Vmax=36.90 umole min -1

0 0.02 0.04 0.06 0.08 0.1 0.12

-1 -0.5 0 0.5 1 1.5 2 2.5

1/[S] (g/L) 1/V (umole min

-1

)

圖十三、Yen 洋菜酶與 low-melting-point agarose 作用之 Lineweaver-Burk 圖

Fig. 13. Lineweaver-Burk plot of Yen- agarases using low-melting-point agarose as substrate.

(Yen- agarases 濃度調整成約 10 U/mL 。各洋菜酶分別在洋菜 (受質)濃度 0.5、1 、1.5、 2、 2.5 g/L 下反應30分鐘，然後依 還原糖酵素活性測定之方法測還原糖產生量，以受質濃度及反應 速率倒數作圖求 Km 和Vmax 值。 (單位為 g/L ; 速率( V ) 單 位為µmole min^-1。)

圖十四、以 TLC 分析 Yen- agarases 水解洋菜糖之水解產物

Fig. 14. Thin layer chromatography of the products of agarose hydrolysis by Yen-agarases

( NA2：neoagarobiose；NA6：neoagarohexaose；G︰galactose。

lane 2 ~ 5為濃度 5U/mL 的Yen-agarases與 0.2 % agarose 於35

℃下，分別作用0.5、1、5、24 小時之水解產物。

M：mark；S：galactose；Lane 1：0.5 hr；Lane 2：1 hr；Lane 3：

5 hr；Lane 4：24 hr )

表一、Alcaligenes sp. strain Yen 生化特徵表

Table 1. Biochemical characterization of Alcaligenes sp. strain Yen.

Symbols: +,positive reaction ; -,negative reaction; m,motile; S:susceptible;

R:resistible

Characteristic tested Result

Motility m Hydrogen Sulfide -

Indole - Lysine - Arginine -

Characteristic tested Result

Ornithine - Tryptophan Deaminase -

Esculin

表二、 Yen 洋菜酶之純化表

Table 2. Summary of the purification of Yen-agarases

Procedure Volume

1: 1 unit (U)/mL = 1 μmole D-galactose equivalent /min/mL.

表三、Yen-agarases 對九種多糖之基質特異性

Table 2. Substrate specificity of Yen-agarases against nine polysaccharides

Substrate Yen-agarases

High-melting-point agarose 51.45¹ Low-melting-point agarose 42.60

Difco agar 30.72

Agar power 24.84

Agar A 32.68

PCH-D agar 24.78

Sodium alginate 9.28

ι-Carrageenan ---²

κ-Carrageenan ---

1: µmole D-galactose equivalent/min/mL.

2: Could not be determined.

表四、金屬離子對 Yen-agarases 耐受性之影響

Table 3. Effect of metal ions on the tolerance of Yen-agarases

Metal ions 0.1 X¹ 1 X 10 X 1 mM

None 100 100 100 100

KCl 120.24 101.02 82.43 127.72

MgCl₂ 89.06 98.87 110.12 99.05

SrCl₂ 106.37 106.73 105.88 102.97

MnCl2 120.01 127.77 122.52 151.13

ZnCl2 100.17 111.02 130.65 128.95

CoCl₂ 110.46 112.86 85.83 61.67

CuCl₂ 95.07 120.20 27.72 118.20

HgCl2 100.20 110.20 112.45 96.06

FeCl3 98.97 115.25 121.47 112.02

1: X = KCl: 10.00 mM; MgCl₂: 4.90 mM; SrCl₂: 0.09 mM; MnCl₂:

1.60 mM; ZnCl₂: 0.45 mM; CuCl₂: 4.00 mM; HgCl₂: 6.00 pM; CoCl₂: 16.00 pM;FeCl3: 1.30 mM.

The activity was measured using 10 U/mL of the enzyme to the 0.2%

substrate which containing prior metal ions at 35℃ for 30 min.Each point represents the average of three experiments.

Activity without added metal ions was taken as 100% activity.

附錄一、中性洋菜凝膠機制

Appendix 1. The gelling mechanism of agarose (李,2005)

(A)

(B)

附錄二、 Agarose 和 agaropectin 的基本結構( Araki, 1956; Anderson and Rees, 1965)

Appendix 2. The structure of (A) agarose (B) agaropectin

附錄三、 Agarases 的生產微生物、種類及分離來源 (陳，2006) Appendix 3. The list of agarase-producing microorganisms, agarases

types and their isolation sources Bacterium Strain

GJ1B α-agarase marine environments

Potin et al., 1993;

Hassairi et al., 2001 Alteromonas sp. E-1 β-agarase soil Kirimura et al., 1999 Alteromonas sp. BR6/3 agarase soil Shome et al., 2000 Alteromonas sp. C-1 β-agarase marine

environments

Leon et al., 1992

Arthrobacter sp. S-22 agarase seaweed Osumi et al., 1997 Bacillus cereus ASK202 β-agarase marine

environments

Kim et al., 1999

Bacillus sp. MK03 α-NAOS hydrolase

soil Suzuki et al., 2002

Bacillus sp. MK03 β-agarase soil Suzuki et al., 2003 Cytophaga NCMB1327 β-agarase marine

environments

Duckworth and Turvey, 1969 Cytophaga diffluens agarase seaweed Nikolaeva et al., 1999 Cytophaga flevensis β-agarase freshwater

lakes

van der Meulen and Harder, 1975 Cytophaga flevensis NABase

NATase

seaweed Whitehead et al., 2001 Microscilla sp. PRE1 agarase marine

sediment

Zhong et al., 2001;

Ensor et al., 1999 Microscilla sp. SB1-4 agarase marine

sediment

Naganuma et al., 1993 Paenibacillus agarase soil Hosoda et al., 2003

Appendix 3. (Continued)

Pasteurella pneumotropica

SCC21 agarase marine environments

吳，1999

Pseudoalteromonas antarctica

N-1 β-agarase marine environments

agarase mussels Ivanova et al., 1998

Pseudoalteromonas gracilis

B9 β-agarase seaweed Schroeder et al., 2003

Pseudomonas

β-agarase seaweed Araki and Arai, 1954

Pseudomonas sp. β-agarase wastewater laboratories

Malmqvist, 1978

Pseudomonas sp. PT-5 agarase seaweed Yamaura et al., 1991 Pseudomonas sp. SK38 β-agarase seaweed Kang et al., 2003;

Ryu, et al., 2001 Pseudomonas sp. W7 β-agarase marine

environments

agarase sea water Ivanova et al., 2001

Streptomyces coelicolor

A3(2) β-agarase marine environments

Hodgson and Chater, 1981

Streptomyces coelicolor

NB3443 agarase marine environments

Stanier, 1942

Appendix 3. (Continued)

β-agarase seaweed Ishimatsu, 1953

Vibrio harvryi FLB-17 agarase marine environments

Fukasawa and Kobayashi, 1987 Vibrio sp. AP-2 β-agarase marine

environments

Aoki et al., 1990a, b

Vibrio sp. LX-3 agarase soil Li et al., 2003 Vibrio sp. PO-303 agarase marine

environments

Araki et al., 1998

Vibrio sp. JT0107 α-NAOS hydrolase

seaweed Sugano et al., 1994

Vibrio sp. JT0107 β-agarase seaweed Sugano et al., 1993a Zobellia

galactanivorans Dsij

β-agarase Allouch et al., 2003

1: Agarase: means still not purification and characterization; NABase: neoagaro-biase; NAB hydrolase:

neoagaro-biose hydrolase; NATase: neoagaro-tetraase; β-NAT hydrolase: β-neoagaro-tetraose hydrolase;α-NAOS hydrolase: α-neoagarooligosaccharide hydrolase.

附錄四、 微生物所產洋菜酶分解洋菜之產物 (陳，2006)

Appendix 4. The oligosaccharides derived from various substrates by the degradation of microbial agarases

Bacterium Enzyme Substances Products Reference Arthrobacter sp.

S-22

Agarase¹ Neo- agarotetraose

neoagarobiose Osumi et al., 1997

β-agarase agarose and

neoagarooligo-saccharides

neoagarobiose Kirimura et al., 1999

Bacillus cereus ASK202

β-agarase agarose neoagarohexaose, neoagarotetraose

β-agarase agarose neoagarotetraose and

neoagarobiose van der Meulen and Harder,1976 Cytophaga

flevensis

NAT neoagarobiose D-galactose and 3,6-anhydro-

β-agarase agar Neoagarohexaose, neoagarotetraose

Vera et al., 1998

Appendix 4. (Continued)

Pseudomonas atlantica

NAB hydrolase

neoagarobiose D-galactose and 3,6-anhydro-

β-agarase Ib neoagarooctaose neoagarotetraose

Malmqvist, 1978

Vibrio sp. AP-2 β-agarase neoagarooligosac charides and neoagarotetraose

neoagarobiose Aoki et al., 1990a

Appendix 4. (Continued)

Vibrio sp. strain JT0107

β-agarase agarose neoagarotetraose and neoagarobiose

Sugano et al., 1993a

Vibrio sp. strain JT0107

α-NAOS hydrolase

Agaro- oligosaccharides

agaropentaose, agarotriose, agarobiose and

D-galactose

Sugano et al., 1994

1: Refer to Appendix 2.

附錄五、 洋菜酶的生化特性 (陳，2006)

Appendix 5. The biochemical properties of agarases Enzyme

Appendix 5. (Continued)

附錄六、海水中各離子基準濃度表(陳，2006) Metal Ion

Type

Conc. Metal Ion Type

Conc.

K⁺ 10 mM Cs⁺ 64 µM

Mg²⁺ 4.9 mM Sr²⁺ 0.09 mM

Mn²⁺ 1.6 mM Zn²⁺ 0.45 mM

Cu²⁺ 4 mM Hg²⁺ 6 pM

Co²⁺ 16 pM Fe³⁺ 1.30 mM

在文檔中 新種洋菜分解菌 Alcaligenes sp. Strain Yen 所產洋菜酶純化與部分特性 (頁 77-93)