Table 1.

Biochemical data of the characterized mutants. The reaction from sucrose only at 30 °C with 1 unit ml⁻¹ of pure enzyme and 50 mm sodium acetate buffer, pH 5.75, is shown. The specific activity of ASRΔ2 was 30.2 ± 1.0 units mg⁻¹. Specific activity was determined in triplicate. The T_m was determined by differential scanning fluorimetry

	Residual activity	ΔTm with the WT enzyme	NMRa		HPSEC		Hydrolysis
			α-1,3-Linkages	α-1,6-Linkages	Polymer formed (area)	tR polymer
	%	°C	%	%	%	min	%
WT
ASRΔ2	100 ± 3.3	0	35	65	31.5 ± 1.6	37 ± 0.2	4.4 ± 0.4
Mutations in domain V
ASRΔ2 Y158A (pocket V-A)	80.2 ± 3.0	+0.1	34	66	27.2	36.8	4.7
ASRΔ2 Y241A (pocket V-B)	78.4 ± 3.2	+0.2	35	65	28.0	36.6	4.7
ASRΔ2 Y158A/Y241A (pockets V-A + V-B)	77.5 ± 2.4	+0.2	33	67	27.6	36.5	4.7
ASRΔ5	79.1 ± 2.7	−2	30	70	4.5	38.2	5.8
Mutations in SBS-A1
ASRΔ2 N703A	95.3 ± 6.0	−0.1	35	65	33.5	36.9	4.3
ASRΔ2 S713A	95.8 ± 3.3	−0.3	35	65	31.7	36.6	4.3
ASRΔ2 W716A	76.4 ± 7.0	−1.0	35	65	30.0	36.4	5.0
ASRΔ2 Q700A	80.5 ± 2.8	−0.2	35	65	20.4	35.7	5.6
ASRΔ2 Y717A	80.2 ± 2.4	−0.1	33	67	18.8	35.6	5.2
ASRΔ2 Q700A/Y717A	26.1 ± 7.7	−0.9	35	65	18.3	35.5	5.8
Mutations in domain V + SBS-A1
ASRΔ2 Y158A/Y717A	62.9 ± 3.9	−0.1	32	68	13.3	35.3	5.5
ASRΔ2 Y241A/Y717A	78.3 ± 5.8	−0.2	31	69	8.5	35.4	5.9
ASRΔ5 Y717A	63.1 ± 3.6	−2.4	30	70	2.3	36.4	6.2

^a NMR was performed on crude reaction medium.