Opportunities For Biocatalysis Research Articles

Bi-directionalized promoter systems allow methanol-free production of hard-to-express peroxygenases with Komagataella Phaffii

BackgroundHeme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii.ResultsIn initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system.ConclusionsIn this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis.

Characterization of thermoadaptive serine metalloprotease and application in waste management

Thermoadaptive enzymes offer new opportunities for bio-catalysis and bio-transformations owing to their ability to function over wide temperature ranges. Microbial alkaline proteases are important constituents of commercial enzyme preparations, accounting for more than 60% of the total industrial enzyme market. Fibrous proteins, such as horn, feathers etc. are abundantly available in nature as recalcitrant solid wastes. Extracellular alkaline proteases derived from microorganisms can be used for degradation of proteinaceous wastes and convert them into value-added products e.g. peptide concentrates or amino acids. The present study relates to the isolation of an extracellular, bacterial thermoadaptive serine metalloprotease from the soil. The purified protease showed a remarkable increase in thermostability (4 °C–100 °C) that was enhanced in the presence of Ca2+. The protease (+Ca2+) was utilized to efficiently degrade fish scales and feather to produce amino acids, as declogging agent and for removing blood stains from cloth with a short reaction time. The ability of the protease could be utilized in bulk for solid waste management providing for an eco-friendly and effective process.

Extremozymes: A Potential Source for Industrial Applications.

Extremophilic microorganisms have established a diversity of molecular strategies in order to survive in extreme conditions. Biocatalysts isolated by these organisms are termed extremozymes, and possess extraordinary properties of salt allowance, thermostability, and cold adaptivity. Extremozymes are very resistant to extreme conditions owing to their great solidity, and they pose new opportunities for biocatalysis and biotransformations, as well as for the development of the economy and new line of research, through their application. Thermophilic proteins, piezophilic proteins, acidophilic proteins, and halophilic proteins have been studied during the last few years. Amylases, proteases, lipases, pullulanases, cellulases, chitinases, xylanases, pectinases, isomerases, esterases, and dehydrogenases have great potential application for biotechnology, such as in agricultural, chemical, biomedical, and biotechnological processes. The study of extremozymes and their main applications have emerged during recent years.

Synthesis of Imidazolidin-4-ones via a Cytochrome P450-Catalyzed Intramolecular C–H Amination

Expanding Nature’s catalytic repertoire to include reactions important in synthetic chemistry opens new opportunities for biocatalysis. An intramolecular C–H amination route to imidazolidin-4-ones via α-functionalization of 2-aminoacetamides catalyzed by evolved variants of cytochrome P450BM3 (CYP102A1) from Bacillus megaterium has been developed. Screening of a library of ca. 100 variants based on four template mutants with enhanced activity for the oxidation of unnatural substrates and preparative scale reactions in vitro and in vivo show that the enzymes give up to 98% isolated yield of cyclization products for diverse substrates. 2-Aminoacetamides with one- and two-ring cyclic amines bearing substituents and aliphatic, alicyclic, and substituted aromatic amides are cyclized. Regiodivergent C–H amination was achieved at benzylic and nonbenzylic positions in a tetrahydroisoquinolinyl substrate by the use of different mutants. This C–H amination reaction offers a scalable route to imidazolidin-4-ones wit...

Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications.

The bioprocess engineering with biocatalysts broadly spans its development and actual application of enzymes in an industrial context. Recently, both the use of bioprocess engineering and the development and employment of enzyme engineering techniques have been increasing rapidly. Importantly, engineering techniques that incorporate unnatural amino acids (UAAs) in vivo has begun to produce enzymes with greater stability and altered catalytic properties. Despite the growth of this technique, its potential value in bioprocess applications remains to be fully exploited. In this review, we explore the methodologies involved in UAA incorporation as well as ways to synthesize these UAAs. In addition, we summarize recent efforts to increase the yield of UAA engineered proteins in Escherichia coli and also the application of this tool in enzyme engineering. Furthermore, this protein engineering tool based on the incorporation of UAA can be used to develop immobilized enzymes that are ideal for bioprocess applications. Considering the potential of this tool and by exploiting these engineered enzymes, we expect the field of bioprocess engineering to open up new opportunities for biocatalysis in the near future.

The phylogenetic analysis and putative function of lysine 2,3-aminomutase from methanoarchaea infers the potential biocatalysts for the synthesis of β-lysine

β-amino acids play important biological roles as precursors in the biosynthesis of antibiotics, anticancer agents, neurotransmitters, and other high molecular weight polymers. Microbial cells and enzymes from extreme environments offer new opportunities for biocatalysis and biotransformations as a result of their extreme stability. Lysine 2,3-aminomutase catalyzes the interconversion of L-α-lysine and L-β-lysine. L-β-lysine is a precursor in the bacterial biosynthesis of several antibiotics, and also is a precursor in the biosynthesis of osmolyte N(ε)-acetyl-β-lysine for salt stress and adaptation in methanoarchaea. Lysine 2,3-aminomutase (AblA) genes from the marine Methanosarcina mazei N2M9705, halotolerant Methanocalculus chunghsingensis K1F9705b(T), and halophilic Methanohalophilus portucalensis FDF1(T) were cloned by PCR and southern hybridization. Both nucleotide and amino acid sequences of AblAs were analyzed and phylogenetic comparisons performed. Additionally, the functional motifs and 3D structure of aminomutases were aligned and compared. The deduced amino acid sequences of AblAs from methanoarchaea share high identity with the known clostridial and Bacillus lysine 2,3-aminomutase. The conserved amino acid residues for cofactors, such as the iron-sulfur cluster, S-adenosylmethionine (SAM), pyridoxal 5'-phosphate (PLP) and zinc-binding sites in methanoarchaeal AblAs suggested that they were lysine 2,3-aminomutases. AblAs from methanoarchaea are lysine 2,3-aminomutases that may function as potential biocatalysts for the synthesis of β-lysine in vivo and in vitro.

Stimulation of novel thermostable extracellular lipolytic enzyme in cultures of Thermus sp.

Selected organisms from the genus Thermus ( T. aquaticus YT1, T. thermophilus HB8 and HB27) offer new opportunities for biocatalysis and biotransformations as a result of the extreme stability of their enzymes. In order to favour the secretion of extracellular lipolytic enzymes the effect of temperature and carbon source has been studied. All strains were able to grow within a wide temperature range (from 60 to 80 °C) with an optimum value of 70 °C for extracellular lipolytic activity. On the other hand, several sugars were used as carbon source in the culture medium at different concentrations (from 0.5 to 14 g dm −3). Supplementation of monosaccharides (glucose, fructose) and disaccharides (maltose, sucrose) was carried out in an attempt to increase biomass and lipolytic enzyme production. The influence of carbon source was variable, depending on the strain. Nevertheless, the addition of low concentrations of mono- or disaccharides helped to improve the productivity of the process in most cases. The most significant effects on extracellular lipolytic activity were detected in T. thermophilus HB27 and T. aquaticus YT1 cultures, using sucrose and fructose as carbon source at an initial concentration of 0.5 g dm −3. Moreover, the effect of addition of lipids (olive oil, coconut oil and tributyrin) was tested. In T. thermophilus HB27 cultures, the highest extracellular lipolytic activities were obtained when olive oil was added (70% higher than those obtained in control cultures).

Extremozymes

Extremozymes offer new opportunities for biocatalysis and biotransformations as a result of their extreme stability. From recent work, major approaches to extending the range of applications of extremozymes have emerged. Both the discovery of new extremophilic species and the determination of genome sequences provide a route to new enzymes, with the possibility that these will lead to novel applications. Of equal importance, protein engineering and directed evolution provide approaches to improve enzyme stability and modify specificity in ways that may not exist in the natural world.