Supercharged Cellulases Show Reduced Non-Productive Binding, But Enhanced Activity, on Pretreated Lignocellulosic Biomass

Non-productive adsorption of cellulolytic enzymes to various plant cell wall components, such as lignin and cellulose, necessitates high enzyme loadings to achieve efficient conversion of pretreated lignocellulosic biomass to fermentable sugars. Carbohydrate-binding modules (CBMs), appended to various catalytic domains (CDs), promote lignocellulose deconstruction by increasing targeted substrate-bound CD concentration but often at the cost of increased non-productive enzyme binding. Here, we demonstrate how a computational protein design strategy can be applied to a model endocellulase enzyme (Cel5A) from Thermobifida fusca to allow fine-tuning its CBM surface charge, which led to increased hydrolytic activity towards pretreated lignocellulosic biomass (e.g., corn stover) by up to ~330% versus the wild-type Cel5A control. We established that the mechanistic basis for this improvement arises from reduced non-productive binding of supercharged Cel5A mutants to cell wall components such as crystalline cellulose (up to 1.7-fold) and lignin (up to 1.8-fold). Interestingly, supercharged Cel5A mutants that showed improved activity on various forms of pretreated corn stover showed increased reversible binding to lignin (up to 2.2-fold) while showing no change in overall thermal stability remarkably. In general, negative supercharging led to increase hydrolytic activity towards both pretreated lignocellulosic biomass and crystalline cellulose whereas positive supercharging led to a reduction of hydrolytic activity. Overall, selective supercharging of protein surfaces was shown to be an effective strategy for improving hydrolytic performance of cellulolytic enzymes for saccharification of real-world pretreated lignocellulosic biomass substrates. Future work should address the implications of supercharging cellulases from various families on inter-enzyme interactions and synergism.

Chemical Properties of 15-year-old Teak (Tectona grandis L.f) from Different Seed Sources

Fifteen year-old teak wood samples planted in Ciamis FMU (Perhutani Enterprise) were evaluated for their chemical properties. Three seed sources such as conventional seed, clone, and superior wood and radial positions namely sapwood, outer heartwood, and inner heartwood were the observed factors. The specimens were taken from the bottom parts of their sources. Completely randomized design was used. Cell wall components were analyzed by various gravimetric methods.Analysis of variance and Duncan’s test were performed for data analysis. The results showed that no significant difference in the quantity of cell wall components (cellulose, hemicellulose, and lignin), extractives (ethanol-toluene and hot-water solubles), ash, and silica content among the seed sources. Superior teakwood or Jati Plus Perhutani, which has the highest growth rate (2.1~3.6 cm/year) among others, showed a comparative higher average pH values (7.08~7.38) and solubility in 1% NaOH (17.22~17.83%) than other sources. Radial factors significantly affected ethanol-toluene extractive and lignin content. The ethanol-toluene extractive had the highest content (9.30~11.54%) at the outer part of heartwood while lignin content was the lowest (28.12~30.10%) in the inner part. The result indicated some good characteristics of young teak trees compared to the mature ones in relation to wood processing.

Cell biology of primary cell wall synthesis in plants

Building a complex structure such as the cell wall, with many individual parts that need to be assembled correctly from distinct sources within the cell, is a well-orchestrated process. Additional complexity is required to mediate dynamic responses to environmental and developmental cues. Enzymes, sugars and other cell wall components are constantly and actively transported to and from the plasma membrane during diffuse growth. Cell wall components are transported in vesicles on cytoskeletal tracks composed of microtubules and actin filaments. Many of these components, and additional proteins, vesicles, and lipids are trafficked to and from the cell plate during cytokinesis. In this review, we first discuss how the cytoskeleton is initially organized to add new cell wall material or to build a new cell wall, focusing on similarities during these processes. Next, we discuss how polysaccharides and enzymes that build the cell wall are trafficked to the correct location by motor proteins and through other interactions with the cytoskeleton. Finally, we discuss some of the special features of newly formed cell walls generated during cytokinesis.