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Publication Abstract from PubMed

The family 81 glycoside hydrolase (GH81) from Clostridium thermocellum is a beta-1,3-glucanase belonging to cellulosomal complex. The gene encoding GH81 from Clostridium thermocellum (CtLam81A) was cloned and expressed displaying a molecular mass of ~82kDa. CtLam81A showed maximum activity against laminarin (100U/mg), followed by curdlan (65U/mg), at pH7.0 and 75 degrees C. CtLam81A displayed Km, 2.1+/-0.12mg/ml and Vmax, 109+/-1.8U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca(2+) or Mg(2+) ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 degrees C to 96 degrees C by Ca(2+) or Mg(2+) ions and decreased to 82 degrees C by EDTA, indicating that Ca(2+) and Mg(2+) ions may be involved in catalysis and in maintaining structural integrity. TLC and MALDI-TOF analysis of beta-1,3-glucan hydrolysed products released initially, showed beta-1,3-glucan-oligosaccharides degree of polymerization (DP) from DP2 to DP7, confirming an endo-mode of action. The catalytically inactive mutant CtLam81A-E515A generated by site-directed mutagenesis was co-crystallized and tetragonal crystals diffracting up to 1.4A resolution were obtained. CtLam81A-E515A contained 15 alpha-helices and 38 beta-strands forming a four-domain structure viz. a beta-sandwich domain I at N-terminal, an alpha/beta-domain II, an (alpha/alpha)6 barrel domain III, and a small 5-stranded beta-sandwich domain IV.

Novel insights into the degradation of beta-1,3-glucans by the cellulosome of Clostridium thermocellum revealed by structure and function studies of a family 81 glycoside hydrolase.,Kumar K, Correia MAS, Pires VMR, Dhillon A, Sharma K, Rajulapati V, Fontes CMGA, Carvalho AL, Goyal A Int J Biol Macromol. 2018 Oct 1;117:890-901. doi: 10.1016/j.ijbiomac.2018.06.003., Epub 2018 Jun 2. PMID:29870811^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.