Fusion of a family 9 cellulose-binding module improves catalytic potential of Clostridium thermocellum cellodextrin phosphorylase on insoluble cellulose

Abstract

Clostridium thermocellum cellodextrin phosphorylase (CtCDP), a single-module protein without an apparent carbohydrate-binding module, has reported activities on soluble cellodextrin with a degree of polymerization (DP) from two to five. In this study, CtCDP was first discovered to have weak activities on weakly water-soluble celloheptaose and insoluble regenerated amorphous cellulose (RAC). To enhance its activity on solid cellulosic materials, four cellulose binding modules, e.g., CBM3 (type A) from C. thermocellum CbhA, CBM4-2 (type B) from Rhodothermus marinus Xyn10A, CBM6 (type B) from Cellvibrio mixtus Cel5B, and CBM9-2 (type C) from Thermotoga maritima Xyn10A, were fused to the C terminus of CtCDP. Fusion of any selected CBM with CtCDP did not influence its kinetic parameters on cellobiose but affected the binding and catalytic properties on celloheptaose and RAC differently. Among them, addition of CBM9 to CtCDP resulted in a 2.7-fold increase of catalytic efficiency for degrading celloheptaose. CtCDP-CBM9 exhibited enhanced specific activities over 20% on the short-chain RAC (DP = 14) and more than 50% on the long-chain RAC (DP = 164). The chimeric protein CtCDP-CBM9 would be the first step to construct a cellulose phosphorylase for in vitro hydrogen production from cellulose by synthetic pathway biotransformation (SyPaB).

New insights into enzymatic hydrolysis of heterogeneous cellulose by using carbohydrate-binding module 3 containing GFP and carbohydrate-binding module 17 containing CFP

“…CBM17 is a family 17 carbohydrate-binding module, belonging to type B [16,17]. This module contains clefts that accommodate single polysaccharide chains that are associated with amorphous cellulose areas [16,18,19]. Therefore, it can specifically bind to the surface of amorphous cellulose but not to that of crystalline cellulose [16,17].…”

C-Terminal carbohydrate-binding module 9_2 fused to the N-terminus of GH11 xylanase from Aspergillus niger

“…These modules were later recognized as CBMs (Dias et al 2004;Sunna et al 2000), and the thermostabilizing effects of CBM deletions were regarded as lacking discrete linker peptides separating from the catalytic domains (Dias et al 2004;Sunna et al 2000). The V max and K m values on oat spelt xylan were 1.55 and 2.03 lmol/min, and 6.98 and 7.76 mg/ml for the enzyme C2-Xyn, respectively made (Furtado et al 2015;Khan et al 2013;Kittur et al 2003;Liu et al 2011Liu et al , 2012aMai-Gisondi et al 2015;Mangala et al 2003;Tang et al 2014;Voutilainen et al 2014;Ye et al 2011), and fusion seems better for elucidating modular functions. 3 Kinetics of the C2-Xyn.…”

“…T opt and t 1/2 are related but different parameters with the former indicating enzyme adaptivity to a higher temperature and the later, enzyme longevity at a certain temperature. Other CBM fusions also improved catalytic activities of the related enzymes (Furtado et al 2015;Khan et al 2013;Kittur et al 2003;Li et al 2015;Mai-Gisondi et al 2015;Mangala et al 2003;Ye et al 2011). 3; Table 1).…”

“…Presently, the terminus is intuitively selected consisting with a CBM natural terminal location (Furtado et al 2015;Li and Shao 2006;Liu et al 2011Liu et al , 2012bMai-Gisondi et al 2015;Ye et al 2011). Presently, the terminus is intuitively selected consisting with a CBM natural terminal location (Furtado et al 2015;Li and Shao 2006;Liu et al 2011Liu et al , 2012bMai-Gisondi et al 2015;Ye et al 2011).…”

Fusing a Carbohydrate-Binding Module into the Aspergillus usamii β-Mannanase to Improve Its Thermostability and Cellulose-Binding Capacity by In Silico Design

“…The K m value (0.66 mg/mL) of the reAuMan5A-CBM was much lower than that (1.36 mg/mL) of the reAuMan5A, implying that the former has a higher affinity towards locust bean gum. The cellulose-binding capacity of the reAuMan5A-CBM with Avicel PH-101 was up to 92.3%, suggesting that it can be gathered around the natural substrate (that is, mannan cross-linked with cellulose, xylan and arabinan) by exclusively binding cellulose [26].…”