950 微生物学通报 2009, Vol.36, No.7 [3] ; 996 [4] Halobacterium sp. H237 ; 2004 [5] Haloferax mediterranei R4, 6S rrna, Halorubrum, Halorubrum sp. CY, CY [6

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1 微生物学通报 JUL 20, 2009, 36(7): 949~955 Microbiology 2009 by Institute of Microbiology, CAS 研究报告嗜盐古菌 Halorubrum sp. CY 的分离鉴定及胞外淀粉酶特性初步研究 * 陈绍兴刘朝茂杨建刘晔邱成书 (. 6600) ( ) 2 谢志雄摘要 : 本研究从云南一平浪盐矿分离到一株产胞外淀粉酶的嗜盐古菌, 通过形态观察, 生理生化特性实验, 并结合 6S rrna 序列分析, 初步鉴定为嗜盐古菌 Halorubrum 属, 命名为 Halorubrum sp. CY 另外对该菌产生的胞外淀粉酶的性质进行初步研究, 结果显示其胞外淀粉酶发挥最大活性的 ph 值和温度分别为 6.0 和 60ºC, Zn 2+ Cu 2+ Al 3+ 2 SO 3 对胞外淀粉酶的活性有抑制作用, 而 Mn 2+ 则有促进作用关键词 : 嗜盐古菌, Halorubrum, 胞外淀粉酶, 分离鉴定 Identification of Halorubrum sp. CY Strain and Primary Study on the Basic Characteristics of Extracellular Amylase CHEN Shao-Xing * LIU Chao-Mao YANG Jian LIU Ye QIU Cheng-Shu XIE Zhi-Xiong 2 (. College of Life Sciences, Honghe University, Mengzi, Yunnan 6600, China) (2. College of Life Sciences, Wuhan University, Wuhan, Hubei , China) Abstract: An extracelluar amylase production extremely halophilic archaen was isolated from a salt ore of Yipinglang of Yunnan province. Based on the morphology, characteristics of biologic and chemical and analyzing of 6S rrna sequence, the strain was identified as genus of Halorubrum, and named Halorubrum sp. CY. The rude characteristics of the extracelluar amylase determined. It had optimal activity at ph 6.0 and 60ºC. Zn 2+, Cu 2+, Al 3+, SO 3 2 could promoted the activity of amylase and Mn 2+ might inhibit the activity of extracelluar amylase. Keywords: Archaeon, Halorubrum, Extracelluar amylase, Isolation and identification [] 5%~30%, 20%~25%, [2], 0% [3,4] [5], [6] [7] [8] [9] [0] [] [2] 970 Good 基金项目 :(No. 0700) * 通讯作者 :Tel: ; : shaoxinchen@yahoo.com.cn 收稿日期 : ; 接受日期 :

2 950 微生物学通报 2009, Vol.36, No.7 [3] ; 996 [4] Halobacterium sp. H237 ; 2004 [5] Haloferax mediterranei R4, 6S rrna, Halorubrum, Halorubrum sp. CY, CY [6] CYpH EDTA 材料与方法. 菌种,.2 培养基及培养条件 Offner [7] (g/l): NaCl 75, MgSO 4 7H 2 O 37, KCl 3.7, Tryptone 5, Yeast extract 3, Tris Cl ( mol/l, ph 7.2) 25 ml, CaCl 2 2H 2 O (0%) 5 ml, MnCl 2 (00 mmol/l) 7 µl 2% 0 5 Pa 20 min : 40 C 80 r/min,.3 形态观察及生理生化特性 Halorubrum sp. CY, 40 C, [8].4 6S rrna 序列分析 [9]: F: 5 -ATTCCGGTTGA TCCTGC-3 ; R: 5 -TTTAAGTTTCATCCTTG-3 ; F/ R bp~859 bp; F2: 5 -AACCGGATTAGATA CCC-3 ; R2: 5 -GTGATCCAGCCGCAGATTCC-3 ; F2/R2 723 bp~472 bp PCR(DTC-PCR ): 94 C 5 min; 94 C min, 55 C 2 min, 72 C min; 30 ; 72 C 5 min PCR Tiangen, pmd-8t ( TaKaRa), ( ) NCBI Blast, MEGA 胞外淀粉酶活性的平板检测 CY, 0.5%, 40 C, 0.4 mol/l (KI-I 2 ),.6 胞外淀粉酶活性测定.6. 淀粉酶活性测定方法 : [6], 5 ml 0.5% 0.5 ml, ph C 5 min, 0. mol/l H 2 SO 4 5 ml, 0.5 ml, 5 ml 0.4 mmol/l (KI-I 2 ), 620 nm 5 min mg.6.2 胞外淀粉酶最适 ph 值温度测定 : [6] Yoo [20], ph,, ph ;, ph, 35 C 40 C 45 C 50 C 55 C 60 C 65 C 70 C 75 C 9,.6.3 金属离子及 EDTA 对初酶活性的影响 : (Zn 2+ Cu 2+ Ca 2+ Al 3+ Mn 2+ SO 2 3 Fe 3+ ) EDTA 0.5% 5, 0 mmol/l 20 mmol/l 30 mmol/l 40 mmol/l 50 mmol/l; ph, EDTA 2 结果与分析 2. 菌落形态观察 A, CY,,,,,, [2], 图菌落形态观察 Fig. Morphology of CY : A: ; B:. Note: A: Colony of CY; B: Gram staining.

3 : Halorubrum sp. CY 95, B, CY, CY, Halorubrum sp. CY, 2.2 生理生化特性 ( ) 7 (), CY, Halorubrum sp. CY ph 9%~20% 7.0~7.2 38ºC~40ºC, Mg 2+ 2 Halorubrum sp. CY 2.3 6S rrna 序列同源性分析 6S rrna, 6S rrna PCR,, pmd-8t, 470 bp 6S rrna, Blast Halorubrum Halorubrum litoreum 99%, Halorubrum 表 Halorubrum sp. CY 的基本特性 Table Basic characteristics of Halorubrum sp. CY Characteristics NaCl Optimum NaCl concentration NaCl NaCl Range for growth ph Optimum ph ph ph range for growth Optimum temperature Temperature for growth Mg 2+ Mg 2+ required concentration Range 9%~20% %~34% 7.0~ ~7.4 38ºC~40ºC 22ºC~ 55ºC mol/l 表 2 Halorubrum sp. CY 的生理生化特征 Table 2 Characteristics of physiology and chemistry of Halorubrum sp. CY Characteristics Results Characteristics Extracelluar protease D-Arabinose Urease Fructose Oxidase Glycine Catalase Alanine Reduction of nitrate Serine -80 Hydrolysis of Tween-80 Cysteine Casein Hydrolysis of Casein Threonine Hydrolysis of gelatin Valine Production of acid Leucine Production of gas Isoleucine H 2 S Production of H 2 S Phenylalanine V-PV V-P Test Tryptophan Anaerobic growth Tyrosine Arg Anaerobic growth with L-Arg Aspartate D-Mannose Asparaginase Lactose Glutamate Glucose Glutamine D-Xylose Lysine D-Galactose Arginine D-Ribose Histidine Maltose Methionine Sucrose Proline : : ; : ; :. Notes: +: Positive; + : Positive but not obviously; : Negative. Results

4 952 微生物学通报 2009, Vol.36, No.7 图 2 基于 6S rrna 序列采用邻接法构建菌株 CY 和 Halorubrum 属相关菌株系统发育树状图 Fig. 2 Neighbor-joining tree constructed showing the phylogenetic relationships among CY and other related Halorubrum strains based on 6S rrna gene sequence Note: The sequence data used were obtained from GenBank database (accession numbers are given in parentheses). Numberals on branches are the supporting percentage (>50%) by 000 replicate. Bar, nucleodide substitution per 00 nucleotides of 6S rrna gene sequence.

5 : Halorubrum sp. CY 953 Halorubrum sp. CY 6S rrna GenBank, FJ 系统发育树的构建 6S rrna Blast,, Halorubrum litoreum Halorubrum xinjiangense, 99% 98%, Halorubrum MEGA 4.0 Neighbor-joining, 50% ( 2) 2, Halorubrum sp. CY Halorubrum distributum, Halorubrum litoreum, GenBank, Blast, MEGA 4.0, [22] 6S rrna,, MEGA 胞外淀粉酶平板检测 [5], CY, 3 0.5%,, Halorubrum sp. CY,, CY,, CY :,, 2.6 胞外淀粉酶最适 ph 值温度 [6] Yoo [20], 4, ph 6.0 ; 5, 60ºC Halorubrum sp. CY ph, ph 6.4~7.2 38ºC~ 40ºC,,, 图 4 ph 值对胞外淀粉酶的影响 Fig. 4 Influence of ph to amylase 图 5 Fig. 5 温度对胞外淀粉酶的影响 Influence of temperature to amylase 图 3 Halorubrum sp. CY 产胞外淀粉酶平板检测 Fig. 3 Production of α-amylase of Halorubrum sp. CY 2.7 金属离子和 EDTA 对淀粉酶活性的影响 ph ºC 00%,,, 00%,, 00%, 00%

6 954 微生物学通报 2009, Vol.36, No.7 6A, Zn 2+ Cu 2+ Al 3+ SO 2 3, SO 2 3, Ca 2+ Fe 3+ EDTA, 0 mmol/l 20 mmol/l, ( 6B); Mn 2+, ( 6C) 3 讨论, 6S rrna, Halorubrum litoreum Halorubrum xinjiangense 99% 98%,, Halorubrum, Halorubrum sp. CY Halorubrum sp. CY ph ºC, Halobacterium halobium ph (6.4 55ºC) [20],, Al 3+ Fe 3+ SO 2 3 Cu 2+ EDTA [23],, Fe 3+ EDTA, Al 3+ SO 2 3 Cu 2+, SO 2 3 Ca 2+ Mn 2+ Zn 2+ [7],, Ca mmol/l, 20 mmol/l; Zn mmol/l, 40 mmol/l ; Mn 2+ Halorubrum sp. CY,,,, 参考文献图 6 金属离子和 EDTA 对淀粉酶活性的影响 Fig. 6 Influence of metallic ion and EDTA to the activity of amylase : A: ; B: ; C:. Note: A: Inhibiting effect; B: No obvious effect; C: Positive effect. [],,,.., 2005, 25(2): [2],,,.., 2003, 30(4): 5 9. [3],,,.., 2002, 24(5): [4],,,. NaCl., 2008, 35(8): [5],..

7 : Halorubrum sp. CY 955, 99, 3(5): [6],. α-. [6],,. α-, 996, 23(6): , 998, 7(9): [7] Offner S. Functional stuides of the gvpacno operon of [7] Bayramoglu G, Yilmaz M, Arica MY. Immobilization of a thermostable α-amylase onto reactive membranes: kinetics characterization and application to continuous starch hydrolysis. Food Chemistry, 2004, 4(84): Halobacterium salinarium reveal that the GvpC protein shapes gas vesicles. Journal of Bacteriology, 996, 78(5): [8] Lingfei Hu, Hailian Pan, Yanfen Xue, et al. Halorubrum [8],.. luteum sp. nov., isolated from Lake Chagannor, Inner, 2002, 2(23): [9],,. α-., 2007, 28(8): Mongolia, China. Int J Syst Evol Microbiol, 2008, 58(7): [9] Kunio Ihara, Satoshi Watanabe, Takeshi Tamura. Haloarcula [0] Van Ee JH, van Rijswijk WC, Bollier M. Enzymatic automated dishwash detergents. Chim Oggi, 992, 0(): [].., 2004, (): 4. [2]. α-. argentinensis sp. nov. and Haloarcula mukohataei sp. nov., two new extremely halophilic archaea collected in Argentina. Int J Syst Bacteiol, 997, 47(): [20] Yoo YJ, Hong J, Hatch RT. Comparison of -amylase activities from different assay methods. Biotechnol Bioengineer, 987, 30(): 47 5., 200, 3(): [2] Dussault HP. An improved technique for staining red halophilic [3] Good WA, Hartan PA. Properties of the Amylase from bacteria. J Biol Chem, 955, 70(4): Halobacterium halobium. Journal of Bacteriology, 970, [22],,,. 04(): , [4], , 35(9): , 30(2): [23] Mohsen FN, Dileep D, Deepti D, et al. Purification and [5],,,. Haloferax mediterranei characterization of an extracellular α- amylase from Bacillus R4., 2004, 26(4): subtilis AX20. Protein Expression and Purification, 2005, 4(22): 年中科院微生物所期刊联合编辑部联合征订全面启动! 征订启事 ( 4 ), 55.00, 660 ISSN ; CODEN WSHPA82-504; BM67 ( 25 ), 65.00, 780 ISSN ; CODEN SGXUED82-3; BM5608 ( 20 ), 48.00, 576 ISSN ; CODEN WSWPDI2-87; BM43 ( 5 ), 80, 480 ISSN ; CODEN JXUUAE2-499; Q723,. (000) 3 B40 ; (00) ; bjb@im.ac.cn

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Microsoft Word - 崔宝臣.doc 微生物学通报 DEC 20, 2009, 36(12): 1871~1875 Microbiology tongbao@im.ac.cn 2009 by Institute of Microbiology, CAS 简报正交实验优选光合细菌混合菌群产氢的最佳条件 * 崔宝臣张国欣侯博邵姝媛 ( 163318) 摘要 : 对光合细菌混合菌群产氢影响因子进行了实验研究通过单因素实验和正交实验, 系统考察了碳源