In today's world, there is an increasing trend towards the use of renewable, cheap and readily available biomass in the production of a wide variety of fine and bulk chemicals in different biorefineries. Biorefineries utilize the activities of microbial cells and their enzymes to convert biomass into target products. Many of these processes require enzymes which are operationally stable at high temperature thus allowing e.g. easy mixing, better substrate solubility, high mass transfer rate, and lowered risk of contamination. Thermophiles have often been proposed as sources of industrially relevant thermostable enzymes. Here we discuss existing and potential applications of thermophiles and thermostable enzymes with focus on conversion of carbohydrate containing raw materials. Their importance in biorefineries is explained using examples of lignocellulose and starch conversions to desired products. Strategies that enhance thermostablity of enzymes both in vivo and in vitro are also assessed. Moreover, this review deals with efforts made on developing vectors for expressing recombinant enzymes in thermophilic hosts.

https://link.springer.com/content/pdf/10.1186%2F1475-2859-6-9.pdf

Potential and utilization of thermophiles and thermostable enzymes in biorefining

Marine biofouling--the colonization of small marine microorganisms on surfaces that are directly exposed to seawater, such as ships' hulls--is an expensive problem that is currently without an environmentally compatible solution. Biofouling leads to increased hydrodynamic drag, which, in turn, causes increased fuel consumption and greenhouse gas emissions. Tributyltin-free antifouling coatings and paints based on metal complexes or biocides have been shown to efficiently prevent marine biofouling. However, these materials can damage the environment through metal leaching (for example, of copper and zinc) and bacteria resistance. Here, we show that vanadium pentoxide nanowires act like naturally occurring vanadium haloperoxidases to prevent marine biofouling. In the presence of bromide ions and hydrogen peroxide, the nanowires catalyse the oxidation of bromide ions to hypobromous acid (HOBr). Singlet molecular oxygen ((1)O(2)) is formed and this exerts strong antibacterial activity, which prevents marine biofouling without being toxic to marine biota. Vanadium pentoxide nanowires have the potential to be an alternative approach to conventional anti-biofouling agents.

Vanadium pentoxide nanoparticles mimic vanadium haloperoxidases and thwart biofilm formation

Enzyme-mediated oxidations for the chemist

The amino acid sequence of a protein affects both its structure and its function. Thus, the ability to modify the sequence, and hence the structure and activity, of individual proteins in a systematic way, opens up many opportunities, both scientifically and (as we focus on here) for exploitation in biocatalysis. Modern methods of synthetic biology, whereby increasingly large sequences of DNA can be synthesised de novo, allow an unprecedented ability to engineer proteins with novel functions. However, the number of possible proteins is far too large to test individually, so we need means for navigating the ‘search space’ of possible protein sequences efficiently and reliably in order to find desirable activities and other properties. Enzymologists distinguish binding (Kd) and catalytic (kcat) steps. In a similar way, judicious strategies have blended design (for binding, specificity and active site modelling) with the more empirical methods of classical directed evolution (DE) for improving kcat (where natural evolution rarely seeks the highest values), especially with regard to residues distant from the active site and where the functional linkages underpinning enzyme dynamics are both unknown and hard to predict. Epistasis (where the ‘best’ amino acid at one site depends on that or those at others) is a notable feature of directed evolution. The aim of this review is to highlight some of the approaches that are being developed to allow us to use directed evolution to improve enzyme properties, often dramatically. We note that directed evolution differs in a number of ways from natural evolution, including in particular the available mechanisms and the likely selection pressures. Thus, we stress the opportunities afforded by techniques that enable one to map sequence to (structure and) activity in silico, as an effective means of modelling and exploring protein landscapes. Because known landscapes may be assessed and reasoned about as a whole, simultaneously, this offers opportunities for protein improvement not readily available to natural evolution on rapid timescales. Intelligent landscape navigation, informed by sequence-activity relationships and coupled to the emerging methods of synthetic biology, offers scope for the development of novel biocatalysts that are both highly active and robust.

https://pubs.rsc.org/en/content/articlepdf/2015/CS/C4CS00351A

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

Naturally produced halogenated compounds are ubiquitous across all domains of life where they perform a multitude of biological functions and adopt a diversity of chemical structures. Accordingly, a diverse collection of enzyme catalysts to install and remove halogens from organic scaffolds has evolved in nature. Accounting for the different chemical properties of the four halogen atoms (fluorine, chlorine, bromine, and iodine) and the diversity and chemical reactivity of their organic substrates, enzymes performing biosynthetic and degradative halogenation chemistry utilize numerous mechanistic strategies involving oxidation, reduction, and substitution. Biosynthetic halogenation reactions range from simple aromatic substitutions to stereoselective C–H functionalizations on remote carbon centers and can initiate the formation of simple to complex ring structures. Dehalogenating enzymes, on the other hand, are best known for removing halogen atoms from man-made organohalogens, yet also function naturally,...

/pdf/enzymatic-halogenation-and-dehalogenation-reactions-5a5tbnfi0d.pdf

Enzymatic Halogenation and Dehalogenation Reactions: Pervasive and Mechanistically Diverse

Laboratory-evolved Vanadium Chloroperoxidase Exhibits 100-Fold Higher Halogenating Activity at Alkaline pH CATALYTIC EFFECTS FROM FIRST AND SECOND COORDINATION SPHERE MUTATIONS

Acid phosphatases are enzymes with a broad substrate specificity showing hydrolytic activity towards several different organic phosphate monoesters, such as nucleotides and sugar phosphates. The acid phosphatase from Salmonella enterica ser. typhimurium LT2 (PhoN-Se) is able to hydrolyze O-phospho-dl-threonine to yield L-threonine with a very high enantioselectivity (E>200). When O-phospho-dl-serine was hydrolyzed by PhoN-Se, D-serine was formed, however, the ee values rapidly dropped to 50 %. Random mutagenesis by error-prone PCR was performed on the phosphatase in order to increase its enantioselectivity in the formation of D-serine. Two variants with increased selectivity from a library of 9600 mutants have been found, N151D and V78L showing E values of 18.1 and 4.1, respectively, compared to 3.4 for the wild-type (WT) enzyme.

Optimization of the Kinetic Resolution of the dl-Phosphomonoesters of Threonine and Serine by Random Mutagenesis of the Acid Phosphatase from Salmonella enterica

Harald J. Ruijssenaars

Papers

Laboratory-evolved Vanadium Chloroperoxidase Exhibits 100-Fold Higher Halogenating Activity at Alkaline pH CATALYTIC EFFECTS FROM FIRST AND SECOND COORDINATION SPHERE MUTATIONS

Optimization of the Kinetic Resolution of the dl-Phosphomonoesters of Threonine and Serine by Random Mutagenesis of the Acid Phosphatase from Salmonella enterica