4i5u
From Proteopedia
Crystal structure of a fungal chimeric cellobiohydrolase Cel6A
Structural highlights
Function[GUX6_HUMIN] Plays a central role in the recycling of plant biomass. The biological conversion of cellulose to glucose generally requires three types of hydrolytic enzymes: (1) Endoglucanases which cut internal beta-1,4-glucosidic bonds; (2) Exocellobiohydrolases that cut the dissaccharide cellobiose from the non-reducing end of the cellulose polymer chain; (3) Beta-1,4-glucosidases which hydrolyze the cellobiose and other short cello-oligosaccharides to glucose.[1] Publication Abstract from PubMedThermostability is an important feature in industrial enzymes: it increases biocatalyst lifetime and enables reactions at higher temperatures, where faster rates and other advantages ultimately reduce the cost of biocatalysis. Here we report the thermostabilization of a chimeric fungal family 6 cellobiohydrolase (HJPlus) by directed evolution using random mutagenesis and recombination of beneficial mutations. Thermostable variant 3C6P has a half-life of 280 min at 75 degrees C and a T50 of 80.1 degrees C, a approximately 15 degrees C increase over the thermostable Cel6A from Humicola insolens (HiCel6A) and a approximately 20 degrees C increase over that from Hypocrea jecorina (HjCel6A). Most of the mutations also stabilize the less-stable HjCel6A, the wild-type Cel6A closest in sequence to 3C6P. During a 60-h Avicel hydrolysis, 3C6P released 2.4 times more cellobiose equivalents at its optimum temperature (Topt ) of 75 degrees C than HiCel6A at its Topt of 60 degrees C. The total cellobiose equivalents released by HiCel6A at 60 degrees C after 60 h is equivalent to the total released by 3C6P at 75 degrees C after approximately 6 h, a 10-fold reduction in hydrolysis time. A binary mixture of thermostable Cel6A and Cel7A hydrolyzes Avicel synergistically and released 1.8 times more cellobiose equivalents than the wild-type mixture, both mixtures assessed at their respective Topt . Crystal structures of HJPlus and 3C6P, determined at 1.5 and 1.2 A resolution, indicate that the stabilization comes from improved hydrophobic interactions and restricted loop conformations by introduced proline residues. Biotechnol. Bioeng. (c) 2013 Wiley Periodicals, Inc. Engineered thermostable fungal Cel6A and Cel7A cellobiohydrolases hydrolyze cellulose efficiently at elevated temperatures.,Wu I, Arnold FH Biotechnol Bioeng. 2013 Feb 12. doi: 10.1002/bit.24864. PMID:23404363[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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