<p>Supplementary data for</p><p>Cross-linked enzyme aggregates of β-glucosidase from Prunus domestica seeds</p><p>Lei Chen,1 Ying-Dan Hu,2 Ning Li,*,1Min-Hua Zong*,1</p><p>1 State Key Laboratory of Pulp and Paper Engineering, College of Light Industry and Food Sciences,</p><p>South China University of Technology, Guangzhou 510640, China</p><p>2 College of Biosciences and Bioengineering, South China University of Technology, Guangzhou</p><p>510006, China</p><p>* Corresponding authors</p><p>Dr. N. Li, Tel/Fax: +86 20 2223 6669; Email: [email protected].</p><p>Prof. M. H. Zong, Tel: +86 20 8711 1452; Fax: +86 20 2223 6669; Email: [email protected].</p><p>Preparation of CLEAs</p><p>Precipitation. To 50 μL enzyme solution, an appropriate amount of precipitant (the corresponding solvents and concentrations listed in Table 1) was added, and the mixture was mechanically shaken at 4</p><p>C for 5 min, and then centrifuged (14,000 g) for 5 min. The supernatant was collected; the pellets were re-dissolved in 100 μL citric acid-Na2HPO4 buffer (50 mM, pH 5.5). The activities of both fractions were measured.</p><p>Cross-linking. To the mixture of 1 mL enzyme solution (approximately 4 mg protein) and 2 mL 1- propanol, 12-60 μL glutaraldehyde (2.5 M) was added with mechanically shaking at 4 C. At different time intervals, samples (50 μL) were withdrawn, and quenched with 450 μL citric acid-Na2HPO4 buffer</p><p>(50 mM, pH 5.0). After centrifugation (14,000 g, 5 min), CLEAs were washed three times. The activities in the supernatant and the aggregates (re-dispersed in buffer) were measured. </p><p>Effect of reducing agent</p><p>To 1 mL enzyme solution (around 4 mg protein), 2 mL 1-propanol and 12 μL glutaraldehyde (2.5 M) were added. After mechanically shaking at 4 C for 1.5 h, a certain amount of NaBH4 solution was added to make its final concentrations to be 10-50 mM. After incubation of 30 min, CLEAs were collected by centrifugation (14,000 g, 5 min), and washed three times with 50 mM citric acid-Na2HPO4 buffer (pH 5.0).The activities of CLEAs were measured. The residual activities were assayed after incubating at 60 C for 30 min. </p><p>After CLEAs prepared under the optimum conditions were subject to reduction by 20 mM NaBH 4 for</p><p>30 min, they were washed three times and re-suspended in 50 mM citric acid-Na 2HPO4 buffer (pH 5.0) for use.</p><p>Fig. S1 Effect of different precipitants on the activity recovery in the aggregates 50 μl enzyme solution, 450 μl precipitants.</p><p>Fig. S2 Effects of the precipitants and concentrations on the activity recovery in the aggregates and the supernatant. (The number on the X-axis represents the volume ratio of organic solvent to enzyme solution)</p><p>Fig. S3 Effect of the precipitants on the redispersibility of CLEAs</p><p>1) 1-Propanol-CLEAs; 2) 2-propanol-CLEAs; 3) acetone-CLEAs; 4) ethanol-CLEAs</p><p>Fig. S4 Effect of NaBH4 concentrations on activity recovery and thermostability of CLEAs Conditions: 1 mL enzyme solution (around 4 mg protein), 2 mL 1-propanol, 10 mM glutaraldehyde and</p><p>10-50 mM NaBH4, cross-linking time of 2 h; Residual activity was assayed after incubating at 60 C for 30 min. </p><p>Fig. S5 Effects of pH (a) and temperature (b) on enzyme activity </p><p>Conditions: a) the total reaction volume of 3 mL, 50 μL free enzyme/CLEAs, 1 mM pNPG, 50 mM</p><p> citric acid-Na2HPO4 buffer (pH 4.0-8.0) at 45 C; b) the total reaction volume of 3 mL, 50 μL free enzyme/CLEAs, 1 mM pNPG, 50 mM citric acid-Na2HPO4 buffer (pH 5.5 for free enzyme; pH 5.0 for</p><p>CLEAs), at 30-70 C.</p><p>Table S1 Effects of precipitants and concentrations on activity recovery</p><p>Precipitant Concentration Total relative activity CLEAs activity</p><p>(%, v/v) a (%) b recovery (%) - - 100 - Ethanol 67% 83 32 2-Propanol 80% 78 28 Acetone 67% 39 17 1-Propanol 67% 76 63 Cross-linking conditions: 40 mM glutaraldehyde at 4 C for 4 h. a Optimal concentration for enzyme precipitation. b The relative activity of the mixture including the supernatant and CLEAs.</p><p>Table S2 Effects of glutaraldehyde concentrations and cross-linking time on activity recovery</p><p>Cross-linking CLEAs activity recovery (%) 10 mM 20 mM 30 mM 40 mM 50 mM time (h) 1 76 84 78 73 59 2 84 82 76 70 57 3 80 77 71 68 44 After the mixture of 1 mL enzyme solution (around 4 mg protein) and 2 mL 1-propanol was shaken at 4 C for 10 min, glutaraldehyde was added. Table S3 Synthesis of various alkyl glycosides by using prune seed meal Substrate Product Time Yield (%) 52 78 a</p><p>52 68 a</p><p>52 51 a</p><p>52 19 a</p><p>42 21 b</p><p>42 17 b</p><p>68 28 b</p><p>72 22 b</p><p>50 26 b</p><p>50 22 b</p><p>50 15 b</p><p>50 15 b</p><p> a Reaction conditions: 0.5 mmol glucose and 2.5 U prune seed meal in 2 ml mixture of 10% (v/v) phosphate buffer (pH 6.0, 50 mM) and the corresponding alcohol at 50 °C and 200 rpm (unpublished data). b Reaction Conditions: 0.5 mmol glucose, 5 mmol alcohol, 5.5 U prune seed meal, ethylene glycol diacetate-10% (v/v) [BMIm]I-10% (v/v) phosphate buffer (pH 6.0, 50 mM), total volume 2 ml, 50 °C, 200 rpm (The results were published on J. Mol. Catal. B: Enzym. 2012, 74: 24-28).</p>