Detrimental effect of cellulose oxidation on cellulose hydrolysis by cellulase

To effectively convert complex and recalcitrant biomass carbohydrates to simple platform sugars useful for fuel and chemicals production, mechanical or chemical pre-treatments are often required to make the carbohydrates more accessible for enzymatic hydrolysis. Due to their harsh conditions, some pre-treatments might negatively affect enzymatic hydrolysis because of events such as cellulose oxidation. To study how oxidative modification may impact cellulose's reactivity toward hydrolysis by cellulases, we prepared three cellulose substrates by cupric ion and hypochlorite oxidations, and subjected the derived celluloses to hydrolysis by various cellobiohydrolases from glycoside hydrolase families 6 and 7, and one cellulolytic Hypocrea jecorina extracellular enzyme mixture. We observed a profound decrease of enzymatic hydrolysis on the oxidized celluloses. The effect was attributed to the interference, from oxidized functional groups in cellulose, on its binding/activation in the active pocket/tunnel of cellobiohydrolases. Potential implication of the observed effect from cellulose oxidation on pre-treatment optimization and cellulase improvement was discussed.     

细菌纤维素的研究进展

细菌纤维素是由醋酸杆菌属、根瘤菌属、土壤杆菌属、八叠球菌属等的某些细菌在一定条件下产生的,其中最有代表性的细菌是木醋杆菌。与传统植物纤维素相比,细菌纤维素具有很高的化学纯度。主要介绍细菌纤维素性质、生物合成的方法及其在食品工业、造纸工业和作为一种生物材料在医学工程等方面的应用。     

The cellulose paradox--simple molecule, complex biosynthesis

Cellulose is the most abundant biopolymer on earth. Despite its simple structure, omnipresence in the plant kingdom, and ever increasing global importance as industrial raw material, the genetic and biochemical regulation of cellulose biosynthesis continues to be unclear. Over the past ten years, the advances in functional genomics have significantly improved our understanding of the processes of cellulose biosynthesis in higher plants. However, for each question answered myriad new unanswered ones have arisen.     

Biodegradable and biocompatible polyampholyte microgels derived from chitosan, carboxymethyl cellulose and modified methyl cellulose

Two biocompatible and biodegradable polyampholyte microgels, namely chitosan-carboxymethyl cellulose (CS-CMC) and chitosan-modified methyl cellulose (CS-ModMC) were synthesized by an inverse microemulsion technique. The CS-CMC microgel system was pH-responsive while the CS-ModMC system possessed both pH and thermo-responsive properties. For CS-CMC system, the number of -OCH₂COOH and -NH₂ groups was determined to be 1.5 and 1.1 meq/g of microgel, respectively. In the pH range of 4-9, the zeta potential values varied from +10 to −40 mV, while the hydrodynamic radius varied from 160 nm in the swollen state (acidic and basic pH) to 110 nm in the “collapse” state (neutral pH). Furthermore, TEM micrographs confirmed the swelling/deswelling behaviour of CS-CMC microgel particles at acidic, neutral and basic conditions. For CS-ModMC system, the number of -OCH₂COOH and -NH₂ groups was determined to be 0.8 and 0.6 meq/g microgel, respectively. In the pH range of 4-9, the surface charge on the microgels varied from +25 to −60 mV and the hydrodynamic radii were 190 nm at low pH, 80 nm at neutral pH, to 120 nm at a high pH. In vitro drug release studies confirmed that CS-CMC microgels could encapsulate and release a model drug, thus they could potentially be used as biocompatible and biodegradable drug carriers.     

Kinetic study on enzymatic hydrolysis of cellulose by cellulose from Trichoderma viride

Pure cellulose (Avicel) was hydrolyzed batchwise at 50 degrees C and pH 4.8 by cellulase from Trichoderma viride (Meicelase CEP). Then the effects of the crystallinity of cellulose as well as the thermal deactivation and product (cellubiose and glucose) inhibition to cellulose on the hydrolysis rate were quantitatively investigated. While these factor had evidently retarded the enzymatic hydrolysis of cellulose to a significant extent, the hydrolysis rates observed could not be explained. For practical purposes, an empirical, simple rate expression was developed which included only one parameter: a overall rate retardation constant. This empirical rate expression held for the hydrolysis of at least two kind of cellulosic materials: Avicel and tissue paper.     

Gravity effects on cellulose assembly

The effect of microgravity on cellulose synthesis using the model system of Acetobacter xylinum was the subject of recent investigations using The National Aeronautics and Space Administration's Reduced Gravity Laboratory, a modified KC-135 aircraft designed to produce 20 sec of microgravity during the top of a parabolic dive. Approximately 40 parabolas were executed per mission, and a period of 2 x g was integral to the pullout phase of each parabola. Cellulose biosynthesis was initiated on agar surfaces, liquid growth medium, and buffered glucose during parabolic flight and terminated with 2.0% sodium azide or 50.0% ethanol. While careful ground and in-flight controls indicated normal, compact ribbons of microbial cellulose, data from five different flights consistently showed that during progression into the parabola regime, the cellulose ribbons became splayed. This observation suggests that some element of the parabola (the 20 sec microgravity phase, the 20 sec 2 x g phase, or a combination of both) was responsible for this effect. Presumably the cellulose I alpha crystalline polymorph normally is produced under strain, and the microgravity/hypergravity combination may relieve this stress to produce splayed ribbons. An in-flight video microscopy analysis of bacterial motions during a parabolic series demonstrated that the bacteria continue to synthesize cellulose during all phases of the parabolic series. Thus, the splaying may be a reflection of a more subtle alteration such as reduction of intermicrofibrillar hydrogen bonding. Long-term microgravity exposures during spaceflight will be necessary to fully understand the cellulose alterations from the short-term microgravity experiments.     

Immunoisotachophoresis on cellulose acetate film

Acetate-cellulose strips of "Cellogel" type have been shown to be a suitable maintenance medium for performance of isotachophoresis. For immuno-isotachophoresis antigen (from 0.5 to 20 microliter) is applied to a strip of acetate-cellulose film. 1--2 microliter of ampholine solution is placed in front of the antigen zone. All the components present on the strip are made in 0.06 M tris-HCl buffer (pH 6.7), and 0.012 M tris-glycine (pH 8.3) is used as an electrode buffer. Electrophoresis produces migrating Kolraush boundary, which at first is the area of antigen concentration into a narrow starting zone, and then of antigens separation with ampholites. The antigens separated on a cellogel strip are subject to cross-electrophoresis on a film saturated with the respective antiserum, with formation of precipitation peaks for each individual antigen. The method permits to operate with low antigen concentrations since electrophoresis ensures their preliminary concentration and the width of the zones is independent of the time of separation.     

L-谷氨酸氧化酶与CBD的融合表达及其在微晶纤维素上固定化分析

酶的固定化作为一种重要的技术,已在生物催化领域得到了广泛的应用。现将来源于普拉特链霉菌3304(Streptomyces platensis NTU3304)产生的胞外L-谷氨酸氧化酶(L-glutamate oxidase,Gox)基因gox融合到来源于粪碱纤维单胞菌Cellulomonas fimi的纤维素结合域(CBDcex)的基因上,构建表达载体p ETM10-Gox-CBD,并在大肠杆菌中表达。通过蛋白纯化获得融合蛋白,并命名为Gox-CBD。利用CBD对微晶纤维素特异性吸附的特性将其固定在微晶纤维素上,并对固定化酶的制备条件、结合量、酶学性质及其微晶纤维素结合稳定性等进行了研究。在4℃条件下结合约1 h,融合蛋白Gox-CBD结合在纤维素上的结合量即可达到9.0 mg/g。通过对重组型、融合表达游离的以及固定化在微晶纤维素上的谷氨酸氧化酶的酶学性质进行比较发现,固定化酶的比酶活有所降低;但固定化酶的热稳定性相对于游离酶有了很大的提高,在60℃孵育30 min后还保留有约70%的活性,而游离的重组Gox在相同条件下几乎完全失去活性。当固定化结合蛋白在p H10或者盐浓度5 mmol/L的Na Cl条件下可以牢固结合。并且可以通过一步纯化方法固定化融合蛋白Gox-CBD于微晶纤维素上。因此,L-谷氨酸氧化酶与纤维素结合域融合表达的研究为蛋白的纯化及酶的固定化提供了一种新策略。     

Tracing cellulose elements adsorbed on composite cellulose biomaterials by a new labeling method

In view of tracing the fate of cellulose fine elements added to a suspension of cellulose fibers, a novel method for specific labeling of polysaccharides in a composite material was developed. The purpose was to visualize a given cellulose material within a cellulose mixture. The method consists of generating aldehyde groups in the chain by mild periodic acid oxidation followed by biotinylation of the carbonyls. Once added to the composite, the biotinylated molecules are labeled with streptavidin conjugated to a fluorescent probe for confocal microscopy, or streptavidin-gold for electron microscopy observations. In the present work, the fate of fresh fines (never-dried) and dead fines (dried) when they were added to a purified suspension of fibers was followed by observation of the labeling in confocal and electron microscopy. The differential mode of interaction of fresh fines and dead fines with the fibers was correlated to the mechanical characteristics measured on the corresponding papers. The versatility of the new labeling method and its possible generalization to other polysaccharides incorporated to a polysaccharide or nonpolysaccharide material should be of potential interest for the study of composite microstructure.     

Formation, derivatization and applications of bacterial cellulose

Acetobacter xylinum produces highly crystalline cellulose extracellulary using glucose as a carbon source. The polymer formed is free of other biogenic compounds, separable in a simple way and characterized by its high water-absorption capacity. Stepwise solvent exchange from water to unpolar solvents leads to a drastic decrease of the water content of the bacterial cellulose without decrease of the highly swollen and activated state. Heterogeneous as well as homogeneous derivatizations, e.g. carboxymethylation, silylation and acetylation, were performed on the wet or dried biopolymer. Furthermore, different methods for formation of hollow fibres during biosynthesis were investigated. Such tubes may have applications as biocompatible material in medicine.     

In vivo degradation of oxidized, regenerated cellulose

Oxidized, regenerated cellulose (ORC) was surgically implanted on the uterine horns of rabbits, and its biodegradation was studied in vivo. Samples of peritoneal lavages, serum, and urine were collected during the degradation process and analyzed for carbohydrate components utilizing high-performance liquid chromatography with pulsed amperometric detection (h.p.l.c.-p.a.d.). Degradation was rapid, and oligomeric products were evident primarily in the peritoneal fluid from the implantation site, with no apparent accumulation in either the serum or the urine. The size distribution and the amount of the oligomeric products decreased after day one, and by day four peritoneal lavages were essentially free of oligomers. The structure of the products formed was consistent with the lability of the polymer in solution, and the kinetics of degradation paralleled the results of the previously reported in vitro studies. Rabbit peritoneal macrophages, when incubated with ORC in vitro were observed to readily ingest and hydrolyze the polymeric material. A mechanism of degradation consisting of chemical depolymerization, followed by enzymatic hydrolysis mediated by glycosidases endogenous to peritoneal macrophages, is proposed.     

Effects of cellulose on growth,enzyme production,and ultrastructure of a Cellulomonas species

Cellulomonas sp. (NRCC 2406) was grown on complex medium (peptone-tryptone-yeast extract) alone, or with the addition of different celluloses (solka floc, avicel, CF 11 cellulose or Whatman No. 1 filter paper) and/or glucose. Cultures growing on the complex medium without cellulose produced low levels of endo- and exo-cellulases and very little -glucosidase. Adding cellulose stimulated growth, as measured by cellular protein or by viable counts, and also stimulated production of cellulases. Adding glucose in the prescene of cellulose inhibited growth and cellulose breakdown. Glucose also inhibited attachment of growing cells to cellulose fibres. Electron microscope studies showed that Cellulomonas sp. adhered to the cellulose fibers. In the presence of cellulose in the media, the cells developed a thicker outer layer which probably helps in the adhesion process.Abbreviations PTYE peptone, tryptone, yeast extract medium - DNS dinitrosalicylic acid - CMC carboxymethyl cellulose - cfu/ml colony-forming units per ml     

Effect of surfactants on cellulose hydrolysis

The effect of surfactants on the heterogeneous enzymatic hydrolysis of Sigmacell 100 cellulose and of steam-exploded wood was studied. Certain biosurfactants (sophorolipid, rhamnolipid, bacitracin) and Tween 80 increased the rate of hydrolysis of Sigmacell 100, as measured by the amount of reducing sugar produced, by as much as seven times. The hydrolysis of steam-exploded wood was increased by 67% in the presence of sophorolipid. At the same time, sophorolipid was found to decrease the amount of enzyme adsorbed onto the cellulose at equilibrium. Sophorolipid had the greatest effect on cellulose hydrolysis when it was present from the beginning of the experiment and when the enzyme/cellulose ratio was low. (c) 1993 John Wiley & Sons, Inc.     

Isolation of transfer RNA isoacceptors by chromatography on dihydroxyboryl-substituted cellulose,polyacrylamide, and glass

Polyacrylamide and porous-glass supports containing the dihydroxyborylphenyl group can be prepared by a method similar to that used in the synthesis of N-[N′-(m-dihydroxyborylphenyl)succinamyl]aminoethylcellulose. The reaction of aminoethylpolyacrylamide or amino-substituted glass with N-(m-dihydroxyborylphenyl)succinamic acid in the presence of N-cyclohexyl-N′-β-(4-methyl-morpholinium) ethylcarbodiimide yields products which, together with the cellulose derivative, are all capable of binding tRNA dissolved in buffers at pH 8.7. The demonstration that bound tRNA can be released with sorbitol solutions or with low pH buffers, together with studies on the binding of tRNA species that contain chemically modified 3′-terminals, indicate that the predominant binding mechanism consists of cyclic complex formation between the immobilized dihydroxyboryl groups and the 3′-terminal cis-diol groups of the tRNA molecules. Aminoacylated tRNA does not bind under the conditions necessary to bind tRNA and this permits the isolation of specific tRNA isoacceptors. The purification of tRNA^Phe and the partial purification of tRNA^Glu and tRNA^Trp are described.     

Utilization of carbohydrates by Thermomonospora sp. Grown on glucose,cellobiose, and cellulose

Thermomonospora sp. was grown on glucose, cellobiose, and in order to study its growth characteristics with different carbohydrate substrates and to assess the validity of some of the assumptions made in a previously proposed model for the cellulose fermentation with this microorganism. It was observed that the nitrogen and protein contents of the cells are essentially constant during the fermentation and independent of the carbon source when glucose or cellobiose are utilized. Under oxygen starvation conditions it was shown that unidentification organic compound(s) accumulate(s) in the culture broth. Culture fluorescence was shown to be an excellent variable for monitoring and control of the fermentation process. This microorganism showed a preference for crystalline cellulose (Avicel) as substrate although it grows readily on a more amorphous cellulose (Solka Floc). The production of extra cellular protein is shown to be growth related. Data were obtained confirming the decrease in the number of active adsorption sites as the cause for the decrease in the cellulose digestion rate. It is suggested that a future model should account for the time change of surface characteristics of the cellulose particles.