Supplementary Table 1. Purification of Chi-V from E. coli

Step 　　 Protein 　 Activity 　 Specific activity Yield 　　　　 (mg) (mmol/min) 　 (mmol/min/mg) (%)

Crude extract 2982.0 　 159.4 　　　　 0.05 　 100

SP-Sepharose 　 53.24 　 62.3 　　　　 1.17 　 39.1

Sephacryl 28.76 34.4 1.20 21.6 S-100 HR

66.4

39.2

26.1

14.3

22.8

1 2

(kDa)

Supplementary Fig. 1.

Supplementary Fig. 1. SDS–PAGE was done by the method of Laemmli. Lane 1, marker proteins and lane 2, purified NtChiV. The gel was stained with Coomassie brilliant blue R250.

A

B

C

D

pH Temperature (oC)

0 2 4 6 8 10 12 20 30 40 50 60 70 80

80

40

60

20

100

80

40

60

20

100

0

0

Rel

ativ

e ac

tivity

(%

)

Supplementary Fig. 2.

Supplementary Fig. 2. Effects of pH and temperature on activity and stability of NtChiV. A, The optimum pH of the chitinase was measured after incubation with glycolchitin in the buffer at various pHs at 37 oC for 15 min. B and D, The pH and thermal stabilities of the enzyme were measured from the residual activities after incubation in the buffer at various pHs at 37 oC for 6 h and in 50 mM sodium acetate buffer, pH 5.0, at various temperatures for 6 h, respectively. C, The optimum temperature was determined by measuring the activities in 50 mM sodium acetate buffer, pH 5.0, at various temperatures.

012345678

0 20 40 60 80 100 120

0

1

2

3

4

5

6

7

8

0 20 40 60 80 100 120 140 160 180

Con

cent

ratio

n (m

M)

Reaction Time (min)

(NAG)6

(NAG)2

(NAG)3

(NAG)4

(NAG)4

(NAG)2

6

0

1

2

3

4

5

0 20 40 60 80 100 120

(NAG)5 (NAG)2

(NAG)3

A

B

C

Supplementary Fig. 3. Time-courses of the enzymatic hydrolysis of (NAG)n. A, substrate (NAG)6; B, substrate (NAG)5; C, substrate (NAG)4. Enzyme concentration was 0.16 M. The enzymatic reaction was conducted in 20 mM sodium acetate buffer pH 5.0 at 40 oC. Determinations of the products were performed by a gel-filtration HPLC using a column of TSK-GEL G2000PW (Tosoh). Symbols: squares, (NAG)2; triangles, (NAG)3; diamonds, (NAG)4; circles, (NAG)6. Lines were obtained by roughly following the experimental data points.

100

Mass (m/z)

A

B

C

(NAG)6

(NAG)3 (NAG)4(NAG)2(NAG)5

(NAG)6

(NAG)6

(NAG)4

(NAG)3

(NAG)2

(NAG)7 (NAG)8

100

0

0

100

Inte

ncity

(%

)In

tenc

ity (

%)

Inte

ncity

(%

)

0200 400 800 1000 1200600 1400 1600

1400 1600

1400 16000

10

10(NAG)7

(NAG)8

0

Supplementary Fig. 4. MALDI-TOF-MS analysis of the enzymatic products. A, standards, (NAG)2~(NAG)6; B, substrate (NAG)6; C, enzymatic products from the substrate (NAG)6. The reaction mixture, consisting of 0.16 M NtChiV and 5.0 mM (NAG)6 in 20 mM sodium acetate buffer, pH 5.0, was incubated for 10 min at 40 oC. The reaction mixture was mixed with an equal volume of 2,5-dihydroxy benzoic acid (2,5-DHB) and the resultant solution was employed for mass measurements using a MALDI micro MX (Waters). (NAG)2-6 oligosaccharides were used as standards for m/z.