The enzymatic hydrolysis of cellulose encounters various limitations that are both substrate- and enzyme-related. Although the crystallinity of pure cellulosic Avicel plays a major role in ...determining the rate of hydrolysis by cellulases from Trichoderma reesei, we show that it stays constant during enzymatic conversion. The mode of action of cellulases was investigated by studying their kinetics on cellulose samples. A convenient method for reaching intermediate degrees of crystallinity with Avicel was therefore developed and the initial rate of the cellulase-catalyzed hydrolysis of cellulose was demonstrated to be linearly proportional to the crystallinity index of Avicel. Despite correlation with the adsorption capacity of cellulases onto cellulose, at a given enzyme loading, the initial enzymatic rate continued to increase with a decreasing crystallinity index, even though the bound enzyme concentration stayed constant. This finding supports the determinant role of crystallinity rather than adsorption on the enzymatic rate. Thus, the cellulase activity and initial rate data obtained from various samples may provide valuable information about the details of the mechanistic action of cellulase and the hydrolysable/reactive fractions of cellulose chains. X-ray diffraction provides insight into the mode of action of Cel7A from T. reesei. In the conversion of cellulose, the (021) face of the cellulose crystal was shown to be preferentially attacked by Cel7A from T. reesei.
The enzymatic hydrolysis of cellulose by cellulases is one of the major steps in the production of ethanol from lignocellulosics. However, cellulosic biomass is not particularly susceptible to ...enzymatic attack and crystallinity of the substrates is one of the key properties that determine the hydrolysis rates. In this work, by quantifying the respective contributions of amorphous and crystalline cellulose to the X-ray diffraction spectra of cellulose with intermediate degrees of crystallinity, a new method to obtain consistent crystallinity index values was developed. Multivariate statistical analysis was applied to spectra obtained from phosphoric acid pretreated cellulose samples of various intermediate (but undetermined) crystallinity indices to reduce their dimensionality. The crystallinity indices obtained were found to be linearly related to the enzymatic hydrolysis rates. The method was validated by predicting the degree of crystallinity of samples containing various ratios of microcrystalline cellulose and amorphous cellulose, both of known crystallinity indices. Dimensionality reduction of the spectra was also used to predict the enzymatic hydrolysis rates of various cellulose samples from X-ray data. The method developed in this work could be generalized to accurately assess the degree of crystallinity for a wide range of varieties of cellulose.
The enzymatic oxidative decarboxylation of linear short‐chain fatty acids (C4:0–C9:0) employing the P450 monooxygenase OleT, O2 as the oxidant, and NAD(P)H as the electron donor gave the ...corresponding terminal C3 to C8 alkenes with product titers of up to 0.93 g L−1 and TTNs of >2000. Key to this process was the construction of an efficient electron‐transfer chain employing putidaredoxin CamAB in combination with NAD(P)H recycling at the expense of glucose, formate, or phosphite. This system allows for the biocatalytic production of industrially important 1‐alkenes, such as propene and 1‐octene, from renewable resources for the first time.
Biocatalysis: The oxidative decarboxylation of fatty acids using the P450 monooxygenase OleT in combination with the CamAB electron‐transfer system and NAD(P)H recycling gave terminal alkenes (e.g., propene, 1‐butene) with excellent conversion. The reaction may become applicable to the transformation of biomass into chemical compounds for organic synthesis.
The review compiles artificial cascades involving enzymes with a focus on the last 10 years. A cascade is defined as the combination of at least two reaction steps in a single reaction vessel without ...isolation of the intermediates, whereby at least one step is catalyzed by an enzyme. Additionally, cascades performed in vivo and in vitro are discussed separately, whereby in vivo cascades are defined here as cascades relying on cofactor recycling by the metabolism or on a metabolite from the living organism. The review introduces a systematic classification of the cascades according to the number of enzymes in the linear sequence and differentiates between cascades involving exclusively enzymes and combinations of enzymes with non-natural catalysts or chemical steps. Since the number of examples involving two enzymes is predominant, the two enzyme cascades are further subdivided according to the number, order, and type of redox steps. Furthermore, this classification differentiates between cascades where all reaction steps are performed simultaneously, sequentially, or in flow.
► Activated CC bonds bearing electron-withdrawing groups are efficiently reduced by flavoproteins from the OYE family. ► The application of ene-reductases for the pharma- and perfumery industry has ...been demonstrated. ► Access to both stereoisomeric products is feasible by choice of stereo-complementary enzymes or via proper substrate engineering.
Ene-reductases from the ‘Old Yellow Enzyme’ family of flavoproteins catalyze the asymmetric reduction of various α,β-unsaturated compounds at the expense of a nicotinamide cofactor. They have been applied to the synthesis of valuable enantiopure products, including chiral building blocks with broad industrial applications, terpenoids, amino acid derivatives and fragrances. The combination of these highly stereoselective biocatalysts with a cofactor recycling system has allowed the development of cost-effective methods for the generation of optically active molecules, which is strengthened by the availability of stereo-complementary enzyme homologues.
Display omitted
•A bi-functional redox biocatalyst was obtained by co-immobilization of ene-reductase and glucose dehydrogenase.•The bi-functional biocatalyst showed increased conversion compared to ...the combined use of individual catalysts.•Excellent reusability of the co-immobilized biocatalyst was shown over 10 cycles.•Preparative-scale synthesis allowed access to important chemical building blocks in high enantiopurity.
An immobilized bi-functional redox biocatalyst was designed for the asymmetric reduction of alkenes by nicotinamide-dependent ene-reductases. The biocatalyst, which consists of co-immobilized ene-reductase and glucose dehydrogenase, was implemented in biotransformations in the presence of glucose as source of reducing equivalents and catalytic amounts of the cofactor. Enzyme co-immobilization employing glutaraldehyde activated Relizyme HA403/M as support material was performed directly from the crude cell-free extract obtained after protein overexpression in E. coli and cell lysis, avoiding enzyme purification steps. The resulting optimum catalyst showed excellent level of activity and stereoselectivity in asymmetric reduction reactions using either OYE3 from Saccharomyces cerevisiae or NCR from Zymomonas mobilis in the presence of organic cosolvents in up to 20 vol%. The bi-functional redox biocatalyst, which demonstrated remarkable reusability over several cycles, was applied in preparative-scale synthesis at 50 mM substrate concentration and provided access to three industrially relevant chiral compounds in high enantiopurity (ee up to 97 %) and in up to 42 % isolated yield. The present method highlights the potential of (co-)immobilization of ene-reductases, notorious for their poor scalability, and complements the few existing methods available for increasing productivity in asymmetric bioreduction reactions.
The enzymatic hydrolysis of cellulose to glucose by cellulases is one of the major steps involved in the conversion of lignocellulosic biomass to yield biofuel. This hydrolysis by cellulases, a ...heterogeneous reaction, currently suffers from some major limitations, most importantly a dramatic rate slowdown at high degrees of conversion. To render the process economically viable, increases in hydrolysis rates and yields are necessary and require improvement both in enzymes (via protein engineering) and processing, i.e. optimization of reaction conditions, reactor design, enzyme and substrate cocktail compositions, enzyme recycling and recovery strategies. Advances in both areas in turn strongly depend on the progress in the accurate quantification of substrate–enzyme interactions and causes for the rate slowdown. The past five years have seen a significant increase in the number of studies on the kinetics of the enzymatic hydrolysis of cellulose. This review provides an overview of the models published thus far, classifies and tabulates these models, and presents an analysis of their basic assumptions. While the exact mechanism of cellulases on lignocellulosic biomass is not completely understood yet, models in the literature have elucidated various factors affecting the enzymatic rates and activities. Different assumptions regarding rate-limiting factors and basic substrate–enzyme interactions were employed to develop and validate these models. However, the models need to be further tested against additional experimental data to validate or disprove any underlying hypothesis. It should also provide better insight on additional parameters required in the case that more substrate and enzyme properties are to be included in a model.
Native and promiscuous catalytic activities of flavin-dependent Old Yellow Enzymes (OYEs) reported to date are initiated by the reduced flavin upon electron transfer. As a rare exception, the ...isomerization of a nonactivated CC bond was shown to be hydride-independent with two nonstereoselective yeast OYEs. Here, we report the asymmetric isomerization of a prochiral model substrate, γ-methyl β,γ-butenolide, to the corresponding (R)- and (S)-enantiomers of the γ-methyl α,β-butenolide in up to >99% ee by two stereocomplementary OYEs of algal and fungal origin, respectively, which operate by asymmetric proton transfer. Mechanistic studies based on two newly solved crystal structures, along with soaking experiments and site-directed mutagenesis, support the crucial role of partially nonconserved tyrosine residues for the activity and stereoselectivity of both (R)- and (S)-isomerases. This study offers a unique view on the potential of flavoproteins in nonredox catalysis and provides hints for scouting olefin isomerases in likely stereodivergent classes of OYEs.
Enzymes, at the turn of the 21st century, are gaining a momentum. Especially in the field of synthetic organic chemistry, a broad variety of biocatalysts are being applied in an increasing number of ...processes running at up to industrial scale. In addition to the advantages of employing enzymes under environmentally friendly reaction conditions, synthetic chemists are recognizing the value of enzymes connected to the exquisite selectivity of these natural (or engineered) catalysts. The use of hydrolases in enantioselective protocols paved the way to the application of enzymes in asymmetric synthesis, in particular in the context of biocatalytic (dynamic) kinetic resolutions. After two decades of impressive development, the field is now mature to propose a panel of catalytically diverse enzymes for (i) stereoselective reactions with prochiral compounds, such as double bond reduction and bond forming reactions, (ii) formal enantioselective replacement of one of two enantiotopic groups of prochiral substrates, as well as (iii) atroposelective reactions with noncentrally chiral compounds. In this review, the major enzymatic strategies broadly applicable in the asymmetric synthesis of optically pure chiral compounds are presented, with a focus on the reactions developed within the past decade.
Asymmetric synthesis achieved with enzymes for stereoselective reduction and bond forming reactions, enantioselective and atroposelective reactions.
PDF
