Two novel cyanobacterial α-dioxygenases for the biosynthesis of fatty aldehydes

Abstractα-Dioxygenases (α-DOXs) are known as plant enzymes involved in the α-oxidation of fatty acids through which fatty aldehydes, with a high commercial value as flavor and fragrance compounds, are synthesized as products. Currently, little is known about α-DOXs from non-plant organisms. The phylogenic analysis reported here identified a substantial number of α-DOX enzymes across various taxa. Here, we report the functional characterization and Escherichia coli whole-cell application of two novel α-DOXs identified from cyanobacteria: CalDOX from Calothrix parietina and LepDOX from Leptolyngbya sp. The catalytic behavior of the recombinantly expressed CalDOX and LepDOX revealed that they are heme-dependent like plant α-DOXs but exhibit activities toward medium carbon fatty acids ranging from C10 to C14 unlike plant α-DOXs. The in-depth molecular investigation of cyanobacterial α-DOXs and their application in an E. coli whole system employed in this study is useful not only for the understanding of the molecular function of α-DOXs, but also for their industrial utilization in fatty aldehyde biosynthesis.Key points• Two novel α-dioxygenases from Cyanobacteria are reported• Both enzymes prefer medium-chain fatty acids• Both enzymes are useful for fatty aldehyde biosynthesis Graphical abstract

Plant Therapeutic Proteases: Chemical Aspects, Applications and Pharmaceutical Formulations

Background: Plants are important sources of therapeutic proteases with expressive activity, stability, specificity, and efficiency. These proteases are employed at low concentrations and produce lesser side effects. They have complex tridimensional structures whose maintenance is a challenge, requiring specific conditions to guarantee the biological and pharmacological activities of these compounds. Aims: To conduct a literature review about plant therapeutic proteases, their principal biochemical aspects, potentials and clinical applications, and main pharmaceutical formulations. Materials and Methods: The present study consisted of a bibliographic survey of the major plant therapeutic proteases. An investigation was performed in the PUBMED, SciELO, ScienceDirect and Academic Google databases using the keywords plant enzymes, therapeutic protease, immobilization, formulation. Results: Some plant therapeutic proteases, such as papain and bromelain, are employed to treat many diseases and conditions, but the complexity of their structures is an important limitation of their uses. Thus, the structure and activities of their formulations need to be stabilized and protected against degradation, with improved pharmacokinetics, a prolonged time of action, reduced toxic effects, and proper direction towards their therapeutic target. Nanotechnology has made it possible to manufacture drug carriers such as polymeric nano- and microparticles, hydrogels, dendrimers and liposomes which are able to increase their efficacy and clinical applicability, as well as patient compliance. Sustainability initiatives that use Green Chemistry together with nanobiotechnology have managed to reduce the risks of toxicity to organisms and the environment. Green synthesis uses lower concentrations of metal ions, water-soluble, biocompatible and non-toxic compounds, as well as seeking energy efficiency and using renewable sources of raw materials. Conclusions: Investigations about new formulations of plant therapeutic proteases using biodegradable and biocompatible polymers is of great biomedical interest because they generate less toxic new biopharmaceuticals, in addition to protecting and stabilizing the enzymatic structure.

Improved Production of Recombinant Myrosinase in Pichia pastoris

The effect of the deletion of a 57 bp native signal sequence, which transports the nascent protein through the endoplasmic reticulum membrane in plants, on improved AtTGG1 plant myrosinase production in Pichia pastoris was studied. Myrosinase was extracellularly produced in a 3-liter laboratory fermenter using α-mating factor as the secretion signal. After the deletion of the native signal sequence, both the specific productivity (164.8 U/L/h) and volumetric activity (27 U/mL) increased more than 40-fold compared to the expression of myrosinase containing its native signal sequence in combination with α-mating factor. The deletion of the native signal sequence resulted in slight changes in myrosinase properties: the optimum pH shifted from 6.5 to 7.0 and the maximal activating concentration of ascorbic acid increased from 1 mM to 1.5 mM. Kinetic parameters toward sinigrin were determined: 0.249 mM (Km) and 435.7 U/mg (Vmax). These results could be applied to the expression of other plant enzymes.

REVIEW ON HERBICIDES RESISTANCE AND THEIR MODE OF ACTION

Herbicides play a vital role in the reduction of crop yield losses by the use of effective weed control and weed interference capability, their ability to improving soil conservation practices. This review covering herbicide's resistance to specific weed and the mode of action of herbicides in the crops of agronomic fields. In most countries, input-intensive agriculture is adopted. For a better understanding, it is necessary to know about the mode of action of herbicides, management, organization, classification, and weed control capacity of herbicides. On other conditions, it also contributes insight into herbicide resistance; therefore it is a big problem of sustainable agricultural management. However, more use of herbicides, pesticides, and insecticides cause resistance among the weeds and it causing injury in plants and also removes beneficial plants in agricultural fields, industries, and management of lands. This review mainly focuses on the thorough determination of different modes of action of different classes of herbicides. The mechanism of action of various herbicides is as variable as their complex composition as they concentrate on managing the susceptible weeds utilizing different biochemical means. Herbicides based on their specific mode of action, they may involve plant enzymes or a biological system. However, these herbicides may break up thus injury or disturb the uniform plant growth and development, the result affects uneven plant death. For proper weed control, it is essential to know the mode of action of specific herbicides and it is important to choose specific herbicides for a specific crop, also understood the symptoms of injury, and using proper crop management practices.

Using Continuous Directed Evolution to Improve Enzymes for Plant Applications

Continuous directed evolution of enzymes and other proteins in microbial hosts is capable of outperforming classical directed evolution by executing hypermutation and selection concurrently in vivo, at scale, with minimal manual input. Provided that a target enzyme’s activity can be coupled to growth of the host cells, the activity can be improved simply by selecting for growth. Like all directed evolution, the continuous version requires no prior mechanistic knowledge of the target. Continuous directed evolution is thus a powerful new way to modify plant or non-plant enzymes for use in plant metabolic research and engineering. Here, we first describe the basic features of the Saccharomyces cerevisiae OrthoRep system for continuous directed evolution and compare it briefly with other systems. We then give a step-by-step account of three ways in which OrthoRep can be deployed to evolve primary metabolic enzymes, using a THI4 thiazole synthase as an example and illustrating the mutational outcomes obtained. We close by outlining applications of OrthoRep that serve growing demands (i) to change the characteristics of plant enzymes destined for return to plants, and (ii) to adapt (‘plantize’) enzymes from prokaryotes – especially exotic prokaryotes – to function well in mild, plant-like conditions.One-sentence summaryContinuous directed evolution using the yeast OrthoRep system is a powerful new way to improve enzymes for use in plant engineering as illustrated by ‘plantizing’ a bacterial thiamin synthesis enzyme..

Recent Developments in Enzymatic Antioxidant Defence Mechanism in Plants with Special Reference to Abiotic Stress

The stationary life of plants has led to the evolution of a complex gridded antioxidant defence system constituting numerous enzymatic components, playing a crucial role in overcoming various stress conditions. Mainly, these plant enzymes are superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), glutathione peroxidase (GPX), glutathione reductase (GR), glutathione S-transferases (GST), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR), which work as part of the antioxidant defence system. These enzymes together form a complex set of mechanisms to minimise, buffer, and scavenge the reactive oxygen species (ROS) efficiently. The present review is aimed at articulating the current understanding of each of these enzymatic components, with special attention on the role of each enzyme in response to the various environmental, especially abiotic stresses, their molecular characterisation, and reaction mechanisms. The role of the enzymatic defence system for plant health and development, their significance, and cross-talk mechanisms are discussed in detail. Additionally, the application of antioxidant enzymes in developing stress-tolerant transgenic plants are also discussed.

Heterologous expression of methylxanthine synthesis enzymes in mammalian cells and their use as reporter proteins

This work demonstrates the reconstitution of active methylxanthine synthesis enzymes in human cells and their potential use as inducible reporter enzymes. A variety of plant enzymes involved in caffeine synthesis have been characterized in vitro and several of these methylxanthine synthesis enzymes have been heterologously-expressed in yeast or bacteria. In this work, enzymes from Coffea arabica, Camellia sinensis, and Paullinia cupana have been heterologously-expressed in human cells. We demonstrate that the enzymes tested exhibit similar patterns of activity with a set of xanthine substrates in human cells when compared to previous reports of in vitro activity. We demonstrate that the activity of these enzymes can be used as a reporter for juxtacrine signaling using synNotch-induced expression in the presence of an appropriate substrate. When used in combination with synthetic caffeine receptors, this work has potential for use as an in vivo reporter (e.g. enabling non-invasive monitoring of cell-cell interactions after a cellular transplant) or in synthetic intercellular signaling a methylxanthine, such as caffeine, acting as a synthetic paracrine hormone.