Characterization of two endoglucanases for the classification of the earthworm, Eisenia fetida Waki

Figures & data

Fig. 1. Phylogenetic analysis of the COI region in earthworms.

Notes: The COI sequences were retrieved by the BLAST program using several databases. Multiple alignment was carried out using Clustal W, and a phylogenetic tree was constructed using neighbor joining by TreeView. The reliability of cluster formation was analyzed by bootstrap resampling 100 times, and the tree was generated with C. oncophora COI serving as the out-group. The scale bar represents the number of changes per nucleic acid position.

Fig. 2. Morphology and cut parts of E. fetida Waki.

Table 1. Enzyme activity of crude extract from each segment of E. fetida Waki.

Substrate	Segment 1	Segment 2	Segment 3	Segment 4	Segment 5
CMC	++	+++	++	+	+
Lichenan	++	++	++	+	+
Laminarin	++	++	++	–	–
Birchwood xylan	++	+++	++	–	–
Oat spelt xylan	++	+++	++	–	–
Xyloglucan	–	–	–	–	–
AZCL-β-glucan	+++	+++	+++	–	–
AZCL-β-1,3-glucan	–	+++	–	–	–
AZCL-amylose	++	+++	++	–	–
AZCL-arabinan	–	–	–	–	–
AZCL-galactan	–	–	–	–	–
AZCL-mannan	++	+++	+++	–	–
AZCL-xylan	–	–	–	–	–

Notes: Protein content of each sample was 9.51 µg. Activity: +++, clear or colored circle diameter >10 mm; ++, diameter 5–10 mm; +, diameter under 5 mm; –, not detected.

Fig. 3. Enzymatic saccharification with E. fetida Waki (A) and T. reesei QM9414 (B) cell-free extracts.

Notes: Wheat bran was used as a substrate. The reducing sugar was determined by the Somogyi–Nelson method. The reaction mixture included 1.0 g wheat bran and 30 units of CMCase equivalent. The white circles, black circles, and triangles represent 30, 40, and 50 °C, respectively.

Fig. 4. Detection of saccharification products in E. fetida Waki (A) and T. reesei QM9414 (B).

Notes: Wheat bran enzymatic saccharification products were determined using high-performance liquid chromatography. The white circles, black circles, white triangles, black triangles, white squares, black squares, and crosses represent xylose, glucose, arabinose, mannose, maltose, and cellobiose products, respectively.

Fig. 5. Elution profile of EG isozymes in E. fetida Waki from the DEAE-Toyopearl column.

Note: CMC (1.0%) was used as a substrate.

Table 2. Elution profile of EG isozymes in E. fetida Waki from a DEAE-Toyopearl column.

Enzyme	Purification step	Total activity (U)	Total protein (mg)	Specific activity (U/mg protein)	Purification (fold)	Yield (%)
	Cell extract	2030	8260	0.246	1	100
	Ammonium sulfate (20–60% saturation)	1360	4300	0.316	1.3	67
EfEG1	DEAE-Toyopearl	315	42.1	7.48	30.4	16
	HiTrapCapto MMC	162	3.36	48.2	196	8
	Bio-ScaleMini CHT Type I	42.7	0.883	48.4	197	2.1
	Toyopearl HW55S	44.8	0.536	83.6	340	2.2
	Resource Q	1.69	0.0146	116	472	0.08
EfEG2	DEAE-Toyopearl	612	4.31	1.42	5.8	0.3
	HiTrapCapto MMC	3.18	2.84	1.12	4.6	0.16
	Toyopearl HW55S	1.52	0.423	3.59	14.6	0.07
	Resource Q	1.23	0.0208	59.1	240	0.06

Note: CMC (1.0%) was used as a substrate.

Fig. 6. SDS-PAGE of purified EfEG1 and EfEG2.

Notes: Lane 1, standard proteins; lane 2, EfEG1; lane 3, EfEG2. Standard proteins were phosphorylase b (97 kDa), albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), and trypsin inhibitor (20 kDa) (all from GE Healthcare). The protein was stained with Coomassie Brilliant Blue R-250 (Sigma-Aldrich).

Fig. 7. Effect of pH on the activity (A) and stability (B) of EfEG1 and EfEG2.

Notes: Enzyme activity was measured using the following buffers: 0.1 M McIlvaine buffer (pH 2.2–8.0) and glycine–NaOH buffer (pH 8.0–12.0). CMC (1.0%) was used as a substrate. The white circles and black circles represent EfEG1 and EfEG2 activity, respectively.

Fig. 8. Effect of temperature on the activity (A) and thermostability (B) of EfEG1 and EfEG2.

Notes: Enzyme activity was measured using 25 mM sodium phosphate buffer (pH 7.0). CMC (1.0%) was used as a substrate. The white circles and black circles represent EfEG1 and EfEG2 activity, respectively.

Table 3. Properties of EGs from animal and T. reesei QM9414.

Origin	Enzyme names	GH Family	Molecular mass (kDa)	Optimal pH	Optimal temp. (°C)	Specific activity (U/mg)	References
Eisenia fetida Waki	EfEG1	GHF9	53	6.0	45	116	This study
(Earthworm)	EfEG2	–	58	5.5	40	59.1	This study
Eisenia fetida Nagane	rEF-EG2	GHF9	50	5.5	40	10.8	^12^)
(Earthworm)	EF-CMCase25	–	25	5.0	40	7.3	^12^)
Trichoderma reesei QM9414	EGI	GHF7	54	6.0	50	100	^28^)
(Filamentous fungus)	EGII	GHF5	50	6.0	50	53	^29^)
	EGIII	GHF12	25	6.3	38	22	^30^)
Reticulitermes speratus Kolbe	YEG1	GHF9	42	6.0	50	73.6	^31^)
(Termite)	YEG2	GHF9	41	6.0	50	83.4	^31^)
Haliotis discus hannai	HdEG66	GHF9	66	6.3	38	–	^32^)
Teleogryllus emma	TeEG-I	GHF9	47	5.0	40	948.1	^33^)
(Cricket)
Apriona germari	Ag-EGase I	GHF45	29	6.0	50	993	^34^)
(Longicorn beetle)	Ag-EGase II	GHF45	36	6.0	50	812	^35^)

Notes: CMC (1.0%) was used as a substrate in this study and Ref.³⁰⁾. CMC (2.0%) was used as a substrate in Refs.^31,33–35). CMC (0.5%) was used as a substrate in Refs.^29,32). CMC (0.4%) was used as a substrate in Ref.¹²⁾. CMC (unknown) was used as a substrate in Ref.²⁸⁾.

Table 4. Substrate specificity of purified EfEG1 and EfEG2 from E. fetida Waki.

Substrate	EfEG1	EfEG2
CMC	+++	+++
Lichenan	++	++
Laminarin	–	±
Birchwood xylan	–	–
Oat spelt xylan	+	–
Xyloglucan	–	–
AZCL-β-glucan	+++	+++
AZCL-β-1,3-glucan	–	±
AZCL-amylose	–	–
AZCL-arabinan	–	–
AZCL-galactan	–	–
AZCL-mannan	–	–
AZCL-xylan	–	–
4MU-G	++	–
4MU-G2	–	–

Notes: Protein content of each sample was 1.60 × 10⁻¹ µg. Activity: +++, clear or colored circle diameter >10 mm; ++, diameter 5–10 mm; +, diameter under 5 mm; ±, present but not measurable; –, not detected.

Fig. 9. TLC analysis of the reaction products of purified rEfEG1.

Notes: Lane 1, standard oligosaccharides; lane 2, reaction products for 0 min; lane 3, 15 min; lane 4, 30 min; lane 5, 1 h; lane 6, 3 h; lane 7, 6 h; lane 8, 12 h; lane 9, 24 h. Lane 1 indicates the standard oligosaccharides (G1, glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose; G5, cellopentaose).

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