REAL-TIME OBSERVATION OF CELLULOSE BIODEGRADATION BY ATOMIC FORCE MICROSCOPY

Cellulose, the major structural component of plant cell walls, is a homopolymer of β-1,4-linked glucose residues. As cellulose is the most abundant biopolymer on Earth comprising approx. 50% of the bioshpere, it has attracted renewed interest as a potential source of energy through its biodegradation and fermentation to biofuels. The biodegradation of cellulose involves the concerted action of three types of enzymes, cellulases (EC 3.2.1.4, endo-β-1,4-glucanases), cellobiohydrolases (EC 3.2.1.91; cellulose 1,4-β-cellobiosidase), and β-glucosidases (EC 3.2.1.21; β-d-glucoside glucohydrolase). The former two classes of enzymes function to hydrolyze insoluble cellulose into soluble oligosaccharides which then serve as substrates for β-glucosidases to release free glucose. In many cases, these enzymes are multi-modular, being comprised of distinct catalytic and carbohydrate-binding modules (CBMs). The CBMS appear to aid in both the adsorption of the enzymes to the insoluble cellulose substrate and the destabilization of the hydrogen-bonding network within the crystalline substrate. An understanding of this latter process is extremely important because it has been demonstrated that binding of the enzymes to the insoluble cellulose represents the rate-limiting step in its hydrolysis. To this end, we have developed a protocol for the direct and real-time observation of cellulose biodegradation by atomic force microscopy (AFM). Working electrodes for AFM experiments consisted of a 200 nm thick gold film vapor deposited onto a glass slide pre-treated with a deposition of a 2 nm thick layer of chromium. After annealing in a muffle furnace at 700°C for 60 s, the slides were treated with thioglucose to provide a highly-ordered monolayer of hydrophilic glucose for the stable adsorption of cellulose. Thin films of bacterial microcrystalline cellulose on these electrodes were prepared using the Langmuir-Blodgett technique. Optimized conditions were established to involve a dispersion of a 2 mg/ml suspension of cellulose in methanol/chloroform (1:5) on aqueous phosphate buffer using a compression of 5 mN/m. With this protocol, drying of the cellulose film thereby precluding any associated structural alterations. AFM images were captured using a Pico SPM Microscope with AFMS 182 scanner and Pico-scan 5.2 software system using silicon nitride tips which had a nominal spring constant of 0.06 N m-1 for contact mode, and magnetically coated silicon tips for MAC mode. Under these conditions, the diameters of the microfibrils in a 50 nm fiber were observed to be 3 - 4 nm, which is smaller than the 7.5 nm previously reported by others. Homogeneous samples of the cellulase CenA from the bacterium Cellulomonas fimi were introduced into the liquid cell through capillary ports for the in situ imaging of cellulose disruption and hydrolysis. This activity was monitored over the course of 19 hours and initial evidence of degradation of the fibers was observed within three minutes of enzyme addition. In addition, details of the process of fiber fraying could be readily discerned. Genetic engineering was used to provide a mutant form of CenA involving a replacement of its catalytic aspartate nucleophile with alanine. Studies with this catalytically inactive enzyme derivative permit the analysis of cellulose fibril destabilization prior to hydrolysis

Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data

Novel carbohydrate binding modules in the surface anchored α‐amylase of Eubacterium rectale provide a molecular rationale for the range of starches used by this organism in the human gut

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141499/1/mmi13881.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141499/2/mmi13881_am.pd

Molecular details of a starch utilization pathway in the human gut symbiont Eubacterium rectale

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110609/1/mmi12859.pd

An efficient arabinoxylan-debranching α-L-arabinofuranosidase of family GH62 from Aspergillus nidulans contains a secondary carbohydrate binding site.

An α-L-arabinofuranosidase of GH62 from Aspergillus nidulans FGSC A4 (AnAbf62A-m2,3) has an unusually high activity towards wheat arabinoxylan (WAX) (67 U/mg; k cat = 178/s, K m = 4.90 mg/ml) and arabinoxylooligosaccharides (AXOS) with degrees of polymerisation (DP) 3-5 (37-80 U/mg), but about 50 times lower activity for sugar beet arabinan and 4-nitrophenyl-α-L-arabinofuranoside. α-1,2- and α-1,3-linked arabinofuranoses are released from monosubstituted, but not from disubstituted, xylose in WAX and different AXOS as demonstrated by NMR and polysaccharide analysis by carbohydrate gel electrophoresis (PACE). Mutants of the predicted general acid (Glu(188)) and base (Asp(28)) catalysts, and the general acid pK a modulator (Asp(136)) lost 1700-, 165- and 130-fold activities for WAX. WAX, oat spelt xylan, birchwood xylan and barley β-glucan retarded migration of AnAbf62A-m2,3 in affinity electrophoresis (AE) although the latter two are neither substrates nor inhibitors. Trp(23) and Tyr(44), situated about 30 Å from the catalytic site as seen in an AnAbf62A-m2,3 homology model generated using Streptomyces thermoviolaceus SthAbf62A as template, participate in carbohydrate binding. Compared to wild-type, W23A and W23A/Y44A mutants are less retarded in AE, maintain about 70 % activity towards WAX with K i of WAX substrate inhibition increasing 4-7-folds, but lost 77-96 % activity for the AXOS. The Y44A single mutant had less effect, suggesting Trp(23) is a key determinant. AnAbf62A-m2,3 seems to apply different polysaccharide-dependent binding modes, and Trp(23) and Tyr(44) belong to a putative surface binding site which is situated at a distance of the active site and has to be occupied to achieve full activity.This work is supported by the Danish Council for Independent Research|Natural Sciences (FNU) [grant number 09-072151], by 1/3 PhD fellowship from the Technical University of Denmark (to CW) and by a Hans Christian Ørsted postdoctoral fellowship from DTU (to DC).This is the author accepted manuscript. The final version is available from Springer via http://dx.doi.org/10.1007/s00253-016-7417-

A Bacterial Glucanotransferase Can Replace the Complex Maltose Metabolism Required for Starch to Sucrose Conversion in Leaves at Night

Peer reviewedPublisher PD

P. falciparum and P. vivax Epitope-Focused VLPs Elicit Sterile Immunity to Blood Stage Infections

In order to design P. falciparum preerythrocytic vaccine candidates, a library of circumsporozoite (CS) T and B cell epitopes displayed on the woodchuck hepatitis virus core antigen (WHcAg) VLP platform was produced. To test the protective efficacy of the WHcAg-CS VLPs, hybrid CS P. berghei/P. falciparum (Pb/Pf) sporozoites were used to challenge immunized mice. VLPs carrying 1 or 2 different CS repeat B cell epitopes and 3 VLPs carrying different CS non-repeat B cell epitopes elicited high levels of anti-insert antibodies (Abs). Whereas, VLPs carrying CS repeat B cell epitopes conferred 98% protection of the liver against a 10,000 Pb/Pf sporozoite challenge, VLPs carrying the CS non-repeat B cell eptiopes were minimally-to-non-protective. One-to-three CS-specific CD4/CD8 T cell sites were also fused to VLPs, which primed CS-specific as well as WHcAg-specific T cells. However, a VLP carrying only the 3 T cell domains failed to protect against a sporozoite challenge, indicating a requirement for anti-CS repeat Abs. A VLP carrying 2 CS repeat B cell epitopes and 3 CS T cell sites in alum adjuvant elicited high titer anti-CS Abs (endpoint dilution titer \u3e1x106) and provided 80–100% protection against blood stage malaria. Using a similar strategy, VLPs were constructed carrying P. vivax CS repeat B cell epitopes (WHc-Pv-78), which elicited high levels of anti-CS Abs and conferred 99% protection of the liver against a 10,000 Pb/Pv sporozoite challenge and elicited sterile immunity to blood stage infection. These results indicate that immunization with epitope-focused VLPs carrying selected B and T cell epitopes from the P.falciparum and P. vivax CS proteins can elicit sterile immunity against blood stage malaria. Hybrid WHcAg-CS VLPs could provide the basis for a bivalent P. falciparum/P. vivax malaria vaccine

Evaluating the efficacy of non-thermal microbial load reduction treatments of heat labile food components for in vitro fermentation experiments.

Increasingly, in vitro simulated colon fermentations are being used as a pre-clinical step to assess the impacts of foods and drugs on the gut microbiota in a cost-effective manner. One challenge in such systems is that they are potentially susceptible to the influences of contaminating microbes in test materials. Simulated gastric and intestinal digestion can relieve some of these concerns, however, live microbes may remain that can confound analysis. Autoclave treatment of test materials is the surest way to eliminate these microbes but presents problems when using heat labile components such as resistant starch. In this study, liquid chemical sterilant alternatives to moist heat sterilization were explored for treating pulse flours for use during in vitro simulated colon fermentation. Key attributes considered in chemical selection were accessibility, impact on treated food components, and effectiveness of the treatments for reducing microbial load. Three chemicals were selected for evaluation, bleach, alcohol, and hydrogen peroxide, at varying concentrations. Flours chosen for testing were from green lentil, field pea, chickpea, or sprouted green lentil. All treatments significantly reduced microbial loads, though there were still detectable levels of microbes after alcohol treatments. Furthermore, in vitro simulated colon fermentations of the treated pulses showed minimal difference from the untreated control both in terms of microbial composition and short chain fatty acid production. Scanning electron microscopy showed minimal impact of sterilization treatments on the gross structure of the pulse flours. Together these results suggest that bleach and hydrogen peroxide treatments can be effective nonthermal treatments to eliminate contaminating microbes in pulse flours without causing significant damage to starch and other fermentable substrates. This is thus also a promising treatment method for other starchy food substrates, though further testing is required

Viable microbial counts of controls and treated pulse flours plated on RUM media and incubated anaerobically for 24 h at 37°C.

Viable microbial counts of controls and treated pulse flours plated on RUM media and incubated anaerobically for 24 h at 37°C.</p