Enzymatic synthesis of chiral amino‐alcohols by coupling transketolase and transaminase‐catalyzed reactions in a cascading continuous‐flow microreactor system

Rapid biocatalytic process development and intensification continues to be challenging with currently available methods. Chiral amino‐alcohols are of particular interest as they represent key industrial synthons for the production of complex molecules and optically pure pharmaceuticals. (2S,3R)‐2‐amino‐1,3,4‐butanetriol (ABT), a building block for the synthesis of protease inhibitors and detoxifying agents, can be synthesized from simple, non‐chiral starting materials, by coupling a transketolase‐ and a transaminase‐catalyzed reaction. However, until today, full conversion has not been shown and, typically, long reaction times are reported, making process modifications and improvement challenging. In this contribution, we present a novel microreactor‐based approach based on free enzymes, and we report for the first time full conversion of ABT in a coupled enzyme cascade for both batch and continuous‐flow systems. Using the compartmentalization of the reactions afforded by the microreactor cascade, we overcame inhibitory effects, increased the activity per unit volume, and optimized individual reaction conditions. The transketolase‐catalyzed reaction was completed in under 10 min with a volumetric activity of 3.25 U ml^?1. Following optimization of the transaminase‐catalyzed reaction, a volumetric activity of 10.8 U ml^?1 was attained which led to full conversion of the coupled reaction in 2 hr. The presented approach illustrates how continuous‐flow microreactors can be applied for the design and optimization of biocatalytic processes.

     

Babassu palm (Attalea speciosa Mart.) super-dominance shapes its surroundings via multiple biotic,soil chemical,and physical interactions and accumulates soil carbon: a case study in eastern Amazonia

Plant and Soil - Aggressive ruderal plant species pose an increasing problem in anthropically degraded lands. They both affect and are affected by other vegetation and by soil fertility in their...     

A low molecular weight proteome comparison of fertile and male sterile 8 anthers of Zea mays

During maize anther development, somatic locular cells differentiate to support meiosis in the pollen mother cells. Meiosis is an important event during anther growth and is essential for plant fertility as pollen contains the haploid sperm. A subset of maize male sterile mutants exhibit meiotic failure, including ms8 (male sterile 8) in which meiocytes arrest as dyads and the locular somatic cells exhibit multiple defects. Systematic proteomic profiles were analysed in biological triplicates plus technical triplicates comparing ms8 anthers with fertile sibling samples at both the premeiotic and meiotic stages; proteins from 3.5 to 20 kDa were fractionated by 1‐D PAGE, cleaved with Lys‐C and then sequenced using a LTQ Orbitrap Velos MS paradigm. Three hundred and 59proteins were identified with two or more assigned peptides in which each of those peptides were counted at least two or more times (0.4% peptide false discovery rate (FDR) and 0.2% protein FDR); 2761 proteins were identified with one or more assigned peptides (0.4% peptide FDR and 7.6% protein FDR). Stage‐specific protein expression provides candidate stage markers for early anther development, and proteins specifically expressed in fertile compared to sterile anthers provide important clues about the regulation of meiosis. 49% of the proteins detected by this study are new to an independent whole anther proteome, and many small proteins missed by automated maize genome annotation were validated; these outcomes indicate the value of focusing on low molecular weight proteins. The roles of distinctive expressed proteins and methods for mass spectrometry of low molecular weight proteins are discussed.     

Carvedilol Decrease IL-1β and TNF-α, Inhibits MMP-2, MMP-9, COX-2, and RANKL Expression,and Up-Regulates OPG in a Rat Model of Periodontitis

Periodontal diseases are initiated primarily by Gram-negative, tooth-associated microbial biofilms that elicit a host response that causes osseous and soft tissue destruction. Carvedilol is a β-blocker used as a multifunctional neurohormonal antagonist that has been shown to act not only as an anti-oxidant but also as an anti-inflammatory drug. This study evaluated whether Carvedilol exerted a protective role against ligature-induced periodontitis in a rat model and defined how Carvedilol affected metalloproteinases and RANKL/RANK/OPG expression in the context of bone remodeling. Rats were randomly divided into 5 groups (n = 10/group): (1) non-ligated (NL), (2) ligature-only (LO), and (3) ligature plus Carvedilol (1, 5 or 10 mg/kg daily for 10 days). Periodontal tissue was analyzed for histopathlogy and using immunohistochemical analysis characterized the expression profiles of MMP-2, MMP-9, COX-2, and RANKL/RANK/OPG and determined the presence of IL-1β, IL-10 and TNF-α, myeloperoxidase (MPO), malonaldehyde (MDA) and, glutathione (GSH). MPO activity in the group with periodontal disease was significantly increased compared to the control group (p<0.05). Rats treated with 10 mg/kg Carvedilol presented with significantly reduced MPO and MDA concentrations (p<0.05) in addition to presenting with reduced levels of the pro-inflammatory cytokines IL-1 β and TNF-α (p<0.05). IL-10 levels in Carvedilol-treated rats remained unaltered. Immunohistochemical analysis demonstrated reduced expression of MMP-2, MMP-9, RANK, RANKL, COX-2, and OPG in rats treated with 10 mg/kg Carvedilol. This study demonstrated that Carvedilol affected bone formation/destruction and anti-inflammatory activity in a rat model of periodontitis.     

The Investigation of Protein Diffusion via H-Cell Microfluidics

In this study, we developed a microfluidics method, using a so-called H-cell microfluidics device, for the determination of protein diffusion coefficients at different concentrations, pHs, ionic strengths, and solvent viscosities. Protein transfer takes place in the H-cell channels between two laminarly flowing streams with each containing a different initial protein concentration. The protein diffusion coefficients are calculated based on the measured protein mass transfer, the channel dimensions, and the contact time between the two streams. The diffusion rates of lysozyme, cytochrome c, myoglobin, ovalbumin, bovine serum albumin, and etanercept were investigated. The accuracy of the presented methodology was demonstrated by comparing the measured diffusion coefficients with literature values measured under similar solvent conditions using other techniques. At low pH and ionic strength, the measured lysozyme diffusion coefficient increased with the protein concentration gradient, suggesting stronger and more frequent intermolecular interactions. At comparable concentration gradients, the measured lysozyme diffusion coefficient decreased drastically as a function of increasing ionic strength (from zero onwards) and increasing medium viscosity. Additionally, a particle tracing numerical simulation was performed to achieve a better understanding of the macromolecular displacement in the H-cell microchannels. It was found that particle transfer between the two channels tends to speed up at low ionic strength and high concentration gradient. This confirms the corresponding experimental observation of protein diffusion measured via the H-cell microfluidics.     

Dihydrochalcones, coumarins and alkaloids from Metrodorea nigra

Two novel dihydrochalcones, 2′,3,4′,6′-tetrahydroxy-4-methoxy-3′,5-di-(3,3-dimethylallyl)-dihydrochalcone and 2′,.3,6′-trihydroxy-4-methoxy-5-(3,3-dimethylallyl)-3′,4′-(2″,2″-dimethyldihydropyran)-dihydrochalcone, have been isolated from fresh fruits of Metrodorea nigra. Stems and leaves showed a similar composition and we have isolated common steroids, simple coumarins, several furocoumarins, furoquinoline alkaloids and a furofuran lignan. From stems, we have also isolated the pentacyclic 6-C-monoterpenyl-5,7-dioxycoumarin, deoxybruceol. Structures of the isolated compounds were elucidated on the basis of spectral data.     

Using antagonistic soil bacteria and their cell‐free filtrates to control the black rot pathogen Xanthomonas campestris pv. campestris

Xanthomonas campestris pv. campestris (Xcc) is a phytopathogenic bacteria, and it is the causative agent of black rot in crucifers. Recent studies have shown that Bacillus species have strong biological control on Xanthomonas. One of the mechanisms of this control is secondary metabolites production. A collection of 257 bacteria isolated from a suppressive soil was evaluated for in vitro antagonistic activity against X. campestris, and 92 isolates (44.6%) were able to inhibit its growth. Among the 92 isolates evaluated in the double‐layer technique, 51 (55.43%) inhibited Xcc growth on the inhibition tests with cell‐free filtrates (CFF) in liquid medium. Thirteen of these isolates presented 50% or more growth inhibition, and five isolates presented 100% growth inhibition of Xcc. The CFF of the isolate TCDT‐08, which belongs to the Paenibacillus genus, was used for in vivo tests with kale crops. The artificial inoculation of kale with Xcc‐629IBSBF pretreated with CFF from the isolate TCDT‐08 demonstrated that the bacterium loses the ability of colonizing kale and of causing black rot. A Paenibacillus sp. isolate has strong inhibitory activity against X. campestris pv. campestris, and further studies can result in the use of this isolate to protect kale from Xcc infection.     

Control of the growth of Alicyclobacillus acidoterrestris in industrialized orange juice using rosemary essential oil and nisin

The use of rosemary essential oil (RO) and its combination with nisin (RO+N) in preventing the multiplication of Alicyclobacillus acidoterrestris in orange juice was evaluated. The minimum inhibitory and bactericidal concentrations (MIC and MBC) for RO were both 125 μg ml⁻¹ while RO+N displayed a synergistic effect. The use of RO and RO+N at concentrations of 1, 4 and 8× MIC in orange juice for 96 h was evaluated in terms of their sporicidal effectiveness. With regard to the action against A. acidoterrestris spores, RO at 8× MIC was sporostatic, whereas RO+N at 1× MIC was sporicidal. Morphological changes in the structure of the micro-organism after treatment were also observed by microscopy. Furthermore, flow cytometric analysis showed that most cells were damaged or killed after treatment. In general, the antioxidant activity after addition of RO+N decreased with time. The results demonstrate that using the combination of RO and nisin can prevent the A. acidoterrestris growth in orange juice.