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.
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 k cat values of LAX and HAX for oat-spelt xylan at 40°C, pH 6.5, were 95,000 and 9900 min-1 and the K m 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 ∆H # 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 (∆H # = 306 kJ/mol) compared with HAX (∆H # = 264 kJ/mol) was owing to more noncovalent 52 Afzal et al.
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.
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 k cat values of LAX and HAX for oat-spelt xylan at 40°C, pH 6.5, were 95,000 and 9900 min-1 and the K m 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 ∆H # 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 (∆H # = 306 kJ/mol) compared with HAX (∆H # = 264 kJ/mol) was owing to more noncovalent 52 Afzal et al.
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