Enzymatic hydrolysis of lignocellulosic biomass using a novel, thermotolerant recombinant xylosidase enzyme from Clostridium clariflavum: a potential addition for biofuel industry
2022
Zafar, Asma | Hamid, Attia | Peng, Liangcai | Wang, Yanting | Aftab, Muhammad Nauman
The present study describes the cloning, expression, purification and characterization of the xylosidase gene (1650 bp) from a thermophilic bacterium Clostridium clariflavum into E. coli BL21 (DE3) using the expression vector pET-21a(+) for utilization in biofuel production. The recombinant xylosidase enzyme was purified to homogeneity by heat treatment and immobilized metal ion affinity chromatography. SDS-PAGE determined that the molecular weight of purified xylosidase was 60 kDa. This purified recombinant xylosidase showed its maximum activity at a temperature of 37 °C and pH 6.0. The purified recombinant xylosidase enzyme remains stable up to 90 °C for 4 h and retained 54.6% relative activity as compared to the control. The presence of metal ions such as Ca²⁺ and Mg²⁺ showed a positive impact on xylosidase enzyme activity whereas Cu²⁺ and Hg²⁺ inhibit its activity. Organic solvents did not considerably affect the stability of the purified xylosidase enzyme while DMSO and SDS cause the inhibition of enzyme activity. Pretreatment experiments were run in triplicate for 72 h at 30 °C using 10% NaOH. Saccharification experiment was performed by using 1% substrate (pretreated plant biomass) in citrate phosphate buffer of pH 6.5 loaded with 150 U mL⁻¹ of purified recombinant xylosidase enzyme along with ampicillin (10 μg mL⁻¹). Subsequent incubation was carried out at 50 °C and 100 rpm in a shaking incubator for 24 h. Saccharification potential of the recombinant xylosidase enzyme was calculated against both pretreated and untreated sugarcane bagasse and wheat straw as 9.63% and 8.91% respectively. All these characteristics of the recombinant thermotolerant xylosidase enzyme recommended it as a potential candidate for biofuel industry.
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