Mélanie Hall

Cellulose crystallinity--a key predictor of the enzymatic hydrolysis rate.

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.

Modeling cellulase kinetics on lignocellulosic substrates

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.

Multivariate statistical analysis of X-ray data from cellulose: A new method to determine degree of crystallinity and predict hydrolysis rates

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.

Asymmetric bioreduction of activated alkenes to industrially relevant optically active compounds.

Highlights ► Activated C 000000000000 000000000000 000000000000 111111111111 000000000000 111111111111 000000000000 000000000000 000000000000 C 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.

Artificial Biocatalytic Linear Cascades for Preparation of Organic Molecules

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.

Oxidative Decarboxylation of Short‐Chain Fatty Acids to 1‐Alkenes

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.

Asymmetric Bioreduction of Alkenes Using Ene–Reductases YersER and KYE1 and Effects of Organic Solvents

Asymmetric trans-bioreduction of activated alkenes by KYE1 from Kluyveromyces lactis and Yers-ER from Yersinia bercovieri, two ene-reductases from the Old Yellow Enzyme family, showed a broad substrate spectrum with a moderate to excellent degree of stereoselectivity. Both substrate- and enzyme-based stereocontrols were observed to furnish opposite stereoisomeric products. The effects of organic solvents on enzyme activity and stereoselectivity were outlined in this study, where two-phase systems hexane and toluene are shown to sustain bioreduction efficiency even at high organic solvent content.

Elucidation of cellulose accessibility, hydrolysability and reactivity as the major limitations in the enzymatic hydrolysis of cellulose

The precipitous decline in the rates of enzymatic hydrolysis of cellulose with conversion is one of the major limitations to the commercialization of second-generation biofuel. In this work, various rate-limiting factors (fractal kinetics, changes in crystallinity, accessibility, reactivity and hydrolysable fraction, enzyme clogging, and degree of polymerization) were investigated employing experimental as well as computational studies. Model-guided experiments showed cellulose accessibility and the hydrolysable fraction of accessible substrate (a previously undefined and unreported quantity) to decrease steadily until a conversion level of nearly 70%, while cellulose reactivity, defined in terms of hydrolytic activity per amount of actively adsorbed cellulase, remained constant. Substrate depletion, accessibility and hydrolysability decrease accounted for approximately 90% of rate retardation up to 70% conversion. Faster restart rates were observed on partially converted cellulose as compared to uninterrupted hydrolysis rates, supporting an enzyme clogging phenomenon that could possibly be responsible for the additional rate decrease.

Biological pretreatment of cellulose: Enhancing enzymatic hydrolysis rate using cellulose-binding domains from cellulases

In this study, cellulose-binding domains (CBDs) of cellulases from Trichoderma reesei were used in a pretreatment step and were found to effectively reduce the crystallinity of cellulose (both Avicel and fibrous cellulose). This, in turn, led to higher glucose concentrations (up to 25% increase) in subsequent hydrolysis of cellulose using a mixture of cellulases and without the need for any intermediate purification step. CBDs were shown to be active in a range of temperatures (up to 50°C), while cellulase hydrolytic activity was greatly reduced after incubation at 50°C. This was explained by retention of full binding capacity after incubation at 50°C for 15 h. Our findings suggest that CBDs may be a valuable tool in pretreating cellulose and eventually afford faster enzymatic conversion of cellulose to glucose, thus contributing to more affordable processes in the production of biofuels.

Characterization of xenobiotic reductase A (XenA): study of active site residues, substrate spectrum and stability

Xenobiotic reductase A (XenA) has broad catalytic activity and reduces various α,β-unsaturated and nitro compounds with moderate to excellent stereoselectivity. Single mutants C25G and C25V are able to reduce nitrobenzene, a non-active substrate for the wild type, to produce aniline. Total turnover is dominated by chemical rather than thermal instability.