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.

     

Influence of glutathione on the catalytic properties of leucine aminopeptidase]

1. Leucine aminopeptidase does not catalyze the hydrolysis of glutathione. 2. Glutathione inhibits the hydrolysis of the substrates leucine hydrazide and leucine-p-nitroanilide by leucine aminopeptidase. 3. By means of kinetic experiments the type of the inhibition has been determined as noncompetitive. The inhibition constant Ki for the Mg2+-activated enzyme is five times higher than for the non-activated enzyme. 4. The degree of inhibition caused by glutathione depends on the pH value indicating a competition between glutathione and OH- ions. Mg2+-activated enzyme is invariably inhibited in the investigated pH range of 7.2 to 9.8. 5. A preincubation of the enzyme with glutathione changes the degree of activity enhancement by metal ions.     

Spin-labeling studies of urea-treated leucine aminopeptidase.

1. Leucine aminopeptidase (EC 3-4-11-1) from bovine eye lens was spin-labeled at the most reactive thiol groups with 2,2,6,6-tetramethyl-4-[2-iodoacetamido]-piperidine-1-oxyl. 2. Electron spin resonance spectra show two spectral parts corresponding to two local conformational states in the environment of bound label. One state (A) exhibits a strong immobilizing effect on the mobility of the bound label whereas the other one (B) immobilizes weakly. Independently on the degree of labeling a ratio of A:B approximately 4:1 was estimated. In B a hydrophobic environment of label was observed. 3. Treatment of leucine aminopeptidase by 6.2 M urea leads to the following structural changes. a) An additional weakly immobilizing conformational state (B') with reduced hydrophobic interactions and increased mobility representing an unfolded conformational state appears. B' shows a time-dependent increase of its extent at the expense of B and A' (half conversion time about 0.5 h). The extent of this conformational change is larger, if the enzyme is additionally complexed with Mn2+. b) Mn2+ complexed with the protein is partly released producting hydrated Mn2+. c) After withdrawal of urea the observed conformational changes in leucine aminopeptidase are fully reversible, giving the initial ratio of A:B approximately 4:1 even after long incubation. 4. 6.2 M urea is not able to destroy the strongly immobilizing conformational state A completely.     

The Maloti minnow Pseudobarbus quathlambae (Barnard, 1938) is not extinct in South Africa

Despite concerted surveys, the Maloti minnow Pseudobarbus quathlambae (Barnard 1938) had not been recorded in South African waters for almost eighty years since the original collections were made at the type locality in the upper uMkhomazana River in 1938. The species was therefore declared extinct in South Africa, whereas extant populations were considered confined to various rivers in the Lesotho highlands. In April 2017, however, this species was rediscovered in the Mzimkhulu River system in KwaZulu-Natal. The rediscovery of a species that was considered locally extinct supports the need for extensive surveys to determine its distribution range, estimate population sizes, assess conservation status and implement effective strategies to ensure its continued existence in KwaZulu-Natal.