Andreas S. Bommarius

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.

Biocatalysis to amino acid-based chiral pharmaceuticals : examples and perspectives

Abstract The search for and development of new pharmaceutically active structures drives the need for new enantiomerically pure compounds (EPC). Many N-containing structures can be derived beneficially from either l - or d -amino acids [K. Drauz, Chimia 51 (1997) 310–314.]. The largest growth occurs in the area of unnatural amino acids. Two examples discussed from the Degussa portfolio concern (i) d -amino acids [A.S. Bommarius, M. Kottenhahn, H. Klenk, K. Drauz, NATO ASI Series C 381 (1992) 161–174.] as components of LHRH antagonists of which the Degussas Cetrorelix is a prime example as well as (ii) l - tert -leucine, occurring in a fast-growing number of pharmaceutical compounds under development [A.S. Bommarius, M. Schwarm, K. Stingl, M. Kottenhahn, K. Huthmacher, K. Drauz, Tetrahedron Asymmetry 6 (1995) 2851–2888.]. For d -amino acids, results of the hydantoinase/carbamoylase route will be presented while redox catalysis by way of reductive amination is a suitable process to l - tert -leucine. The number of biocatalytic applications is growing and an updated list is discussed. The presentation will also cover comparisons of biocatalysis with potentially competitive technologies such as enantioselective crystallization, chemical asymmetric synthesis, or chromatographic separation of racemates. Future trends relevant to the perspective for biocatalysis include the need for ever more complex chiral molecules as well as shortened development times in the pharmaceutical industry.

Status of protein engineering for biocatalysts: how to design an industrially useful biocatalyst

Recent advances in the development of both experimental and computational protein engineering tools have enabled a number of further successes in the development of biocatalysts ready for large-scale applications. Key tools are first, the targeting of libraries, leading to far smaller but more useful libraries than in the past, second, the combination of structural, mechanistic, and sequence-based knowledge often based on prior successful cases, and third, the advent of structurally based algorithms allowing the design of novel functions. Based on these tools, a number of improved biocatalysts for pharmaceutical applications have been presented, such as an (R)-transaminase for the synthesis of active pharmaceutical ingredients (APIs) of sitagliptin (Januvia®) and ketoreductases, glucose dehydrogenases, and haloalkane dehalogenases for the API synthesis toward atorvastatin (Lipitor®) and montelukast (Singulair®).

SYNTHESIS AND USE OF ENANTIOMERICALLY PURE TERT-LEUCINE

Abstract Owing to its bulky, inflexible and hydrophobic tert -butyl side chain, tert -leucine (Tle) finds increased use in templates or catalysts in asymmetric synthesis as well as in peptidic medicinal compounds. ( S )-Tle, available through a large-scale enzymatic reductive amination process, has been incorporated into a variety of anti-AIDS and -cancer compounds. With two new routes to ( R )-Tle coming available, the number of applications of both ( R )- and ( S )-Tle can be expected to increase considerably.

Biocatalysis, Fundamentals and Applications

The whole range of biocatalysis, from a firm grounding in theoretical concepts to in-depth coverage of practical applications and future perspectives. The book not only covers reactions, products and processes with and from biological catalysts, but also the process of designing and improving such biocatalysts. One unique feature is that the fields of chemistry, biology and bioengineering receive equal attention, thus addressing practitioners and students from all three areas.

Cellulase kinetics as a function of cellulose pretreatment

Microcrystalline cellulose (Avicel) was subjected to three different pretreatments (acid, alkaline, and organosolv) before exposure to a mixture of cellulases (Celluclast). Addition of beta-glucosidase, to avoid the well-known inhibition of cellulase by cellobiose, markedly accelerated cellulose hydrolysis up to a ratio of activity units (beta-glucosidase/cellulase) of 20. All pretreatment protocols of Avicel were found to slightly increase its degree of crystallinity in comparison with the untreated control. Adsorption of both cellulase and beta-glucosidase on cellulose is significant and also strongly depends on the wall material of the reactor. The conversion-time behavior of all four states of Avicel was found to be very similar. Jamming of adjacent cellulase enzymes when adsorbed on microcrystalline cellulose surface is evident at higher concentrations of enzyme, beyond 400 U/L cellulase/8 kU/L beta-glucosidase. Jamming explains the observed and well-known dramatically slowing rate of cellulose hydrolysis at high degrees of conversion. In contrast to the enzyme concentration, neither the method of pretreatment nor the presence or absence of presumed fractal kinetics has an effect on the calculated jamming parameter for cellulose hydrolysis.

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.

Development of an Amine Dehydrogenase for Synthesis of Chiral Amines

A leucine dehydrogenase has been successfully altered through several rounds of protein engineering to an enantioselective amine dehydrogenase. Instead of the wild-type α-keto acid, the new amine dehydrogenase now accepts the analogous ketone, methyl isobutyl ketone (MIBK), which corresponds to exchange of the carboxy group by a methyl group to produce chiral (R)-1,3-dimethylbutylamine.

Enzymatic reduction of α-keto acids leading to l-amino acids, d- or l-hydroxy acids

Abstract The substrate range of leucine dehydrogenase and phenylalanine dehydrogenase isolated from different organisms was investigated using a range of hydrophobic α -keto acids. Several aliphatic l -amino acids with bulky side chains were synthesized by reductive amination, l -neopentylglycine was produced on 30 kg scale. Besides the reductive amination of the α -keto acids these compounds were also investigated as substrates for hydroxyisocaproate dehydrogenase from Lactobacillus confusus and Lactobacillus casei , respectively.