Papers by khawar siddiqui
Two isoenzymes of endo-1,4-β-xylanase (EC 3.2.1.8) from Scopulariopsis sp. were purified by a com... more Two isoenzymes of endo-1,4-β-xylanase (EC 3.2.1.8) from Scopulariopsis sp. were purified by a combination of ammonium sulfate precipitation, hydrophobic interaction, and anion-exchange and gel filtration chromatography. The native mol wts of the least acidic xylanase (LAX) and the highly acidic xylanase (HAX) were 25 and 144 kDa and the subunit mol wts were 25 and 36 kDa, respectively. The kcat values of LAX and HAX for oat-spelt xylan at 40°C, pH 6.5, were 95,000 and 9900 min–1 and the Km values of LAX and HAX were 30 and 3.3 mg/mL. The thermodynamic activation parameters of xylan hydrolysis showed that the high activity of LAX when compared
with HAX was not owing to a reduction in ΔH# but was entropically driven. High-performance liquid chromatography analysis of the degradation products showed that LAX formed both xylotrioses and xylobioses, but HAX predominantly formed xylotrioses. The half-lives of LAX and HAX at 50°C in 50 mM 2-N-morpholino ethanesulfonic acid (MES), pH 6.5 buffer
were 267 and 69 min, respectively. Thermodynamic analysis showed that at lower temperatures, the increased thermostability of LAX (ΔH# = 306 kJ/mol) compared with HAX (ΔH# = 264 kJ/mol) was owing to more noncovalent surface interactions. At higher temperatures, LAX (ΔS* = –232 J/[mol·K])
was more thermostable than HAX (ΔS* = 490 J/[mol·K]) owing to a more ordered transition-state conformation. An energy-activity diagram was introduced showing that kcat/Km does not successfully explain the true kinetic behavior of both xylanase isoenzymes. The simultaneously thermostable and highly active LAX could be utilized in biotechnological processes involving xylan hydrolysis.
The full biotechnological exploitation of enzymes is still hampered by their low activity, low st... more The full biotechnological exploitation of enzymes is still hampered by their low activity, low stability and high cost. Temperature-dependent catalytic properties of enzymes are a key to efficient and cost-effective translation to commercial applications. Organisms adapted to temperature extremes are a rich source of enzymes with broad ranging thermal properties which, if isolated, characterized and their structure–function–stability relationship elucidated, could underpin a variety of technologies. Enzymes from thermally-adapted organisms such as psychrophiles (low-temperature) and thermophiles (high-temperature) are a vast natural resource that is already under scrutiny for their biotechnological potential. However, psychrophilic and thermophilic enzymes show an activity–stability trade-off that necessitates the use of various genetic and chemical modifications to further improve their properties to suit various industrial applications. This review describes in detail the properties and biotechnological applications of both cold-adapted and thermophilic enzymes. Furthermore, the review critically examines ways to improve their value for biotechnology, concluding by proposing an integrated approach involving thermally-adapted, genetically and magnetically modified enzymes to make biocatalysis more efficient and cost-effective.
tMetalloproteases represent the largest fraction of the global enzyme market. For biotechnologica... more tMetalloproteases represent the largest fraction of the global enzyme market. For biotechnological pur-poses the accumulation of product (i.e. productivity) provides the best measure of assessing enzymeperformance because it takes into account the interplay between activity, stability, activation and inhi-bition. Studies assessing the productivity of alkaline metalloproteases and chemicals that improve theirproductivity have not previously been reported. In this study we report the specificity, productiv-ity, kinetic and thermodynamic properties of an extracellular protease, purified from a new strain ofPseudomonas sp. isolated from refrigerated milk. Mass spectrometry analysis revealed the enzyme is aserralysin-type alkaline metalloprotease, with broad cleavage-site specificity. By studying the effects ofCa2+ion removal (using a chelator) and Ca2+ion addition, conditions were identified that led to an increasein productivity by 300% (6.3 vs 1.9 mg azopeptide g−1enzyme at 40◦C). The basis for the enhanced pro-ductivity was linked to elevated melting temperatures of secondary (Tm47 vs 38◦C) and tertiary structure(Tm50 vs 44◦C), increased half-life of inactivation (t1/230 vs 4.9 min), increased optimum temperature(44 vs 36◦C), and changes in both catalytic activity (kcat3.3 vs 2.2 min−1) and substrate affinity (Km3.9vs 2.5 mg mL−1). Thermodynamic data were indicative of Ca2+-binding causing the transition-state to bemore ordered (less entropy) relative to the folded-state, thereby resisting a transition to an unfoldedstate. The specificity, kinetics and response to calcium of this AMP illustrate its potential usefulness forindustrial applications, and the research highlights the broader potential for using calcium to enhancethe productivity of proteases.
The use of plant biomass as feedstock for biomaterial and biofuel production is relevant in the c... more The use of plant biomass as feedstock for biomaterial and biofuel production is relevant in the current bio-based economy scenario of valorizing renewable resources. Fungi, which degrade complex and recalcitrant plant polymers, secrete different enzymes that hydrolyze plant cell wall polysaccharides. The present review discusses the current research trends on fungal, as well as extremophilic cell wall hydrolases that can withstand extreme physico-chemical conditions required in efficient industrial processes. Secretomes of fungi from the phyla Ascomycota, Basidiomycota, Zygomycota and Neocallimastigomycota are presented along with metabolic cues (nutrient sensing, coordination of carbon and nitrogen metabolism) affecting their composition. We conclude the review by suggesting further research avenues focused on the one hand on a comprehensive analysis of the physiology and epigenetics underlying cell wall degrading enzyme production in fungi and on the other hand on the analysis of proteins with unknown function and metagenomics of extremophilic consortia. The current advances in consolidated bioprocessing, altered secretory pathways and creation of designer plants are also examined. Furthermore, recent developments in enhancing the activity, stability and reusability of enzymes based on synergistic, proximity and entropic effects, fusion enzymes, structure-guided recombination between homologous enzymes and magnetic enzymes are considered with a view to improving saccharification.
The full biotechnological exploitation of enzymes is still hampered by their low activity, low st... more The full biotechnological exploitation of enzymes is still hampered by their low activity, low stability and high cost. Temperature-dependent catalytic properties of enzymes are a key to efficient and cost-effective translation to commercial applications. Organisms adapted to temperature extremes are a rich source of enzymes with broad ranging thermal properties which, if isolated, characterized and their structure–function–stability relationship elucidated, could underpin a variety of technologies. Enzymes from thermally-adapted organisms such as psychrophiles (low-temperature) and thermophiles (high-temperature) are a vast natural resource that is already under scrutiny for their biotechnological potential. However, psychrophilic and thermophilic enzymes show an activity–stability trade-off that necessitates the use of various genetic and chemical modifications to further improve their properties to suit various industrial applications. This review describes in detail the properties and biotechnological applications of both cold-adapted and thermophilic enzymes. Furthermore, the review critically examines ways to improve their value for biotechnology, concluding by proposing an integrated approach involving thermally-adapted, genetically and magnetically modified enzymes to make biocatalysis more efficient and cost-effective.
PLoS ONE, 2014
Calcium (Ca(2+)) has an important structural role in guaranteeing the integrity of the outer lipo... more Calcium (Ca(2+)) has an important structural role in guaranteeing the integrity of the outer lipopolysaccharide layer and cell walls of bacterial cells. Extracellular DNA (eDNA) being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca(2+) and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca(2+) binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca(2+) had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca(2+) at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (ΔG) values in iTC data confirmed that the interaction between DNA and Ca(2+) is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca(2+) alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca(2+) to eDNA thereby mediating bacterial aggregation and biofilm formation.
Methods in Microbiology, 2006
World Journal of Microbiology & Biotechnology, 1996
Removal of non-covalently attached polysaccharides from carboxymethylcellulase (CMCase) of Asperg... more Removal of non-covalently attached polysaccharides from carboxymethylcellulase (CMCase) of Aspergillus niger improved its activity but decreased its thermostability and protease resistance. The activation energy profile of the hydrolysis of carboxymethylcellulose (CMC) was triphasic with increasing values of 17,-55 and-562 kJ/mol for polysaccharide-free and 19, -21 and -207 kJ/mol for polysaccharide-complexed CMCase. The specificity constant (Vmax/Km) of polysaccharide-free CMCase was 1.41 compared to polysaccharide-complexed CMCase which was only 0.68. The polysaccharide free CMCase had lower thermostability ('melting point' = 82°C) and higher protease susceptibility compared to polysaccharide-complexed CMCase ('melting point'>100°C).
Journal of Bacteriology, 2007
Archaea are abundant and drive critical microbial processes in the Earth's cold biosphere. Despit... more Archaea are abundant and drive critical microbial processes in the Earth's cold biosphere. Despite this, not enough is known about the molecular mechanisms of cold adaptation and no biochemical studies have been performed on stenopsychrophilic archaea (e.g., Methanogenium frigidum). This study examined the structural and functional properties of cold shock proteins (Csps) from archaea, including biochemical analysis of the Csp from M. frigidum. csp genes are present in most bacteria and some eucarya but absent from most archaeal genome sequences, most notably, those of all archaeal thermophiles and hyperthermophiles. In bacteria, Csps are small, nucleic acid binding proteins involved in a variety of cellular processes, such as transcription. In this study, archaeal Csp function was assessed by examining the ability of csp genes from psychrophilic and mesophilic Euryarchaeota and Crenarchaeota to complement a cold-sensitive growth defect in Escherichia coli. In addition, an archaeal gene with a cold shock domain (CSD) fold but little sequence identity to Csps was also examined. Genes encoding Csps or a CSD structural analog from three psychrophilic archaea rescued the E. coli growth defect. The three proteins were predicted to have a higher content of solvent-exposed basic residues than the noncomplementing proteins, and the basic residues were located on the nucleic acid binding surface, similar to their arrangement in E. coli CspA. The M. frigidum Csp was purified and found to be a single-domain protein that folds by a reversible two-state mechanism and to exhibit a low conformational stability typical of cold-adapted proteins. Moreover, M. frigidum Csp was characterized as binding E. coli single-stranded RNA, consistent with its ability to complement function in E. coli. The studies show that some Csp and CSD fold proteins have retained sufficient similarity throughout evolution in the Archaea to be able to function effectively in the Bacteria and that the function of the archaeal proteins relates to cold adaptation. The initial biochemical analysis of M. frigidum Csp has developed a platform for further characterization and demonstrates the potential for expanding molecular studies of proteins from this important archaeal stenopsychrophile.
IUBMB Life (International Union of Biochemistry and Molecular Biology: Life), 2004
In most studies of enzyme kinetics it has been found sufficient to use the classical Transition S... more In most studies of enzyme kinetics it has been found sufficient to use the classical Transition State Theory (TST) of Eyring and others. This theory was based on the solvent being an ideal dilute substance treated as a heat bath. However, enzymes found in organisms adapted to very low (psychrophiles) and very high (thermophiles) temperatures are also subjected to variable solute concentrations and viscosities. Therefore, the TST may not always be applicable to enzyme reactions carried out in various solvents with viscosities ranging from moderate to very high. There have been numerous advances in the theory of chemical reactions in realistic non-ideal solvents such as Kramers Theory. In this paper we wish to propose a modified thermodynamic equation, which have contributions from k cat , K m and the viscosity of the medium in which the enzyme reaction is occurring. These could be very useful for determining the thermodynamics of enzymes catalyzing reactions at temperature extremes in the presence of substrate solutions of different compositions and viscosities.
Folia Microbiologica, 1996
The removal of noncovalently bound polysaccharide coating from the extracellular enzymes of Asper... more The removal of noncovalently bound polysaccharide coating from the extracellular enzymes of Aspergillus niger, by the technique of compartmental eleetrophoresis, had a very dramatic effect on the stability of 13-glucosidase. The polysaceharide-~-glucosidase complex was extremely resistant to proteinases and far more stable against urea and temperature as compared with polysaccharide-free ~giucosidase. The I$-glucosidase-polysaccharide complex was 18-, 36-, 40-and 82-fold more stable against chymotrypsin, 3 mol/L urea, total thermal denaturation and irreversible thermal denaturation, respectively, as compared with polysaccharide-free Igglucosidase. The activation energy of polysaccharide-complexed 13-glucosidase (55 kJ/mol) was lower than polysaccharide-free enzyme (61 kJ/moi), indicating a slight activation of the enzyme by the polysaccharide. No significant difference could be detected in the specificity constant (V/Km) for 4-nitrophenyi ~-D-glucopyranoside between polysaccharide-free and polysaccharide-complexed 15-glucosidase. We suggest that the function of these polysaccharides secreted by fungi including A. niger might be to protect the extraceUular enzymes from proteolytic degradation, hence increasing their life span.
Folia Microbiologica, 1997
Purified ~glucosidase from Ceilulomonas biazotea had an apparent Km and V for 2-nitrophenyl ~I>-g... more Purified ~glucosidase from Ceilulomonas biazotea had an apparent Km and V for 2-nitrophenyl ~I>-glucopyranoside (oNPG) of 0A16 mmol/L and 0.22 U/rag protein, respectively. The activation energy for the hydrolysis of pNPG of I$-glueo6idase was 65 kJ/mol. The inhibition by Mn 2+ vs. oNPG of parental ~glucosidase was of mixed type with apparent inhibition constants of 0.19 and 0.60 lamol/L for the enzyme and enzyme-substrate complex, respectively. Ethanol at lower concentrations activated while at higher concentrations it inhibited the enzyme. The determination of apparent pKa's at different temperatures and in the presence of 30 % dioxane indicated two carboxyt groups which control the Vvalue. The thermal stability of 13-glucosidase decreased in the presence of 10 % ethanol. The half-life of 13-glucosidase in 1.75 moi/L urea at 35 ~ was 145 rain, as determined by 0-9 mol/L transverse urea gradient-PAGE.
Extremophiles, 2005
In order to improve the thermal stability (t 1/2 ) and activity of lipase B from cold-adapted Can... more In order to improve the thermal stability (t 1/2 ) and activity of lipase B from cold-adapted Candida antarctica (CALB), amino groups of the enzyme were chemically linked to a range of oxidized polysaccharides using a range of reducing agents. By chemically modifying CALB using 0.1% dextran (250 kDa) at pH 8.6 for 10 days using borane-pyridine complex as reducing agent, increased thermal stability (t 1/2 , 168 min at 70°C) and activity (65% higher specific activity) was achieved compared to the unmodified enzyme (t 1/2 , 18 min at 70°C). Improvements in thermostability were generally better with high molecular weight polymers such as dextran (40 and 250 kDa) or ficoll (70 and 400 kDa) in comparison to low molecular weight inulin (5 kDa). The shape of the polymer also appeared to be important with elongated, elipsoidal-shaped dextran providing better thermostabilization than spherical-shaped ficoll. Borane-pyridine complex was found to be a good, non-toxic reducing agent for improving thermostability, compared with sodium borohydride and sodium cyanoborohydride. An interesting finding was that, in all cases, specific activity of the modified enzymes increased with a concomitant increase in thermostability. This response defies the general principle of a trade-off between activity and stability, and demonstrates that chemical modification provides new avenues for improving the thermal stability of enzymes from psychrophiles without sacrificing their activity.
Enzyme and Microbial Technology, 2000
Carboxymethylcellulase (CMCase) from Aspergillus niger NIAB280 was purified by a combination of a... more Carboxymethylcellulase (CMCase) from Aspergillus niger NIAB280 was purified by a combination of ammonium sulphate precipitation, ion-exchange, hydrophobic interaction and gel filtration chromatography on FPLC with 9-folds increase in specific activity. Native and subunit molecular weights were found to be 36 kDa each. The purified CMCase was modified by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) in the presence of glycinamide for 15 min (GAM15) and glycinamide plus cellobiose for 75 min (GAM75). Similarly, the enzyme was modified by EDC in the presence of ethylenediamine dihydrochloride plus cellobiose for 75 min (EDAM75). The neutralization (GAM15 and GAM75) and reversal (EDAM75) of negative charges of carboxyl groups of CMCase had profound effect on the specificity constant (k cat /K m ), pH optima, pK a 's of the active-site residues and thermodynamic parameters of activation. The specificity constants of native, GAM15, GAM75, and EDAM75 were 143, 340, 804, and 48, respectively. The enthalpy of activation (⌬H # ) of Carboxymethylcellulose (CMC) hydrolysis of native (50 and 15 kJ mol Ϫ1 ) and GAM15 (41 and 16 kJ mol Ϫ1 ) were biphasic whereas those of GAM75 (43 kJ mol Ϫ1 ) and EDAM75 (41 k J mol Ϫ1 ) were monophasic. Similarly, the entropy of activation (⌬S # ) of CMC hydrolysis of native (Ϫ61 and Ϫ173 J mol Ϫ1 K Ϫ1 ) and GAM15 (Ϫ91 and Ϫ171 J mol Ϫ1 K Ϫ1 ) were biphasic whereas those of GAM75 (Ϫ82 J mol Ϫ1 K Ϫ1 ) and EDAM75 (Ϫ106 J mol Ϫ1 K Ϫ1 ) were monophasic. The pH optima/pK a 's of both acidic and basic limbs of charge neutralized CMCases increased compared with those of native enzyme. The CMCase modification in the presence of glycinamide and absence of cellobiose at different pH's periodically activated and inhibited the enzyme activity indicating conformational changes. We believe that the alteration of the surface charges resulted in gross movement of loops that surround the catalytic pocket, thereby inducing changes in the vicinity of active site residues with concomitant alteration in kinetic and thermodynamic properties of the modified CMCases.
Enzyme and Microbial Technology, 1998
from any protein or enzyme. 0 1998 Elsevier Science Inc.
Current Opinion in Biotechnology, 2002
Psychrophilic (cold-adapted) organisms and their products have potential applications in a broad ... more Psychrophilic (cold-adapted) organisms and their products have potential applications in a broad range of industrial, agricultural and medical processes. In order for growth to occur in lowtemperature environments, all cellular components must adapt to the cold. This fact, in combination with the diversity of Archaea, Bacteria and Eucarya isolated from cold environments, highlights the breadth and type of biological products and processes that might be exploited for biotechnology. Relative to this undisputed potential, psychrophiles and their products are under-utilised in biotechnology; however, recent advances, particularly with cold-active enzymes, herald rapid growth for this burgeoning field.
Biotechnology Progress, 2002
We wish to report the attainment of the highest ever T opt by introducing approximately two aroma... more We wish to report the attainment of the highest ever T opt by introducing approximately two aromatic rings through chemical modification of surface carboxyl groups in carboxymethylcellulase from Scopulariopsis sp. with concomitant decrease in V max , K m , and optimum pH! This extraordinary enhancement in thermophilicity of anilinecoupled CMCase (T opt ) 122°C) by a margin of 73°C as compared with the native enzyme (T opt ) 49°C) is the highest reported for any mesophilic enzyme that has been modified either through chemical modification or site-directed mutagenesis. It is also reported for the first time that aniline coupled CMCase (ACC) is simultaneously thermostable in aqueous as well as water-miscible organic solvents. The T opt of native CMCase and ACC were 25 and 90°C, respectively, in 40% (v/v) aqueous dioxan. The modified enzyme was also stabilized against irreversible thermal denaturation. Therefore, at 55°C, ACC had a half-life of 136 min as compared with native CMCase whose half-life was only 5 min. We believe that the reasons for this elevated thermostability and thermophilicity are surface aromatic-aromatic interactions and aromatic interactions with the sugar backbone of the substrate, respectively.
Biotechnology and Bioengineering, 2009
The alkaline protease, savinase was chemically modified to enhance the productivity of the enzyme... more The alkaline protease, savinase was chemically modified to enhance the productivity of the enzyme at low temperatures on a complex polymeric protein (azocasein) substrate. At 5 and 158C, savinase modified with ficol or dextran hydrolyzed fivefold more azocasein than the unmodified savinase. Kinetic studies showed that the catalytic improvements are associated with changes in uncompetitive substrate inhibition with K i values of modified savinases sixfold higher than the unmodified savinase. Modeling of small-angle scattering data indicates that two substrate molecules bind on opposing sides of the enzyme. The combined kinetic and structural data indicate that the polysaccharide modifier sterically blocks the allosteric site and reduces substrate inhibition. In contrast to the properties of cold-active enzymes that generally manifest as low activation enthalpy and high flexibility, this study shows that increased activity and productivity at low temperature can be achieved by reducing uncompetitive substrate inhibition, and that this can be achieved using chemical modification with an enzyme in a commercial enzyme-formulation.
Applied Biochemistry and Biotechnology, 2005
Two isoenzymes of endo-1,4-beta-xylanase (EC 3.2.1.8) from Scopulariopsis sp. were purified by a ... more Two isoenzymes of endo-1,4-beta-xylanase (EC 3.2.1.8) from Scopulariopsis sp. were purified by a combination of ammonium sulfate precipitation, hydrophobic interaction, and anion-exchange and gel filtration chromatography. The native mol wts of the least acidic xylanase (LAX) and the highly acidic xylanase (HAX) were 25 and 144 kDa and the subunit mol wts were 25 and 36 kDa, respectively. The kcat values of LAX and HAX for oat-spelt xylan at 40 degrees C, pH 6.5, were 95,000 and 9900 min-1 and the Km values of LAX and HAX were 30 and 3.3 mg/mL. The thermodynamic activation parameters of xylan hydrolysis showed that the high activity of LAX when compared with HAX was not owing to a reduction in DeltaH# but was entropically driven. High-performance liquid chromatography analysis of the degradation products showed that LAX formed both xylotrioses and xylobioses, but HAX predominantly formed xylotrioses. The half-lives of LAX and HAX at 50 degrees C in 50 mM 2-N-morpholino ethanesulfonic acid (MES), pH 6.5 buffer were 267 and 69 min, respectively. Thermodynamic analysis showed that at lower temperatures, the increased thermostability of LAX (DeltaH#=306 kJ/mol) compared with HAX (DeltaH#=264 kJ/mol) was owing to more noncovalent surface interactions. At higher temperatures, LAX (DeltaS*=-232 J/[mol.K]) was more thermostable than HAX (DeltaS*=490 J/[mol.K]) owing to a more ordered transition-state conformation. An energy-activity diagram was introduced showing that kcat/Km does not successfully explain the true kinetic behavior of both xylanase isoenzymes. The simultaneously thermostable and highly active LAX could be utilized in biotechnological processes involving xylan hydrolysis.
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Papers by khawar siddiqui
with HAX was not owing to a reduction in ΔH# but was entropically driven. High-performance liquid chromatography analysis of the degradation products showed that LAX formed both xylotrioses and xylobioses, but HAX predominantly formed xylotrioses. The half-lives of LAX and HAX at 50°C in 50 mM 2-N-morpholino ethanesulfonic acid (MES), pH 6.5 buffer
were 267 and 69 min, respectively. Thermodynamic analysis showed that at lower temperatures, the increased thermostability of LAX (ΔH# = 306 kJ/mol) compared with HAX (ΔH# = 264 kJ/mol) was owing to more noncovalent surface interactions. At higher temperatures, LAX (ΔS* = –232 J/[mol·K])
was more thermostable than HAX (ΔS* = 490 J/[mol·K]) owing to a more ordered transition-state conformation. An energy-activity diagram was introduced showing that kcat/Km does not successfully explain the true kinetic behavior of both xylanase isoenzymes. The simultaneously thermostable and highly active LAX could be utilized in biotechnological processes involving xylan hydrolysis.
with HAX was not owing to a reduction in ΔH# but was entropically driven. High-performance liquid chromatography analysis of the degradation products showed that LAX formed both xylotrioses and xylobioses, but HAX predominantly formed xylotrioses. The half-lives of LAX and HAX at 50°C in 50 mM 2-N-morpholino ethanesulfonic acid (MES), pH 6.5 buffer
were 267 and 69 min, respectively. Thermodynamic analysis showed that at lower temperatures, the increased thermostability of LAX (ΔH# = 306 kJ/mol) compared with HAX (ΔH# = 264 kJ/mol) was owing to more noncovalent surface interactions. At higher temperatures, LAX (ΔS* = –232 J/[mol·K])
was more thermostable than HAX (ΔS* = 490 J/[mol·K]) owing to a more ordered transition-state conformation. An energy-activity diagram was introduced showing that kcat/Km does not successfully explain the true kinetic behavior of both xylanase isoenzymes. The simultaneously thermostable and highly active LAX could be utilized in biotechnological processes involving xylan hydrolysis.