Cloning, molecular modeling, and docking analysis of alkali-thermostable [beta]-mannanase from Bacillus nealsonii PN-11

• Published in: Applied microbiology and biotechnology Vol. 99; no. 21; pp. 8917 - 8925
• Main Authors: Chauhan, Prakram Singh, Tripathi, Satya Prakash, Sangamwar, Abhays T, Puri, Neena, Sharma, Prince, Gupta, Naveen
• Format: Journal Article
• Language: English
• Published: Heidelberg Springer 01-11-2015; Springer Nature B.V
• Subjects: Amino acids; Analysis; Bacillus; Bacteria; Biosynthesis; Cellulose; Cloning; E coli; Enzymes; Escherichia coli; Genomics; Hydrolases; Hydrophobic surfaces; Influence; Physiological aspects; Proteins; Studies; Work stations
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-015-6613-2

An alkali-thermostable [beta]-mannanase gene from Bacillus nealsonii PN-11 was cloned by functional screening of E. coli cells transformed with pSMART/HaeIII genomic library. The ORF encoding mannanase consisted of 1100 bp, corresponding to protein of 369 amino acids and has a catalytic domain belonging to glycoside hydrolase family 5. Cloned mannanase was smaller in size than the native mannanase by 10 kDa. This change in molecular mass could be because of difference in the glycosylation. The tertiary structure of the [beta]-mannanase (MANPN11) was designed and it showed a classical ([alpha]/[beta]) TIM-like barrel motif. Active site of MANPN11 was represented by 8 amino acid residues viz., Glu152, Trp189, His217, Tyr219, Glu247, Trp276, Trp285, and Tyr287. Model surface charge of MANPN11 predicted that surface near active site was mostly negative, and the opposite side was positive which might be responsible for the stability of the enzymes at high pH. Stability of MANPN11 at alkaline pH was further supported by the formation of a hydrophobic pocket near active site of the enzyme. To understand the ability of MANPN11 to bind with different substrates, docking studies were performed and found that mannopentose fitted properly into active site and form stable enzyme substrate complex.