Characterization of $beta$-xylosidase and $alpha$-L-Arabinofuranosidase activities from Thermomonospora fusca BD25

Termofilik bir aktinomiset olan Thermomonospora fusca BD25, diğer organizmalara oranla daha yüksek seviyede β-ksilozidaz ve α-L-arabinofuranozidaz enzimleri üretmektedir. Bu çalışmada, karbon ve enerji kaynağı olarak yulaf ksileni kullanılan kültür ortamında T. fusca tarafından üretilen β- ksilozidaz ve α-L-arabinofuranozidaz enzimlerinin bazı karakteristik özelliklerinin belirlenmesi amaçlanmıştır. β-ksilozidaz ve α-L-arabinofuranozidaz enzimlerinin spesifik aktivitelerini belirlemek amacı ile yapılan çalışmalarda, substrat olarak sırası ile p-NPX ve p-NPA kullanılmıştır. β-ksilozidaz enziminin spesifik aktivitesi 4,01 U mg-1 protein, α-L-arabinofuranozidaz enziminin spesifik aktivitesi ise 0,35 U mg-1 protein olarak saptanmıştır. β-ksilozidaz ve α-L-arabinofuranozidaz enzimleri için optimum sıcaklıklar ise sırası ile 65 °C ve 55 °C olarak tesbit edilmiştir. β-ksilozidaz ve α-L- arabinofuranozidaz enzimleri (substrat yokluğunda ve pH 7’de) 50 °C’ta 9 saat süre ile inkübe edildikten sonra sırası ile aktivitelerinin % 86 ve % 83 ünü korudukları saptanmıştır. β-ksilozidaz pH 4,5 ile 10 arasında ise, pH 8’de göstermiş olduğu maksimum aktivitenin % 50 sini göstermiştir. α-L- arabinofuranozidaz ise pH 4,5 ile 11 arasında, pH 9’de göstermiş olduğu maksimum aktivitenin % 54-55 ini göstermiştir. β-ksilozidaz ve α-L-arabinofuranozidaz için substrat olarak sırası ile p-NPX ve p- NPA kullanıldığında görünür Km değerleri 0,5 mM ve 0,18 mM olarak tesbit edilirken, Vmax değerleri 0,83 µmol ml -1 dak-1 ve 0,04 µmol ml -1 dak-1 olarak tesbit edilmiştir. Son ürün inhibitörü olarak D- ksilozun (10 mM) β-ksilozidaz enziminin reaksiyon ortamına ilavesi ise enzimin aktivitesinde yaklaşık olarak % 10 azalmaya neden olduğu saptanırken α-L-arabinofuranozidazın L-arabinoz (10 mM) tarafından %15 oranında inhibe edildiği saptanmıştır.

Thermomonospora fusca bd25' ten elde edilen $beta$-Ksilozidaz ve $alpha$-L- Arabinofuranozidaz aktivitelerinin karakterizasyonları

Thermomonospora fuscaBD25 produces relatively high levels of activity of $beta$-xylosidase and $alpha$-L-arabinofuranosidase. The aim of the work described in this study was to characterize some properties of $beta$-xylosidase and $alpha$ -L-arabinofuranosidase produced by T. fusca BD25 when growing on oat spelt xylan as the main carbon and energy sources. The substrates p-NPX and p-NPA reacted with b-xylosidase and $alpha$-L-arabinofuranosidase with specific activities of 4.01 U $mg^{ -1}$ protein and 0.35 U $mg^{ -1}$ protein, respectively. The b-xylosidase remained stable at up to 65 °C, but $alpha$-L-arabinofuranosidase lost 10 % of its maximum activity at 55 °C. $beta$-xylosidase and a-L-arabinofuranosidase activities remained at 86 % and 83 % of their maximum activities after 9 h of incubation at 50 °C. The maximum relative b-xylosidase activit occurred (0.82 U $mg^{ -1}$ protein) at pH 8.0 with a 50 % decrease of maximum relative activity occurring at pH 4.5 and 10. $alpha$-L-arabinofuranosidase exhibited maximum relative activity (0.136 U mg -1 protein) at pH 9.0 with 54 % and 55 % activities remaining at pH of 4.5 and 11, respectively. The apparent $K_ m$ values for the crude b-xylosidase and $alpha$-L-arabinofuranosidase preparations were 0.5 mM of p-NPX and 0.18 mM of p-NPA, while the $V_{ max}$ values were 0.83 $mu$mol p-nitrophenol $ml^{ -1}$ $min^{ -1}$ and 0.04 mmol p-nitrophenol $ml^{ -1}$ $min^{ -1}$ , respectively. The addition of D-xylose (10 mM) to the reaction mixture caused a 10 % reduction in $beta$-xylosidase activity as the end-product inhibitor. However, a 15 % reduction in $alpha$-L-arabinofuranosidase activity was detected when L-arabinose (10 mM) was added to the reaction mixture.

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