Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: A review

Removal of synthetic dyes from wastewaters: a review.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced in many places in living cells and at an increased rate during biotic or abiotic stress. ROS and RNS participate in signal transduction, but also modify cellular components and cause dam- age. We first look at the most common ROS and their properties. We then consider the ways in which the cell can regulate their pro- duction and removal. We critically assess current knowledge about modifications of polyunsaturated fatty acids (PUFAs), DNA, carbo- hydrates, and proteins and illustrate this knowledge with case stories wherever possible. Some oxidative breakdown products, e.g., from PUFA, can cause secondary damage. Other oxidation products are secondary signaling molecules. We consider the fate of the modi- fied components, the energetic costs to the cell of replacing such components, as well as strategies to minimize transfer of oxidatively damaged components to the next generation.

Oxidative Modifications to Cellular Components in Plants

http://www.loewenlabs.com/peter/wp-content/themes/atahualpa/Manuscripts/BA_29_675.pdf

Biomass pretreatment: fundamentals toward application

Proteolytic enzymes are ubiquitous in occurrence, being found in all living organisms, and are essential for cell growth and differentiation. The extracellular proteases are of commercial value and find multiple applications in various industrial sectors. Although there are many microbial sources available for producing proteases, only a few are recognized as commercial producers. A good number of bacterial alkaline proteases are commercially available, such as subtilisin Carlsberg, subtilisin BPN′ and Savinase, with their major application as detergent enzymes. However, mutations have led to newer protease preparations with improved catalytic efficiency and better stability towards temperature, oxidizing agents and changing wash conditions. Many newer preparations, such as Durazym, Maxapem and Purafect, have been produced, using techniques of site-directed mutagenesis and/or random mutagenesis. Directed evolution has also paved the way to a great variety of subtilisin variants with better specificities and stability. Molecular imprinting through conditional lyophilization is coming up to match molecular approaches in protein engineering. There are many possibilities for modifying biocatalysts through molecular approaches. However, the search for microbial sources of novel alkaline proteases in natural diversity through the "metagenome" approach is targeting a hitherto undiscovered wealth of molecular diversity. This fascinating development will allow the biotechnological exploitation of uncultured microorganisms, which by far outnumber the species accessible by cultivation, regardless of the habitat. In this review, we discuss the types and sources of proteases, protease yield-improvement methods, the use of new methods for developing novel proteases and applications of alkaline proteases in industrial sectors, with an overview on the use of alkaline proteases in the detergent industry.

Bacterial alkaline proteases: molecular approaches and industrial applications

Currently, the relatively high cost of enzymes such as glycoside hydrolases that catalyze cellulose hydrolysis represents a barrier to commercialization of a biorefinery capable of producing renewable transportable fuels such as ethanol from abundant lignocellulosic biomass. Among the many families of glycoside hydrolases that catalyze cellulose and hemicellulose hydrolysis, few are more enigmatic than family 61 (GH61), originally classified based on measurement of very weak endo-1,4-beta-d-glucanase activity in one family member. Here we show that certain GH61 proteins lack measurable hydrolytic activity by themselves but in the presence of various divalent metal ions can significantly reduce the total protein loading required to hydrolyze lignocellulosic biomass. We also solved the structure of one highly active GH61 protein and find that it is devoid of conserved, closely juxtaposed acidic side chains that could serve as general proton donor and nucleophile/base in a canonical hydrolytic reaction, and we conclude that the GH61 proteins are unlikely to be glycoside hydrolases. Structure-based mutagenesis shows the importance of several conserved residues for GH61 function. By incorporating the gene for one GH61 protein into a commercial Trichoderma reesei strain producing high levels of cellulolytic enzymes, we are able to reduce by 2-fold the total protein loading (and hence the cost) required to hydrolyze lignocellulosic biomass.

Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family

Directed evolution of industrial enzymes: an update

Enzymes play a critical role in the conversion of lignocellulosic waste into fuels and chemicals, but the high cost of these enzymes presents a significant barrier to commercialization. In the simplest terms, the cost is a function of the large amount of enzyme protein required to break down polymeric sugars in cellulose and hemicellulose to fermentable monomers. In the past 6 years, significant effort has been expended to reduce the cost by focusing on improving the efficiency of known enzymes, identification of new, more active enzymes, creating enzyme mixes optimized for selected pretreated substrates, and minimization of enzyme production costs. Here we describe advances in enzyme technology for use in the production of biofuels and the challenges that remain.

Progress and challenges in enzyme development for biomass utilization.

The Coprinus cinereus (CiP) heme peroxidase was subjected to multiple rounds of directed evolution in an effort to produce a mutant suitable for use as a dye-transfer inhibitor in laundry detergent. The wild-type peroxidase is rapidly inactivated under laundry conditions due to the high pH (10.5), high temperature (50 degrees C), and high peroxide concentration (5-10 mM). Peroxidase mutants were initially generated using two parallel approaches: site-directed mutagenesis based on structure-function considerations, and error-prone PCR to create random mutations. Mutants were expressed in Saccharomyces cerevisiae and screened for improved stability by measuring residual activity after incubation under conditions mimicking those in a washing machine. Manually combining mutations from the site-directed and random approaches led to a mutant with 110 times the thermal stability and 2.8 times the oxidative stability of wild-type CiP. In the final two rounds, mutants were randomly recombined by using the efficient yeast homologous recombination system to shuffle point mutations among a large number of parents. This in vivo shuffling led to the most dramatic improvements in oxidative stability, yielding a mutant with 174 times the thermal stability and 100 times the oxidative stability of wild-type CiP.

Directed evolution of a fungal peroxidase

DNA family shuffling of 26 protease genes was used to create a library of chimeric proteases that was screened for four distinct enzymatic properties. Multiple clones were identified that were significantly improved over any of the parental enzymes for each individual property. Family shuffling, also known as molecular breeding, efficiently created all of the combinations of parental properties, producing a great diversity of property combinations in the progeny enzymes. Thus, molecular breeding, like classical breeding, is a powerful tool for recombining existing diversity to tailor biological systems for multiple functional parameters.

Joel R. Cherry

Papers

Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family

Directed evolution of industrial enzymes: an update

Progress and challenges in enzyme development for biomass utilization.

Directed evolution of a fungal peroxidase

DNA shuffling of subgenomic sequences of subtilisin.