Purification And Characterization Of β-glucosidase From Rubber(Hevea Brasiliensis) Seeds

In recent years, β-glucosidase is studied on its application prospect in cellulose degradation, aroma enhancement and relationship with cyanogenic degradation. The high level cyanogenic glycoside content in rubber (Hevea brasiliensis) seed is hardly removed. An efficient decyanation method in rubber seeds could be established for there is a relevance to cyanogenic glycoside degradation and β-glucosidase in rubber (Hevea brasiliensis) seeds. The optimal assay condition, extraction process, purification and primary charaterization of β-glucosidase from rubber seed was investigated in this article.To find out the optimal assay condition, the effect of temperature, time, substrate concentration, pH and buffer type on the assay of P-glucosidase from rubber seed was investigated. The results suggest that enzyme activity is assayed by0.1mL crude P-glucosidase extract,0.3mL Na2HPO4-KH2PO4buffer (pH6.0) and0.1ml10mmol/L pNPG at42℃for30min, the product p-nitrophenol is assayed by the UV at400nm.Three notable factors in β-glucosidase extracting, pH, solid-liquid ratio, homogenizing time were elcted from9possible factors with a Plackett Burman design. Total activity and specific activity were taken together to find the best extraction conditions with a central composite design based on single factor experiment. The results suggested that the optimal extract conditions are pH8.6, solid-liquid ratio12.6:1, homogenizing time63.6s.Preliminary purification methods on salt out, PEG6000precipitation, organic solvent precipitation, isoelectric precipitation were compared. The results suggest that acetone precipitation is the proper methord. The β-glucosidase was further purified from rubber seeds by acetone precipitation, ion exchange chromatography and hydrophobic chromatography. The molecular mass of the β-glucosidase is approximately65kDa. The specific activity of β-glucosidase in the purification is increased to173.21U/mg from3.03U/mg. The purification fold is57.16.The activity of β-glucosidase is inhibited by most of the metal ion, AgT affects most, then Ba2+, Zn2+while Na+, K+, Fe2+affect little. The activity of β-glucosidase is inhibited by ethanol, carbinol, glycerin, formaldehyde, acetone, pyridine, ethyl acetate. Urea and vitamin C inhibit the activity. The purfied β-glucosidase from rubber seed inactivates rapidly at60-65℃, crude β-glucosidase extract tolerate50℃while β-glucosidase in rubber seed powder tolerate90℃, while β-glucosidase activity changes little in room temperature ventilated storage of rubber seeds. The purfied β-glucosidase remain stable at pH5-9. The β-glucosidase from rubber seeds hydrolysis pNPG, amygdalin, salicin, while it can not hydrolysis IPTG. Disaccharide is weakly hydrolysis by β-glucosidase. There is a relevance to cyanogenic glycoside degradation and P-glucosidase in rubber seed. The cyanogenic glycoside in rubber seed has a good temperaturesistant. The cyanogenic glycoside is linamarin. The P-glucosidase activity is higher in germ than in endosperm.