toxic residue from the biodiesel industry: production of two distinct pool of lipases from Penicillium simplicissimum in castor bean waste.

In countries with a strong agricultural base, such as Brazil, the generation of solid residues is very high. In some cases, these wastes present no utility due to their toxic and allergenic compounds, and so are an environmental concern. The castor bean (Ricinus communis) is a promising candidate for biodiesel production. From the biodiesel production process developed in the Petrobras Research Center using castor bean seeds, a toxic and alkaline waste is produced. The use of agroindustrial wastes in solid-state fermentation (SSF) is a very interesting alternative for obtaining enzymes at low cost. Therefore, in this work, castor bean waste was used, without any treatment, as a culture medium for fungal growth and lipase production. The fungus Penicillium simplicissimum was able to grow and produce an enzyme in this waste. In order to maximize the enzyme production, two sequential designs-Plackett-Burman (variable screening) followed by central composite rotatable design (CCRD)-were carried out, attaining a considerable increase in lipase production, reaching an activity of 155.0 U/g after 96 h of fermentation. The use of experimental design strategy was efficient, leading to an increase of 340% in the lipase production. Zymography showed the presence of different lipases in the crude extract. The partial characterization of such extract showed the occurrence of two lipase pools with distinct characteristics of pH and temperature of action: one group with optimal action at pH 6.5 and 45°C and another one at pH 9.0 and 25°C. These results demonstrate how to add value to a toxic and worthless residue through the production of lipases with distinct characteristics. This pool of enzymes, produced through a low cost methodology, can be applied in different areas of biotechnology.

Fungal and enzymatic bio-depolymerization of waste post-consumer poly(ethylene terephthalate) (PET) bottles using Penicillium species.

Poly(ethylene terephthalate) (PET) is a petroleum-based plastic that is massively produced and used worldwide. A promising PET recycling process to circumvent petroleum feedstock consumption and help to reduce environmental pollution is microbial or enzymatic biodegradation of post-consumer (PC) PET packages to its monomers-terephthalic acid (TPA) and ethylene glycol (EG)-or to key intermediates in PET synthesis-such as mono- and bis-(2-hydroxyethyl) terephthalate (MHET and BHET). Two species of filamentous fungi previously characterized as lipase producers (Penicillium restrictum and P. simplicissimum) were evaluated in submerged fermentation for induction of lipase production by two inducers (BHET and amorphous PET), and for biodegradation of two substrates (BHET and PC-PET). BHET induced lipase production in P. simplicissimum, achieving a peak of 606.4 U/L at 49 h (12.38 U/L.h), representing an almost twofold increase in comparison to the highest peak in the control (without inducers). Microbial biodegradation by P. simplicissimum after 28 days led to a 3.09% mass loss on PC-PET fragments. In contrast, enzymatic PC-PET depolymerization by cell-free filtrates from a P. simplicissimum culture resulted in low concentrations of BHET, MHET and TPA (up to 9.51 µmol/L), suggesting that there are mechanisms at the organism level that enhance biodegradation. Enzymatic BHET hydrolysis revealed that P. simplicissimum extracellular enzymes catalyze the release of MHET as the predominant product. Our results show that P. simplicissimum is a promising biodegrader of PC-PET that can be further explored for monomer recovery in the context of feedstock recycling processes.

Evaluation of cell recycle on Thermomyces lanuginosus Xylanase A production by Pichia pastoris GS 115.

This work aims to evaluate cell recycle of a recombinant strain of Pichia pastoris GS115 on the Xylanase A (XynA) production of Thermomyces lanuginosus IOC-4145 in submerged fermentation. Fed-batch processes were carried out with methanol feeding at each 12 h and recycling cell at 24, 48, and 72 h. Additionally, the influence of the initial cell concentration was investigated. XynA production was not decreased with the recycling time, during four cell recycles, using an initial cell concentration of 2.5 g/L. The maximum activity was 14,050 U/L obtained in 24 h of expression. However, when the initial cell concentration of 0.25 g/L was investigated, the enzymatic activity was reduced by 30 and 75% after the third and fourth cycles, respectively. Finally, it could be concluded that the initial cell concentration influenced the process performance and the interval of cell recycle affected enzymatic production.

Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst).

Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/ß protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required.