Biochemical and mutational analyses of a multidomain cellulase/mannanase from Caldicellulosiruptor bescii

Abstract

Thermophilic cellulases and hemicellulases are of significant interest to the biofuel industry due to their perceived advantages over their mesophilic counterparts. We describe here biochemical and mutational analyses of Caldicellulosiruptor bescii Cel9B/Man5A (CbCel9B/Man5A), a highly thermophilic enzyme. As one of the highly secreted proteins of C. bescii, the enzyme is likely to be critical to nutrient acquisition by the bacterium. CbCel9B/Man5A is a modular protein composed of three carbohydrate-binding modules flanked at the N terminus and the C terminus by a glycoside hydrolase family 9 (GH9) module and a GH5 module, respectively. Based on truncational analysis of the polypeptide, the cellulase and mannanase activities within CbCel9B/Man5A were assigned to the N- and C-terminal modules, respectively. CbCel9B/Man5A and its truncational mutants, in general, exhibited a pH optimum of ∼5.5 and a temperature optimum of 85°C. However, at this temperature, thermostability was very low. After 24 h of incubation at 75°C, the wild-type protein maintained 43% activity, whereas a truncated mutant, TM1, maintained 75% activity. The catalytic efficiency with phosphoric acid swollen cellulose as a substrate for the wild-type protein was 7.2 s(-1) ml/mg, and deleting the GH5 module led to a mutant (TM1) with a 2-fold increase in this kinetic parameter. Deletion of the GH9 module also increased the apparent k(cat) of the truncated mutant TM5 on several mannan-based substrates; however, a concomitant increase in the K(m) led to a decrease in the catalytic efficiencies on all substrates. These observations lead us to postulate that the two catalytic activities are coupled in the polypeptide.

Fig 1

(A) Schematic structures of the CbCel9B/Man5A wild type and its truncation mutants. The signal peptide is shown in filled rectangle. GH9, family 9 glycoside hydrolase domain; GH5, family 5 glycoside hydrolase domain; CBM3c, family 3 type C carbohydrate binding module; CBM3b, family 3 type B carbohydrate binding module. (B) SDS-PAGE of the CbCel9B/Man5A wild type and its truncation mutants. Lane 1, protein molecular mass marker; lane 2, CbCel9B/Man5A wild type; lane 3, CbCel9B/Man5ATM1; lane 4, CbCel9B/Man5ATM2; lane 5, CbCel9B/Man5ATM3; lane 6, CbCel9B/Man5ATM4; lane 7, CbCel9B/Man5ATM5; lane 8, CbCel9B/Man5ATM6; lane 9, CbCel9B/Man5ATM7. Portions (2 μg) of each enzyme were analyzed on an SDS–12% polyacrylamide gel.

Fig 2

Hydrolysis patterns of CbCel9B/Man5A wild type, TM1, and TM5 on PASC (A), and reactions of CbCel9B/Man5A wild type, TM1, and TM5 with glucose and cello-oligosaccharides (B) and with mannose and manno-oligosaccharides (C). (A) Time course hydrolysis of PASC by CbCel9B/Man5A WT (i), TM1 (ii), and TM5 (iii). PASC (2.5 mg/ml) was incubated with 0.5 μM CbCel9B/Man5A WT, TM1, and TM5 at 75°C. At different time intervals (0 min, 2 min, 10 min, 60 min, 4 h, and 24 h), samples were taken out and applied to HPAEC-PAD analysis. (B and C) TLC analysis of glucose and cello-oligosaccharides (B) and mannose and manno-oligosaccharides (C) reacted with CbCel9B/Man5A WT, TM1, and TM5. The reactions were carried out by incubating 0.1 μM (each) CbCel9B/Man5A WT, TM1, and TM5 with 1 mg of glucose and cello-oligosaccharides/ml or 1 mg of mannose and manno-oligosaccharides/ml in a total volume of 40 μl. The incubation was kept at 75°C for 14 h. The reaction products were dried and added with 3.5 μl of H₂O. A 1-μl portion of the product was subjected to TLC analysis. G1, glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose; G5, cellopentaose; G6, cellohexaose. M1, mannose; M2, mannobiose; M3, mannotriose; M4, mannotetraose; M5, mannopentaose; M6, mannohexaose. S, cello-oligosaccharides (B) or manno-oligosaccharides (C) standard.

Fig 3

Amino acid sequence alignment of the GH9 domain of CbCel9B/Man5A with those of CloceCel9G (Clostridium cellulolyticum Cel9G, GenBank accession number AAA73868) (36) and ThefuCel9A (Thermobifida fusca Cel9A, GenBank accession number AAB42155) (45). CloceCel9G (nonprocessive) and ThefuCel9A (processive) represent the two types of family 9 theme B1 endoglucanases whose enzyme–cello-oligosaccharide complex structures have been solved. The asterisks indicate the identical or similar amino acid residues within the three sequences. The filled triangles indicate nonconserved residues. The numbers under a specific amino acid residue indicate the subsites of the cello-oligosaccharides interacting with this amino acid residue based on the CloceCel9G and ThefuCel9A enzyme-substrate complex structures.

Fig 4

Qualitative binding of the CbCel9B/Man5A wild type and its truncation mutants to Avicel (A) and phosphoric acid swollen cellulose (PASC) (B). Portions (30 μg) of each protein were incubated with 40 mg of Avicel cellulose/ml or 2.5 mg of PASC/ml in 50 mM Tris buffer–150 mM NaCl (pH 7.5). The mixture was shaken end over end at 4°C for 1 h. Then the bound and unbound proteins were separated by centrifugation of the mixture at 25,000 × g for 3 min. The cellulose pellet was washed four times with 1 ml of buffer (50 mM Tris buffer, 150 mM NaCl [pH 7.5]). The pellet was then added with 70 μl of 1× SDS-PAGE loading buffer and boiled for 5 min. The protein corresponding to a one-tenth volume of each fraction was subjected to SDS–12% PAGE.