Mechanisms of laccase-mediator treatments improving the enzymatic hydrolysis of pre-treated spruce

Background

The recalcitrance of softwood to enzymatic hydrolysis is one of the major bottlenecks hindering its profitable use as a raw material for platform sugars. In softwood, the guaiacyl-type lignin is especially problematic, since it is known to bind hydrolytic enzymes non-specifically, rendering them inactive towards cellulose. One approach to improve hydrolysis yields is the modification of lignin and of cellulose structures by laccase-mediator treatments (LMTs).

Results

LMTs were studied to improve the hydrolysis of steam pre-treated spruce (SPS). Three mediators with three distinct reaction mechanisms (ABTS, HBT, and TEMPO) and one natural mediator (AS, that is, acetosyringone) were tested. Of the studied LMTs, laccase-ABTS treatment improved the degree of hydrolysis by 54%, while acetosyringone and TEMPO increased the hydrolysis yield by 49% and 36%, respectively. On the other hand, laccase-HBT treatment improved the degree of hydrolysis only by 22%, which was in the same order of magnitude as the increase induced by laccase treatment without added mediators (19%). The improvements were due to lignin modification that led to reduced adsorption of endoglucanase Cel5A and cellobiohydrolase Cel7A on lignin. TEMPO was the only mediator that modified cellulose structure by oxidizing hydroxyls at the C6 position to carbonyls and partially further to carboxyls. Oxidation of the reducing end C1 carbonyls was also observed. In contrast to lignin modification, oxidation of cellulose impaired enzymatic hydrolysis.

Conclusions

LMTs, in general, improved the enzymatic hydrolysis of SPS. The mechanism of the improvement was shown to be based on reduced adsorption of the main cellulases on SPS lignin rather than cellulose oxidation. In fact, at higher mediator concentrations the advantage of lignin modification in enzymatic saccharification was overcome by the negative effect of cellulose oxidation. For future applications, it would be beneficial to be able to understand and modify the binding properties of lignin in order to decrease unspecific enzyme binding and thus to increase the mobility, action, and recyclability of the hydrolytic enzymes.

     

Polarized-light-stimulated enzymatic hydrolysis of xylan

The 1- to 2-h illumination of xylanase with visible polarized light (PL) prior to the action of that enzyme upon beechwood xylan significantly increased its activity. The activity only negligibly decreased on 3 months storage. The hydrolysis of xylan proceeded in three well-distinguished stages. In the first and fastest stage the effect of illumination was only slightly positive. The effect of the stimulation was noted in the second, slower stage. Enzyme stimulated with PL, preferably by means of the 2-h illumination, performed better than enzyme stimulated with nonpolarized light and non-stimulated enzyme. In the last, the slowest stage, the rates of the reaction were nearly the same using either stimulated or non-stimulated enzyme.     

Semicontinuous enzymatic hydrolysis of lignocelluloses

Lignocelluloses (steamed hardwood and hardwood kraft pulp) were semicontinuously hydrolyzed on a large scale [2-2. 5 kg of substrate vs. 20, 000 IU filter paperase (FPase)] using a 10-L hydrolysis reactor with an ultrafiltration unit for the recovery and reuse of cellulases. The substrate was added to the reactor at appropriate intervals to keep a concentration of approximately 5% (w/v). All of the enzyme was added at the beginning and no further addition was done. The ultrafiltration unit was operated intermittently rather than continuously due to its enough capacity (dilution rate of 2.5 h(-1)) and making the enzyme durable. The enzyme required to produce one gram of reducing sugar in this reactor was 27.3 FPase IU/g RS for steamed hardwood and 7.4 FPase IU/g RS for hardwood kraft pulp. The sugar composition of hydrolyzate was unaltered virtually from beginning to end of the hydrolysis in spite of the progressive loss of enzyme activities. The analysis of the enzyme composition in the hydrolyzate during hydrolysis revealed that an exo-beta-D-glucanase component was adsorbed selectively at the stages of advanced hydrolysis extent.     

魔芋葡甘聚糖的pH触发酶解

利用高效凝胶排阻色谱技术研究并实现了以溶液pH值为简易调节开关,对魔芋葡甘聚糖的酶解反应进行触发调控：结果表明：pH为4时底物分子量不发生变化,而当pH调至7时,酶解被触发进行,底物分子量随之快速下降。分析表明：酶在pH变化过程中发生部分复性的可能原因是pH由7调至4的过程中发生沉淀的酶分子维持了天然构象,当pH反调回7时重新溶解,并对KGM进行剪切。     

Polymer-coated liposomes resistant to enzymatic hydrolysis

A polymerizable electrolyte, 2-aminoethyl 1,6-heptadien-4-yl phosphate (AEHDP), which has the same hydrophilic head group as naturally occurring phospholipids, was prepared. Five equivalents of AEHDP were added to a suspension of liposomes (closed bilayer vesicles made of phospholipids) and layered on the liposomes. After polymerization by UV irradiation, the resulting polymer-coated liposomes were resistant to hydrolysis of their constituent phospholipids by phospholipase A2.     

New approaches to enzymatic glycoside synthesis through directed evolution

The expanding field of glycobiology requires tools for the synthesis of structurally defined oligosaccharides and glycoconjugates, while any potential therapeutic applications of sugar-based derivates would require access to substantial quantities of such compounds. Classical chemical approaches are not well suited for such large-scale syntheses, thus enzymatic approaches are sought. Traditional routes to the enzymatic assembly of oligosaccharides have involved the use of either Nature’s own biosynthetic enzymes, the glycosyl transferases, or glycosidases run in transglycosylation mode. However, each approach has drawbacks that have limited its application. Glycosynthases are mutant glycosidases in which the catalytic nucleophile has been replaced by mutation, inactivating them as hydrolases. When used in conjunction with glycosyl fluorides of the opposite anomeric configuration to that of the substrate, these enzymes function as highly efficient transferases, frequently giving stoichiometric yields of products. Further improvements can be obtained through directed evolution of the gene encoding the enzyme in question, but this requires the ability to screen very large libraries of catalysts. In this review we survey new screening methods for the formation of glycosidic linkages using high-throughput techniques, such as FACS, chemical complementation, and robot-assisted ELISA assays. Enzymes were evolved to have higher catalytic activity with their natural substrates, to show altered substrate specificities or to be promiscuous for efficient application in oligosaccharide, glycolipid, and glycoprotein synthesis.     

Real-time monitoring of enzymatic hydrolysis of galactomannans

Enzymatic hydrolysis was monitored in real-time using time dependent static light scattering (TDSLS) for a variety of galactomannans from native Brazilian flora. alpha-Galactosidase, which strips only the (1-6)alpha-D galactose side groups, and beta-mannanase, which hydrolyses only the (1-4)beta-D mannan main chain into oligosaccharides were investigated separately and in combination. The time-dependent signatures matched those describing side-chain stripping for galactosidase, whereas those resulting from the action of mannanase followed the signature typical of random backbone cleavage. Use of both enzymes together required that the TDSLS theory of polymer degradation be extended to the case where random backbone cleavage sites appear as side chains are stripped by the first enzyme. Whereas galactosidase allowed mannanase to access more backbone cleavage sites as time passes, leading to a higher degree of hydrolysis, there was no increase in rate constants. The distribution of random fragments in the case of mannanase digestion alone followed reasonably well the predictions for random cleavage of a single-strand polymer with a restricted number of cleavage sites. The fragment distributions were evaluated by size exclusion chromatography.     

Assay of the enzymatic hydrolysis of pantetheine

Four rapid, independent assays of enzymatic pantetheine hydrolysis are described and compared using an enzyme partially purified from pig kidney. Two assays detect specifically the hydrolysis products: cysteamine (2-aminoethanethiol) is measured by the absorbance of its fluoropyruvate adduct at 300 nm and pantothenate is measured by radioimmunoassay. Methods of [¹⁴C]pantethine synthesis are discussed and the labeled substrate employed in a third enzymatic assay. A fourth assay continuously monitors the absorbance of mercaptide ion at 240 nm. The mercaptide ion concentration increases proportionally with hydrolysis at a buffered pH because of a difference in pK(-SH) between pantetheine (9.9) and cysteamine (8.1) at 37°C. The enzyme shows a pH optimum of ca. 9 and an apparent K_m of 20 μm.     

Kinetics of the enzymatic hydrolysis of cellulose

Enzymatic hydrolysis of cellulose for sugar production offers advantages of higher conversion, minimal by-product formation, low energy requirements, and mild operating conditions over other chemical conversions. The development of a kinetic model, based on observable, macroscopic properties of the overall system, is helpful in design and economic evaluation of processes for sugar conversion and ethanol production. A kinetic model is presented, incorporating enzyme adsorption, product inhibition, and considers a multiple enzyme and substrate system. This model was capable of simulating saccharification of a lignocellulosic material, rice straw, at high substrate (up to 333 g/L) and enzyme concentrations (up to 9.2 FPU/mL) that are common to proposed process designs.     

Modeling intrinsic kinetics of enzymatic cellulose hydrolysis

A multistep approach was taken to investigate the intrinsic kinetics of the cellulase enzyme complex as observed with hydrolysis of noncrystalline cellulose (NCC). In the first stage, published initial rate mechanistic models were built and critically evaluated for their performance in predicting time-course kinetics, using the data obtained from enzymatic hydrolysis experiments performed on two substrates: NCC and alpha-cellulose. In the second stage, assessment of the effect of reaction intermediates and products on intrinsic kinetics of enzymatic hydrolysis was performed using NCC hydrolysis experiments, isolating external factors such as mass transfer effects, physical properties of substrate, etc. In the final stage, a comprehensive intrinsic kinetics mechanism was proposed. From batch experiments using NCC, the time-course data on cellulose, cello-oligosaccharides (COS), cellobiose, and glucose were taken and used to estimate the parameters in the kinetic model. The model predictions of NCC, COS, cellobiose, and glucose profiles show a good agreement with experimental data generated from hydrolysis of different initial compositions of substrate (NCC supplemented with COS, cellobiose, and glucose). Finally, sensitivity analysis was performed on each model parameter; this analysis provides some insights into the yield of glucose in the enzymatic hydrolysis. The proposed intrinsic kinetic model parametrized for dilute cellulose systems forms a basis for modeling the complex enzymatic kinetics of cellulose hydrolysis in the presence of limiting factors offered by substrate and enzyme characteristics.     

High consistency enzymatic hydrolysis of hardwood substrates

The feasibility of using a laboratory peg mixer to carry out high consistency enzymatic hydrolysis of lignocellulosic substrates was investigated. Two hardwood substrates, unbleached hardwood pulp (UBHW) and organosolv pretreated poplar (OPP), were used in this study. Hydrolysis of UBHW and OPP at 20% substrate consistency led to a high glucose concentration in the final hydrolysate. For example, a 48 h enzymatic hydrolysis of OPP resulted in a hydrolysate with 158 g/L of glucose. This is the highest glucose concentration ever obtained from enzymatic hydrolysis of lignocellulosic substrates. Fermentation of UBHW and OPP hydrolysates with high glucose content led to high ethanol concentrations, 50.4 and 63.1 g/L, respectively after fermentation. Our results demonstrate that using common pulping equipment to carry out high consistency hydrolysis can overcome the rheological problems and greatly increase the sugar and ethanol concentrations after the hydrolysis and fermentation.