Sequential Co-Immobilization of Enzymes on Magnetic Nanoparticles for Efficient l-Xylulose Production.

Multi-enzymatic strategies have shown improvement in bioconversion during cofactor regeneration. In this study, purified l-arabinitol 4-dehydrogenase (LAD) and nicotinamide adenine dinucleotide oxidase (Nox) were immobilized via individual, mixed, and sequential co-immobilization approaches on magnetic nanoparticles, and were evaluated to enhance the conversion of l-arabinitol to l-xylulose. Initially, the immobilization of LAD or Nox on the nanoparticles resulted in a maximum immobilization yield and relative activity of 91.4% and 98.8%, respectively. The immobilized enzymes showed better pH and temperature profiles than the corresponding free enzymes. Furthermore, co-immobilization of these enzymes via mixed and sequential methods resulted in high loadings of 114 and 122 mg/g of support, respectively. Sequential co-immobilization of these enzymes proved more beneficial for higher conversion than mixed co-immobilization because of better retaining Nox residual activity. Sequentially co-immobilized enzymes showed a high relative conversion yield with broader pH, temperature, and storage stability profiles than the controls, along with high reusability. To the best of our knowledge, this is the first report on the mixed or sequential co-immobilization of LAD and Nox on magnetic nanoparticles for l-xylulose production. This finding suggests that selecting a sequential co-immobilization strategy is more beneficial than using individual or mixed co-immobilized enzymes on magnetic nanoparticles for enhancing conversion applications.

Manipulating Microbial Cell Morphology for the Sustainable Production of Biopolymers.

The total rate of plastic production is anticipated to surpass 1.1 billion tons per year by 2050. Plastic waste is non-biodegradable and accumulates in natural ecosystems. In 2020, the total amount of plastic waste was estimated to be 367 million metric tons, leading to unmanageable waste disposal and environmental pollution issues. Plastics are produced from petroleum and natural gases. Given the limited fossil fuel reserves and the need to circumvent pollution problems, the focus has shifted to biodegradable biopolymers, such as polyhydroxyalkanoates (PHAs), polylactic acid, and polycaprolactone. PHAs are gaining importance because diverse bacteria can produce them as intracellular inclusion bodies using biowastes as feed. A critical component in PHA production is the downstream processing procedures of recovery and purification. In this review, different bioengineering approaches targeted at modifying the cell morphology and synchronizing cell lysis with the biosynthetic cycle are presented for product separation and extraction. Complementing genetic engineering strategies with conventional downstream processes, these approaches are expected to produce PHA sustainably.

Encapsulation of Methanotrophs within a Polymeric Matrix Containing Copper- and Iron-Based Nanoparticles to Enhance Methanol Production from a Simulated Biogas.

The production of renewable energy or biochemicals is gaining more attention to minimize the emissions of greenhouse gases such as methane (CH4) and carbon dioxide for sustainable development. In the present study, the influence of copper (Cu)- and iron (Fe)-based nanoparticles (NPs), such as Cu, Fe3O4, and CuFe2O4, was evaluated during the growth of methanotrophs for inoculum preparation and on the development of a polymeric-matrix-based encapsulation system to enhance methanol production from simulated biogas (CH4 and CO2). The use of simulated biogas feed and the presence of NP-derived inoculums produce a remarkable enhancement in methanol production up to 149% and 167% for Methyloferula stellata and Methylocystis bryophila free-cells-based bioconversion, respectively, compared with the use of pure CH4 as a control feed during the growth stage. Furthermore, these methanotrophs encapsulated within a polymeric matrix and NPs-based systems exhibited high methanol production of up to 156%, with a maximum methanol accumulation of 12.8 mmol/L over free cells. Furthermore, after encapsulation, the methanotrophs improved the stability of residual methanol production and retained up to 62.5-fold higher production potential than free cells under repeated batch reusability of 10 cycles. In the presence of CH4 vectors, methanol production by M. bryophila improved up to 16.4 mmol/L and retained 20% higher recycling stability for methanol production in paraffin oil. These findings suggest that Cu and Fe NPs can be beneficially employed with a polymeric matrix to encapsulate methanotrophs and improve methanol production.

Coriolus versicolor laccase-based inorganic protein hybrid synthesis for application in biomass saccharification to enhance biological production of hydrogen and ethanol.

In this study, a bio-friendly inorganic protein hybrid-based enzyme immobilization system using partially purified Coriolus versicolor laccase (CvLac) was successfully applied to biomass hydrolysis for the enhancement of sugar production aimed at generating biofuels. After four days of incubation, the maximum CvLac production was achieved at 140 U/mg of total protein in the presence of inducers such as copper and wheat bran after four days of incubation. Crude CvLac immobilized through inorganic protein hybrids such as nanoflowers (NFs) using zinc as Zn3(PO4)2/CvLac hybrid NFs (Zn/CvLac-NFs) showed a maximum encapsulation yield of 93.4% and a relative activity of 265% compared to free laccase. The synthesized Zn/CvLac-NFs exhibited significantly improved activity profiles and stability compared to free enzymes. Furthermore, Zn/CvLac-NFs retained a significantly high residual activity of 96.2% after ten reuse cycles. The saccharification of poplar biomass improved ∼2-fold in the presence of Zn/CvLac-NFs, with an 8-fold reduction in total phenolics compared to the control. The Zn/CvLac-NFs treated biomass hydrolysate showed high biological hydrogen (H2) production and ethanol conversion efficiency of up to 2.68 mol/mol of hexose and 79.0% compared to the control values of 1.27 mol of H2/mol of hexose and 58.4%, respectively. The CvLac hybrid NFs are the first time reported for biomass hydrolysis, and a significant enhancement in the production of hydrogen and ethanol was reported. The synthesis of such NFs based on crude forms of diverse enzymes can potentially be extended to a broad range of biotechnological applications.

Characterization of a xylitol dehydrogenase from Aspergillus flavus and its application in l-xylulose production.

An NAD+-dependent xylitol dehydrogenase from A. flavus (AfXDH) was cloned and successfully expressed in Escherichia coli. AfXDH gene sequence revealed an open reading frame of 1,110 bp, encoding a polypeptide of 369 amino acids with a calculated molecular mass of 38,893 Da. Among various polyols, sorbitol and xylitol were preferred substrates of AfXDH with Km values of 16.2 and 16.9 mM, respectively. AfXDH showed the highest activity in Tris-glycine-NaOH buffer (pH 9.5) at 50°C; it required Zn2+ or Mn2+ for enzyme activity. The half-life at 40°C and half denaturation temperature (T1/2) was 200 min and 45°C, respectively. Bioinformatic analyses along with biochemical properties confirmed that AfXDH belonged to the medium-chain dehydrogenase/reductase family. AfXDH exhibits higher thermostability and k cat values than those of other XDHs. The feasibility of using AfXDH in l-xylulose production was demonstrated. AfXDH, when coupled with Streptococcus pyogenes NADH oxidase, efficiently converted xylitol to l-xylulose with 97% yield, suggesting its usefulness for the industrial l-xylulose production from xylitol.

Immobilization of Laccase Through Inorganic-Protein Hybrids Using Various Metal Ions.

In this study, the inorganic-protein hybrid strategy was employed for immobilization of laccase from Rhus vernicifera (Rvlac) using various metals calcium, cobalt, copper, and zinc (Zn). The efficient synthesis of hybrids for Rvlac immobilization was noted at 4 °C for incubation of 24 h. Among these hybrids, the maximum encapsulation yields (EY) of 90.1% and relative activity (RA) of 225% to free enzyme were recorded for Zn and Rvlac based inorganic-protein hybrids as Zn3(PO4)2-Rvlac. The upper optimum pH, and temperature values were observed of 4.0, and 45 °C after immobilization as compared to 3.5, and 40 °C for the free enzyme, respectively. After encapsulation, Rvlac showed a significant improvement up to 11.4-fold in pH and 5.7-fold in temperature the activity profiles. Free enzyme completely lost its activity at 60 °C after 2 h of incubation, whereas Zn3(PO4)2-Rvlac retained its residual activity of 56.7% under similar conditions. After ten cycles of reusability, Zn3(PO4)2-Rvlac possessed high residual activity of 90.8%. This study showed that the variation in the metal ions for immobilization of Rvlac as inorganic-protein hybrids significantly altered EY and RA. Also, Zn3(PO4)2-Rvlac proved more efficient as compared to free laccase that can be beneficially employed for biotechnological applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01000-5.

Green Synthesis of Silver-Decorated Magnetic Particles for Efficient and Reusable Antimicrobial Activity.

Metal and metal hybrid nanostructures have shown tremendous application in the biomedical and catalytic fields because of their plasmonic and catalytic properties. Here, a green and clean method was employed for the synthesis of silver nanoparticle (Ag NP)-SiO2-Fe2O3 hybrid microstructures, and biomolecules from green tea extracts were used for constructing the hybrid structures. The SiO2-Fe2O3 structures were synthesized using an ethanolic green tea leaf extract to form Bio-SiO2-Fe2O3 (BSiO2-Fe2O3) structures. Biochemical studies demonstrated the presence of green tea biomolecules in the BSiO2 layer. Reduction of the silver ions was performed by a BSiO2 layer to form Ag NPs of 5-10 nm in diameter in and on the BSiO2-Fe2O3 microstructure. The reduction process was observed within 600 s, which is faster than that reported elsewhere. The antimicrobial activity of the Ag-BSiO2-Fe2O3 hybrid structure was demonstrated against Staphylococcus aureus and Escherichia coli, and the nanostructures were further visualized using confocal laser scanning microscopy (CLSM). The magnetic properties of the Ag-BSiO2-Fe2O3 hybrid structure were used for studying reusable antimicrobial activity. Thus, in this study, we provide a novel green route for the construction of a biomolecule-entrapped SiO2-Fe2O3 structure and their use for the ultra-fast formation of Ag NPs to form antimicrobial active multifunctional hybrid structures.

Biomolecules Production from Greenhouse Gases by Methanotrophs.

Harmful effects on living organisms and the environment are on the rise due to a significant increase in greenhouse gas (GHG) emissions through human activities. Therefore, various research initiatives have been carried out in several directions in relation to the utilization of GHGs via physicochemical or biological routes. An environmentally friendly approach to reduce the burden of significant emissions and their harmful effects is the bioconversion of GHGs, including methane (CH4) and carbon dioxide (CO2), into value-added products. Methanotrophs have enormous potential for the efficient biotransformation of CH4 to various bioactive molecules, including biofuels, polyhydroxyalkanoates, and fatty acids. This review highlights the recent developments in methanotroph-based systems for methanol production from GHGs and proposes future perspectives to improve process sustainability via biorefinery approaches.

Biotin and Zn2+ Increase Xylitol Production by Candida tropicalis.

In this study, the medium requirements to increase the production of xylitol by using Candida tropicalis (CT) have been investigated. The technique of single addition or omission of medium components was applied to determine the nutritional requirements. The addition of amino acids such as Asp, Glu, Gln, Asn, Thr, and Gly had no significant effect on CT growth. However, in the absence of other metal ions, there was a higher concentration of cell growth and xylitol production when only Zn2+ was present in the medium. The analysis of various vitamins unveiled that biotin and thiamine were the only vitamins required for the growth of CT. Surprisingly, when only biotin was present in the medium as a vitamin, there was less growth of CT than when the medium was complete, but the amount of xylitol released was significantly higher. Overall, this study will increase the xylitol production using the single omission or addtion technique.

Tropical Indo-Pacific SST influences on vegetation variability in eastern Africa.

Mechanisms by which tropical Pacific and Indian Ocean sea surface temperatures (SST) influence vegetation in eastern Africa have not been fully explored. Here, we use a suite of idealized Earth system model simulations to elucidate the governing processes for eastern African interannual vegetation changes. Our analysis focuses on Tanzania. In the absence of ENSO-induced sea surface temperature anomalies in the Tropical Indian Ocean (TIO), El Niño causes during its peak phase negative precipitation anomalies over Tanzania due to a weakening of the tropical-wide Walker circulation and anomalous descending motion over the Indian Ocean and southeastern Africa. Resulting drought conditions increase the occurrence of wildfires, which leads to a marked decrease in vegetation cover. Subsequent wetter La Niña conditions in boreal winter reverse the phase in vegetation anomalies, causing a gradual 1-year-long recovery phase. The 2-year-long vegetation decline in Tanzania during an ENSO cycle can be explained as a double-integration of the local rainfall anomalies, which originate from the seasonally-modulated ENSO Pacific-SST forcing (Combination mode). In the presence of interannual TIO SST forcing, the southeast African precipitation and vegetation responses to ENSO are muted due to Indian Ocean warming and the resulting anomalous upward motion in the atmosphere.

Rhus vernicifera Laccase Immobilization on Magnetic Nanoparticles to Improve Stability and Its Potential Application in Bisphenol A Degradation.

In the present study, Rhus vernicifera laccase (RvLac) was immobilized through covalent methods on the magnetic nanoparticles. Fe2O3 and Fe3O4 nanoparticles activated by 3-aminopropyltriethoxysilane followed with glutaraldehyde showed maximum immobilization yields and relative activity up to 81.4 and 84.3% at optimum incubation and pH of 18 h and 5.8, respectively. The maximum RvLac loading of 156 mg/g of support was recorded on Fe2O3 nanoparticles. A higher optimum pH and temperature of 4.0 and 45 °C were noted for immobilized enzyme compared to values of 3.5 and 40 °C for free form, respectively. Immobilized RvLac exhibited better relative activity profiles at various pH and temperature ranges. The immobilized enzyme showed up to 16-fold improvement in the thermal stability, when incubated at 60 °C, and retained up to 82.9% of residual activity after ten cycles of reuses. Immobilized RvLac exhibited up to 1.9-fold higher bisphenol A degradation efficiency potential over free enzyme. Previous reports have demonstrated the immobilization of RvLac on non-magnetic supports. This study has demonstrated that immobilization of RvLac on magnetic nanoparticles is very efficient especially for achieving high loading, better pH and temperature profiles, and thermal- and solvents-stability, high reusability, and higher degradation of bisphenol A.

Characterization of Cellobiohydrolases from Schizophyllum commune KMJ820.

A novel cellobiohydrolase (CBH)-generating fungi have been isolated and categorized as Schizophyllum commune KMJ820 based on morphology and rDNA gene sequence. Cellulose powder was used as carbon source, the total enzyme activity was 11.51 U/ml is noted; which is among the highest amounts of CBH-generating microbes studied. CBH have been purified to homogenize, with pursual of serial chromatography using S. commune supernatants and two different CBHs were found; CBH 1 and 2. The filtered CBHs showed greater activity (V max = 51.4 and 20.8 U/mg) in contrast to CBHs from earlier studies. The MW (molecular weights) of S. commune CBH 1 and 2 were verified to be approximately 50 kDa and 150 kDa, respectively, by size exclusion chromatography. Even though CBHs have been evaluated from other sources, but S. commune CBH is prominent in comparison to other CBHs by its high enzyme activity.

Tyr320 is a molecular determinant of the catalytic activity of ß-glucosidase from Neosartorya fischeri.

ß-Glucosidases (BGL) are key members of the cellulase enzyme complex that determine efficiency of lignocellulosic biomass degradation, which have shown great functional importance to many biotechnological systems. A previous reported BGL from Neosartorya fischeri (NfBGL) showed much higher activity than other BGLs. Screening the important residues based on sequence alignment, analyzing a homology model, and subsequent alteration of individually screened residues by site-directed mutagenesis were carried out to investigate the molecular determinants of the enzyme's high catalytic efficiency. Tyr320, located in the wild-type NfBGL substrate-binding pocket was identified as crucial to the catalytic function of NfBGL. The replacement of Tyr320 with aromatic amino acids did not significantly alter the catalytic efficiency towards p-nitrophenyl ß-d-glucopyranoside (pNPG). However, mutants with charged and hydrophilic amino acids showed almost no activity towards pNPG. Computational studies suggested that an aromatic acid is required at position 320 in NfBGL to stabilize the enzyme-substrate complex formation. This knowledge on the mechanism of action of the molecular determinants can also help rational protein engineering of BGLs.

Inhibition of Microbial Quorum Sensing Mediated Virulence Factors by Pestalotiopsis sydowiana.

Quorum sensing (QS)-mediated infections cause severe diseases in human beings. The control of infectious diseases by inhibiting QS using antipathogenic drugs is a promising approach as antibiotics are proving inefficient in treating these diseases. Marine fungal (Pestalotiopsis sydowiana PPR) extract was found to possess effective antipathogenic characteristics. The minimum inhibitory concentration (MIC) of the fungal extract against test pathogen Pseudomonas aeruginosa PAO1 was 1,000 µg/ml. Sub-MIC concentrations (250 and 500 µg/ml) of fungal extract reduced QS-regulated virulence phenotypes such as the production of pyocyanin, chitinase, protease, elastase, and staphylolytic activity in P. aeruginosa PAO1 by 84.15%, 73.15%, 67.37%, 62.37%, and 33.65%, respectively. Moreover, it also reduced the production of exopolysaccharides (74.99%), rhamnolipids (68.01%), and alginate (54.98%), and inhibited the biofilm formation of the bacteria by 90.54%. In silico analysis revealed that the metabolite of P. sydowiana PPR binds to the bacterial QS receptor proteins (LasR and RhlR) similar to their respective natural signaling molecules. Cyclo(-Leu-Pro) (CLP) and 4-Hydroxyphenylacetamide (4-HPA) were identified as potent bioactive compounds among the metabolites of P. sydowiana PPR using in silico approaches. The MIC values of CLP and 4-HPA against P. aeruginosa PAO1 were determined as 250 and 125 µg/ml, respectively. All the antivirulence assays were conducted at sub-MIC concentrations of CLP (125 µg/ml) and 4-HPA (62.5 µg/ml), which resulted in marked reduction in all the investigated virulence factors. This was further supported by gene expression studies. The findings suggest that the metabolites of P. sydowiana PPR can be employed as promising QS inhibitors that target pathogenic bacteria.

Bioelectrochemical Detoxification of Phenolic Compounds during Enzymatic Pre-Treatment of Rice Straw.

The use of lignocellulosic biomass such as rice straw can help subsidize the cost of producing value-added chemicals. However, inhibitory compounds, such as phenolics, produced during the pre-treatment of biomass, hamper the saccharification process. Laccase and electrochemical stimuli are both well known to reduce phenolic compounds. Therefore, in this study, we implemented a bioelectrochemical detoxification system (BEDS), a consolidated electrochemical and enzymatic process involving laccase, to enhance the detoxification of phenolics, and thus achieve a higher saccharification efficiency. Saccharification of pretreated rice straw using BEDS at 1.5 V showed 90% phenolic reduction (Phr), thereby resulting in a maximum saccharification yield of 85%. In addition, the specific power consumption when using BEDS (2.2 W/Kg Phr) was noted to be 24% lower than by the electrochemical process alone (2.89 W/kg Phr). To the best of our knowledge, this is the first study to implement BEDS for reduction of phenolic compounds in pretreated biomass.

Antimicrobial Activity of Biosynthesized Silver Nanoparticles Decorated Silica Nanoparticles.

The production of cheap and effective compound for medicinal application is a major challenge for scientific community. So, several biological materials have been explored for the possible application in material synthesis which are useful in biomedical uses. Here, biomolecules from green tea leaves were functionalized on the surface of silicon dioxide nanoparticles (GSiO2 NPs). Next, the decoration silver (Ag) NPs on the surface of the GSiO2 NPs was observed in very short time of incubation in aqueous AgNO3. Ultraviolet-visible spectroscopy confirmed the formation of Ag NPs and the high-resolution transmission and scanning electron microscopies confirmed the decoration of spherical Ag NPs of 10 to 15 nm size on the surface of GSiO2 NPs. The antimicrobial activity of the Ag-GSiO2 NPs was determined against Staphylococcus aureus and Escherichia coli. The Ag-GSiO2 NPs displayed sustainable antimicrobial activity compared to Ag ions. The results indicate the potential value of Ag-GSiO2 NPs in surgical material and food processing.

Author Correction: Overcoming NADPH product inhibition improves D-sorbitol conversion to L-sorbose.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cloning, Expression and Characterization of a Highly Active Alcohol Dehydrogenase for Production of Ethyl (S)-4-Chloro-3-Hydroxybutyrate.

A novel alcohol dehydrogenase from Bartonella apis (BaADH) was heterologous expressed in Escherichia coli. Its biochemical properties were investigated and used to catalyze the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), which is a chiral intermediate of the cholesterol-lowering drug atorvastatin. The purified recombinant BaADH displayed 182.4 U/mg of the specific activity using ethyl 4-chloroacetoacetate as substrate under the conditions of 50 °C in pH 7.0 Tris-HCl buffer. It was stable in storage buffers of pH 7 to 9 and retains up to 96.7% of the initial activity after 24 h. The K m and V max values of BaADH were 0.11 mM and 190.4 µmol min-1 mg-1, respectively. Synthesis of (S)-CHBE catalyzed by BaADH was performed with a cofactor regeneration system using a glucose dehydrogenase, and a conversion of 94.9% can be achieved after 1 h reaction. Homology modeling and substrate docking revealed that a typical catalytic triad is in contact with local water molecules to form a catalytic system. The results indicated this ADH could contribute to the further enzymatic synthesis of (S)-CHBE.

Bioelectrogenesis from Raw Algal Biomass Through Microbial Fuel Cells: Effect of Acetate as Co-substrate.

Algae are autotrophic organisms that are widespread in water bodies. Increased pollution in water bodies leads to eutrophication. However, algae growing in lakes undergoing eutrophication could be utilized towards the generation of added-value bio-electricity using microbial fuel cells (MFCs). In the present study, two methods of electricity generation using raw algae (RA) and RA + acetate (AC) as co-substrate were analyzed in single chamber air cathode MFCs. MFCs supplemented with RA and RA + AC clearly showed higher power density, greater current generation, and improved COD (chemical oxygen demand) removal, which demonstrated the feasibility of using AC as substrate for MFC. The MFC-RA + AC (0.48 mA) generated 28% higher current relative to that generated by MFC with RA alone. Notably, the maximum power densities generated by MFC-RA and MFC-RA + AC were 230 and 410 mW/m2, respectively. MFC-RA and MFC-RA + AC exhibited TCOD (total chemical oxygen reduction) removal values of 77% and 86.6%, respectively. Despite the high influent TCOD (758 mg/l) concentration, the MFC-RA + AC exhibited an 8.5% higher COD removal relative to that of MFC-RA (525 mg/l). Our current findings demonstrated effective energy generation using algae biomass with a co-substrate.

Copper Ferrite Magnetic Nanoparticles for the Immobilization of Enzyme.

In this study, novel, hollow superparamagnetic copper ferrite (CuFe2O4) nanoparticles (NPs) were synthesized by a low-temperature hydrothermal method. The hollow magnetic spheres were characterized by field emission scanning electron microscopy and high resolution transmission electron microscopy to confirm their morphology and size. The hollow NPs were demonstrated as the support for biological materials by the immobilization of Thermomyces lanuginosus lipase on the inner and outer surfaces of the hollow spheres. The immobilization of the enzyme was confirmed by Fourier Transform Infra-red spectroscopy and confocal laser scanning microscopy. The immobilized enzyme was shown to have an immobilization efficiency of 84.5%, with approximately 176 mg g-1 of enzyme loading, for the hollow-NPs support. The immobilized enzyme exhibited high storage and temperature stability. The reusability of the immobilized lipase was more than 80% after 10 cycles of repeated use.