Hemicellulases and auxiliary enzymes for improved conversion of lignocellulosic biomass to monosaccharides

Background  

High enzyme loading is a major economic bottleneck for the commercial processing of pretreated lignocellulosic biomass to produce fermentable sugars. Optimizing the enzyme cocktail for specific types of pretreated biomass allows for a significant reduction in enzyme loading without sacrificing hydrolysis yield. This is especially important for alkaline pretreatments such as Ammonia fiber expansion (AFEX) pretreated corn stover. Hence, a diverse set of hemicellulases supplemented along with cellulases is necessary for high recovery of monosaccharides.     

Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification

Background

Non-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification.

Results

In this study, we have utilized an FPLC (fast protein liquid chromatography)-based methodology to quantify free Trichoderma reesei cellulases (namely CBH I, CBH II, and EG I) concentration within a complex hydrolyzate mixture during the varying time course of biomass saccharification. Three pretreated corn stover (CS) samples were included in this study: Ammonia Fiber Expansion^a (AFEX?-CS), dilute acid (DA-CS), and ionic liquid (IL-CS) pretreatments. The relative fraction of bound individual cellulases varied depending not only on the pretreated biomass type (and lignin abundance) but also on the type of cellulase. Acid pretreated biomass had the highest levels of non-recoverable cellulases, while ionic liquid pretreated biomass had the highest overall cellulase recovery. CBH II has the lowest thermal stability among the three T. reesei cellulases tested. By preparing recombinant family 1 carbohydrate binding module (CBM) fusion proteins, we have shown that family 1 CBMs are highly implicated in the non-productive binding of full-length T. reesei cellulases to lignin.

Conclusions

Our findings aid in further understanding the complex mechanisms of non-productive binding of cellulases to pretreated lignocellulosic biomass. Developing optimized pretreatment processes with reduced or modified lignin content to minimize non-productive enzyme binding or engineering pretreatment-specific, low-lignin binding cellulases will improve enzyme specific activity, facilitate enzyme recycling, and thereby permit production of cheaper biofuels.

     

Behavioral and Electrophysiologic Responses of Drosophila melanogaster to Prolonged Periods of Anoxia

Sensitivity to anoxia varies tremendously among phyla and species. Most mammals are exquisitely sensitive to low concentrations of inspired oxygen, while some fish, turtles and crustacea are very resistant. To determine the basis of anoxia tolerance, it would be useful to utilize a model system which can yield mechanistic answers. We studied the fruit fly, Drosophila melanogaster, to determine its anoxia resistance since this organism has been previously studied using a variety of approaches and has proven to be very useful in a number of areas of biology. Flies were exposed to anoxia for periods of 5-240 min, and, after 1-2 min in anoxia, Drosophila lost coordination, fell down, and became motionless. However, they tolerated a complete nitrogen atmosphere for up to 4 h following which they recovered. In addition, a nonlinear relation existed between time spent in anoxia and time to recovery. Extracellular recordings from flight muscles in response to giant fiber stimulation revealed complete recovery of muscle-evoked response, a response that was totally absent during anoxia. Mean O(2) consumption per gram of tissue was substantially reduced in low O(2) concentrations (20% of control). We conclude from these studies that: (1) Drosophila melanogaster is very resistant to anoxia and can be useful in the study of mechanisms of anoxia tolerance; and (2) the profound decline in metabolic rate during periods of low environmental O(2) levels contributes to the survival of Drosophila. Copyright 1997 Elsevier Science Ltd. All rights reserved     

Entomopathogenic Nematodes Are Not an Alternative to Fenamiphos for Management of Plant-Parasitic Nematodes on Golf Courses in Florida

With the cancellation of fenamiphos in the near future, alternative nematode management tactics for plant-parasitic nematodes (PPN) on golf courses need to be identified. The use of entomopathogenic nematodes (EPN) has been suggested as one possible alternative. This paper presents the results of 10 experiments evaluating the efficacy of EPN at managing PPN on turfgrasses and improving turf performance. These experiments were conducted at various locations throughout Florida over the course of a decade. In different experiments, different EPN species were tested against different species of PPN. Separate experiments evaluated multiple rates and applications of EPN, compared different EPN species, and compared single EPN species against multiple species of PPN. In a few trials, EPN were associated with reductions in certain plant-parasite species, but in other trials were associated with increases. In most trials, EPN had no effect on plant parasites. Because EPN were so inconsistent in their results, we conclude that EPN are not acceptable alternatives to fenamiphos by most turf managers in Florida at this time.     

Isolation of Mitochondria from Orobanche, a "Total" Root Angiospermic Parasite

Mitochondria were isolated from the scapes of Orobanche cernua. On the basis of comparative studies with four media, a mannitol grinding medium was selected at a slightly alkaline pH and supplemented with EDTA, metabisulphite, BSA and soluble PVP. Various intermediates of the Krebs cycle, pyruvate, glutamate and NADH were oxidized by the isolated particles, though with different efficiencies. The rate of oxidation was steady for every intermediate tested over a 60 min period, except for NADH. In the majority of cases, the oxidation was accompanied by efficient coupling with phosphorylation. Antimycin A led to significant reduction in the oxygen uptake in the presence of succinate and complete suppression of phosphate-esterification. The amount of protein recovered in the mitochondrial fraction was comparable with the reported recoveries from a number of plant tissues. The biochemical integrity of mitochondria in the particulate fraction appeared possible from: (a) the absence of respiratory response to added cytochrome c during the oxidation of succinate, (b) insignificant oxidation of exogenously added pyruvate in the absence of sparker and (c) the ability to satisfactorily couple phosphorylation to the oxidation of a number of substrates.     

Changes in o-Diphenol Oxidase During Fibre Development in Cotton

Quantitative and electrophoretic changes in o-diphenol oxidase(o-diphenol: O₂ oxidoreductase, E.C. 1 10,3.1) were studiedduring the entire period of cotton (Gossypium arboreum L. cv.Sanjay) fibre development. A significant increase in o-diphenoloxidase activity was recorded during the fibre initiation phaseand it is suggested that a shift in redox balance towards oxidationmay play an important role in fibre initiation. Low o-diphenoloxidase activity during elongation and its high activity duringthe phase of secondary thickening, together with isoenzyme patterns,suggest an important role of this enzyme in cotton fibre development.The role of o-diphenol oxidase in relation to auxin turnoverand redox balance is discussed. Gossypium arboreum, cotton, fibre development, o-diphenol oxidase, redox balance, auxin turnover