Applied Biochemistry and Biotechnology (v.185, #3)

Cre/loxP-Mediated Multicopy Integration of the Mevalonate Operon into the Genome of Methylobacterium extorquens AM1 by Wei-Fan Liang; Ming-Yang Sun; Lan-Yu Cui; Chong Zhang; Xin-Hui Xing (565-577).
Methylobacterium extorquens AM1 is the model strain for methylotrophic bacteria that metabolize methanol as the sole carbon and energy source. Genetically modified M. extorquens AM1 is used as a methylotrophic cell factory (MeCF) for high value-added chemical production. We tested the Cre-loxP recombination system for its ability to mediate multicopy gene integration of the mvt3 operon (mvt3) in M. extorquens AM1. mvt3 controls the expression of the first three enzymes of the mevalonate synthesis pathway. We assayed for Cre-mediated multigene integration by screening for multicopy mutants via their survival in culture with a high kanamycin concentration (600 μg/mL). We identified mutant strains in which the mevalonate titer was increased by up to 1.9-fold compared with M2 (M. extorquens AM1ΔcelABCΔattTn7::mvt3::loxP) and confirmed mvt3 integration at 2–3 copies per genome. This result demonstrates the feasibility of multicopy integration in M. extorquens AM1 mediated by Cre-loxP recombination and its potential for improving the output of M. extorquens AM1 metabolic pathways, e.g., optimization of terpenoid synthesis.
Keywords: Methylobacterium extorquens AM1; Cre-loxP; Multicopy; mvt3 operon; Mevalonate

The original version of this article unfortunately contained a mistake in Eq. 3.Optically pure 3-substituted glutarates can be prepared from the alcoholic ring-opening of cyclic anhydride derivatives, esterification of 3-substituted glutaric acid, and hydrolysis, alcoholysis, aminolysis, and ammonolysis of the diester derivatives via hydrolases or organocatalysts. Unfortunately, most of them mainly focus on the first-step desymmetrization, leading to the difficulty on producing optically pure enantiomers. As a general trend in lipase-catalyzed desymmetrization of 3-methylglutarates, poorer enantiomeric excesses with lower chemical yields were found, as the methyl substituent is relatively small to induce a high enzyme stereodiscrimination. The two-step desymmetrization for CALB-catalyzed alcoholysis of 3-methylglutaric di-1,2,4-triazolide 1a in anhydrous MTBE is first developed to increase the enzyme activity in each reaction step. The enantioselectivity for the second-step kinetic resolution is furthermore improved by using 3-methylglutaric dipyrazolide 1b as the substrate. The kinetic and thermodynamic analysis is, moreover, addressed for shedding insights into the desymmetrization process.
Keywords: Two-step desymmetrization; Kinetic and thermodynamic analysis; CALB; 3-Methylglutaric diazolide

The original version of this article unfortunately contained a mistake in Eq. 3.

Medium-chain fatty acids (MCFA) are saturated monocarboxylic acids and can be used as antimicrobials, corrosion inhibitors, precursors in biodiesel, and bioplastic production. In the present study, MCFA production was evaluated with acetate and ethanol using the bacteria Clostridium kluyveri. Effects of substrate, electron donor, and methane inhibitor on MCFA production were evaluated. Bacteria successfully converted the ethanol and acetate to butyrate (C4), caproate (C6), and caprylate (C8) by chain elongation process. The highest concentrations of butyrate (4.6 g/l), caproate (3.2 g/l), and caprylate (0.5 g/l) were obtained under methane inhibition conditions than other conditions. The productions of butyrate and caproate were 1.6 and 1.48 times higher under methane inhibition conditions, respectively. Results denoted that the bacteria C. kluyveri can be used for conversion of acetate and ethanol into useful products like butyrate and caproate.
Keywords: Clostridium kluyveri ; Medium-chain fatty acids (MCFA); Butyrate; Antimicrobials; Caproate

Immobilization of Lipase by Ionic Liquid-Modified Mesoporous SiO2 Adsorption and Calcium Alginate-Embedding Method by Bin Zou; Yanhong Chu; Jiaojiao Xia; Xueshan Chen; Shuhao Huo (606-618).
Porcine pancreatic lipase (PPL) was immobilized onto functionalized ionic liquid-modified silica carrier using gelatinization and physical adsorption. The immobilized lipase was characterized with N2 adsorption–desorption, X-ray diffraction (XRD), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR) before and after modification and immobilization. The results showed that the modification of the ionic liquid and the introduction of lipase had been successfully approved. The rate of enzymatic reaction and its influencing factors was primarily studied by enzymatic reaction kinetics. K m values of PPL-SiO2@CA and PPL-IM/BF4-SiO2@CA were 4.9 and 3.7 mg/ml, respectively. It indicated that the modification of the functionalized ionic liquid enhanced the affinity between the immobilized enzyme and the substrate. The immobilization efficiency, specific activity, optimum temperature, optimum pH, thermal stability, reusability, and storage stability of the immobilized enzyme were investigated. We found that the stability of the immobilized enzyme was significantly higher than that of the unmodified immobilized enzyme. Specially, PPL-IM/BF4-SiO2@CA maintained good thermal stability and retained more than 92% of its activity at 65 °C after preheating 3 h. Graphical AbstractAbove, the immobilized lipase maintained more than 92% of its initial activity after incubating at 65 °C for 3 h.
Keywords: Immobilization; Mesoporous; Ionic liquid; Embedding; Lipase

The Purification and Characterization of Lipases from Lasiodiplodia theobromae, and Their Immobilization and Use for Biodiesel Production from Coconut Oil by Balaji Venkatesagowda; Ebenezer Ponugupaty; Aneli M. Barbosa-Dekker; Robert F. H. Dekker (619-640).
The coconut kernel-associated fungus, Lasiodiplodia theobromae VBE1, was grown on coconut cake with added coconut oil as lipase inducer under solid-state fermentation conditions. The extracellular-produced lipases were purified and resulted in two enzymes: lipase A (68,000 Da)—purified 25.41-fold, recovery of 47.1%—and lipase B (32,000 Da)—purified 18.47-fold, recovery of 8.2%. Both lipases showed optimal activity at pH 8.0 and 35 °C, were activated by Ca2+, exhibited highest specificity towards coconut oil and p-nitrophenyl palmitate, and were stable in iso-octane and hexane. Ethanol supported higher lipase activity than methanol, and n-butanol inactivated both lipases. Crude lipase immobilized by entrapment within 4% (w/v) calcium alginate beads was more stable than the crude-free lipase preparation within the range pH 2.5–10.0 and 20–80 °C. The immobilized lipase preparation was used to catalyze the transesterification/methanolysis of coconut oil to biodiesel (fatty acyl methyl esters (FAMEs)) and was quantified by gas chromatography. The principal FAMEs were laurate (46.1%), myristate (22.3%), palmitate (9.9%), and oleate (7.2%), with minor amounts of caprylate, caprate, and stearate also present. The FAME profile was comparatively similar to NaOH-mediated transesterified biodiesel from coconut oil, but distinctly different to petroleum-derived diesel. This study concluded that Lasiodiplodia theobromae VBE1 lipases have potential for biodiesel production from coconut oil.
Keywords: Endophytic plant seed oil-associated fungus; Coconut cake/meal; Solid-state fermentation; Transesterification/methanolysis; Calcium alginate-immobilized lipases; Fatty acyl methyl esters

Endonuclease I is a widely distributed periplasmic or extracellular enzyme. A method for the high-level production of recombinant AsEndI (endonuclease I from Aliivibrio salmonicida) in Escherichia coli with secretion expression is investigated. The coding sequence of AsEndI gene was assembled according to the E. coli codon usage bias, and AsEndI was expressed in the periplasm of E. coli TOP10 with a C-terminal 6× His-tagged fusion. The recombinant AsEndI (His-AsEndI) was purified by Ni-NTA resin with a yield of 1.29 × 107 U from 1-L LB medium. His-AsEndI could be classified into Ca2+/Mg2+-dependent nucleases and showed highest nuclease activity to dsDNA at pH 8.0 and 37 °C. His-AsEndI is highly active in a broad range of salt concentration range up to 1.0 M with optimal NaCl concentration at 0.4 M. His-AsEndI can effectively remove DNA contamination in RNA sample or in PCR reagents to the level that cannot be detected by highly sensitive nested PCR and without adverse effects on the subsequent PCR reaction. His-AsEndI can remove DNA contamination at high salt conditions, especially for the DNA that may be shielded by DNA-binding protein at low salt conditions.
Keywords: Codon optimization; Purification; Nuclease activity; High salt; DNA contamination

UM-164, a potent Src/p38 inhibitor, is a promising lead compound for developing the first targeted therapeutic strategy against triple-negative breast cancer (TNBC). However, lack of understanding of conformational features of UM-164 in complex with Src serves a challenge in the rational design of novel Src dual inhibitors. Herein, we provide an in-depth insight into conformational features of Src-UM-164 using different computational approaches. This involved molecular dynamics (MD) simulation, principal component analysis (PCA), thermodynamics calculations, dynamic cross-correlation (DCCM) analysis, and hydrogen bond formation. Findings from this study revealed that (1) the binding of UM-164 to Src induces a more stable and compact conformation; (2) the binding of UM-164 results in increased correlation among the active site residue; (3) the presence of multiple phenyl rings and fluorinated phenyl group in UM-164 contributes to the steric effect; (4) a relatively high-binding free energy estimated for the Src-UM-164 system is affirmative of its experimental potency; (5) hydrophobic packing contributes significantly to the drug binding in Src-UM-164; and (6) observed increase in H-bond distance of interacting residue atoms and Dasatinib compared to UM-164. Findings from this study can serve as a baseline in the design of novel Src inhibitors with dual inhibitory properties.
Keywords: Src; Molecular dynamics; TNBC; UM-164; Dual kinase inhibitor and DFG-out

The alpha-pinene oxide lyase (Prα-POL) from Pseudomonas rhodesiae CIP107491 belongs to catabolic alpha-pinene degradation pathway. In this study, the gene encoding Prα-POL has been identified using mapping approach combined to inverse PCR (iPCR) strategy. The Prα-POL gene included a 609-bp open reading frame encoding 202 amino acids and giving rise to a 23.7 kDa protein, with a theoretical isoelectric point (pI) of 5.23. The amino acids sequence analysis showed homologies with those of proteins with unknown function from GammaProteobacteria group. Identification of a conserved domain in amino acid in positions 18 to 190 permitted to classify Prα-POL among the nuclear transport factor 2 (NTF2) protein superfamily. Heterologous expression of Prα-POL, both under its native form and with a histidin tag, was successfully performed in Escherichia coli, and enzymatic kinetics were analyzed. Bioconversion assay using recombinant E. coli strain allowed to reach a rate of isonovalal production per gramme of biomass about 40-fold higher than the rate obtained with P. rhodesiae.
Keywords: Alpha-pinene oxide lyase; Isonovalal; Bioconversion; Pseudomonas ; Orphan enzyme

Considering the importance of methylotrophs in industrial wastewater treatment, focus of the present study was on utilization of a methylotrophic bacterial consortium as a microbial seed for biotreatment of a variety of industrial effluents. For this purpose, a mixed bacterial methylotrophic AC (Ankleshwar CETP) consortium comprising of Bordetella petrii AC1, Bacillus licheniformis AC4, Salmonella subterranea AC5, and Pseudomonas stutzeri AC8 was used. The AC consortium showed efficient biotreatment of four industrial effluents procured from fertilizer, chemical and pesticide industries, and common effluent treatment plant by lowering their chemical oxygen demand (COD) of 950–2000 mg/l to below detection limit in 60–96 h in 6-l batch reactor and 9–15 days in 6-l continuous reactor. The operating variables of wastewater treatment, viz. COD, BOD, pH, MLSS, MLVSS, SVI, and F/M ratio of these effluents, were also maintained in the permissible range in both batch and continuous reactors. Therefore, formation of the AC consortium has led to the development of an efficient microbial seed capable of treating a variety of industrial effluents containing pollutants generated from their respective industries.
Keywords: Chemical oxygen demand; Industrial effluents; Methylotrophs

Production of FAME and FAEE via Alcoholysis of Sunflower Oil by Eversa Lipases Immobilized on Hydrophobic Supports by Daniela Remonatto; J. Vladimir de Oliveira; J. Manuel Guisan; Débora de Oliveira; Jorge Ninow; Gloria Fernandez-Lorente (705-716).
The performance of two new commercial low-cost lipases Eversa® Transform and Eversa® Transform 2.0 immobilized in different supports was investigated. The two lipases were adsorbed on four different hydrophobic supports. Interesting results were obtained for both lipases and for the four supports. However, the most active derivative was prepared by immobilization of Eversa® Transform 2.0 on Sepabeads C-18. Ninety-nine percent of fatty acid ethyl ester was obtained, in 3 h at 40 °C, by using hexane as solvent, a molar ratio of 4:1 (ethanol/oil), and 10 wt% of immobilized biocatalyst. The final reaction mixture contained traces of monoacylglycerols but was completely free of diacylglycerols. After four reaction cycles, the immobilized biocatalyst preserved 75% of activity. Both lipases immobilized in Sepabeads C-18 were very active with ethanol and methanol as acceptors, but they were much more stable in the presence of ethanol.
Keywords: Eversa® Transform; Eversa® Transform 2.0; Transesterification; Fatty acid ethyl ester; Fatty acid methyl ester

Immobilization of Recombinant Human Catalase on Gold and Silver Nanoparticles by Agnieszka Małgorzata Pudlarz; Ewa Czechowska; Katarzyna Ranoszek-Soliwoda; Emilia Tomaszewska; Grzegorz Celichowski; Jarosław Grobelny; Janusz Szemraj (717-735).
Human catalase cDNA was cloned into a pEX-C-His vector. Purified recombinant catalase was immobilized on nanoparticles. Gold and silver nanoparticles were synthesized in a variety of sizes by chemical reduction; no agglomerates or aggregates were observed in any of the colloids during dynamic light scattering or scanning transmission electron microscopy analysis. After immobilization on gold nanoparticles, recombinant catalase activity was found to be lower than that of the same amount of enzyme in aqueous solution. However, after 10 days of storage at room temperature, the activity of catalase immobilized on gold nanoparticles (AuNPs) of 13 and 20 nm and coverage of 133% was 68 and 83% greater than catalase in aqueous solution, respectively. During 10 days of experiment, percentage activity of catalase immobilized on those gold nanoparticles was higher in comparison to CAT in aqueous solution. Catalase immobilized on silver nanoparticles did not lose activity as significantly as catalase immobilized on AuNPs. Those results confirm the ability to produce recombinant human enzymes in a bacterial expression system and its potential use while immobilized on silver or gold nanoparticles.
Keywords: Catalase; Gold nanoparticles; Silver nanoparticles; Immobilization; Expression

Tyrosinases catalyze oxidation of phenols with a formation of biphenols, quinones, and highly polymerized melanins. Tyrosinases have prospects for industrial use to remove phenols, also in biosensors, in bioorganic synthesis, and for a production of biocompatible adhesives (medical glues). Despite growing fields of potential applications, a selection of commercially available tyrosinases are currently limited to a single enzyme which is isolated from fruiting bodies of mushrooms. This article describes a preparation of recombinant tyrosinase from a bacterium Verrucomicrobium spinosum using a heterologous expression in Escherichia coli. Recombinant V. spinosum tyrosinase has high specific activity (13,200 U/mg). A resistance of the enzyme was investigated to chemical agents used to denature proteins and keep poorly solvable proteins in a solution. The enzyme preserves activity in the presence of urea and retains at least a fraction of its enzymatic activity at concentrations of urea up to 4.5 M. An addition of sodium lauroyl sarcosinate to 1 or 2% activates the tyrosinase. Novel means of quantitatively expressing tyrosinase activity is described in this article. The method uses a set of parameters obtained from non-linear estimation of the progress curves and is suitable for enzymatic reactions which do not comply with Michaelis–Menten kinetics. Tyrosinase may be used to introduce into proteins a post-translational modification which is a conversion of tyrosine residues (Tyr) into residues of 3,4-dioxyphenylalanine (DOPA). The presence of DOPA provides the polypeptides with a capability of strong molecular adhesion. Co-expression of tyrosinase and a recombinant protein mimicking marine mussel-encoded adhesive proteins resulted in obtaining of the protein in which at least a part of Tyr residues had been converted to DOPA. The DOPA-containing protein had high adhesion strength (2.5 MPa).
Keywords: Tyrosinase; Verrucomicrobium ; Denaturing agents; Activity; DOPA; Adhesive protein

Phylogenetic Analysis and Biological Evaluation of Marine Endophytic Fungi Derived from Red Sea Sponge Hyrtios erectus by Mervat Morsy Abbas Ahmed El-Gendy; Shaymaa M. M. Yahya; Ahmed R. Hamed; Maha M. Soltan; Ahmed Mohamed Ahmed El-Bondkly (755-777).
Forty-four endophytic fungal isolates obtained from marine sponge, Hyrtios erectus, were evaluated and screened for their hydrolase activities. Most of the isolates were found to be prolific producers of hydrolytic enzymes. Only 11 isolates exhibited maximum cellular contents of lipids, rhamnolipids, and protein in the fungal isolates under the isolation numbers MERVA5, MERVA22, MERVA25, MERVA29, MERVA32, MERVA34, MERV36, MERVA39, MERVA42, MERVA43, and MERVA44. These isolate extracts exhibit the highest reducing activities against carbohydrate-metabolizing enzymes including α-amylase, α-glucosidase, β-glucosidase, β-glucuronidase, and tyrosinase. Consequently, based on morphological and cultural criteria, as well as sequence information and phylogenetic analysis, these isolates could be identified and designated as Penicillium brevicombactum MERVA5, Arthrinium arundinis MERVA22, Diaporthe rudis MERVA25, Aspergillus versicolor MERVA29, Auxarthron alboluteum MERVA32, Dothiorella sarmentorum MERVA34, Lophiostoma sp. MERVA36, Fusarium oxysporum MERVA39, Penicillium chrysogenum MERVA42, Penicillium polonicum MERVA43, and Trichoderma harzianum MERVA44. The endophytic fungal species, D. rudis MERVA25, P. polonicum MERVA43, Lophiostoma sp. MERVA36, A. alboluteum MERVA32, T. harzianum MERVA44, F. oxysporum MERVA39, A. versicolor MERVA29, and P. chrysogenum MERVA42 extracts, showed significant hepatitis C virus (HCV) inhibition. Moreover, D. sarmentorum MERVA34, P. polonicum MERVA43, and T. harzianum MERVA44 extracts have the highest antitumor activity against human hepatocellular carcinoma cells (HepG2).
Keywords: Marine endophytic fungi; Phylogenetic analysis; Hydrolyse enzymes; Antimicrobial; Anti-HCV; Antiproliferative and cell lines

Decoding Selection Bias Imparted by Unpaired Cysteines: a Tug of War Between Expression and Affinity by B. Vijayalakshmi Ayyar; Stephen Hearty; Richard O’Kennedy (778-785).
In a recombinant antibody scFv format, the presence of an unpaired cysteine (Cys) is implicated in reduced soluble expression and inefficient presentation in phage display. Compared to other species, antibodies derived from rabbits are more likely to contain this unpaired Cys residue at position 80 (Cys80), when generated in a scFv format. In a screening campaign to isolate rabbit scFv against cardiac troponin I (cTnI), it was found that, a large proportion of isolated cTnI-specific clones contained unpaired Cys80. To analyze the factors that led to the selection of anti-cTnI Cys80 scFv, after five rounds of biopanning, the biopanning experiments were repeated with a Cys80 scFv (MG4Cys), its alanine variant (MG4Ala), and an irrelevant high expressing scFv control. It was found that the selection and subsequent enrichment of MG4Cys scFv was ousted by the superior expressing variant MG4Ala, indicating that the Cys80 scFv was selected primarily due to its affinity. It is evident that phage-based selection is influenced by specific sequence characteristics affecting the expression as well as the binding specificity and this needs to be taken into account for selection of optimal antibody derivatives.
Keywords: Rabbit antibodies; scFv; Unpaired cysteine; Phage display; Biopanning; Expression bias

Inhibition of Human Immunodeficiency Type 1 Virus (HIV-1) Life Cycle by Different Egg White Lysozymes by Mandana Behbahani; Mokhtar Nosrati; Hassan Mohabatkar (786-798).
Lysozyme is a relatively small enzyme with different biological activities, which is found in tears, saliva, egg white, and human milk. In the study, the anti-HIV-1 activity of lysozymes purified from quail, Meleagris, and hen egg white has been determined. For this end, a time-of-drug-addition assay was performed to identify the target of anti-HIV-1 agents and for determination of probable anti HIV-1 mechanism of the studied lysozyme, the binding affinity of the lysozymes to the human CD4 receptor was studied by molecular docking method. To define structural differences between studied lysozymes, structural motifs of them were predicted by MEME tool. Quail, hen, and Meleagris lysozymes showed potent anti-HIV-1 activity with EC50 of 7.5, 10, and 55 nM, respectively. The time-of-drug-addition study demonstrated that the inhibitory effect of all purified lysozymes is before HIV-1 infection. The frequency and intensity of CD4 expression in PBMCs decreased in the presence of all mentioned lysozymes. Also, the expression level of C-C chemokine receptor type 5 (CCR5) and chemokine receptor type 4 (CXCR4) on CD4+ T cells was not changed in cells treated with these lysozymes. The results of in silico study confirmed that the binding energy of quail lysozyme with CD4 was more than that of other studied lysozymes. The results revealed that these lysozymes restrict HIV-1 attachment to host cell CD4.
Keywords: HIV-1; Antiviral activity; Lysozymes; Molecular docking

A precipitous increase in the number of flaviviral infections has been noted over the last 5 years. Despite these outbreaks, treatment protocols for infected individuals remain ambiguous. Numerous studies have identified NITD008 as a potent flavivirus inhibitor; however, clinical testing was dismissed due to undesirable toxic effects. The binding landscape of NITD008 in complex with five detrimental flaviviruses at the RNA-dependent RNA polymerase active sites was explored. An “all-in-one” pharmacophore model was created for the design of small molecules that may inhibit a broad spectrum of flaviviruses. This pharmacophore model approach serves as a robust cornerstone, thus assisting medicinal experts in the composition of multifunctional inhibitors that will eliminate cross-resistance and toxicity and enhance patient adherence.
Keywords: Flaviviridae ; Pharmacophore model; Free-binding energy; Binding landscape; NITD008; RdRp

Mycobacterium tuberculosis, the causative agent of tuberculosis is now causing death of more than 10 million people. Because of the development of drug-resistant TB, drug delivery to the infected site through nanoparticle had been studied for long time. Nanoparticles indicate different sorts of association with the natural particles of the body. Nanoparticles can be used as controlled or specific drug delivery system. It can be through temporal controlled or can be distribution controlled. Glucose polymer-based nanoparticles might play an important role as drug delivery system in case of targeted drug delivery in the infected site of the body or in infected macrophages, as they are biodegradable so there should not be any side effects of these particles in the body and also they show very slow immune response. CD4, Beta 1, TGFb-1, IL-2, IL-13 SEC14L1, GUSB, BPI, and CCR7 are major biomarkers secreted after infection of this bacterium by the macrophages which can be used for targeted drug delivery in infected macrophages. As these markers can be used for delivery of drugs at destined position, they can be very beneficial in reducing toxicities of antituberculer drugs to the other uninfected sites and in operating only the infected macrophages.
Keywords: Mycobacterium tuberculosis ; Alveolar macrophages; Minimal inhibitory concentration; Nanoparticles; Phagolysosome

Polyhydroxybutyrate (PHB) Synthesis by Spirulina sp. LEB 18 Using Biopolymer Extraction Waste by Cleber Klasener da Silva; Jorge Alberto Vieira Costa; Michele Greque de Morais (822-833).
The reuse of waste as well as the production of biodegradable compounds has for years been the object of studies and of global interest as a way to reduce the environmental impact generated by unsustainable exploratory processes. The conversion of linear processes into cyclical processes has environmental and economic advantages, reducing waste deposition and reducing costs. The objective of this work was to use biopolymer extraction waste in the cultivation of Spirulina sp. LEB 18, for the cyclic process of polyhydroxybutyrate (PHB) synthesis. Concentrations of 10, 15, 20, 25, and 30% (v/v) of biopolymer extraction waste were tested. For comparison, two assays were used without addition of waste, Zarrouk (SZ) and modified Zarrouk (ZM), with reduction of nitrogen. The assays were carried out in triplicate and evaluated for the production of microalgal biomass and PHB. The tests with addition of waste presented a biomass production statistically equal to ZM (0.79 g L−1) (p < 0.1). The production of PHB in the assay containing 25% of waste was higher when compared to the other cultivations, obtaining 10.6% (w/w) of biopolymer. From the results obtained, it is affirmed that the use of PHB extraction waste in the microalgal cultivation, aiming at the synthesis of biopolymers, can occur in a cyclic process, reducing process costs and the deposition of waste, thus favoring the preservation of the environment.
Keywords: Cyanobacteria; Cyclic process; Polyhydroxybutyrate (PHB); Waste; Spirulina

A novel sandwich-structured nanocomposite based on Ti2NbO7 nanosheets and cobalt porphyrin (CoTMPyP) was fabricated through electrostatic interaction, in which CoTMPyP has been successfully inserted into the lamellar spacing of layered titanoniobate. The resultant Ti2NbO7/CoTMPyP nanocomposite was characterized by XRD, SEM, TEM, EDS, FT-IR, and UV-vis. It is demonstrated that the intercalated CoTMPyP molecules were found to be tilted approximately 63° against Ti2NbO7 layers. The glass carbon electrode (GCE) modified by Ti2NbO7/CoTMPyP film showed a fine diffusion-controlled electrochemical redox process. Furthermore, the Ti2NbO7/CoTMPyP-modified electrode exhibited excellent electrocatalytic oxidation activity of ascorbic acid (AA). Differential pulse voltammetric studies demonstrated that the intercalated nanocomposite detects AA linearly over a concentration range of 4.99 × 10−5 to 9.95 × 10−4 mol L−1 with a detection limit of 3.1 × 10−5 mol L−1 at a signal-to-noise ratio of 3.0.
Keywords: Intercalation; Electrostatic self-assembly; Layered titanoniobate; Cobalt porphyrin; Electrocatalysis

A Comparative Study on Immobilization of Fructosyltransferase in Biodegradable Polymers by Electrospinning by Jakub Gabrielczyk; Thilo Duensing; Stefanie Buchholz; Alexander Schwinges; Hans-Joachim Jördening (847-862).
Commercial application of biocatalysts depends on the efficiency of the immobilization method and residual enzyme activity. Electrospinning offers a simple and versatile route to immobilize enzymes in submicron-sized fibers and thus improved mass transfer characteristics. Performance of encapsulation of fructosyltransferase from Bacillus subtilis by emulsion, suspension, and coaxial electrospinning was compared. We particularly focused on the effect of hydrophilic properties of a set of biodegradable polymers on support’s activity. Bioactivity of electrospun support in aqueous medium increased in order of the matrix hydrophilicity. Additionally, the efficiency of electrospun fibers was compared with Sepabeads®, commercial epoxy-activated resins. In fibers, enzyme loading of 68.1 mg/g and specific enzyme activity of 5.5 U/mg was achieved compared to 49.5 mg/g and 2.2 U/mg on Sepabeads. Fructosyltransferase exhibited high sensitivity towards organic solvents and covalent attachment, respectively. Immobilization of native enzyme in coaxial fibers increased the specific activity to approx. 30 U/mg which corresponds to 24% of that of the free enzyme. Finally, operational stability of fiber supports was examined in a plug-flow reactor and 5% of initial substrate conversion remained after > 2000 cycles. The efficiency of core-shell immobilizates compared to one-dimensional fibers was both in batch and continuous reaction at least 4.4-fold higher.
Keywords: Fructosyltransferase; Electrospinning; Coaxial electrospinning; Sepabeads; Enzyme immobilization; Plug-flow reactor; Biodegradable polymers