( H2 H4 H5), 1% 20 ml, 28, 150 r/min, 12 h, 620 nm 540 nm 1.4 褐藻酸裂解酶酶活的测定, DNS [10] 1.0 ml 10 ml, 1.0 ml ph %, min, 1.5 ml DNS, 5 min,,

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1 一株高效褐藻酸降解菌的筛选鉴定及其发酵条件的优化侯士昌 1, 温少红 1, 唐志红 1, 崔玉琳 2 2, 秦松 (1., ; 2., ) 摘要 : 以褐藻酸钠为唯一碳源, 从腐烂海带中筛选得到褐藻酸钠降解能力较强的菌株 H4, 经生理生化和 16S rdna 序列分析鉴定该菌株属于交替单胞菌属 (Alteromonas) 对菌株 H4 的发酵条件优化研究表明, H4 的较优培养基组成为 (w/v): 褐藻酸钠 0.6% 酵母粉 0.5% 蛋白胨 0.25% NaCl 3%; 较优培养条件为 : 培养温度 28, 初始 ph7.5, 接种量 1.5%(v/v), 培养时间 48 h Fe 3+ 对交替单胞菌 H4 的产酶有明显的促进作用, 这在其他褐藻酸裂解酶生产菌株中未见报道, 而 Cu 2+ 对褐藻酸裂解酶的抑制作用高达 45.78% 在优化后的培养条件下, 粗酶液酶活达到 U/mL, 较优化前提高了 39.4% 关键词 : 褐藻酸钠降解菌 ; 筛选 ; 鉴定 ; 酶活优化中图分类号 : Q935 文献标识码 : A 文章编号 : (2014) doi: /hykx ,, 24%~31%, Stanford Ascophyllum Macrocystis Laminaria Ascophyllum 3 [1], -D-1,4- ( M) -L-1,4- ( G) C-1,4 [2],,,,, [3] [4] [5], [6],,,,, C4 C5 [7],,,, [8-9] H4,, H4,, 1 材料与方法 1.1 培养基 (1) : 5 g/l, 10 g/l, NaCl 10 g/l (2) : (NH 4 ) 2 SO 4 5 g/l, K 2 HPO 4 2 g/l, NaCl 30 g/l, MgSO 4 7H 2 O 1g/L, FeSO 4 7H 2 O 0.01 g/l, 5g/L, ph 7.5 (3) : 5g/L, 1g/L, 5g/L, NaCl 30g/L, ph 种子液的制备, 1%, 28, 150 r/min 24 h, 1.3 菌种的分离与筛选, , 28 4 d 4, : ; : : ( ); ( ); (JQ200914) : (1988-),,,,, : , houshichang5@163. com;,, sqin@yic.ac.cn 20 / 2014 / 38 / 7

2 ( H2 H4 H5), 1% 20 ml, 28, 150 r/min, 12 h, 620 nm 540 nm 1.4 褐藻酸裂解酶酶活的测定, DNS [10] 1.0 ml 10 ml, 1.0 ml ph %, min, 1.5 ml DNS, 5 min,,, 540 nm (U), l μg (U/mL) : 4, r/min, 10 min, 1.5 菌株的鉴定 [10] S rdna [11] [12] 16S rdna GenBank BLAST,, CLUSTAL MEGA4. 0, 1.6 菌株发酵条件的优化 50 ml 20 ml, 1.5%(v/v), 28, 150 r/min 48 h 3, 2 结果与分析 2.1 最佳褐藻酸裂解酶菌株的确定及酶活的测定, 3 H2 H4 H5 3 ( 1a 1b), 3 24 h, 36 h, 48h, H2, H4, H4, 48 h 2.2 菌株 H4 的鉴定 H4 1 1 H2 H4 H5 Fig.1 The growth and enzyme activity curves of H2 H4 H5 a. 3 ; b. 3 a. The growth curves of H2, H4 and H5: b. The enzyme activity curves of H2, H4 and H5 表 1 菌株 H4 的生理生化特征 Tab.1 The physiological and biochemical characters of strain H4 G M.R. + VP D- + + : +, Marine Sciences / Vol. 38, No. 7 /

3 S rdna H4 16S rdna 1494 bp, H4 (Alteromonas), 16S rdna 99%( 2), (Alteromonas), H4(Alteromonas sp. H4) 2.3 发酵条件的优化 H4 H4, 3, 0.5%~1.5%,, 1.5 %, H4 H4 4, H4 28, 24~28, 30,, U/mL, 2 H4 16S rdna Fig.2 Phylogenetic tree of 16S rdna from H4 and other strains 3 H4 Fig.3 Effects of inoculum concentration on biomass and Fig.4 4 H4 Effects of culture temperature on biomass and 22 / 2014 / 38 / 7

4 2.3.3 ph H4 ph,,, [10] ph H4 5 H4 ph 7.0~8.0,, ph 7.5,,, [13] NaCl 3%, H4, NaCl, H4,,,, (0.15%), H4 7, H4,, H4, H4,, [14] 5 ph H4 H4 Fig.5 Effects of initial ph on biomass and enzyme production of strain H NaCl H4 NaCl H4 6, NaCl NaCl 0,,, NaCl, NaCl 3% NaCl 7 H4 H4 Fig.7 Effects of different carbon sources on biomass and, H4 ( 8) 0.3%~0.6%, H4, 0.6%,,,,, 6 NaCl H4 H4 Fig.6 Effects of NaCl on biomass and enzyme production of strain H4, 0.5%~0.7%, 0.6% Marine Sciences / Vol. 38, No. 7 /

8 H4 H4 Fig.8 Effects of sodium alginate content on biomass and 2.3.6 H4,, (0.5%), H4 9, H4,,, [15] 9 H4 H4 Fig.9 Effects of different nitrogen sources on biomass and, ( 10) : 5 g/l 2.

5 8 H4 H4 Fig.8 Effects of sodium alginate content on biomass and H4,, (0.5%), H4 9, H4,,, [15] 9 H4 H4 Fig.9 Effects of different nitrogen sources on biomass and, ( 10) : 5 g/l 2.5 g/l, H4, 5 g/l 2.5 g/l H4,, H4 ( 2) : H4, Cu 2+, 26.65%; Fe 3+ K +, Ca 2+ Mg 2+ Cu 2+, Cu 2+, 45.78% Fig H4 H4 Effects of Yeast Extraction and Tryptone content on biomass and 24 / 2014 / 38 / 7

6 表 2 不同金属离子对 H4 的菌株 H4 生长和产酶的影响 Tab.2 Effects of different ions on biomass and enzyme production of strain H4 (5 mmol/l) (%) (U/mL) Fe Ca Mg Cu K Fe 3+ H4(Alteromonas sp.h4), Fe 3+, K + Ca 2+ Mg 2+, Ca 2+ Mg 2+,, [16] 3, H4, H4, H U/mL, 39.4% 3 结论 (1), 3 H2 H4 H5, H4, 16S rdna, H4(Alteromonas sp.h4)( KF358313),,,, [17] (Alteromonas espejiana) (2), H4,,, H4 (w/v): 0.6 %, 0.5%, 0.25%, NaCl 3%; 28, ph7.5, 1.5%(v/v), 48 h U/mL, 39.4% K +, Fe 3+, Ca 2+ Mg 2+ Cu 2+, Cu % 4 前景展望,,,,,,, : [1] Skiak B G, Martinsen A. Applications of some algal polysaccharides in biotechnology[m]. Seaweed Resources in Europe: Uses and potential,1991: [2] Gacesa P. Enzymic degradation of alginates[j]. The International journal of biochemistry, 1992, 24(4): [3] Kawada A, Hiura N, Shiraiwa M, et al. Stimulation of human keratinocyte growth by alginate oligosaccharides, a possible co-factor for epidermal growth factor in cell culture[j]. Federation of European Biochemical Societies,1997, 408(1): [4] Schaeffer D J, Krylov V S. Anti-HIV activity of extracts and compounds from algae and cyanobacteria[j]. Ecotoxicology and Environmental Safety, 2000, 45(3): [5]. [J]., 1992, 1: 4-6. [6] Tomoda Y, Nagaki A, Kono T. Method for Improving Marine Sciences / Vol. 38, No. 7 /

7 Freeze-resistance of Farm Corp[M]. Tokyo: OMei Seika Kaisha LTD, 1995: [7] Wong T Y, Preston L A, Schiller N L. Alginate lyase: review of major sources and enzyme characteristics, structure-function analysis, biological roles, and applications[j]. Annual Reviews in Microbiology, 2000, 54(1): [8] Kim D E, Lee E Y, Kim H S. Cloning and characterization of alginate lyase from a marine bacterium Streptomyces sp. ALG-5[J]. Marine Biotechnology, 2009, 11(1): [9] Sim S J, Baik K S, Park S C, et al. Characterization of alginate lyase gene using a metagenomic library constructed from the gut microflora of abalone[j]. Journal of Industrial Microbiology & Biotechnology, 2012, 39(4): [10],,,., [J]., 2012, 38(7): [11] Weisburg W G, Barns S M, Pelletier D A, et al. 16S ribosomal DNA amplification for phylogenetic study[j]. Journal of Bacteriology, 1991, 173(2): [12] Cello D F, Bevivino A, Chiarini L, et al. Biodiversity of a Burkholderia cepacia population isolated from the maize rhizosphere at different plant growth stages[j]. Applied and Environmental Microbiology, 1997, 63 (11): [13],,,. LJ1 [J]., 2009, 49(8): [14] Momma K, Okamoto M, Mishima Y, et al. A novel bacterial ATP-binding cassette transporter systerm allows uptake of macromolecules[j]. Journal of Bacteriology,2000, 182(14): [15]. S3 [J]., 2010, 38(10): [16],,,. [J]., 2006, 30(2): [17],. :. [J]., 1997, 19(5): The screening, identification of alginate degrading bacteria and optimization of fermentation conditions HOU Shi-chang 1,WEN Shao-hong 1, TANG Zhi-hong 1, CUI Yu-lin 2, QIN Song 2 (1. College of Life Sciences, Yantai University, Yantai , China; 2. Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai , China) Received: Dec.,8,2013 Key words: alginate degrading bacteria; screening; identification; optimization Abstract: A screening work was carried out to obtain a bacterium with high alginate lyase activity by using alginate as sole source of carbon. The strain was identified as Alteromonas based on physiology and biochemistry analysis, 16S rdna sequence analysis, and was named Alteromonas sp. H4. It was shown that the optimal culture medium composition of producing alginate lyase was as follows (w/v): alginate 0.6%, yeast extracts 0.5%, peptone 0.25% and NaCl 3%. The optimized culture condition was as follows: culture temperature 28, initial ph value 7.5, inoculum size 1.5%(v/v) and optimal harvest time 48 h. The activity of alginate lyase was activated by Fe 3+, which hasn t been reported in other alginate lyase production strains, and the inhibitory effect by Cu 2+ was up to 45.78%. Under optimized conditions, the alginate lyase activity of the Alteromonas sp. H4 was U/ml, which was increased by 39.4% than that before optimization. ( 本文编辑 : 梁德海 ) 26 / 2014 / 38 / 7

L 8 0 g L ~0 700 g L % 6 00% 1 50% 1 3 NaCl 0 50% NH 4 2 SO % KH 2 PO % CaCO % 0 03% ~ 0 04% 2 24 h 0 02

L 8 0 g L ~0 700 g L % 6 00% 1 50% 1 3 NaCl 0 50% NH 4 2 SO % KH 2 PO % CaCO % 0 03% ~ 0 04% 2 24 h 0 02 38 5 Vol 38 No 5 2014 9 Journal of Jiangxi Normal UniversityNatural Science Sep 2014 1000-5862201405-0501-05 1 1* 2 2 1 3300222 330052 Streptomyces lincolnensis 13 8 6% 12 4% Q 93 A 0 1 2 Streptomyces