Screening of β-glucoside kinase directed evolution library to form a sulfur-phosphorus bond

• Main Authors: Bellag, Oualid Abdelkader, Ben Belgacem, Farah Fadwa, Noor Batcha, Ibrahim Ali, Mohd Salleh, Hamzah
• Format: Conference or Workshop Item
• Language: English
• Published: Kulliyah of Engineering, International Islamic University Malaysia 2016
• Subjects: TP248.13 Biotechnology
• Online Access: http://irep.iium.edu.my/51693/1/51693.pdf; http://irep.iium.edu.my/51693/; http://www.iium.edu.my/icbioe/2016/index.php/78-the-4th-international-conference-on-biotechnology-engineering

Glycoscience is a field that explores the structures and functions of glycans promising great advances in an array of applications. The objective of this research is to form a sulfur-phosphorus bond at the position 6’ of the sugar involved in many biological processes. A directed evolution strategy was adopted with error-prone PCR technique to create random mutagenesis of recombinant plasmid library of the β-glucoside kinase (βglk) recloned into a pTrcHisB plasmid and transformed into Escherichia coli. Following the creation of libraries, high throughput screening by 96-well microplate using fluorescent activated cell sorting (FACS) techniques were carried out. Positive clones were selected, sequenced for identification and enzyme expression and characterization were carried out.