Molecular cloning of carbohydrate binding module (cbm40) from vibrio cholerae non o1 neuraminidase in Escherichia coli

• Main Authors: Shadariah, Mamat, Nadiawati, Alias
• Format: Conference or Workshop Item
• Language: English; English
• Published: 2016
• Subjects: HD28 Management. Industrial Management; QH301 Biology; T Technology (General)
• Online Access: http://eprints.unisza.edu.my/1076/1/FH03-FBIM-16-07585.pdf; http://eprints.unisza.edu.my/1076/2/FH03-FBIM-16-07586.pdf; http://eprints.unisza.edu.my/1076/

Carbohydrate binding modules (CBMs) are a contiguous amino acid sequence within a carbohydrate-active enzyme and are discrete, non-catalytic modules that primarily exist to target parent enzyme to its substrate for efficient hydrolysis. Based on Carbohydrate-Active Enzyme database (CaZY), CBMs are divided into 80 defined families based on amino acid sequence similarity, binding specificity and structure. Interesting to study family 40 of CBM domain, a few bacteria have been screened include Pseudomonas aeruginosa ATCC 27853, Bacillus cereus ATCC 14579, Staphylococcus aureus ATCC 29213, Salmonella thypii ATCC 14028 and Vibrio cholerae Non O1. A gene encoding CBM40 domain was screened from all the DNA samples and subjected to PCR amplification. PCR components used were 1X PCR buffer, 2mM MgCl2, 10μM dNTP’s, 1μg Template DNA, 1μM Forward Primer (5’- GTC CAC TTT TTG ACT ATA ACG C-3’), 1μM Reverse Primer (5’-CGG CTA GTC GCC TTG AAT TTC AAA C-3’) and 1.25U Taq polymerase. While, parameters for PCR amplification cycles were as followed; 95⁰C for 2 minutes (pre denaturation), 95⁰C for 1 minute (denaturation), 58⁰C for 1 minute (annealing), 72⁰C for 5 minutes (extension) and 72⁰C for 5 minutes (final extension). From all the samples tested, only Vibrio Cholerae Non O1 showed an amplified PCR product size of 530 bp. Data from the sequence and Blast analysis have shown 99% similarity with the target Vibrio cholerae neuraminidase, NanH (M83562). Prior to that, the confirmed CBM40 gene was further ligated into pGEMT Easy Vector system and transformed into E. coli JM109 host.