2.5 Preparation of curde laccase from Stenotrophomonas YBX1
The harvest Stenotrophomonas sp. YBX1 cells grown on 250ml of nutrient broth after 24 hrs of incubation followed suspended in 20 ml of 50 mM sodium aceatate buffer pH 6, and sonicated for 5 minutes at 4°C. The extract was centrifuged (9500xg, 30 min, at 4°C) and the resulting supernatant was filtered first by passing 0.45µ filter membrane. Clear filtrate was used for enzymatic assays and further subjected to purification steps followed by salting out with 80% ammonium sulfate preciptetion .
2.4. Purification of MCO like laccase from strain YBX1
The cell free extract precipitation with 80% ammounium sulfate and incubate ice bath about an hour and followed by centrifugation 9500x g for 10min at 4?C and decard superant , Use the residue containing MCO like laccase bonded with salt ammonium sulfate redisloved in sodium aceate buffer pH 5.
keep diaylisis using spectra pore membrane molecular cuff of 10 KDa for two time for 6-8 hrs. The dialysed curde laccase extract was subjected to ion exchange chromatography HiTrap HP Q (GE Healthcare) column (0.
7 x 2.5cm) equilibrated with sodium acetate buffer at a flow rate of 0.5ml /min and the fractions of 1.5 ml were collected using -100 fraction collector eucppied with AKTA pur Protein Purification System ver UNICORN 7 system. Subsequently, the column was washed with Same buffer, until the absorbance of the eluting fractions read zero at 280 (spectrophotometer senor). The ionic bound laccase was eluted with sodium acetate buffer (50 mM, pH 5.0) containing 1 M NaCl. All the operations of ion exchange column chromatography were carried out at 4°C. Fractions containing laccase activity were pooled and dialyzed extensively against same buffer and stored at -20°C for further purification by gel filtration chromtography.
Gel fitration chromatography carriedout in AKTA pu rProtein Purification System ver UNICORN 7 using HiPrep Sephacryl S 100 16/60 column (16/60) of (GE Healthcare) equilibrated with sodium aceate buffer pH 6. Fractions of 2.0 ml were collected at a flow rate of 0.3ml/min, and the elution of the protein was monitored by measuring the absorbance at 280 nm. After calibration with sodium aceate buffer pH6 . 5ml of ion exchange purified laccase fraction filter through 0.45µ membrane and load to column using manual method. The elution of load laccase by isocratic by same buffer and elute fraction collected fraction collector read at 280nm.
2.5.Enzymatic aassay
MCO like laccase activity determine by spectrophotometrically (WTS-200,Shanghai) using ABTS (2,2?-azinobis-(-3-ethylbenzothiazoline-6- sulfononic acid) diammonium salt) at absorbance at 420nm, enzymatic reaction volume 3ml; 2.6 ml of 50 mM sodium acetate buffer pH6 and add 0.2ml of substrate(4.2mM) reaction initiated with adding 0.2ml of cell free extract, molar coefficient of extinction of ABTS 3600cm-1/ml.
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