One-step utilization of non-detoxified pretreated lignocellulose for enhanced cellulolytic enzyme production using recombinant Trichoderma reesei RUT C30 carrying alcohol dehydrogenase and nicotinate phosphoribosyltransferase.

Cell growth of Trichoderma reesei is greatly inhibited by furan derivatives (furfural and HMF) generated during pretreatment of lignocellulose, and the cellulase production is hence suppressed. In this study, a novel recombinant strain of T. reesei with high tolerance to furans was constructed by homologously co-expressing nicotinate phosphoribosyltransferase and alcohol dehydrogenase. We observed that furfural had a stronger inhibitory effect than HMF and cellulase production was decreased by 35% in T. reesei with the stress of 2.5 mM furfural. The activities of nicotinate phosphoribosyltransferase and alcohol dehydrogenase increased 8.6-fold and 2.9-fold in the recombinant strain, respectively. Furfural was effectively converted into furfuryl alcohol which was then depleted, thus the production of cellulase could be recovered when the recombinant strain was grown in 5% (w/v) two-step stem explosion pretreated rice straw without detoxification. This work presents an important strategy for efficient enzyme production in T. reesei from non-detoxified pretreated lignocellulose feedstocks.

Advances in improving the performance of cellulase in ionic liquids for lignocellulose biorefinery.

Ionic liquids (ILs) have been considered as a class of promising solvents that can dissolve lignocellulosic biomass and then provide enzymatic hydrolyzable holocellulose. However, most of available cellulases are completely or partially inactivated in the presence of even low concentrations of ILs. To more fully exploit the benefits of ILs to lignocellulose biorefinery, it is critical to improve the compatibility between cellulase and ILs. Various attempts have been made to screen natural IL-tolerant cellulases from different microhabitats. Several physical and chemical methods for stabilizing cellulases in ILs were also developed. Moreover, recent advances in protein engineering have greatly facilitated the rational engineering of cellulases by site-directed mutagenesis for the IL stability. This review is aimed to provide the first detailed overview of the current advances in improving the performance of cellulase in non-natural IL environments. New ideas from the most representative progresses and technical challenges will be summarized and discussed.

Dipeptide-Based Carbohydrate Receptors and Polymers for Glycopeptide Enrichment and Glycan Discrimination.

Glycoproteomics identifies and catalogs protein glycosylation and explores its impact on protein conformations and biofunctions. However, these studies are restricted by the bottleneck to enrich low-abundance glycopeptides from complex biosamples and the difficulties in analyzing glycan structures by mass spectrometry. Here, we report dipeptide as a simple but promising carbohydrate binding platform to tackle these problems. We build a hydropathy-index-based strategy for sequence optimization and screen out three optimal dipeptide sequences from 54 types of dipeptides. The optimized dipeptide-based homopolymers display excellent performance (e.g., selectivity up to ∼70% for real biosamples and strong anti-interference capacity capable of resisting 1000-fold bovine serum albumin interference) in glycopeptide enrichment. Meanwhile, our polymers exhibit high-efficiency chromatographic separation toward oligosaccharides with different compositions, polymerization degrees and even their linkage isomers. This brings another attractive feature that our materials can discriminate subtly variable glycan structures of glycopeptides, especially, isomeric glycosidic linkages. These features provide a solid foundation to analyze the complex glycan structures and glycosites simultaneously, which will benefit future development of glycoproteomics and glycobiology.

Enhancement in ionic liquid tolerance of cellulase immobilized on PEGylated graphene oxide nanosheets: Application in saccharification of lignocellulose.

The objective of the present work was to improve ionic liquid (IL) tolerance of cellulase based on the exploration of functional nanoscale carriers for potential application in lignocellulosic biorefinery. PEGylated graphene oxide (GO) composite was successfully fabricated by chemical binding of 4-arm-PEG-NH2 and GO and applied to the immobilization of cellulase. The PEGylated GO-Cellulase retained 61% of the initial activity in 25% (w/v) 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]) while free cellulase only retained 2%. The IL stability was enhanced more than 30 times. The relatively minor change in Km value (from 2.7 to 3.2mgmL(-1)) after the immobilization suggested that PEGylated GO-Cellulase was capable of closely mimicking the performance of free enzyme. After treating rice straw with [Bmim][Cl] and dilution to a final IL concentration of 15% (w/v), the slurry was directly hydrolyzed using PEGylated GO-Cellulase without IL removing and a high hydrolysis rate of 87% was achieved.

Surface Stiffness--a Parameter for Sensing the Chirality of Saccharides.

Surface stiffness is considered a key parameter for designing high-performance implantable materials and artificial extracellular matrices because of its substantial effects on cell behavior. How to transform biomolecule recognition events, particularly chiral recognition, into stiffness change on material surfaces is biologically essential but very challenging for chemists. Here, we report a chirality-triggered stiffness transition on a smart polymer film, which consists of flexible polyethylenimine (PEI) main chains grafted with dipeptide units capable of discriminating chiral monosaccharides. The polymer film became substantially softer after interacting with L-ribose and became more rigid after interacting with D-ribose (the basic building block of DNA and RNA). This chiral effect provides a new method for determining the enantiomeric purity of an L/D-ribose mixture and facilitates the chiral separation of deoxyribose racemates as well as the separation of diverse mono-, di-, and oligosaccharides. These are three puzzle problems in carbohydrate chemistry. Furthermore, taking advantage of the significant differences in the surface stiffness, the proliferation of fibroblast cells on the polymeric surfaces can also be regulated by chiral biomolecules.