Cellulase immobilization on superparamagnetic nanoparticles for reuse in cellulosic biomass conversion

Current cellulosic biomass hydrolysis is based on the one-time use of cellulases. Cellulases immobilized on magnetic nanocarriers offer the advantages of magnetic separation and repeated use for continuous hydrolysis. Most immobilization methods focus on only one type of cellulase. Here, we report co-immobilization of two types of cellulases, β-glucosidase A (BglA) and cellobiohydrolase D (CelD), on sub-20 nm superparamagnetic nanoparticles. The nanoparticles demonstrated 100% immobilization efficiency for both BglA and CelD. The total enzyme activities of immobilized BglA and CelD were up to 67.1% and 41.5% of that of the free cellulases, respectively. The immobilized BglA and CelD each retained about 85% and 43% of the initial immobilized enzyme activities after being recycled 3 and 10 times, respectively. The effects of pH and temperature on the immobilized cellulases were also investigated. Co-immobilization of BglA and CelD on MNPs is a promising strategy to promote synergistic action of cellulases while lowering enzyme consumption

Fungal cellulase; production and applications: minireview

Cellulose is the most abundant biomaterial derived from the living organisms on the earth; plant is the major contributor to the cellulose pool present in the biosphere. Cellulose is used in variety of applications ranging from nanomaterials to biofuel production. For biofuel production, cellulose has first to be broken-down into its building blocks; β-D-glucosyl unit which subsequently can be fermented to different product such as ethanol, acetic acids, among others. Cellulase is the enzymatic system, which degrades cellulose chains to glucose monomers. Cellulase is a group of three enzymes endoglucanase, exoglucanases and β-glucosidases which act together to hydrolyze cellulose to glucose units. Cellulases are found in bacteria, fungi, plants, and some animals. Fungi are the preferred source of cellulase for industrial applications since they secrete large quantities of cellulase to culture medium. Despite a remarkable number of fungi found to produce cellulase enzymes, few have been extensively investigated because they produce large quantities of these enzymes extracellularly. In this mini-review, the production of cellulase from fungi and the parameters affecting cellulase production are discussed briefly on light of recent publications. Furthermore, potential applications of cellulase enzymes are highlighted

Cellulases from extremely thermophilic bacteria

Cellulose is the most abundant biopolymer on earth, and is the major component of urban waste. Thus cellulose must be seen as a very significant renewable source of chemical foodstocks when fossil fuels become restricted

Comparison of Family 9 Cellulases from Mesophilic and Thermophilic Bacteria

Cellulases containing a family 9 catalytic domain and a family 3c cellulose binding module (CBM3c) are important components of bacterial cellulolytic systems. We measured the temperature dependence of the activities of three homologs: Clostridium cellulolyticum Cel9G, Thermobifida fusca Cel9A, and C. thermocellum Cel9I. To directly compare their catalytic activities, we constructed six new versions of the enzymes in which the three GH9-CBM3c domains were fused to a dockerin both with and without a T. fusca fibronectin type 3 homology module (Fn3). We studied the activities of these enzymes on crystalline cellulose alone and in complex with a miniscaffoldin containing a cohesin and a CBM3a. The presence of Fn3 had no measurable effect on thermostability or cellulase activity. The GH9-CBM3c domains of Cel9A and Cel9I, however, were more active than the wild type when fused to a dockerin complexed to scaffoldin. The three cellulases in complex have similar activities on crystalline cellulose up to 60°C, but C. thermocellum Cel9I, the most thermostable of the three, remains highly active up to 80°C, where its activity is 1.9 times higher than at 60°C. We also compared the temperature-dependent activities of different versions of Cel9I (wild type or in complex with a miniscaffoldin) and found that the thermostable CBM is necessary for activity on crystalline cellulose at high temperatures. These results illustrate the significant benefits of working with thermostable enzymes at high temperatures, as well as the importance of retaining the stability of all modules involved in cellulose degradation

Secretion and assembly of functional mini-cellulosomes from synthetic chromosomal operons in Clostridium acetobutylicum ATCC 824.

Background: Consolidated bioprocessing (CBP) is reliant on the simultaneous enzyme production, saccharification of biomass, and fermentation of released sugars into valuable products such as butanol. Clostridial species that produce butanol are, however, unable to grow on crystalline cellulose. In contrast, those saccharolytic species that produce predominantly ethanol, such as Clostridium thermocellum and Clostridium cellulolyticum, degrade crystalline cellulose with high efficiency due to their possession of a multienzyme complex termed the cellulosome. This has led to studies directed at endowing butanol-producing species with the genetic potential to produce a cellulosome, albeit by localising the necessary transgenes to unstable autonomous plasmids. Here we have explored the potential of our previously described Allele-Coupled Exchange (ACE) technology for creating strains of the butanol producing species Clostridium acetobutylicum in which the genes encoding the various cellulosome components are stably integrated into the genome. Results: We used BioBrick2 (BB2) standardised parts to assemble a range of synthetic genes encoding C. thermocellum cellulosomal scaffoldin proteins (CipA variants) and glycoside hydrolases (GHs, Cel8A, Cel9B, Cel48S and Cel9K) as well as synthetic cellulosomal operons that direct the synthesis of Cel8A, Cel9B and a truncated form of CipA. All synthetic genes and operons were integrated into the C. acetobutylicum genome using the recently developed ACE technology. Heterologous protein expression levels and mini-cellulosome self-assembly were assayed by western blot and native PAGE analysis. Conclusions: We demonstrate the successful expression, secretion and self-assembly of cellulosomal subunits by the recombinant C. acetobutylicum strains, providing a platform for the construction of novel cellulosomes. © 2013 Kovács et al.; licensee BioMed Central Ltd

A family of thermostable fungal cellulases created by structure-guided recombination

SCHEMA structure-guided recombination of 3 fungal class II cellobiohydrolases (CBH II cellulases) has yielded a collection of highly thermostable CBH II chimeras. Twenty-three of 48 genes sampled from the 6,561 possible chimeric sequences were secreted by the Saccharomyces cerevisiae heterologous host in catalytically active form. Five of these chimeras have half-lives of thermal inactivation at 63°C that are greater than the most stable parent, CBH II enzyme from the thermophilic fungus Humicola insolens, which suggests that this chimera collection contains hundreds of highly stable cellulases. Twenty-five new sequences were designed based on mathematical modeling of the thermostabilities for the first set of chimeras. Ten of these sequences were expressed in active form; all 10 retained more activity than H. insolens CBH II after incubation at 63°C. The total of 15 validated thermostable CBH II enzymes have high sequence diversity, differing from their closest natural homologs at up to 63 amino acid positions. Selected purified thermostable chimeras hydrolyzed phosphoric acid swollen cellulose at temperatures 7 to 15°C higher than the parent enzymes. These chimeras also hydrolyzed as much or more cellulose than the parent CBH II enzymes in long-time cellulose hydrolysis assays and had pH/activity profiles as broad, or broader than, the parent enzymes. Generating this group of diverse, thermostable fungal CBH II chimeras is the first step in building an inventory of stable cellulases from which optimized enzyme mixtures for biomass conversion can be formulated

Methods Of Treating A Biomass For Enzymatic Hydrolysis

The present invention is a process for treating a feedstock comprising holocellulose. The process comprises mixing the feedstock with a solution comprising cellulose binding domains to form a mixture. The mixture is then subjected to conditions sufficient to reduce the crystallinity of holocellulose. Subsequent enzymatic hydrolysis may show an improved rate and/or fermentable sugar yield as compared to processes which do not employ the process.Georgia Tech Research Corporatio

Molecular characterization of Portuguese populations of the pinewood nematode Bursaphelenchus xylophilus using cytochrome b and cellulase genes

Bursaphelenchus xylophilus is the causal agent of pine wilt disease and a worldwide pest with high economic impact. Since its first diagnosis in Portugal in 1999, it has been subjected to quarantine measures with impact on forest health and ecosystem stability, significantly affecting international trade of wood products. The disease was detected in the north and centre of continental Portugal and, since 2008, the whole country has been considered an affected area. Recently, it was detected in Madeira Island. In order to avoid new outbreaks, it has become of major importance to understand the patterns of spread, introduction points and to characterize the new populations from continental Portugal and Madeira Island. Mitochondrial cytochrome b (cytb) and parasitic cellulase gene sequences were used to evaluate the genetic relationships among isolates that could indicate possible origins of the new outbreaks. Portuguese isolates were compared with isolates from USA, China, Japan and South Korea, in order to investigate possible infection pathways and disease spread patterns in Portugal. Phylogenetic trees based on both genes show that Portuguese isolates group with Asian isolates. Isolates from USA are in a separate position in both gene trees. However, the phylogenetic tree based on the cellulase gene sequences shows higher differentiation among Portuguese isolates than that of cytb. These results agree with those previously obtained using inter-simple sequence repeats (ISSR). This was the first study to use cytb and cellulase genes to characterize pinewood nematode (PWN) populations. This study suggests that cellulase is a better marker than cytb to study genetic diversity in B. xylophilus