Time Course Of Hydrolysis Research Articles

Time-resolved β-lactam cleavage by L1 metallo-β-lactamase

Serial x-ray crystallography can uncover binding events, and subsequent chemical conversions occurring during enzymatic reaction. Here, we reveal the structure, binding and cleavage of moxalactam antibiotic bound to L1 metallo-β-lactamase (MBL) from Stenotrophomonas maltophilia. Using time-resolved serial synchrotron crystallography, we show the time course of β-lactam hydrolysis and determine ten snapshots (20, 40, 60, 80, 100, 150, 300, 500, 2000 and 4000 ms) at 2.20 Å resolution. The reaction is initiated by laser pulse releasing Zn2+ ions from a UV-labile photocage. Two metal ions bind to the active site, followed by binding of moxalactam and the intact β-lactam ring is observed for 100 ms after photolysis. Cleavage of β-lactam is detected at 150 ms and the ligand is significantly displaced. The reaction product adjusts its conformation reaching steady state at 2000 ms corresponding to the relaxed state of the enzyme. Only small changes are observed in the positions of Zn2+ ions and the active site residues. Mechanistic details captured here can be generalized to other MBLs.

Product Inhibition and the Catalytic Destruction of a Nerve Agent Simulant by Zirconium-Based Metal-Organic Frameworks.

Rapid degradation/destruction of chemical warfare agents, especially ones containing a phosphorous-fluorine bond, is of notable interest due to their extreme toxicity and typically rapid rate of human incapacitation. Recent studies of the hydrolytic destruction of a key nerve agent simulant, dimethyl 4-nitrophenylphosphate (DMNP), catalyzed by Zr6-based metal-organic frameworks (MOFs), have suggested deactivation of the active sites due to inhibition by the products as the reaction progresses. In this study, the interactions of two MOFs, NU-1000 and MOF-808, and two hydrolysis products, dimethyl phosphate (DMP) and ethyl methyl phosphonate (EMP), from the hydrolysis of the simulant (DMNP) and nerve agent ethyl methylphosphonofluoridate (EMPF), resembling the hydrolysis degradation product of the G-series nerve agent, Sarin (GB), have been investigated to deconvolute the effect of product inhibition from other effects on catalytic activity. Kinetic studies via in situ nuclear magnetic resonance spectroscopy indicated substantial product inhibition upon catalyst activity after several tens to several thousand turnovers, depending on specific conditions. Apparent product binding constants were obtained by fitting initial reaction rates at pH 7.0 and pH 10.5 to a Langmuir-Freundlich binding/adsorption model. For the fits, varying amounts/concentrations of candidate inhibitors were introduced before the start of catalytic hydrolysis. The derived binding constants proved suitable for quantitatively describing product inhibition effects upon reaction rates over the extended time course of simulant hydrolysis by aqua-ligand-bearing hexa-zirconium(IV) nodes.

(Invited) Ligand-Functionalised Conducting Polymer Films for Analysis of Metal Ions in Solution

Innumerable technological devices and applications utilise metals and metal ions that ultimately find their way into the environment. While many metals are required at trace level for good health, their presence at higher levels is generally detrimental to human health and to the environment at large. For example, higher levels of copper are associated with liver and kidney disease; of nickel with cancer, cardiovascular disease, neurological conditions and infant developmental disorders; and of cobalt with respiratory conditions. This motivates the need to detect and determine these species at trace level; a related goal (not addressed directly here) is their extraction for the purposes of remediation. Although numerous analytical techniques exist for the determination of metal ions, electrochemically-based devices have a number of advantages, including portability for remote application. This work explores underpinning fundamental electrochemistry and interfacial characterisation associated with the fabrication and performance of carboxylate ligand-functionalised conducting polymer films as metal ion sensing moieties immobilised on electrode surfaces.Typical chelating ligands for metal ions are relatively bulky compared to the electropolymerizable monomer units, for example based on pyrrole or thiophene, commonly used to generate electroactive polymer films. As a consequence, electropolymerization of ligand-functionalised monomers is not generally effective: steric hindrance simply impedes coupling of the monomer units. We therefore adopt the alternative approach of electropolymerizing monomers functionalised with relatively small labile groups, followed by hydrolysis of the labile groups and re-functionalisation with the desired ligand. Here we explore this strategy for pyrrole-, thiophene- and aniline-based monomers functionalised with 9-fluorenylmethoxycarbonyl (fmoc) or pentafluorophenyl groups via ester linkages. Following electrochemically controlled polymerization, the resulting polymer films were treated with base to remove the labile groups, then with Nα,Nα-bis-(carboxymethyl)-L-lysine to couple N-nitrilotriacetic acid functionalities via amide formation.The polymerization process and the subsequent electroactivity of each of the parent films were studied using voltammetric and nanogravimetric (QCM) techniques. The ultimate effectiveness of the surface synthetic sequence, effected by post-deposition functionalisation with the ligand species, was validated using FTIR spectroscopy. This was accomplished by monitoring the bands associated with the carbonyl functionality: this is initially part of an ester, then an acid and finally an amide, each of which has a characteristic frequency. In the case of initially pentafluorophenyl-functionalised monomers, the hydrolysis of this group is associated with the loss of the C-F functionality. Measurements as a function of time permit the dynamics of these processes to be followed. Additionally, nanogravimetric measurements allow the time course of as-deposited polymer hydrolysis and subsequent functionalisation to be followed and quantitatively compared with the reaction stoichiometry.The effectiveness of these ligand-functionalised films was explored in terms of their ability to take up nickel, cobalt and copper ions from aqueous solution. Coulometric and gravimetric (QCM) data at various points in the process and as a function of solution metal ion concentration permit the acquisition of isotherms for metal ion uptake. At high metal ion concentration, the ligand sites are saturated; this permits metal:ligand stoichiometry to be evaluated. The relative merits of Langmuir, Frumkin, Temkin and Freundlich isotherms are discussed and the viability of the films for metal ion sensing evaluated. Unsurprisingly, the Langmuir isotherm provides a relatively poor description of the variation of metal ion uptake with concentration; this is attributed to the rather simplistic assumptions of this model. Of the remaining models, the Frumkin isotherm provides the best description of metal ion uptake for all systems considered. The binding constants evaluated from the polymer isotherms are significantly smaller than for the same ligand in solution; this is presumably a consequence of steric effects in the surface-confined polymer environment. Of relevance to potential applications, the films can be regenerated, i.e. restored to metal-free state, by exposure to EDTA solution.

Efficient conversion of crystalline chitin to N-acetylglucosamine and N,N'-diacetylchitobiose by the enzyme cocktail produced by Paenibacillus sp. LS1

Direct conversion of crystalline chitin to N-acetylglucosamine and the related chitooligomers through enzymatic approaches is gaining importance owing to their various biological applications. Here we report the crystalline chitin degradation ability of chitinolytic cocktail produced by the isolate Paenibacillus sp. LS1. Growth studies of the isolate in presence of different chitin substrates revealed preference for β-chitin and colloidal chitin. FE-SEM micrographs showed formation of visible perforations on the crystalline chitin particles by the isolate. Further, zymogram analysis revealed the presence of six potential chitinase isozymes. The enzyme-cocktail produced by the isolate was optimally active at 50 °C in 50 mM sodium acetate, pH-4.0. Time-course hydrolysis of crystalline α- and β-chitin with the Paenibacillus sp. LS1 enzyme-cocktail produced N-acetylglucosamine and N,N'-diacetylchitobiose as the predominant products. Taken together, our results confirm that the Paenibacillus sp. LS1 enzyme-cocktail would have potential application in eco-friendly enzymatic approaches for efficient saccharification of crystalline chitin.

Time-course of human cholinesterases-catalyzed competing substrate kinetics

Competing substrate kinetic analysis of human butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) from the time-course of enzyme-catalyzed substrate hydrolysis, using spectrophotometric assays is described. This study is based on the use of a chromogenic reporter “visible” substrate (substrate A), whose complete hydrolysis time course is retarded by a competing “invisible” substrate (substrate B). For BChE, four visible substrates were used, two thiocholine esters, benzoylthiocholine and butyrylthiocholine, and two aryl-acylamides, o-nitro trifluoro acetaminide and 3-(acetamido)-N,N,N-trimethylanilinium. Three different competing invisible substrates were used, phenyl acetate, acetylcholine and butyrylcholine. For AChE, two visible substrates were used, acetylthiocholine and 3-(acetamido)-N,N,N-trimethylanilinium. For AChE, acetylcholine was competing with visible substrates.The ratio (R) of bimolecular rate constants, kcat/Km, for all couples of substrates, invisible/visible (B/A) covered all possible limit situations, R ≪ 1, R ≈ 1 and R ≫ 1.The kinetic approach, based on the method developed by Golicnik and Masson allowed determination of binding and catalytic parameters of cholinesterases for both visible and invisible substrates. This analysis was applied to michaelian and non-michaelian catalytic behaviors (activation and inhibition by excess substrate). Reevaluation of catalytic parameters obtained for acetylcholine and butyrylcholine more than 50 years ago was made.The method is fast, reliable, and particularly suitable for poorly soluble substrates and for substrates B when no direct spectrophotometric assays exist. Moreover, replacing substrate B by a reversible inhibitor, mechanism of cholinesterase inhibition was possible to study. It is therefore, useful for screening libraries of new substrates and inhibitors, and/or screening of new cholinesterase mutants. This method can be applied to any other enzymes.

The impact of kinetic parameters on cellulose hydrolysis rates

Abstract Mechanistic kinetic models are vital tools to elucidate rate-limiting interactions and processes hampering complete hydrolysis of cellulose by cellulases. Properly formulated, the models should ultimately simulate experimental cellulose hydrolysis time courses given that model parameters are known a priori. Here, we review reported values of critical parameters in cellulose hydrolysis (catalytic, adsorption/desorption, complexation/decomplexation rate constants and apparent processivity) and examine how estimation methods may impact the magnitudes and interpretation of the values. The sensitivity of the extents and rates of cellulose hydrolysis to the range of reported values were examined by conducting simulations with a general mechanistic model of TrCel7A hydrolysis of cellulose. When the simulation was conducted at enzyme-limiting conditions, initial bursts in cellulose hydrolysis rates were evident and highly sensitive to specific hydrolysis rates of productively bound enzymes and dissociation rates of non-productively bound enzymes. Under substrate limiting conditions, hydrolysis rates were significantly reduced and no initial burst was observed, with more pronounced sensitivity of hydrolysis rates to the range of kinetic values. Enzyme-substrate interaction rate constants reviewed here only capture short-time hydrolysis rate outcomes and future work to elucidate mechanisms of cellulose hydrolysis must also incorporate how substrate-limitations evolve over the course of hydrolysis.

Why does GH10 xylanase have better performance than GH11 xylanase for the deconstruction of pretreated biomass?

One approach to improve biomass deconstruction is to formulate a more efficient cellulase cocktail by adding “accessory enzymes” (e.g. xylanase/LPMO/laccases). Among different xylanases, glycoside hydrolase family 10 endo-xylanase (GH10EX) shows better performance than family 11 endo-xylanase (GH11EX) even though GH11EX has better kinetic activity on various xylan substrates. To better understand this phenomenon, the xylan accessibility of GH10/11 xylanases was assessed on various “model” and realistic cellulosic substrates and their thermostability was also compared during time course of hydrolysis. It showed that GH10EX had higher accessibility towards the xylan backbone within pretreated biomass, especially for these with higher acetyl group content. Acetyl group removal could greatly intensify the synergistic cooperation between GH11EX and cellulases. Additionally, the higher thermostability of GH10EX appeared to be another reason for its outstanding potential during biomass decomposition. This work provides further insights for engineering better biocatalysts to enhance the economic viability of enzyme based biorefinery.

Aggregation and pH Responsive Behavior of Thioester Surfactants and Formation of Disulfide Linkages in Aqueous Solutions.

pH responsive surfactants, [C12H25N(CH3)2(CH2)nSCOCH3]Br (C12nSAc, n = 4, 11, 12), were prepared, and their properties in aqueous solution were examined. The critical micelle concentration (cmc) and critical vesicle concentration (cvc) were determined based on changes in conductivity, as well as by fluorescence measurements, and light scattering methods. A significant increase in the light scattering intensities of the C12nSAc (n=11, 12) systems suggested that the growth of aggregates was accompanied by considerable counterion binding with increasing surfactant concentration. The diameter of C1211SAc, recorded by the dynamic light scattering measurements, was about 9.6 ±1.0 nm, which was slightly smaller than that for didodecyldimethylammonium bromide (DDAB) vesicles. The thioester group was easily hydrolyzed upon the addition of NaOH, while it was hardly hydrolyzed with the addition of HCl. The time course of alkaline hydrolysis was examined by the conductivity measurements and high-performance liquid chromatography analysis. [C12H25N(CH3)2(CH2)11SS(CH2)11N(CH3)2C12H25]2Br (2C1211SS) was generated in the C1211SAc alkaline solution because of air oxidation. The C1211SAc alkaline solution gradually became an opaque blue color with increasing light scattering at 346 nm, indicating the remarkable growth of vesicles. The chemical structure of 2C1211SS was consistent with that of a disulfide linked double tailed surfactant, similar to DDAB. The disulfide linkage between the double tailed surfactants will contribute to the stabilization and growth of vesicles.

Interaction of carbapenems and β-lactamase inhibitors towards CTX-M-15 and CTX-M-15G238C mutant.

The aim of this study was to evaluate the role of residue 238 in CTX-M-15 and CTX-M-15G238C mutant with respect to carbapenems and various β-lactamase inhibitors. A CTX-M-15G238C laboratory mutant was generated by site-directed mutagenesis from CTX-M-15 enzyme by replacing glycine 238 with cysteine. Thiol titration and p-chloromercuribenzoate (PCMB) inactivation assays were used to ascertain the presence of a disulfide bridge in the active site of CTX-M-15G238C. Kinetic parameters were determined both for CTX-M-15 and CTX-M-15G238C enzymes by analysing either the complete hydrolysis time courses or under initial rate conditions. In CTX-M-15G238C mutant, the two cysteines (C69 and C238) located in the enzyme active site were unable to form a disulfide bridge. CTX-M-15 and thermostable CTX-M-15G238C were used to study the kinetic interaction with carbapenems, which behaved as poor substrates for both enzymes. Meropenem and ertapenem acted as transient inactivators for CTX-M-15 and CTX-M-15G238C, and for these compounds the variation of kobs versus the inactivator concentration was linear. Imipenem behaved as a transient inactivator for CTX-M-15 and as an inactivator (with k+3=0) for CTX-M-15G238C. In any case, the k+2/K values for CTX-M-15G238C were higher than those for CTX-M-15. Compared with CTX-M-15, CTX-M-15G238C mutant appears to have a more favourable conformation for carbapenem acylation and higher activity against cefotaxime, which could be due to the presence of free -SH groups in the enzyme active site.

Mechanistic kinetic models of enzymatic cellulose hydrolysis-A review.

Bioconversion of lignocellulose forms the basis for renewable, advanced biofuels, and bioproducts. Mechanisms of hydrolysis of cellulose by cellulases have been actively studied for nearly 70 years with significant gains in understanding of the cellulolytic enzymes. Yet, a full mechanistic understanding of the hydrolysis reaction has been elusive. We present a review to highlight new insights gained since the most recent comprehensive review of cellulose hydrolysis kinetic models by Bansal et al. (2009) Biotechnol Adv 27:833-848. Recent models have taken a two-pronged approach to tackle the challenge of modeling the complex heterogeneous reaction-an enzyme-centric modeling approach centered on the molecularity of the cellulase-cellulose interactions to examine rate limiting elementary steps and a substrate-centric modeling approach aimed at capturing the limiting property of the insoluble cellulose substrate. Collectively, modeling results suggest that at the molecular-scale, how rapidly cellulases can bind productively (complexation) and release from cellulose (decomplexation) is limiting, while the overall hydrolysis rate is largely insensitive to the catalytic rate constant. The surface area of the insoluble substrate and the degrees of polymerization of the cellulose molecules in the reaction both limit initial hydrolysis rates only. Neither enzyme-centric models nor substrate-centric models can consistently capture hydrolysis time course at extended reaction times. Thus, questions of the true reaction limiting factors at extended reaction times and the role of complexation and decomplexation in rate limitation remain unresolved. Biotechnol. Bioeng. 2017;114: 1369-1385. © 2017 Wiley Periodicals, Inc.

A novel thermostable and halotolerant xylanase from Colletotrichum graminicola

A novel endoxylanase from Colletotrichum graminicola (Excg1) was purified. Similar apparent molecular masses were estimated by gel filtration (17.3±1.9kDa) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (20.0±2.4kDa), suggesting that Excg1 is monomeric. The enzyme showed good halotolerance, retaining about 85% and 50% of the control activity in the presence of 0.5molL−1 and 3.0molL−1 NaCl, respectively. The optimum temperature of Excg1 (65°C) was not affected by NaCl, but the optimum pH rose from 5.5 in the absence and presence of 0.5molL−1 NaCl to 6.0, in 2.5molL−1 NaCl. Excg1 was highly thermostable at 50°C, with half-lives around 48h in either water or 0.5molL−1 NaCl and a residual activity of 75% at 2.5molL−1 NaCl. Excg1 was fully stable at pH 3.0–10.0 in the absence of salt, and from pH 4.0–10.0 in the presence of 0.5molL−1 and 2.5molL−1 NaCl. The enzyme hydrolyzed beechwood xylan with maximal velocity and apparent affinity constant of 481.3±34.0Umg−1 and 3.7±0.3mgmL−1, respectively. Similar kinetic parameters were obtained in the presence of 0.5molL−1 NaCl, but a maximum velocity about 34% lower was determined in 2.5molL−1 NaCl. Excg1 was tolerant to various organic solvents at a concentration of 5% (w/v) and also to sodium acetate up to 200mmolL−1. Xylobiose and xylotriose with a 4-O-methylglucuronic acid branching were the main products of beechwood xylan hydrolysis, and the time course of hydrolysis was not affected by NaCl 0.5molL−1 or sea water. To the best of our knowledge, this is the first report of a halotolerant endoxylanase from fungal origin. The properties of Excg1 suggested good potential for use in lignocellulose saccharification processes particularly using sea water or under high salt conditions, or in the presence of residues and/or byproducts of pretreatment steps, contributing to improve the economic viability of 2G ethanol production.

What Are the Major Components in Steam Pretreated Lignocellulosic Biomass That Inhibit the Efficacy of Cellulase Enzyme Mixtures?

Any pretreat process used on lignocellulosic substrates, such as steam pretreatment, although opening up and enhancing access to the cellulose, will typically generate inhibitory compounds (i.e., soluble mono/oligomeric sugars, phenolics, furans, extractives, etc.) that limit or restrict the efficiency of enzyme mediated cellulose hydrolysis. In the work reported here, the major groups of inhibitors derived from various, “real-life” steam pretreated lignocellulosic biomass were identified and their inhibitory effects on the rate and extent of enzymatic hydrolysis were assessed. It was apparent that monomeric sugars and phenolics inhibited the hydrolytic potential of the cellulases most severely, with sugar accounting for the majority of the decrease. The inhibitory effect of the monomeric sugars was consistent and concentration (but not composition) dependent throughout the time course of hydrolysis. In contrast, the influence of the phenolics was substrate dependent and more harmful at the later stage of...

Analysis of the operational strategies for the enzymatic hydrolysis of food proteins in batch reactor

The aim of this work was to validate a mathematical model and evaluate hydrolysis of salmon muscle proteins by Alcalase in batch reactor. The inhibition constants were correlated with conversion and temperature thus improving dramatically the agreement between model predictions and experimental hydrolysis time courses. The standard operation in batch reactor consisting in pre-heating, isothermal stage and inactivation of enzyme was evaluated through simulation of different operational strategies based on increasing temperature gradients. Operational conditions were 7.5%(w/w) protein, 1 g/l Alcalase and different temperatures from 50 °C to 73 °C. The optimal temperature for the standard process was 68 °C with a processing time of 41 min. Different operational strategies, included temperature profiles, resulted in longer operation times. The standard process was the best reactor performance due to the maximization of the enzyme efficiency.

Cloning, Expression, and Characterization of a Thermophilic Endoglucanase, AcCel12B from Acidothermus cellulolyticus 11B.

The gene ABK52392 from the thermophilic bacterium Acidothermus cellulolyticus 11B was predicted to be endoglucanase and classified into glycoside hydrolase family 12. ABK52392 encodes a protein containing a catalytic domain and a carbohydrate binding module. ABK52392 was cloned and functionally expressed in Escherichia coli. After purification by Ni-NTA agarose affinity chromatography and Q-Sepharose® Fast Flow chromatography, the properties of the recombinant protein (AcCel12B) were characterized. AcCel12B exhibited optimal activity at pH 4.5 and 75 °C. The half-lives of AcCel12B at 60 and 70 °C were about 90 and 2 h, respectively, under acidic conditions. The specific hydrolytic activities of AcCel12B at 70 °C and pH 4.5 for sodium carboxymethylcellulose (CMC) and regenerated amorphous cellulose (RAC) were 118.3 and 104.0 U·mg−1, respectively. The Km and Vmax of AcCel12B for CMC were 25.47 mg·mL−1 and 131.75 U·mg−1, respectively. The time course of hydrolysis for RAC was investigated by measuring reducing ends in the soluble and insoluble phases. The total hydrolysis rate rapidly decreased after the early stage of incubation and the generation of insoluble reducing ends decreased earlier than that of soluble reducing ends. High thermostability of the cellulase indicates its potential commercial significance and it could be exploited for industrial application in the future.

Effect of pretreatment severity in continuous steam explosion on enzymatic conversion of wheat straw: Evidence from kinetic analysis of hydrolysis time courses

Focusing on continuous steam explosion, the influence of pretreatment severity due to varied acid loading on hydrolysis of wheat straw by Trichoderma reesei cellulases was investigated based on kinetic evaluation of the saccharification of each pretreated substrate. Using semi-empirical descriptors of the hydrolysis time course, key characteristics of saccharification efficiency were captured in a quantifiable fashion. Not only hydrolysis rates per se, but also the transition point of their bi-phasic decline was crucial for high saccharification degree. After 48h the highest saccharification was achieved for substrate pretreated at relatively low severity (1.2% acid). Higher severity increased enzyme binding to wheat straw, but reduced the specific hydrolysis rates. Higher affinity of the lignocellulosic material for cellulases does not necessarily result in increased saccharification, probably because of lignin modifications occurring at high pretreatment severities. At comparable severity, continuous pretreatment produced a substrate more susceptible to enzymatic hydrolysis than the batch process.

Extracellular production of a novel endo-β-agarase AgaA from Pseudomonas vesicularis MA103 that cleaves agarose into neoagarotetraose and neoagarohexaose.

The gene agaA, of the isolated marine bacterium Pseudomonas vesicularis MA103, comprised 2958-bp nucleotides encoding a putative agarase AgaA of 985 amino acids, which was predicted to contain a signal peptide of 29 amino acids in the N-terminus, a catalytic domain of glycoside hydrolase 16 (GH16) family, a bacterial immunoglobulin group 2 (Big 2), and three carbohydrate binding modules 6 (CBM 6). The gene agaA was cloned and overexpressed in Escherichia coli, and the optimum temperatures for AgaA overexpression were 16, 20 and 24 °C. The agaA was cloned without its signal peptide for cytosolic production overexpression, whereas it was cloned with the heterologous signal peptide PelB and its endogenous signal peptide for periplasmic and extracellular productions, respectively. Extracellular and periplasmic rAgaA showed greater activity than that of cytosolic rAgaA, indicating that membrane translocation of AgaA may encourage proper protein folding. Time-course hydrolysis of agarose by rAgaA was accomplished and the products were analyzed using thin layer chromatography and matrix-assisted laser desorption inoization-time of flight mass spectrometry, indicating that AgaA from P. vesicularis was an endo-type β-1,4 agarase that cleaved agarose into neoagarotetraose and neoagarohexaose as the final products.

Novel modeling methodology for the characterization of enzymatic hydrolysis of proteins

A novel methodology for the modeling and characterization of the enzymatic hydrolysis of proteins is proposed. The hydrolysis time course can be predicted at different operating conditions for protein concentration, enzyme concentration and temperature. The hydrolysis kinetics of salmon muscle proteins and whey protein isolate by Alcalase were studied using a central composite design. A combination of the logarithmic equation P=1/bln (abt+1) to model the hydrolysis time course with the response surface methodology to correlate the kinetic constants a and b with the operating conditions: protein concentration, enzyme concentration and temperature were achieved. The logarithmic equation was a very good fit with the hydrolysis time courses, with both substrates achieving R2>0.995. The kinetic constants a and b were significantly affected by the operating conditions. Empirical models were obtained for a and b as functions of operating conditions. The kinetic constant values were predicted, and a strong correlation between predicted and experimental values was obtained for a (R2=0.949) and b (R2=0.945). The predicted and experimental time courses resulted in good correlation for both salmon muscle proteins (R2>0.987) and whey protein isolate (R2>0.978). The model allowed calculation of the hydrolysis time course of proteins from a given set of operating conditions with good predictability. This methodology can be used with different sources of proteins and enzymes to test the susceptibility of proteins to hydrolysis as well as the catalytic efficiency of proteases. Additionally, the combined model can be used as a design and optimization function.

Visualizing ATP hydrolysis in a viral DNA-packaging molecular motor

Many DNA viruses encode powerful molecular machines to package viral genome into preformed protein shells. These DNA-packaging motors contain an ATPase module that converts the chemical reaction of ATP hydrolysis to physical motion of DNA. We previously determined the structures of the DNA-packaging motor gp2 of Shigella phage Sf6 in the apo form and in complex with ADP and ATP-gamma-S (Zhao et al, 2013, PNAS, 110, 8075). Here we report the structure of gp2 in complex with its substrate ATP at 2.0 Angstrom resolution. To our knowledge, this is the first time to capture, at high resolution, a precatalytic state for ASCE-superfamily ATPases, which include a large group of nucleic acid helicases and translocases involved in a broad range of cellular and viral processes. The structure reveals the precise architecture of the ATP-bound state of the motor immediately prior to catalysis. Comparison with structures of the apo and ADP-complexed forms unveils motions of the Walker A motif coupled with ATP and Mg2+ binding and ATP hydrolysis. In the Walker B motif, residue E118 undergoes a side chain conformational switching coupled with the ATP hydrolysis, whereas residue E119 locks residue R51 side chain to a conformation that is readily reachable to residue E118 side chain. Residue E121 in the Walker B motif deprotonates a water molecule, which acts as a nucleophile to attack the gamma-phosphorous, leading to ATP hydrolysis. The alpha-helix (residue G182-R194) in the linker domain undergoes a translational motion against the ATPase domain triggered by ATP hydrolysis, serving as a mechanism for translating the energy from the chemical reaction into physical movement of DNA. We further observed the time course of ATP hydrolysis by gp2 by determining structures of gp2:ATP complexes captured at various incubation time. These structures have made it possible to delineate, at atomic detail, the complete cycle of ATP hydrolysis of this viral DNA-packaging molecular motor.

Dissecting the effect of chemical additives on the enzymatic hydrolysis of pretreated wheat straw

Chemical additives were examined for ability to increase the enzymatic hydrolysis of thermo-acidically pretreated wheat straw by Trichoderma reesei cellulase at 50°C. Semi-empirical descriptors derived from the hydrolysis time courses were applied to compare influence of these additives on lignocellulose bioconversion on a kinetic level, presenting a novel view on their mechanism of action. Focus was on rate retardation during hydrolysis, substrate conversion and enzyme adsorption. PEG 8000 enabled a reduction of enzyme loading by 50% while retaining the same conversion of 67% after 24h. For the first time, a beneficial effect of urea is reported, increasing the final substrate conversion after 48h by 16%. The cationic surfactant cetyl-trimethylammonium bromide (CTAB) enhanced the hydrolysis rate at extended reaction time (rlim) by 34% and reduced reaction time by 28%. A combination of PEG 8000 and urea increased sugar release more than additives used individually.

A thermo-halo-tolerant and proteinase-resistant endoxylanase from Bacillus sp. HJ14.

A glycosyl hydrolase family 10 endoxylanase from Bacillus sp. HJ14 was grouped in a separated cluster with another six Bacillus endoxylanases which have not been characterized. These Bacillus endoxylanases showed less than 52% amino acid sequence identity with other endoxylanases and far distance with endoxylanases from most microorganisms. Signal peptide was not detected in the endoxylanase. The endoxylanase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant enzyme (rXynAHJ14) was characterized. rXynAHJ14 was apparent optimal at 62.5 °C and pH 6.5 and retained more than 55% of the maximum activity when assayed at 40-75 °C, 23% at 20 °C, 16% at 85 °C, and even 8% at 0 °C. Half-lives of the enzyme were more than 60 min, approximately 25 and 4 min at 70, 75, and 80 °C, respectively. The enzyme exhibited more than 62% xylanase activity and stability at the concentration of 3-30% (w/v) NaCl. No xylanase activity was lost after incubation of the purified rXynAHJ14 with trypsin and proteinase K at 37 °C for 60 min. Different components of oligosaccharides were detected in the time-course hydrolysis of beechwood xylan by the enzyme. During the simulated intestinal digestion phase in vitro, 11.5-19.0, 15.3-19.0, 21.9-27.7, and 28.2-31.2 μmol/mL reducing sugar were released by the purified rXynAHJ14 from soybean meal, wheat bran, beechwood xylan, and rapeseed meal, respectively. The endoxylanase might be an alternative for potential applications in the processing of sea food and saline food and in aquaculture as agastric fish feed additive.