Fig 1.
(A) C18-reversed-phase HPLC elution profile of HrGH45. (B) MALDI-TOF mass spectrum of HrGH45. The doubly charged ion ([M+2H]2+ = 11.772), the molecular ion ([M+H]+ = 23.395), and a protein aggregate ([2M+H]+ = 47.901) were detected. The inset shows the SDS-PAGE pattern of HrGH45. 1, Precision plus protein unstained standards; 2, HrGH45.
Fig 2.
HrGH45 is a glycoprotein and behaves as a trimer.
(A) Gel filtration calibration curve used to estimate the molecular mass of HrGH45 in aqueous systems. The protein standards used for calibration were: 1, Tetrameric glucose isomerase (173 kDa); 2, BSA (66.6 kDa); 3, Agave chitinase (31.9 kDa); 4, Thaumatin (22.2 kDa); 5, Lysozyme (14 kDa). The solid line represents the calculated calibration curve. (B) Zymogram analysis of HrGH45. Left: Migration pattern of HrGH45 on 12% SDS-PAGE gel containing 0.1% (w/v) CMC in the resolving phase. 1, Precision plus protein unstained standards; 2, HrGH45. Right: In-gel assay (zymogram) for cellulase activity. 3, HrGH45. Endoglucanase activity is visualized as a clear-translucent band. (C) 12% SDS-PAGE gel treated with the Pierce glycoprotein staining kit. 1, Horseradish peroxidase (positive control); 2, Bio Basic prestained protein ladder; 3, HrGH45. Degradation of horseradish peroxidase was evident. Glycoproteins are visualized as magenta bands.
Fig 3.
pH and temperature effects on enzyme activity and cellulase mode of action.
(A) pH and temperature effects. Cellulase activity was determined by measuring the amount of reducing sugars released from the hydrolysis of 0.5% (w/v) CMC using the DNS method. Assays were carried out in triplicate, and results were expressed as mean ± standard deviation. (B) Mode of action of HrGH45: cello-oligosaccharides hydrolysis. TLC was performed using the double-ascending method and a mixture of AcOEt/AcOH/H2O (3:2:1) as the mobile phase. After separation, the TLC plate was air-dried, sprayed with 10% (v/v) H2SO4 in EtOH, and heated until visualizing the resolved products. +, Hydrolysis reaction; -, Blank reaction; G1, Glucose; G2, Cellobiose; G3, Cellotriose; G4, Cellotetraose; G5, Cellopentaose. (C) Hypothesized substrate-binding model of HrGH45. Numbers (-2 to +4) represent the putative sugar-binding subsites. The arrow indicates the cleavage point between subsites -1 and +1. Open circles illustrate D-glucosyl moieties.
Fig 4.
HrGH45 is a β-sheet-rich protein and belongs to GH45.
(A) Far-UV CD spectrum of HrGH45. Ellipticities are reported as mean residue molar ellipticity ([θ] deg∙cm2∙dmol-1). Three scans were averaged to obtain the final spectrum of HrGH45. CD data were corrected from solvent contributions. (B) Schematic representation of HrGH45 gene. White and magenta boxes represent exons and introns, respectively. (C) Nucleotide and deduced amino acid sequences of HrGH45. The single-letter amino acid code represents the polypeptide chain below the DNA sequence. Residue numbers for nucleotides and amino acids are indicated to the right of each row. The asterisk denotes the translational stop codon (TAA). In green, catalytic residues; in blue, predicted O-glycosylated amino acids; in red, N-glycosylation sequon; in yellow, the putative signal peptide for secretion.
Table 1.
Amino acid sequences identified by MALDI-TOF MS in protein sequencing experiments.
Fig 5.
Structure and catalytic groove of HrGH45.
(A) AlphaFold model for HrGH45. Secondary structure elements are shown in ribbons. (B) Superposition of EG27II (PDB 5XC8), a GH45 endoglucanase from the snail A.crosssean (magenta), and HrGH45 (cyan). (C) Surface representation of HrGH45 showing the putative active site residues (yellow) and its possible interaction with cellopentaose.
Fig 6.
Trimeric organization of HrGH45.
Overall view of the AlphaFold-Multimer model for HrGH45 showing (A) the buried surface area among monomers (green), (B) the putative location of the C-termini (blue), and (C) the catalytic clefts exposed to the solvent (red). Monomers are shown in white.