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Fatty aldehydes (FALs) can be derived from fatty acids (FAs) and related compounds and are frequently used as flavors and fragrances. Although chemical methods have been conventionally used, their selective biotechnological production aiming at more efficient and eco-friendly synthetic routes is in demand. α-Dioxygenases (α-DOXs) are heme-dependent oxidative enzymes biologically involved in the initial step of plant FA α-oxidation during which molecular oxygen is incorporated into the Cα-position of a FA (Cn) to generate the intermediate FA hydroperoxide, which is subsequently converted into the shortened corresponding FAL (Cn-1). α-DOXs are promising biocatalysts for the flavor and fragrance industries, they do not require NAD(P)H as cofactors or redox partner proteins, and they have a broad substrate scope. Here, we highlight recent advances in the biocatalytic utilization of α-DOXs with emphasis on newly discovered cyanobacterial α-DOXs as well as analytical methods to measure α-DOX activity in vitro and in vivo.
Fast screening of enzyme variants is crucial for tailoring biocatalysts for the asymmetric synthesis of non-natural chiral chemicals, such as amines. However, most existing screening methods either are limited by the throughput or require specialized equipment. Herein, we report a simple, high-throughput, low-equipment dependent, and generally applicable growth selection system for engineering amine-forming or converting enzymes and apply it to improve biocatalysts belonging to three different enzyme classes. This results in (i) an amine transaminase variant with 110-fold increased specific activity for the asymmetric synthesis of the chiral amine intermediate of Linagliptin; (ii) a 270-fold improved monoamine oxidase to prepare the chiral amine intermediate of Cinacalcet by deracemization; and (iii) an ammonia lyase variant with a 26-fold increased activity in the asymmetric synthesis of a non-natural amino acid. Our growth selection system is adaptable to different enzyme classes, varying levels of enzyme activities, and thus a flexible tool for various stages of an engineering campaign.
p-Coumaric acid (p-CA) is a key precursor for the biosynthesis of flavonoids. Tyrosine ammonia lyases (TALs) specifically catalyze the synthesis of p-CA from l-tyrosine, which is a convenient enzymatic pathway. To explore novel and highly active TALs, a phylogenetic tree-building approach was conducted including 875 putative TALs and 46 putative phenylalanine/tyrosine ammonia lyases (PTALs). Among them, 5 TALs and 3 PTALs were successfully characterized and found to exhibit the proposed enzymatic activity. The TAL from Chryseobacterium luteum sp. nov (TALclu) has the highest affinity (Km=0.019 mm) and conversion efficiency (kcat/Km=1631 s−1 ⋅ mm−1) towards l-tyrosine. The reaction conditions for two purified enzymes and their E. coli recombinant cells were optimized and p-CA yields of 2.03 g/L after 8 hours by TALclu and 2.35 g/L after 24 h by TAL from Rivularia sp. PCC 7116 (TALrpc) in whole cells were achieved. These TALs are thus candidates for the construction of whole-cell systems to produce the flavonoid precursor p-CA.
Abstract
Macroalgae species are fast growing and their polysaccharides are already used as food ingredient due to their properties as hydrocolloids or they have potential high value bioactivity. The degradation of these valuable polysaccharides to access the sugar components has remained mostly unexplored so far. One reason is the high structural complexity of algal polysaccharides, but also the need for suitable enzyme cocktails to obtain oligo‐ and monosaccharides. Among them, there are several rare sugars with high value. Recently, considerable progress was made in the discovery of highly specific carbohydrate‐active enzymes able to decompose complex marine carbohydrates such as carrageenan, laminarin, agar, porphyran and ulvan. This minireview summarizes these achievements and highlights potential applications of the now accessible abundant renewable resource of marine polysaccharides.
Entdeckung und Design promiskuitiver Acyltransferase‐Aktivität in Carboxylesterasen der Familie VIII
(2021)
Abstract
Methylation of free hydroxyl groups is an important modification for flavonoids. It not only greatly increases absorption and oral bioavailability of flavonoids, but also brings new biological activities. Flavonoid methylation is usually achieved by a specific group of plant O‐methyltransferases (OMTs) which typically exhibit high substrate specificity. Here we investigated the effect of several residues in the binding pocket of the Clarkia breweri isoeugenol OMT on the substrate scope and regioselectivity against flavonoids. The mutation T133M, identified as reported in our previous publication, increased the activity of the enzyme against several flavonoids, namely eriodictyol, naringenin, luteolin, quercetin and even the isoflavonoid genistein, while a reduced set of amino acids at positions 322 and 326 affected both, the activity and the regioselectivity of the methyltranferase. On the basis of this work, methylated flavonoids that are rare in nature were produced in high purity.
Marine algae produce complex polysaccharides, which can be degraded by marine heterotrophic bacteria utilizing carbohydrate-active enzymes. The red algal polysaccharide porphyran contains the methoxy sugar 6-O-methyl-D-galactose (G6Me). In the degradation of porphyran, oxidative demethylation of this monosaccharide towards D-galactose and formaldehyde occurs, which is catalyzed by a cytochrome P450 monooxygenase and its redox partners. In direct proximity to the genes encoding for the key enzymes of this oxidative demethylation, genes encoding for zinc-dependent alcohol dehydrogenases (ADHs) were identified, which seem to be conserved in porphyran utilizing marine Flavobacteriia. Considering the fact that dehydrogenases could play an auxiliary role in carbohydrate degradation, we aimed to elucidate the physiological role of these marine ADHs. Although our results reveal that the ADHs are not involved in formaldehyde detoxification, a knockout of the ADH gene causes a dramatic growth defect of Zobellia galactanivorans with G6Me as a substrate. This indicates that the ADH is required for G6Me utilization. Complete biochemical characterizations of the ADHs from Formosa agariphila KMM 3901T (FoADH) and Z. galactanivorans DsijT (ZoADH) were performed, and the substrate screening revealed that these enzymes preferentially convert aromatic aldehydes. Additionally, we elucidated the crystal structures of FoADH and ZoADH in complex with NAD+ and showed that the strict substrate specificity of these new auxiliary enzymes is based on a narrow active site.
Enzymes, the driving biocatalysts in living organisms, are typically not suited for large-scale industrial use. In the last decade, enzyme engineering has evolved into the key technology to design tailor-made enzymes for chemical and pharmaceutical applications. We highlight current trends in enzyme engineering and biocatalysis based on outstanding examples from the pharmaceutical industry.
Abstract
Enzyme activity data for biocatalytic applications are currently often not annotated with standardized conditions and terms. This makes it extremely hard to retrieve, compare, and reuse enzymatic data. With advances in the fields of artificial intelligence (AI) and machine learning (ML), the automated usability of data in the form of machine‐readable annotations will play a crucial role for their success. It is becoming increasingly easy to retrieve complex data sets and extract relevant information; however, standardized data readability is a current limitation. In this contribution, we outline an iterative approach to develop standardized terms and create semantic relations (ontologies) to achieve this highly desirable goal of improving the discoverability, accessibility, interoperability, and reuse of digital resources in the field of biocatalysis.