Cellulose-binding domains CBDs

The introduction of a glutamic acid (E bold), after each RADI 6 repeat was made to allow cleavage by endoproteinase Glu-C, which cleaves C-terminal to glutamate. A cellulose binding domain (CBD) (Table 1) was selected as the affinity tag, as CBDs bind strongly and specifically to cellulose which is a relatively cheap and abundant purification matrix. The main problem encountered was the low level of peptide recovered. Theoretically, 1 g of fusion protein should give 267mg of peptide, but only 10.1 mg of peptide was recovered after RP-HPLC. 

An efficient system for the production of recombinant antibodies is cellulose-assisted refolding technology, as described by Berdichevsky et al. [7]. The expressed scFvs were fused to a cellulose-binding domain (CBD) from the bacterium Clostridium thermocellum in the format scFv-CBD. The resulting fusion proteins were obtained in high yield from bacterially produced inclusion bodies that become solubilized and then refold while immobilized on cellulose. The refolded and purified scFv-CBD fusion proteins can be used to form cellulose-based affinity matrices or, as described herein, can be immobilized on a cellulose matrix that makes up part of the immunoelectrochemical sensor device. 

Because free cellulose chain ends are so difficult to quantify, other measurable cellulose characteristics have been shown to be factors correlating to hydrolysis susceptibihty and rate [15,18]. These factors affect how well the cellulose-binding domain, CBD, adheres to the cellulose and how well the catalytic domain acts on the cellulose. Because of what can and cannot be measured easily, models of enzymatic cellulose hydrolysis combine adsorption behavior with kinetic action. 

Many glycosidases which attack insoluble or partly soluble polysaccharides have a modular construction, in which catalytic domains and carbohydrate binding modules (CBMs) are produced in the same polypeptide. Although there may be occasional exceptions, the main function of these domains appears to be to increase the local concentration of enzyme by loosely absorbing the enzyme to the substrate. CBMs have been classified into families on the basis of amino acid sequence, like the catalytic domains and are on CAZy, the continuously updated website in summer 2007 there were 49 families. Early work had concentrated on the cellulose binding domains (CBDs) previous classifications had referred to CBM (or CBD) Types and used Roman numerals and, to concur with this usage, CBM Families 1-13 are the same as CBM Types 1-XIII. 

Overexpression of a cellulose-binding domain (CBD) has been reported to result in increased cellulose produetion in poplar (Levy et al. 2002). It was proposed that the CBD increased the rate of eellulose production by slowing down the rate of crystallization of the gluean ehains into mierofibrils, which is believed to limit the rate of cellulose synthesis in intact cells. This idea is based on the earlier finding that calcofluor white, a fluorescent dye commonly used to deteet cellulose, interfered with the crystallization of the gluean chains into mierofibrils (Haigler et al. 1980). 

Recently, a fusion protein between a cellulose binding domain (CBOeios)> isolated from Clostridium cellulovorans, and OPH was shown to be capable of immobilization onto various cellulose materials (24). The use of cellulose as an immobilization matrix is advantageous due to its low cost, wide spread availability and non-toxic nature. The kinetic parameters of OPH fused to die CBD domain were essentially identical to die soluble protein with a 3.8 fold increase in from 0.058 to 0.220 and a 10.4% decrease in Kd from 3170 to 2840. Additionally, the immobilized fusion protein offered superior stability over that of soluble OPH, retaining over 85% activity over a period of 45 days (24). 

Pinto, R Carvalho, J Mota, M Gama, M. Large-scale production of cellulose-binding domains. Adsorption studies using CBD-FITC conjugates. Cellulose, 2006,13, 557 -69. 

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