β-glucosidase from Sclerotinia sclerotiorum:: a new and efficient purification procedure and use as a suitable marker in immuno-enzymatic assay

被引：0

作者：

Kalifa, Sonia Ben Hadj

Limam, Ferid

Smaali, M. Issam

Maugard, Thierry

Marzouki, M. Nejib

机构：

[1] Natl Int Appl Sci & Technol, Bioengn Unit 99UR09 26, Tunis 1080, Tunisia

[2] Biotechnol Ctr Bordj Cedria, Tunis, Tunisia

[3] La Rochelle Univ, FRE CNRS 2766, Lab Biotechnol & Bioorgan Chem, La Rochelle, France

来源：

WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY | 2007年 / 23卷 / 10期

关键词：

antibody; beta-glu2; enzyme; glutaraldehyde conjugation; ELISA test; enzyme-antibody conjugate; purification;

D O I：

10.1007/s11274-007-9374-y

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

The beta-glucosidase enzyme beta-glu2 isolated from Sclerotinia sclerotiorum was purified and used as tracer in enzyme linked immunosorbent assay. A novel purification procedure of the protein was developed that consists of ammonium sulphate precipitation, gel filtration on Sephacryl S-200-HR column, ion exchange chromatography on DEAE-Toyopearl and polybuffer exchanger PBE 94 TM chromatofocusing. The pI value was 4.45. K (m) and V-max values for the enzyme towards p-nitrophenyl-beta-D-glucopyranoside were respectively 0.45 mM and 0.2 U/mL. Thermal stability showed that beta-glu2 has a half-life of 85 min at 55 degrees C and of 25 min at 65 degrees C. beta-glu2 was conjugated to goat anti-rabbit antibodies with glutaraldehyde as cross linking agent according to the one-step method. Conjugates were purified by HPLC gel filtration on TSK 2000. Enzymatic and immunological activities of the beta-glucosidase conjugate component were tested by the ELISA method.

引用

页码：1363 / 1370

页数：8