Structural and biochemical properties of lichenase from Clostridium thermocellum

Abstract

The recombinant enzyme lichenase of size 30 kDa was over-expressed using E. coli cells and purifi ed by immobilized metal ion affi nity chromatography (IMAC) and size exclusion chromatography. The enzyme displayed high activity towards lichenan and β-glucan. The enzyme showed no activity towards carboxymethyl cellulose, laminarin, galactomannan or glucomannan. Surprisingly, affi nity-gel electrophoresis on native-PAGE showed that the enzyme binds only glucomannan and not lichenan or β-glucan or other manno-confi gured substrates. The enzyme was thermally stable between the temperatures 60°C and 70°C. Presence of Cu 2+ ions at a concentration of 5 mM enhanced enzyme activity by 10% but higher concentrations of Cu 2+ (>25 mM) showed a sharp fall in the enzyme activity. Heavy metal ions Ni 2+ , Co 2+ and Zn 2+ did not affect the activity of the enzyme at low concentrations (0-10 mM) but at higher concentrations (>10 mM), caused a decrease in the enzyme activity. The crystals of lichenase were produced and the 3-dimensional structure of native form of enzyme was previously solved at 1.50 Å. Lichenase displayed (ß/α) 8 -fold a common fold among many glycoside hydrolase families. A cleft was identifi ed that represented the probable location of active site.

A Novel α-L-Arabinofuranosidase of Family 43 Glycoside Hydrolase (Ct43Araf) from Clostridium thermocellum

“…The decrease in activity in presence of EGTA indicated that Ca 2+ ions may be essential for enzyme activity as EGTA specifically binds and chelates calcium ions in 1∶1 molar ratio [35]. The catalytic activity was noticeably increased in the presence of Ca 2+ and Mg 2+ salts elucidating the fact that these metal cations may be needed as co-factors while the heavy metals especially Co 2+ , Hg 2+ , Cu 2+ and Ag + caused decrease in enzyme activity as shown for recombinant cellulases [36], [37].…”

Ultrasound Induced Enhancement of Biological Gas-to-Liquid Process for Methanol Synthesis from Methane Using Methylotuvimicrobium buryatense 5GB1C

“…The cells were lysed using a probe sonicator (Sonics & Materials, Vibra cell, 150 W, pulse rate: 5 s on/10 s off) for 20 min in an ice bath. Subsequently, 20 μL of each sample was separately mixed with 5 μL of loading dye and boiled for 4–5 min before loading onto the SDS-PAGE . Each sample (5 μL) and a pre-stained protein marker were loaded in the wells, and SDS-PAGE was run at 60–80 V. Following electrophoresis, the gel was stained and destained as per the standard protocols.…”

“…Subsequently, 20 μL of each sample was separately mixed with 5 μL of loading dye and boiled for 4−5 min before loading onto the SDS-PAGE. 28 Each sample (5 μL) and a pre-stained protein marker were loaded in the wells, and SDS-PAGE was run at 60−80 V. Following electrophoresis, the gel was stained and destained as per the standard protocols. Gel Documentation system (BioRAD, USA) and Image Lab 5.2.1 software were used to observe and analyze the protein bands based on their respective molecular weights.…”