Kim Borch

Thermochemistry of the specific binding of C12 surfactants to bovine serum albumin

The specific binding to bovine serum albumin (BSA) of anionic and non-ionic surfactants with C12 acyl chains has been studied by high sensitivity isothermal titration calorimetry. This method proved particularly effective in resolving the binding of anionic surfactants into separate classes of sites with different affinity. For sodium dodecylsulfate (SDS) the measured binding curves could be rationalized as association to two classes (high affinity/low affinity) of sites comprising, respectively, three and six similar (i.e. thermodynamically equivalent), independent sites. Changes in the thermodynamic functions enthalpy, standard free energy, standard entropy and heat capacity could be discerned for each class of binding site, as well as for micelle formation. These data suggest that binding to low affinity sites (in analogy with micelle formation) exhibits energetic parameters; in particular, a large negative change in heat capacity, which is characteristic of hydrophobic interactions. The thermodynamics of high affinity binding, on the other hand, is indicative of other dominant forces; most likely electrostatic interactions. Other anionic ligands investigated (laurate and dodecyl benzylsulfonate) showed a behavior similar to SDS, the most significant difference being the high affinity binding of the alkylbenzyl sulfonate. For this ligand, the thermodynamic data is indicative of a more loosely associated complex than for SDS and laurate. BSA was found to bind one or two of the non-ionic surfactants (NIS) hepta- or penta(ethylene glycol) monododecyl ether (C12EO7 and C12EO5) with binding constants about three orders of magnitude lower than for SDS. Hence, the free energy of the surfactant in the weakly bound BSA-NIS complex is only slightly favored over the micellar state. The binding process is characterized by very large exothermic enthalpy changes (larger than for the charged surfactants) and a large, positive increment in heat capacity. These observations cannot be reconciled with a molecular picture based on simple hydrophobic condensation onto non-polar patches on the protein surface.

Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A

Background: The molecular understanding of factors that limit enzymatic hydrolysis of cellulose remains incomplete. Results: Pre-steady-state analysis of cellulolytic activity provides rate constants for basic steps of the overall reaction. Conclusion: Slow dissociation of inactive enzyme-cellulose complexes governs the hydrolytic rate at pseudo-steady state. Significance: Kinetic constants elucidate molecular mechanisms and structure-function relationships for cellulases. The transient kinetic behavior of enzyme reactions prior to the establishment of steady state is a major source of mechanistic information, yet this approach has not been utilized for cellulases acting on their natural substrate, insoluble cellulose. Here, we elucidate the pre-steady-state regime for the exo-acting cellulase Cel7A using amperometric biosensors and an explicit model for processive hydrolysis of cellulose. This analysis allows the identification of a pseudo-steady-state period and quantification of a processivity number as well as rate constants for the formation of a threaded enzyme complex, processive hydrolysis, and dissociation, respectively. These kinetic parameters elucidate limiting factors in the cellulolytic process. We concluded, for example, that Cel7A cleaves about four glycosidic bonds/s during processive hydrolysis. However, the results suggest that stalling the processive movement and low off-rates result in a specific activity at pseudo-steady state that is 10–25-fold lower. It follows that the dissociation of the enzyme-substrate complex (half-time of ∼30 s) is rate-limiting for the investigated system. We suggest that this approach can be useful in attempts to unveil fundamental reasons for the distinctive variability in hydrolytic activity found in different cellulase-substrate systems.

BIOCHEMICAL PROPERTIES OF CLONED LIPASES FROM THE PSEUDOMONAS FAMILY

Three Pseudomonas lipases, representing three subfamilies, were analysed for pH optima, destabilization by EGTA and surfactants, phospholipase and cholesterolesterase side activities. All the Pseudomonas lipases tested showed alkaline pH optima. The Pseudomonas cepacia and the P. pseudoalcaligenes lipases were totally inhibited by EGTA at pH 9, and the latter was also fully inhibited at pH 7. The lipase from P. mendocina was not inhibited by EGTA at any of the pH values tested. These findings indicate that a calcium binding site exists in some of the Pseudomonas lipases. The P. pseudoalcaligenes, P. cepacia and P. mendocina lipases were inhibited by the anionic surfactant SDS at concentrations between 0.01-0.5 mg/ml. The P. pseudoalcaligenes and P. cepacia lipases were not inhibited by the nonionic surfactant Brij35 in concentration up to 1 mg/ml, whereas the lipase from P. mendocina was inhibited at 0.1 mg/ml. The P. pseudoalcaligenes and P. cepacia lipases were found to possess high cholesterol esterase activity. P. pseudoalcaligenes lipase was further found to have high phospholipase activity. Ten Pseudomonas lipase sequences were compared by automatic sequence alignment. On the basis of sequence identity we have classified Pseudomonas lipases into five subfamilies.

A kinetic model for the burst phase of processive cellulases

Cellobiohydrolases (exocellulases) hydrolyze cellulose processively, i.e. by sequential cleaving of soluble sugars from one end of a cellulose strand. Their activity generally shows an initial burst, followed by a pronounced slowdown, even when substrate is abundant and product accumulation is negligible. Here, we propose an explicit kinetic model for this behavior, which uses classical burst phase theory as the starting point. The model is tested against calorimetric measurements of the activity of the cellobiohydrolase Cel7A from Trichoderma reesei on amorphous cellulose. A simple version of the model, which can be solved analytically, shows that the burst and slowdown can be explained by the relative rates of the sequential reactions in the hydrolysis process and the occurrence of obstacles for the processive movement along the cellulose strand. More specifically, the maximum enzyme activity reflects a balance between a rapid processive movement, on the one hand, and a slow release of enzyme which is stalled by obstacles, on the other. This model only partially accounts for the experimental data, and we therefore also test a modified version that takes into account random enzyme inactivation. This approach generally accounts well for the initial time course (approximately 1 h) of the hydrolysis. We suggest that the models will be useful in attempts to rationalize the initial kinetics of processive cellulases, and demonstrate their application to some open questions, including the effect of repeated enzyme dosages and the ‘double exponential decay’ in the rate of cellulolysis.

[19] Protein engineering of microbial lipases of industrial interest

Publisher Summary This chapter shows that protein engineering can be successfully used to produce new, commercially interesting products. An understanding of lipase function in general and under specific application conditions is mandatory to make the correct decisions in a protein-engineering strategy. Lipases have a number of potential industrial applications, such as production of esters and specialty fats, removal of resins from pulp, cleaning of hard surfaces, and use in detergents. Enzymes showing lipolytic activity can in some cases also act as esterases, phospholipases, cholesterolesterases, thioesterases, and cutinases. The specificities of lipases are broad, but each enzyme has a preference. The selectivity for a specific activity of the lipases can be improved by protein engineering. At present, most of the industrially relevant efforts in protein engineering of lipases have been to improve the hydrolytic efficiency, peracid generation, and detergent and protease stability, all aiming at applications for detergents.

EFFECT OF MUTATIONS IN CANDIDA ANTARCTICA B LIPASE

Three variants of the Candida antarctica B lipase have been constructed and characterized. The variant containing the T103G mutation, which introduces the consensus sequence G-X-S-X-G found in most other known lipases, shows an increased thermostability but retains only half the specific activity of the native enzyme. Also in ester synthesis the activity is lowered but the specificity and enantioselectivity remains unchanged. The W104H mutant, in which more space is introduced into the active site, has more dramatically changed properties. Both the thermostability and the specific activity are slightly reduced but the activity and specificity in ester synthesis is highly different from the native enzyme. In general, the activity is very low and the enantioselectivity is, furthermore, highly reduced. Finally, the mutation M72L was introduced to increase the oxidation stability of the enzyme. This variant did exhibit an increased resistance towards oxidation but the thermostability was, unfortunately, also reduced.

Product inhibition of five Hypocrea jecorina cellulases.

Product inhibition of cellulolytic enzymes has been deemed a critical factor in the industrial saccharification of cellulosic biomass. Several investigations have addressed this problem using crude enzyme preparations or commercial (mixed) cellulase products, but quantitative information on individual cellulases hydrolyzing insoluble cellulose remains insufficient. Such knowledge is necessary to pinpoint and quantify inhibitory weak-links in cellulose hydrolysis, but has proven challenging to come by. Here we show that product inhibition of mono-component cellulases hydrolyzing unmodified cellulose may be monitored by calorimetry. The key advantage of this approach is that it directly measures the rate of hydrolysis while being essentially blind to the background of added product. We investigated the five major cellulases from Hypocrea jecorina (anamorph: Tricoderma reesei), Cel7A (formerly CBH1), Cel6A (CBH2), Cel7B (EG1), Cel5A (EG2) and Cel12A (EG3), for their sensitivity to the products glucose and cellobiose. The strongest inhibition was found for Cel7A, which showed a 50% activity-loss in 19 mM cellobiose (IC(50)=19 mM). The other exoglucanase, Cel6A, was much less inhibited by cellobiose, but showed the highest sensitivity to glucose among all investigated enzymes. The endoglucanases Cel12A and Cel7B were moderately inhibited by cellobiose (IC(50)=60-80 mM), and weakly inhibited by glucose (IC(50)=350-380 mM). The highest resistance to both products was found for Cel5A, which retained about 75% of its activity at the highest investigated concentrations (respectively 65 mM cellobiose and 1000 mM glucose).

Transient Kinetics and Rate-Limiting Steps for the Processive Cellobiohydrolase Cel7A: Effects of Substrate Structure and Carbohydrate Binding Domain

Cellobiohydrolases are exoacting, processive enzymes that effectively hydrolyze crystalline cellulose. They have attracted considerable interest because of their role in both natural carbon cycling and industrial enzyme cocktails used for the deconstruction of cellulosic biomass, but many mechanistic and regulatory aspects of their heterogeneous catalysis remain poorly understood. Here, we address this by applying a deterministic model to real-time kinetic data with high temporal resolution. We used two variants of the cellobiohydrolase Cel7A from Hypocrea jecorina , and three types of cellulose as substrate. Analysis of the pre-steady-state regime allowed delineation rate constants for both fast and slow steps in the enzymatic cycle and assessment of how these constants influenced the rate of hydrolysis at quasi-steady state. Processive movement on the cellulose strand advanced with characteristic times of 0.15-0.7 s per step at 25 °C, and the rate was highest on amorphous substrate. The cellulose binding module was found to raise this rate on crystalline, but not on amorphous, substrate. The rapid processive movement signified high intrinsic reactivity, but this parameter had marginal influence on the steady-state rate. This was because dissociation and association were slower and, hence, rate limiting. Specifically, the dissociation from the strand was found to occur with characteristic times of 45-100 s. This meant that dissociation was the bottleneck, except at very low substrate loads (0.5-1 g/L), where association became slower.

Xylan oligosaccharides and cellobiohydrolase I (TrCel7A) interaction and effect on activity.

BackgroundThe well-studied cellulase mixture secreted by Trichoderma reesei (anamorph to Hypocrea jecorina) contains two cellobiohydolases (CBHs), cellobiohydrolase I (TrCel7A) and cellobiohydrolase II (TrCeI6A), that are core enzymes for the solubilisation of cellulose. This has attracted significant research interest because of the role of the CBHs in the conversion of biomass to fermentable sugars. However, the CHBs are notoriously slow and susceptible to inhibition, which presents a challenge for the commercial utilisation of biomass. The xylans and xylan fragments that are also present in the biomass have been suggested repeatedly as one cause of the reduced activity of CHBs. Yet, the extent and mechanisms of this inhibition remain poorly elucidated. Therefore, we studied xylan oligosaccharides (XOSs) of variable lengths with respect to their binding and inhibition of both TrCel7A and an enzyme variant without the cellulose-binding domain (CBM).ResultsWe studied the binding of XOSs to TrCel7A by isothermal titration calorimetry. We found that XOSs bind to TrCel7A and that the affinity increases commensurate with XOS length. The CBM, on the other hand, did not affect the affinity significantly, which suggests that XOSs may bind to the active site. Activity assays of TrCel7A clearly demonstrated the negative effect of the presence of XOSs on the turnover number.ConclusionsOn the basis of these binding data and a comparison of XOS inhibition of the activity of the two enzyme variants towards, respectively, soluble and insoluble substrates, we propose a competitive mechanism for XOS inhibition of TrCel7A with phosphoric swollen cellulose as a substrate.

Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose.

The kinetics of cellulose hydrolysis have longbeen described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies. We have used isothermal calorimetry to monitor the early time course of the hydrolysis of insoluble cellulose by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B, which reaches a maximal turnover of about 20 s−1 at 30 °C and conducts about 1200 catalytic cycles per enzyme molecule in the initial fast phase. For TrCel5A and TrCel12A the extent of the burst is 2–300 cycles per enzyme molecule. The availability of continuous data on EG activity allows an analysis of the mechanisms underlying the initial kinetics, and it is suggested that the slowdown is linked to transient inactivation of enzyme on the cellulose surface. We propose, therefore, that the frequency of structures on the substrate surface that cause transient inactivation determine the extent of the burst phase.