Removal of N-linked glycans in cellobiohydrolase Cel7A from Trichoderma reesei reveals higher activity and binding affinity on crystalline cellulose

Kołaczkowski, Bartłomiej M.; Schaller, Kay S.; Sørensen, Trine Holst; Peters, Günther H. J.; Jensen, Kenneth; Krogh, Kristian B. R. M.; Westh, Peter

doi:10.1186/s13068-020-01779-9

Biotechnology for Biofuels and Bioproducts

Table 3 Steady-state kinetics and binding affinity parameters (50 °C) of TrCel7A WT and the variants with modified N-glycosylation pattern

From: Removal of N-linked glycans in cellobiohydrolase Cel7A from Trichoderma reesei reveals higher activity and binding affinity on crystalline cellulose

TrCel7A	^convMM			^invMM		Kinetic substrate accessibility	Adsorption isotherms
	^convV_max/E₀	^convK_M	η	^invV_max/S₀	^invK_M	Γ_attack	Γ_max	K_d
	(s⁻¹)	(g/ L)	(L/g/s)	(μmol/g/s)	(μM)	(μmol/g)	(μmol/g)	(μM)
Avicel
WT_Ao	0.40 ± 0.01^a	18.1 ± 1.8^a	0.022	0.034 ± 0.001	2.45 ± 0.18	0.08 ± 0.00	0.1 ± 0.01	0.26 ± 0.06
N45Q_Ao	0.34 ± 0.02	11.6 ± 2.5	0.030	0.048 ± 0^a	2.92 ± 0.04^a	0.14 ± 0.01	0.23 ± 0.02^a	0.69 ± 0.21
N270Q_Ao	0.35 ± 0.02	11.6 ± 2.4	0.030	0.031 ± 0.001	2.22 ± 0.2	0.09 ± 0.01	0.13 ± 0.01	1 ± 0.28
N384Q_Ao	0.35 ± 0.02	10.6 ± 1.5	0.033	0.037 ± 0.001^a	2.54 ± 0.25^a	0.11 ± 0.01	0.10 ± 0.01	0.3 ± 0.18
ΔN-glyc_Ao	0.24 ± 0.01^a	6.2 ± 0.8	0.040	0.048 ± 0.002^a	3.54 ± 0.3^a	0.20 ± 0.01	0.27 ± 0.02^a	1.01 ± 0.22^a
WT_Tr	0.32 ± 0.01	9 ± 1.2	0.035	0.02 ± 0.001^a	1.16 ± 0.09^a	0.06 ± 0.00	0.08 ± 0.01^a	0.27 ± 0.08
ΔN-glyc_Tr	0.32 ± 0.01	7.4 ± 0.9	0.043	0.032 ± 0.001	1.71 ± 0.12	0.10 ± 0.00	0.11 ± 0.01	0.4 ± 0.11
RAC
WT_Ao	0.48 ± 0.02^a	1.3 ± 0.2^a	0.37	0.65 ± 0.01	0.95 ± 0.06	1.4 ± 0.1	0.92 ± 0.04^a	0.03 ± 0.01
N45Q_Ao	0.37 ± 0.02	0.9 ± 0.1	0.41	0.74 ± 0.02^a	1.23 ± 0.1^a	2.0 ± 0.1	1.79 ± 0.0 ^a	0.05 ± 0^a
N270Q_Ao	0.38 ± 0.02	1.1 ± 0.2	0.33	0.61 ± 0.01	1.15 ± 0.08	1.6 ± 0.1	1.14 ± 0.05^a	0.03 ± 0.01
N384Q_Ao	0.34 ± 0.01^a	0.8 ± 0.1	0.43	0.62 ± 0.02	1.03 ± 0.08	1.8 ± 0.1	1.2 ± 0.08^a	0.02 ± 0.01
ΔN-glyc_Ao	0.30 ± 0.01^a	0.5 ± 0.0^a	0.40	0.68 ± 0.01^a	0.82 ± 0.05	2.9 ± 0.1	1.58 ± 0.04	0.11 ± 0.01^a
WT_Tr	0.47 ± 0.01^a	1 ± 0.1	0.45	0.46 ± 0.01^a	0.94 ± 0.09	1.0 ± 0.0	1.83 ± 0.18^a	0.01 ± 0.01^a
ΔN-glyc_Tr	0.47 ± 0.01^a	0.8 ± 0.1	0.58	0.59 ± 0.02	0.93 ± 0.1	1.3 ± 0.0	1.59 ± 0.16	0.03 ± 0.01
BMCC
WT_Ao	1.46 ± 0.14^a	2.1 ± 0.4^a	0.71	0.45 ± 0.02^a	1.59 ± 0.15^a	0.31 ± 0.03	1.17 ± 0.12	1.28 ± 0.29
N45Q_Ao	1.33 ± 0.09^a	1.7 ± 0.2^a	0.77	0.52 ± 0.02^a	1.23 ± 0.11	0.39 ± 0.03	1.96 ± 0.35^a	1.2 ± 0.44
N270Q_Ao	1.13 ± 0.06^a	1.1 ± 0.1	1.01	0.44 ± 0.02	1.49 ± 0.14^a	0.39 ± 0.03	0.93 ± 0.15^a	0.99 ± 0.38
N384Q_Ao	1.01 ± 0.03	1 ± 0.1	1.02	0.38 ± 0.02	1.29 ± 0.15	0.38 ± 0.02	1.06 ± 0.04	0.85 ± 0.08
ΔN-glyc_Ao	0.96 ± 0.09	1.6 ± 0.3^a	0.59	0.48 ± 0.01^a	1.38 ± 0.1^a	0.50 ± 0.05	1.2 ± 0.06	0.62 ± 0.09
WT_Tr	0.63 ± 0.03^a	0.7 ± 0.1	0.89	0.29 ± 0.01^a	0.66 ± 0.06^a	0.46 ± 0.03	1.34 ± 0.11	0.48 ± 0.12
ΔN-glyc_Tr	0.76 ± 0.03	0.6 ± 0.1^a	1.33	0.4 ± 0.01	0.82 ± 0.06^a	0.53 ± 0.03	1.71 ± 0.04^a	0.56 ± 0.04

The parameters were derived from ^convMM, ^invMM and binding isotherm using Avicel, RAC and BMCC. The ± values correspond to the error of non-linear fit of Michaelis–Menten curves and binding isotherm curves. The parameters statistically different from the others at the 0.05 level of significance are indicated with letter ‘a’ (Additional file 1: Table S2)

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