Enhanced features of Dictyoglomus turgidum Cellulase A engineered with carbohydrate binding module 11 from Clostridium thermocellum

Abstract

Lignocellulosic biomass (LCB) is a low-cost and abundant source of fermentable sugars. Enzymatic hydrolysis is one of the main ways to obtain sugars from biomass, but most of the polysaccharide-degrading enzymes are poorly efficient on LCB and cellulases with higher performances are required. In this study, we designed a chimeric protein by adding the carbohydrate binding module (CBM) of the cellulosomal enzyme CtLic26A-Cel5E (endoglucanase H or CelH) from Clostridium (Ruminiclostridium) thermocellum to the C-terminus of Dtur CelA, an interesting hyperthermostable endoglucanase from Dictyoglomus turgidum. The activity and binding rate of both native and chimeric enzyme were evaluated on soluble and insoluble polysaccharides. The addition of a CBM resulted in a cellulase with enhanced stability at extreme pHs, higher affinity and activity on insoluble cellulose.

Structural and Functional Studies of a Klebsiella Phage Capsule Depolymerase Tailspike: Mechanistic Insights into Capsular Degradation

“…They are classified into numerous families, based on amino acid sequence similarity. Depending on the family, CBMs may have different carbohydrate specificities and binding cleft locations, either in the concave surface of one of ll Article the b sheets (e.g., CBM11 and CBM15) or on loops connecting b strands (e.g., CBM6, CBM20, CBM32, and CBM35) (Cattaneo et al, 2018;Shinya and Fukamizo, 2017). Despite poor sequence identity, a DALI search shows that the middle domain of KP32gp38 displays closest structural similarity to two family-6 CBMs of Saccharophagus degradans (Henshaw et al, 2006) (PDB: 2CDO, DALI Z = 12.9, RMSD = 2.6 Å , seqid: 6%), followed by the CBM35 domain of a cycloisomaltooligosaccharide glucanotransferase from Bacillus circulans (Suzuki et al, 2014) (PDB: 3WNK, DALI Z = 12.6, RMSD = 2.8 Å , seqid: 14%) and the CBM35 domain of b-(1,4)-mannanases from Podospora anserina (Couturier et al, 2013) (PDB: 3ZM8, DALI Z = 12.0, RMSD = 2.4 Å , seqid: 8%).…”

Analysis of Toll-Like Receptors in Human Milk: Detection of Membrane-Bound and Soluble Forms

“…Before mass spectrometry analysis, protein bands separated on SDS-PAGE, immunoreactive bands detected on PVDF membranes, and eluted fractions from the modified ELISA were submitted to trypsin digestion. Proteins from SDS-gels were in-gel digested as described in [11] with a few modifications. Briefly, the protein bands corresponding to the apparent MW of anti-TLR1/2/4/6 immunoreactive bands were excised from Coomassie-stained gels, destained overnight with 50% methanol and 5% acetic acid.…”

Progress in Ameliorating Beneficial Characteristics of Microbial Cellulases by Genetic Engineering Approaches for Cellulose Saccharification

“…At acidic pH, chimeric DturCelA exhibited higher relative activity (20%) than the native form (0%). It also retained 100% enzymatic activity at 70 • C (Cattaneo et al, 2018). The CBMs are known to bring the catalytic domains close to the target substrate, leading to enhancement in the rate of catalysis (Gilbert et al, 2013).…”