Munir Tuncer

Optimization of extracellular endoxylanase, endoglucanase and peroxidase production by Streptomyces sp. F2621 isolated in Turkey

Aims:  To determine the effect of environmental conditions on the production of extracellular lignocellulose‐degrading enzymes by Streptomyces sp. F2621 and to assess the potential use of these enzymes in the hydrolysis of lignocellulose material.

Co-operative actions and degradation analysis of purified xylan-degrading enzymes from Thermomonospora fusca BD25 on oat-spelt xylan.

Aims: To determine and quantify the products from the degradation of xylan by a range of purified xylan‐degrading enzymes, endoxylanase, β‐xylosidase and α‐l‐arabinofuranosidase produced extracellularly by Thermomonospora fusca BD25.

Production and partial characterization of extracellular peroxidases produced bystreptomyces avermitilis UAH30

The effect of a number of environmental parameters (pH, temperature, carbon and nitrogen ratio of nutrient) on the production of extracellular peroxidase enzymes byStreptomyces avermitilis UAH30 was examined. Maximum specific peroxidase activity (0.12 U/mg of protein) was obtained after 72 hours of 1 incubation at 45‡C in a minimal salt medium (pH 7.5) containing 0.6% (w/v) yeast extract and 0.6% (w/v) xylan corresponding to a C:N ratio of 4 to 1. A study of the effect of incubation on peroxidase activity showed that the enzyme was stable and active for at least one hour after incubation at 50‡C while at higher temperatures the stability and activity of the peroxidase was reduced such that at 60‡C the peroxidase activity has a half life of 20 min while at 80‡C the half life was reduced to 5 min. The activation energy for deactivation as a result of thermal denaturation of the enzyme was calculated to be 80 ±7 kJ/mol. The optimum pH for the activity occurred between a pH range of 6.5–8.5 with pKa1 and pKa2 of 5.1 ±0.1 and 9.7 ±0.1, respectively. The Km and Vmax for the peroxidase activity were determined to be 1.45 mM and 0.31 unit per mg protein respectively using 2,4dicholorophenol (2,4-DCP) as a substrate. Characterization of the peroxidase activity revealed activity against L,3–4 dihydroxyphenylalanine and guaiacol, while no inhibition of peroxidase activity could be detected with the haem inhibitors such as potassium cyanide and sodium azide, suggesting the lack of haem component in the tertiary structure. Aspects of using the crude peroxidase preparation in the pulp and paper industry are discussed.

Production and partial characterization of extracellular peroxidase produced byStreptomyces sp. F6616 isolated in Turkey

Streptomyces sp. F6616 was found to produce higher levels of extracellular peroxidase activity (0.535 U/mL) without any inducers than other actinobacteria which are previously reported. Maximum specific peroxidase activity (6.21 U/mg of protein) was obtained after 72 h of incubation at 30°C in a minimal salt medium (pH 8.0) containing (in wt/v) 0.6% yeast extract and 0.8% ball-milled wheat straw corresponding to a C:N ratio of 4.6:1. Characterization of the peroxidase revealed that the optimal temperature for the enzyme activity, using the standard 2,4-dichlorophenol (2,4-DCP) assay was 50°C, when the enzyme reaction was performed at pH 8.0. A study of the effect of temperature on the stability of peroxidase over time, showed that the enzyme was stable at 50°C, with a half-life of 145 min, while at higher temperature the stability and activity was reduced such that at 60°C the half-life of the enzyme was 30 min. The optimum pH for the activity of the enzyme occurred between pH 9.0 and 10.0. The apparentKm andVmax values for the peroxidase preparations were determined to be 1.52 mmol/L and 1.84 U/mg protein, respectively using 2,4-DCP as a substrate. Characterization of the peroxidase activity revealed activity against 2,4-DCP, L-3,4-dihydroxyphenylalanine (L-DOPA), 2,4,5-trichlorophenol and other chlorophenols in the presence of hydrogen peroxide. However, inhibition of peroxidase activity with the addition of potassium cyanide and sodium azide, suggested the presence of heme component in the tertiary structure of the enzyme.

Purification and partial characterization of a-l-arabinofuranosidase produced by Thermomonospora fusca

Thermomonospora fusca produced a relatively high level of α-l-arabinofuranosidase when growing on oat spelt xylan as the main carbon and energy source. The enzyme exhibited maximum relative activity (0.136 U/g protein) at pH 9.0 with 54 and 55% activity remaining at pH of 4.5 and 11.0, respectively. The apparentKm value for the crude α-l-arabinofuranosidase preparation was 180 µmol/L 4-nitrophenyl α-l-arabinofuranoside; thevlim value was the release of 40 µmol/L 4-nitrophenol per min. Enzyme activity was eluted as a single peak (HPLC gel filtration chromatography) corresponding to molar mass of ≈92 kDa. Native electrophoresis of crude cell lysate confirmed the presence of a single active intracellular α-l-arabinofuranosidase component. SDS-PAGE of this enzyme, developed as zymogram, did not demonstrate any activity; denaturing gel was stained and a protein band of relative molar mass of 46 kDa was revealed. Isoelectric focusing of a purified α-l-arabinofuranosidase yielded a single protein band for the corresponding activity zone with pI 7.9. The enzyme was purified approximately 21-fold the mean overall yield was about 16%.

Production, Characterization and Application of a Xylanase from Streptomyces sp. AOA40 in Fruit Juice and Bakery Industries

ABSTRACT Streptomyces sp. AOA40, which produces halotolerant and thermotolerant xylanase, was isolated from Mersin soil. Various carbon sources were tested for xylanase production with selected fermentation medium. The best carbon source was selected as corn stover. The effect of corn stover concentration and particle size, composition of fermentation medium, fermentation condition such as initial pH and agitation rate on xylanase production was determined. After production, xylanase was partially purified with ion-exchange chromatography and gel filtration chromatography for characterization of xylanase and application in fruit juice and dough improvement. The optimum pH for the activity of xylanase occurred at pH 6.0 in phosphate buffer, while the optimum temperature was 60°C. The relative xylanase activity in the pH ranges of 4–9 remained between 59.93 and 54.43% of the activity at pH 6.0 (100.00%). The xylanase activity showed a half-life of 172 min at 70°C, which was reduced to 75 min at 80°C. The enzyme was highly inhibited by 10–100 mM of Hg+2, EDTA, Mg+2, SDS and 100 mM Cu+2. Clarity of fruit juices increased after enzymatic treatment of apple (17.85%), grape (17.19%) and orange juice (18.36%) with partially purified xylanase and also reducing sugar concentrations of these fruit juices were improved by 17.21, 16.79 and 19.57%, respectively. Also, dough volume was raised 17.06% with using partially purified Streptomyces sp. AOA40 xylanase in bread making.

Purification and Characterization of Cutinase from Bacillus sp. KY0701 Isolated from Plastic Wastes

ABSTRACT A total of approximately 400 bacterial strains were isolated from 73 plastic wastes collected from 14 different regions. Nineteen isolates that form clear zones both on tributyrin and poly ε-caprolactone (PCL) agar, were identified based on 16S rRNA gene sequences. Among these, Bacillus sp. KY0701 that caused the highest weight loss of PCL films in minimal salt medium, was selected for cutinase production. The highest enzyme activity (15 U/mL) was obtained after 4 days of incubation at 50°C, pH 7.0 and 200 rpm in a liquid medium containing 1.5% (w/v) apple cutin and 0.1% (w/v) yeast extract. The purified enzyme was stable at high temperatures (50–70°C) and over a wide pH range (5.5–9.0). The relative activity of cutinase was at least 75% in the percent of various organic solvents. The apparent Km and Vmax values of the cutinase for p-nitrophenyl butyrate were 0.72 mM and 336.8 µmol p-nitrophenol/h/g, respectively. In addition, it showed high stability and compatibility with commercial detergents. These features of cutinase obtained from Bacillus sp. KY0701 make it a promising candidate for application in the detergent and chemical industries. In our best knowledge, this is the first report for cutinase production and characterization produced by a Bacillus strain.

Production, purification, characterization and usage of a detergent additive of endoglucanase from isolated halotolerant Amycolatopsis cihanbeyliensis mutated strain Mut43

Abstract The Amycolatopsis cihanbeyliensis Mut43, which is obtained by UV radiation, exhibited endoglucanase activity of 5.21 U/mL, which was ∼2.3-fold higher than that of the wild strain (2.04 U/mL). The highest enzyme activity was obtained after 3 days of incubation at 32 °C, pH 7.0, 150 rpm, and 6% NaCl in a liquid medium containing 1.5% (w/v) wheat straw (0.25 mm of particle size) and 0.6% (w/v) yeast extract. Enzyme activity was eluted as a single peak (gel filtration chromatography), and Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of the corresponding peak revealed a molar mass of 30 kDa. Zymogram analysis confirmed the presence of a single active endoglucanase component. The enzyme was purified to ∼21-fold, and the mean overall yield was ∼6%. The purified endoglucanase was active up to 80 °C and showed a half-life of 214 min at 60 °C in the absence of substrate at pH 8.0. The apparent Km value for the purified endoglucanase was 0.70 mg/mL, while the Vmax value was 6.20 Units/μg. Endoglucanase activity was reduced (25%) by treatment with 30 U of proteinase K/mg. The addition of Mg+2 and Ca+2 (5 mM) enhanced endoglucanase activity. Additionally, endoglucanase activity in the presence of 5 mM SDS or organic solvents was 75 and 50% of maximum activity, respectively. The high levels of enzyme production from A. cihanbeyliensis Mut43 achieved under batch conditions, coupled with the temperature stability, activity over a broad pH range, relatively high stability (70–80%) in the presence of industrial laundry detergents and storage half-lives of 45 days at +4 °C and 75 days at −20 °C signify the suitability of this enzyme for industrial applications as detergent additive.