Alteromonas sp.nov. SY37-12菌株产生的琼胶酶酶学性质及酶解产物的分析
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摘要
本文以开发海洋活性寡糖为最终目标，以筛选具有优良性状的产琼胶酶菌株以及用酶法降解琼胶获得不同聚合度的寡糖为基本目的和出发点，研究了产酶菌株的生理生化特征，不同因素对菌株发酵产酶的影响和酶的分子量及基本性质，分析了琼胶酶降解琼胶底物产生的寡聚糖产物的结构和组成，对琼胶寡糖的生物活性进行了初步的探讨。
(1) 从自然界中筛选一株具有优良性状、产琼胶酶能力高的菌株Alteromonas sp. nov. SY37-12，长杆状，0.5～0.6μm×1.8～2.1μm。端生单鞭毛。Alteromonas sp. nov. SY 37-12在2216E培养基上培养24h，菌落圆形，乳白色。菌株在增殖的同时，可使菌落周围的琼胶培养基形成较深的釜底形凹陷，并且具有较大的透明圈，直径约为10mm。该菌株氧化酶阳性，过氧化氢酶阳性，不积累聚羟丁酸盐颗粒作为胞内贮存物，生长不需要有机因子，在没有NaCl存在时菌体不能生长。根据伯杰氏手册第九版确定其为交替单胞菌属的一个新种。通过诱变提高其产酶能力。
(2) 研究不同因素对产酶的影响以确定最适的发酵培养条件。最适发酵培养基组成：琼胶0.3％，K_2HPO_4 0.1％，氯化铵0.1％，NaCl 2％，0.05％MgSO_4，0.01％CaCl_2。最适培养条件：250ml三角瓶中装入125ml液体发酵液，最适培养温度、起始pH、转速和培养时间分别为35℃、pH7.0、150rpm在上述条件下发酵培养20h。
(3) 研究所选菌株产生的胞外琼胶酶的基本酶学性质，琼胶酶分子量并确定最佳的酶反应条件。用饱和度80％的硫酸铵盐析法提取粗酶，用离子交换柱层析和凝胶柱层析将酶纯化了27.6倍，SDS-PAGE测定该菌株产生的琼胶酶的分子量为39.5KDa。确定了最适的酶反应条件：酶反应的最适温度为35℃；酶反应的最适pH值为pH7.0；底物的最适NaCl含量为1％。K~+、Ca~(2+)、Na~+、Mg~(2+)、等离子对酶有促进作用；Fe~(3+)、Zn~(2+)、Sn~(2+)、Hg~(2+)、Ba~(2+)等离子对酶有抑制作用。该酶在35℃下具有较好的热稳定性，在50℃以上酶
刀tero，on口;sP.nov.sY37一12菌株所产生琼胶酶的酶学性质及酶解产物的分析
易失活。该酶对琼胶底物的动力学结果为:Km=6.073，Vmax=0.152
(4)研究琼胶酶降解琼胶的工艺，制备琼胶寡糖。通过基体辅助激光解析
离子化飞行质谱确定其分子量和样品的组成。通过’“c一NMR确定琼胶寡糖的
类型，确定了该琼胶酶为p一琼胶酶，降解琼胶的p一1，4糖昔键，产生以D一半
乳糖为还原性末端，以3，6一内醚一L一半乳糖为非还原性末端的新琼寡糖系列。
(5)分析琼胶降解产物的生物学活性。系列琼胶寡糖具有很好的抗氧化活
性。在抗溶血，清除轻基自由基和抑制脂质过氧化方面的活性高于Vc的抗
氧化活性。其中样品H7具有一定的抑菌作用，对铜绿假单胞菌和产气杆菌
有抑菌作用。最小抑菌浓度分别为10mg/mL和2.5m留mL。
本研究为琼胶酶的工业化生产提供了一定的理论基础，为进一步开发海
洋活性寡糖具有一定的指导意义。
In order to gain the ocean oligosaccharides with the bioactivities, we have studied the bacterial biochemical properties, the different effects on producing agarase, agarase characterization and degradation products beginning with the isolation of the bacterium.
1 A bacterium strain SY 37-12 with the high level of agarase was isolated from seaweed and the isolate was named as Alteromonasp. nov. SY 37-12 according to the Bergey's manual of determinative bacteriology (9th ed.). Physical and chemical mutation can increase the bacterial ability in producing the agarase.
2 An optimum culture medium was determinate as: 0.3% agar, 0.1% K2HPO4, 0.1% NH4C1, 2%NaCl, 0.05%MgSO4, 0.01%CaCl2. An optimum culture conditions were determinate as: 125ml liquid culture media in 250ml Erlenmeyer flask, pH7.0, culture temperature 35, rotation rate ISOrpm and culture time 20h.
3 We study the basic properties of agarase produced by Alteromonas sp. nov. SY 37-12. Agarase was extracted by 80% saturation with solid sulfate ammonium. The enzyme was purified 27.6 fold and the molecular weight of the agarase is 39.5KDa by SDS-PAGE. The basic properties of the enzyme were studied as follows: The optimum pH, the optimum temperature and the optimum NaCl concentration are pH7.0, 35 and 1% respectively. The enzyme is activated by K. Ca2+. Na+. Mg2+and inhabited by Fe3+. Zn2+ Sn2+ Hg2+ Ba2+. The enzyme is stable at temperatures up to 35癈.
4 The composition and structure of main degradation product were examined by MALDI-TOF-MS and 13C-NMR. The agarase belongs to |3-agarase and can hydrolyze the (3-1,4-glycosydic linkages of agar, yielding
neoagarotetraose and neoagarohexaose as the main products.
5 The antioxidative activities and antibacterial activities of neoagarooligosaccharide were studied. The neoagarooligosaccharide have the high antioxidative activities in scavenging the lipid peroxidation, removing theOH and H2O2. The neoagarooligosaccharide, H7 shows the antibacterial activity. The MIC of H7 against E.aerogenes and P.aeruginosa are 2.5mg/mL and I Omg/mL respectively.
(1) 从自然界中筛选一株具有优良性状、产琼胶酶能力高的菌株Alteromonas sp. nov. SY37-12，长杆状，0.5～0.6μm×1.8～2.1μm。端生单鞭毛。Alteromonas sp. nov. SY 37-12在2216E培养基上培养24h，菌落圆形，乳白色。菌株在增殖的同时，可使菌落周围的琼胶培养基形成较深的釜底形凹陷，并且具有较大的透明圈，直径约为10mm。该菌株氧化酶阳性，过氧化氢酶阳性，不积累聚羟丁酸盐颗粒作为胞内贮存物，生长不需要有机因子，在没有NaCl存在时菌体不能生长。根据伯杰氏手册第九版确定其为交替单胞菌属的一个新种。通过诱变提高其产酶能力。
(2) 研究不同因素对产酶的影响以确定最适的发酵培养条件。最适发酵培养基组成：琼胶0.3％，K_2HPO_4 0.1％，氯化铵0.1％，NaCl 2％，0.05％MgSO_4，0.01％CaCl_2。最适培养条件：250ml三角瓶中装入125ml液体发酵液，最适培养温度、起始pH、转速和培养时间分别为35℃、pH7.0、150rpm在上述条件下发酵培养20h。
(3) 研究所选菌株产生的胞外琼胶酶的基本酶学性质，琼胶酶分子量并确定最佳的酶反应条件。用饱和度80％的硫酸铵盐析法提取粗酶，用离子交换柱层析和凝胶柱层析将酶纯化了27.6倍，SDS-PAGE测定该菌株产生的琼胶酶的分子量为39.5KDa。确定了最适的酶反应条件：酶反应的最适温度为35℃；酶反应的最适pH值为pH7.0；底物的最适NaCl含量为1％。K~+、Ca~(2+)、Na~+、Mg~(2+)、等离子对酶有促进作用；Fe~(3+)、Zn~(2+)、Sn~(2+)、Hg~(2+)、Ba~(2+)等离子对酶有抑制作用。该酶在35℃下具有较好的热稳定性，在50℃以上酶
刀tero，on口;sP.nov.sY37一12菌株所产生琼胶酶的酶学性质及酶解产物的分析
易失活。该酶对琼胶底物的动力学结果为:Km=6.073，Vmax=0.152
(4)研究琼胶酶降解琼胶的工艺，制备琼胶寡糖。通过基体辅助激光解析
离子化飞行质谱确定其分子量和样品的组成。通过’“c一NMR确定琼胶寡糖的
类型，确定了该琼胶酶为p一琼胶酶，降解琼胶的p一1，4糖昔键，产生以D一半
乳糖为还原性末端，以3，6一内醚一L一半乳糖为非还原性末端的新琼寡糖系列。
(5)分析琼胶降解产物的生物学活性。系列琼胶寡糖具有很好的抗氧化活
性。在抗溶血，清除轻基自由基和抑制脂质过氧化方面的活性高于Vc的抗
氧化活性。其中样品H7具有一定的抑菌作用，对铜绿假单胞菌和产气杆菌
有抑菌作用。最小抑菌浓度分别为10mg/mL和2.5m留mL。
本研究为琼胶酶的工业化生产提供了一定的理论基础，为进一步开发海
洋活性寡糖具有一定的指导意义。
In order to gain the ocean oligosaccharides with the bioactivities, we have studied the bacterial biochemical properties, the different effects on producing agarase, agarase characterization and degradation products beginning with the isolation of the bacterium.
1 A bacterium strain SY 37-12 with the high level of agarase was isolated from seaweed and the isolate was named as Alteromonasp. nov. SY 37-12 according to the Bergey's manual of determinative bacteriology (9th ed.). Physical and chemical mutation can increase the bacterial ability in producing the agarase.
2 An optimum culture medium was determinate as: 0.3% agar, 0.1% K2HPO4, 0.1% NH4C1, 2%NaCl, 0.05%MgSO4, 0.01%CaCl2. An optimum culture conditions were determinate as: 125ml liquid culture media in 250ml Erlenmeyer flask, pH7.0, culture temperature 35, rotation rate ISOrpm and culture time 20h.
3 We study the basic properties of agarase produced by Alteromonas sp. nov. SY 37-12. Agarase was extracted by 80% saturation with solid sulfate ammonium. The enzyme was purified 27.6 fold and the molecular weight of the agarase is 39.5KDa by SDS-PAGE. The basic properties of the enzyme were studied as follows: The optimum pH, the optimum temperature and the optimum NaCl concentration are pH7.0, 35 and 1% respectively. The enzyme is activated by K. Ca2+. Na+. Mg2+and inhabited by Fe3+. Zn2+ Sn2+ Hg2+ Ba2+. The enzyme is stable at temperatures up to 35癈.
4 The composition and structure of main degradation product were examined by MALDI-TOF-MS and 13C-NMR. The agarase belongs to |3-agarase and can hydrolyze the (3-1,4-glycosydic linkages of agar, yielding
neoagarotetraose and neoagarohexaose as the main products.
5 The antioxidative activities and antibacterial activities of neoagarooligosaccharide were studied. The neoagarooligosaccharide have the high antioxidative activities in scavenging the lipid peroxidation, removing theOH and H2O2. The neoagarooligosaccharide, H7 shows the antibacterial activity. The MIC of H7 against E.aerogenes and P.aeruginosa are 2.5mg/mL and I Omg/mL respectively.
引文
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69. Yaphe W., The use of agarase from Pseudomonas atlantica in the identification of agar on marine algae. Can. J.Microbiol. 1957, 3: 143-148.
70. Yarotsky SV., Shashkov A.S. and Usov, A.I.Analysis of 13C-NMR spectra of some red seaweed galactans, Bioorg. Khim. 1977, 3: 1135-1137.
71. Yoshizawa Y, Tsunehiro J, Nomura K, Itoh M, Fukui F, Ametani A, Kaminogawa S., In vivo macrophage-stimulation activity of the enzyme-degraded water-soluble polysaccharide fraction from a marine alga (Gracilaria verrucosa). Biosci Biotechnol Biochem. 1996, 60(10): 1667-1671
72. Yoshizawa, Y., Enomoto, A., Todoh, H., Ametani, A. Kaminogawa, S. Activation of murine macrophages by polysaccharide fractions from marine algae (Porphyra yezoensis). Biosci. Biotech. Biochem.1993, 57: 1862-1866,
73. Young, K.S., S.S.Bhattachrjie and W.Yaphe, Enzymic cleavage of the α-linkages in agarode, to yield agaro-oligosaccharides. Carbohyd. Res. 1978, 66: 207-212.
74. Young, K.S., Hong, K.C, Duckworth, M., Yaphe, W., Enzyme hydrolysis, of agar and properties of bacterial agarase, proc. Int. Seaweed Symp. 1971, 7: 15-22.
75. Zhang Q, Li N, Zhou G, Lu X, Xu Z, Li Z. Zhang et al. In vivo antioxidant activity of polysaccharide fraction from Porphyra haitanesis (Rhodephyta) in aging mice. Pharmacoi Res. 2003, 48(2): 151-155.
76. Zhang Q, Yu P, Li Z, Zhang H, Xu Z, Li P. Antioxidant activities of sulfated polysaccharide fractions from Porphyra haitanesis. J. Appl. Phycol. 2003, 15: 305-310.
77．刘翠平，方积年，质谱技术在糖类结构分析中的应用，分析化学，2001，29(6)：716-720。
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68. Yaphe W., Proposals on the classification of microorganisms which utilize the polysaccharides of marine algae and a definition for agar. Marine Microbiology. 1963, 588-593.
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73. Young, K.S., S.S.Bhattachrjie and W.Yaphe, Enzymic cleavage of the α-linkages in agarode, to yield agaro-oligosaccharides. Carbohyd. Res. 1978, 66: 207-212.
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75. Zhang Q, Li N, Zhou G, Lu X, Xu Z, Li Z. Zhang et al. In vivo antioxidant activity of polysaccharide fraction from Porphyra haitanesis (Rhodephyta) in aging mice. Pharmacoi Res. 2003, 48(2): 151-155.
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77．刘翠平，方积年，质谱技术在糖类结构分析中的应用，分析化学，2001，29(6)：716-720。