: 883, 60 [1],,, [2],,, [3 10], Co( ) Ni( ), Co( ) Ni( ) [11] Cu( ), Cu( ) Zn( ), [12],,,,, Pb Zn Cu Cd,,, 1 材料与方法 1.1 菌种培养基与重金属菌种 :,

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1 微生物学通报 JUN 20, 2008, 35(6): 882~887 Microbiology 研究报告 2008 by Institute of Microbiology, CAS 蛋白酶分泌菌对重金属胁迫的反应 * 马国芳王江张崇邦 ( ) 摘要 : 本文研究了 Pb Zn Cu 和 Cd 对 3 种蛋白酶产生菌 : 碱性地衣芽孢杆菌 2709(Bacillus licheniformis 2709) 中性枯草杆菌 1.398(Bacillus subtilis1.398) 和酸性宇佐美曲霉 537(Aspergillus usamii 537) 生长及其酶活性的影响, 并采用生长抑制皿分析 (GIPA) 方法针对 3 种菌株在 Pb Zn Cu Cd 不同胁迫浓度下的生长量进行打分, 分析其耐性与抗性指标 (MTC 与 MIC) 结果表明 4 种重金属在超过一定临界浓度后, 对 3 种菌株的生长和蛋白酶活性均产生了较大的抑制作用碱性蛋白酶对 4 种重金属均具有较大的适应性, 其次是中性蛋白酶对 Zn 和 Cd 具有一定的适应性, 而酸性蛋白酶对 4 种重金属均表现出被抑制状态 3 种菌株对 Pb 和 Zn 耐性与抗性最高 (2.0 mmol/l~6.0 mmol/l), 其次是对 Cd 也具有一定的抗性 (0.5 mmol/l~0.75 mmol/l) 关键词 : 蛋白酶分泌菌, 重金属, 生长, 酶活性, 耐性与抗性 Responses of Microorganisms Producing Protease to Heavy Metal Stress MA Guo-Fang WANG Jiang ZHANG Chong-Bang * (School of Life Sciences, Taizhou College, Linhai ) Abstract: Effects of Pb, Zn, Cu and Cd on growth and enzyme activities of microorganisms such as Bacillus licheniformis 2709, Bacillus subtilis1.398 and Aspergillus usamii 537, which can individually produce alkaline, neutral and acid protease, were studied, respectively. By determining growth and enzyme activities of three bacterial species, as well as analyzing their tolerance and resistance indicators (MTC and MIC) using growth inhibition plate analysis (GIPA) under different heavy metal gradients, results showed that four heavy metals under higher than a special concentration level significantly inhibited growth and enzyme activities of three species. The alkaline protease exhibited greatest adaptability to four heavy metals, next the neutral protease also exhibited certain adaptibilty to Zn and Cd, whereas the acid protease was totally inhibited under any levels of heavy metal concentrations. Three species not only showed great potential to tolerate and resist toxicity of lead and zinc (2.0 mmol/l 6.0 mmol/l), but also showed certain resistance to cadmium (0.5 mmol/l 0.75 mmol/l). Keywords: Strains producing protease, Heavy metals, Growth, Enzyme activity, Tolerance and resistance 基金项目 : * 通讯作者 :Tel: ; : llhzcb@163.com 收稿日期 : ; 接受日期 :

2 : 883, 60 [1],,, [2],,, [3 10], Co( ) Ni( ), Co( ) Ni( ) [11] Cu( ), Cu( ) Zn( ), [12],,,,, Pb Zn Cu Cd,,, 1 材料与方法 1.1 菌种培养基与重金属菌种 :, 培养基 : , 537 (PDA) [13] 重金属与处理浓度 : A R, mmol/l~6.0 mmol/l 1.2 方法重金属对 3 个菌株生长的影响 : PDA,,,, 3,, 0.01 mmol/l K 2 HPO 4,, ( ) 3 [14,15],,, ++++, Log +++,, ++ +, 重金属对蛋白酶活性的影响 : [16] PDA [17] 3, 5000 r/min 10 min,, 0.6 (ph 10.5); 0.6 (ph 7.0); -HCl 0.6 (ph 3.0) 5 ml 3, 40 2 min, 1 ml 10, min, 5 ml 0.4 mol/l,, min,, 275 nm,,, (100 ), [18] 个菌株对重金属的耐性与抗性 : 1.2.1,,,,, MTC MIC [14,15], MTC, MIC, 1.3 数据处理 3

3 884 微生物学通报 2008, Vol.35, No.6, 2 结果与分析个菌株生长对 Pb Zn Cu 和 Cd 的胁迫反应,,,, [19] 1~3, 3 Pb Zn Cu Cd, 2709 Zn 0.25 mmol/l,, Pb Cu Cd mmol/l Zn 0.01 mmol/l, Pb Cu Cd 0.00 mmol/l 537 Pb Zn 0.75 mmol/l 图 1 地衣芽孢杆菌 2709 生长对 4 种重金属胁迫的反应 Fig. 1 Responses of growth of Bacillus licheniformis 2709 to four heavy metal stress 图 2 枯草杆菌生长对 4 种重金属胁迫的反应 Fig. 2 Responses of growth of Bacillus subtilis1.398 to four heavy metal stress 图 3 宇佐美曲霉 537 生长对 4 种重金属胁迫的反应 Fig. 3 Responses of growth of Aspergillus usamii 537 to four heavy metal stress 0.50 mmol/l, 537 Cu Cd, 0.00 mmol/l, 3 Zn, Pb, Cu Cd 3, 3 1) 3 Pb Zn, Cu Cd ; 2) - - Pb Zn Cu Cd, Pb Zn,,, [20] ; 3) ph, ph [8,21], ph, [22] 个菌株蛋白酶相对活性对 Pb Zn Cu 和 Cd 的胁迫反应, [21,22], Pb Cu, 0.01 mmol/l( 4), Zn Cd 0.00 mmol/l 4, Pb Zn Cu

4 : 885 Cd 2.00 mmol/l 1.00 mmol/l 0.75 mmol/l 0.25 mmol/l, Zn Cd mmol/l 0.01 mmol/l ( 5) Pb Cu 0.00 mmol/l 4 图 4 地衣芽孢杆菌 2709 蛋白酶活性对 4 种重金属胁迫的反应 Fig. 4 Responses of protease activity in Bacillus licheniformis 2709 to four heavy metal stress 图 5 枯草杆菌蛋白酶活性对 4 种重金属胁迫的反应 Fig. 5 Responses of protease activity in Bacillus subtilis1.398 to four heavy metal stress 图 6 宇佐美曲霉 537 蛋白酶活性对 4 种重金属胁迫的反应 Fig. 6 Responses of protease activity in Aspergillus usamii 537 to four heavy metal stress mmol/l, 4 ( 6) 3, 4, Zn Cd, 4 3, [23], ph ph, [24] 个菌株对 Pb Zn Cu 和 Cd 的耐性与抗性 MTC MIC, Richards MTC MIC Frankia, [14,15], 1, Pb,, MTC, 0.75 mmol/l, MIC, 3 Pb, MIC 6.0 mmol/l, 4.0 mmol/l Zn, 3 MTC (0.50 mmol/l)> (0.25 mmol/l)> (0.01 mmol/l), MIC (4.0 mmol/l)> = (2.0 mmol/l) 3 Cu, MTC mmol/l, MTC 0.00 mmol/l 3 Cu Zn, MIC (1.0 mmol/l)> = (0.1 mmol/l) Cd, 3 Cd Cu, (0.005 mmol/l)> = (0.00 mmol/l) 3 Cd Zn Cu, MIC (0.75 mmol/l)> =

5 886 微生物学通报 2008, Vol.35, No.6 Table 1 表 1 3 个菌株生长对重金属的 MTC 与 MIC MTCs and MICs of growth of three microbial strains for four heavy metals Species 2709 Bacillus licheniformis Bacillus subtilis Aspergillus usamii 537 Pb Zn Cu Cd Pb Zn Cu Cd Pb Zn Cu Cd MTC MIC (0.50 mmol/l) 3,, ( ), [25] ; [25,27] 3 结论 Pb Zn Cu Cd,, 3 3 Pb Zn, Cd,, 参考文献 [1],,, , 2002, 8(2): [2] Takii Y, Urata Y. Themmostable neutral protease resembling thermolysin derived from Bacillus brevinoriko. Bioscience and Biotechnology, 1999, 62(5): [3],,,.., 2003, 39(3): [4],,,.., 1979, 19(3): [5],,,.., 1999, 39(2): [6],,,.., 2002, 18(6): [7],,,. (Bacillus sp. HX-318)., 2001, 38(1): [8],,,.., 2002, 1: [9] Jacobs M, Eliasson M, Uhlen M. Cloning, sequence and expression of subtilisin carlsberg from Bacillus licheniformis. Nucleic Acids Research, 1985, 13: [10] Takagi H, Hira K, Maeda Y. Enhance thermostability of the singlecys mutant subtilisin E under oxidizing conditions. Journal of Biochemistry, 2000, 128: [11],,,.., 2000, 45(9): [12],,,. Cu( ) EPR., 2001, 10: [13].. :, 1985, pp [14] Richards JW, Krumholz GD, Chval MS, et al. Heavy metal resistance patterns of Frankia strains. Applied and Environmental Microbiology, 2002, 68: [15],,,. Frankia., 2003, 17(3): [16],,.. ( ), 2006, 23(1): [17].., 2004, p.76.

6 : 887 [18] Tunga R, Shrivastava B, Banerjee R. Purification and 2001, 16: characterization of a protease from solid state cultures of [23] Boyd SA, Mortland MM. Enzyme interactions with clays Aspergillus parasiticus. Process Biochemistry, 2003, 38: and clay-organic matter complexes. In: Bollag JM, Stotzky G (Eds.), Soil Biochemistry. Marcel Dekker, New [19] Canovas D, Cases I, der Lorenzo V. Heavy metal tolerance York, 1990, pp and metal homeostasis in Pseudomonas putida as revealed [24] Lee IS, Kim OK, Chang YY, et al. Heavy metal concentrations and enzyme activities in soil from contaminated by complete genome analysis. Environmental Microbiology, 2003, 5: Korean shooting range. Journal of Biological Science and [20] Vallee BL, Ulmer DD. Biochemical effects of mercury, Bioengineering, 2002, 94: cadmium and lead. Annual Review of Biochemistry, 1972, [25] Brown NL, Rouch DA, Lee BTO. Copper resistance determinants in bacteria. Plasmid, 1992, 27: : [21] Tsai YC, Juang RY, Lin SF, et al. Production and Further [26] Silver S, Phung LT. Bacterial heavy metal resistance: new Characterization of an Alkaline Elastase Produced by Alkalophilic Bacillus Strain Ya-B. Applied and Environ surprises. Annual Review of Microbiology, 1996, 50: mental Microbiology, 1988, 54: [27] Cervantes C, Gutrierrez-Corona F. Copper resistance [22] Obbard JP. Ecotoxicological assessment of heavy metals mechanisms in bacteria and fungi. FEMS Microbiological in sewage sludge amended soils. Applied Geochemistry, Review, 1994, 14: 稿件书写规范高等院校教学栏目简介及撰稿要求,,,,,,,,,,,,,,,,,,,,,,,, 欢迎投稿! 欢迎对本栏目多提宝贵意见!

: 307, [], [2],,,, [3] (Response Surface Methodology, RSA),,, [4,5] Design-Expert 6.0,,,, [6] VPJ33 ph 3,, ph, OD, Design-Expert 6.0 Box-Behnken, VPJ3

: 307, [], [2],,,, [3] (Response Surface Methodology, RSA),,, [4,5] Design-Expert 6.0,,,, [6] VPJ33 ph 3,, ph, OD, Design-Expert 6.0 Box-Behnken, VPJ3 微生物学通报 FEB 20, 2008, 35(2) 306~30 Microbiology 2008 by Institute of Microbiology, CAS tongbao@im.ac.cn 生物实验室响应面分析法优化副溶血性弧菌生长条件刘代新宁喜斌 * 张继伦 2 (. 200090) (2. 20202) 摘要

: 883, 60 [1],,, [2],,, [3 10], Co( ) Ni( ), Co( ) Ni( ) [11] Cu( ), Cu( ) Zn( ), [12],,,,, Pb Zn Cu Cd,,, 1 材料与方法 1.1 菌种 培养基与重金属 菌种 :,

: 883, 60 [1],,, [2],,, [3 10], Co( ) Ni( ), Co( ) Ni( ) [11] Cu( ), Cu( ) Zn( ), [12],,,,, Pb Zn Cu Cd,,, 1 材料与方法 1.1 菌种培养基与重金属菌种 :,