The Purification And Exploration Of Crystallization Conditions For Two Kinds Of β-Glucosidase

Alkyl glycosides is a class of novel non-ionic surfactants and it can be synthesized fromrenewable raw materials.It have many advantages，such as non-toxic, non-irritating to the skinand eyes, good compatibility,and also possess some features of antibiotics and completelybiodegradable. In view of the above advantages, alkyl glycosides have great prospect in theindustrial applications of detergent, food, and daily chemical products. What’s more,compared with chemical method, enzymatic synthesis of alkyl glycosides is more mild,selectivity and specificity are well, product purity is higher. As a result, β-glucosidase whichinvolved in the synthesis of alkyl glycosides were concerned.This paper studies the separation, purification and crystallization conditions of Aspergilluaculeatus β-glucosidase I and Dalbergia cochinchinensis Pierre β-glucosidase, whichexpression in Pichia pastoris. Using different saturation of ammonium sulfate fractionation toseparate ABGL, we confirmed its best salting out method, namely precipitated ABGL using50%-80%ammonium sulfate. By optimizing pH and ion strength of anion-exchangechromatography, we confirmed the best separated method of ABGL whose binding buffer was20mM Tris-HCl pH8.0and three-step gradient elution with0.1M NaCl、0.15M NaCl、1MNaCl. Besides, ABGL was washed in0.15M eluant. Finally, we obtained higher purity ABGLthrough gel filtration chromatography. Using ammonium sulfate precipitation, anion exchangechromatography and gel filtration chromatography, we successfully obtained95%purity anduniform ABGL. What’s more, the recovery rate reached50.8%. Because of the lowconcentration of DCBGL,we concentrated it with VIVA FLOW200ultrafiltration membrane(100kDa) and optimized pH and ion strength of anion-exchange chromatography, thus weconfirmed the best separated method of DCBGL, whose binding buffer was20mM Tris-HClpH6.0and three-step gradient elution with0.05M NaCl、0.15M NaCl、1M NaCl. Besides,DCBGL was washed in0.15M eluant. At last, we purified DCBGL through gel filtrationchromatography and obtained95%purity and uniform DCBGL,whose recovery rate reached39.8%. Screening crystallization conditions of ABGL and DCBGL using sitting Drop methodand the screening kit were Crystal Screen and Crystal Screen2. We successfully screened twoconditions,the first condition was0.1M HEPES, pH7.5,10%polyethylene glycol8000and8%ethylene glycol, the second was0.1M Bicine, pH9.0,2%dioxane,10%polyethyleneglycol20000that ABGL can grow crystals under4℃,15mg/mL of protein concentrationwas dissolved in50mM sodium acetate buffer pH5.0. Using seeding method training wereceived stereo strip type crystalis which was more than0.05mm. By screening crystalconditions of DCBGL, we successfully screened six conditions that can grow DCBGLmicro-crystallin under the condition of20℃,20mg/mL of protein concentration and50mMsodium acetate buffer pH5.0.