Real-time single molecular study of a pretreated cellulose hydrolysis mode and individual enzyme movement

Abstract

BackgroundThe main challenges of large-scale biochemical conversion involve the high costs of cellulolytic enzymes and the inefficiency in enzymatic deconstruction of polysaccharides embedded in the complex structure of the plant cell wall, leading to ongoing interests in studying the predominant mode of enzymatic hydrolysis. In this study, complete enzymatic hydrolysis of pretreated biomass substrates was visualized in situ and in real time by atomic force microscopy (AFM) topography and recognition imaging. Throughout the entire hydrolytic process, a hydrolysis mode for exoglucanase (CBH I) consisting of a peeling action, wherein cellulose microfibrils are peeled from sites on the pretreated cellulose substrate that have cracks sufficiently large for CBH I to immobilize.ResultsWe quantitatively monitored the complete hydrolytic process on pretreated cellulose. The synergetic effect among the different enzymes can accelerate the cellulose hydrolysis rate dramatically. However, the combination of CBH I and β-glucosidases (β-G) exhibited a similar degradation capacity as did whole enzyme (contains the cellobiohydrolases and endoglucanase as its major enzyme components). We developed a comprehensive dynamic analysis for individual cellulase acting on single pretreated cellulose through use of functional AFM topography and recognition imaging. The single crystalline cellulose was divided into different regions based on the cracks on the substrate surface and was observed to either depolymerize or to peel away by the jammed enzyme molecules. After the exfoliation of one region, new cracks were produced for the enzyme molecules to immobilize. The fiber width may have a relationship with the peeling mode of the fibers. We performed a statistical height measure of the generated peaks of the peeled fibers. The height values range from 11 to 24 nm. We assume that the CBH I enzymes stop progressing along the cellulose microfibril when the peeled microfibril height exceeds 11 nm.ConclusionThe combination of CBH I and β-G can achieve an effective hydrolysis of the pretreated biomass substrates. The single-molecule study of the complete hydrolytic process indicates that the hydrolytic mode involves the peeling of the microfibrils and progressive depolymerization, which depend on the size of the cracks on the surface of the pretreated cellulose microfibrils.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0498-x) contains supplementary material, which is available to authorized users.

Single-molecule study of oxidative enzymatic deconstruction of cellulose

“…In general, the travel distance and the exact path of enzyme motion–upward, downward, or horizontal–varied depending on the crystal analyzed. The average velocity of continuous CBH I movement was measured as 3.1 ± 0.9 nm/s, consistent with the literature 26, 27 , however, unresolved into discrete stop and go phases, observed using higher time resolution 25, 28 .…”

Microbial lipid production by oleaginous yeasts grown on Scenedesmus obtusiusculus microalgae biomass hydrolysate

“…Complete chemical hydrolysis (H 2 SO 4 ) have been regularly implemented for the production of hydrolysate from lignocellulosic biomass [ 47 , 48 ]. Lately, two-stage hydrolysis processes starting with mild chemical treatment (dilute sulfuric acid) and followed by enzymatic hydrolysis have gained popularity amongst industrial applications [ 49 ]. Corn-stover biomass hydrolysis required a pretreatment of the biomass with 0.5 M NaOH at 80 °C for 75 min [ 50 ].…”

Plant cell wall hydrolysis process reveals structure–activity relationships

“…Recognition area percentage (RAP) is a measure of percentage of the exposed crystalline cellulose over the whole imaged area. The method has been introduced in the supporting file of a previous publication [ 28 ]. Figure 5 shows RAPs changes measured under hydrolysis of EG.…”

“…The recombinant CBM 3a (translated amino acid sequence: MGVSGNLKVEFYNSNPSDTTNSINPQFKVTNTGSSAIDLSKLTLRYYYTVDGQKDQTFWCDHAAIIGSNGSYNGITSNVKGTFVKMSSSTNNADTYLEISFTGGTLEPGAHVQIQGRFAKNDWSNYTQSNDYSFKSASQFVEWDQVTAYLNGVLVWGKEPGLEHHHHHH) was provided by the Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA. The preparation of recombinant CBM 3a and CBM 3a-AFM tip functionalization procedure has been described previously [ 28 ]. A Ni–NTA-His system was used to provide accurate orientation of biomolecules.…”

“…Because of the specific interaction with crystalline cellulose, CBM molecules (derived from Clostridium thermocellum Scaffoldin CipA) have been chosen as a probe to specifically recognize and map the crystalline cellulose distribution [ 27 ]. According to our previous research, AFM topography and recognition imaging can be successfully applied to investigate the complete enzymatic hydrolysis of pretreated cellulose substrates in situ and in real time [ 28 ]. The s chematic of AFM recognition imaging is presented in Additional file 1 : Fig.…”