Cross-Linked Enzyme Aggregates of Β-Glucosidase from Prunus Domesticaseeds

Supplementary data for

Cross-linked enzyme aggregates of β-glucosidase from Prunus domestica seeds

Lei Chen,1 Ying-Dan Hu,2 Ning Li,*,1Min-Hua Zong*,1

1 State Key Laboratory of Pulp and Paper Engineering, College of Light Industry and Food Sciences,

South China University of Technology, Guangzhou 510640, China

2 College of Biosciences and Bioengineering, South China University of Technology, Guangzhou

510006, China

* Corresponding authors

Dr. N. Li, Tel/Fax: +86 20 2223 6669; Email: [email protected].

Prof. M. H. Zong, Tel: +86 20 8711 1452; Fax: +86 20 2223 6669; Email: [email protected].

Preparation of CLEAs

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

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.

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glutaraldehyde (2.5 M) was added with mechanically shaking at 4 C. At different time intervals, samples (50 μL) were withdrawn, and quenched with 450 μL citric acid-Na2HPO4 buffer

(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.

Effect of reducing agent

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.

After CLEAs prepared under the optimum conditions were subject to reduction by 20 mM NaBH 4 for

30 min, they were washed three times and re-suspended in 50 mM citric acid-Na 2HPO4 buffer (pH 5.0) for use.

Fig. S1 Effect of different precipitants on the activity recovery in the aggregates 50 μl enzyme solution, 450 μl precipitants.

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)

Fig. S3 Effect of the precipitants on the redispersibility of CLEAs

1) 1-Propanol-CLEAs; 2) 2-propanol-CLEAs; 3) acetone-CLEAs; 4) ethanol-CLEAs

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

10-50 mM NaBH4, cross-linking time of 2 h; Residual activity was assayed after incubating at 60 C for 30 min.

Fig. S5 Effects of pH (a) and temperature (b) on enzyme activity

Conditions: a) the total reaction volume of 3 mL, 50 μL free enzyme/CLEAs, 1 mM pNPG, 50 mM

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

CLEAs), at 30-70 C.

Table S1 Effects of precipitants and concentrations on activity recovery

Precipitant Concentration Total relative activity CLEAs activity

(%, 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.

Table S2 Effects of glutaraldehyde concentrations and cross-linking time on activity recovery

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

52 68 a

52 51 a

52 19 a

42 21 b

42 17 b

68 28 b

72 22 b

50 26 b

50 22 b

50 15 b

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).