微生物学通报 APR 20, 2008, 35(4): 481~485 Microbiology 2008 by Institute of Microbiology, CAS 研究报告一种耐热几丁质酶的产生及其稳定性研究郭润芳 1* 史小琴 2 李多川 3 魏青

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1 微生物学通报 APR 20, 2008, 35(4): 481~485 Microbiology 2008 by Institute of Microbiology, CAS 研究报告一种耐热几丁质酶的产生及其稳定性研究郭润芳 1* 史小琴 2 李多川 3 魏青 1 贾英民 1 ( ) ( ) ( ) 摘要 : 疏绵状嗜热丝孢菌 (Thermomyces lanuginosus)sy2 在以胶状几丁质为唯一碳源的诱导培养基中产生了胞外几丁质酶该酶在 50 保温 1 h, 酶活稳定 ; 65 时半衰期为 25 min; 酶液在室温下保存到 12 周, 残余酶活性为 45% 左右该酶有较宽的 ph 范围, 3.0~9.0 之间保持稳定, ph 值为 2.5 时, 仍具有 70% 的剩余酶活性 Ca 2+ 对几丁质酶的活性有显著的激活作用 ; 高浓度变性剂对酶有抑制作用结果表明该酶是一种热稳定性高且耐酸碱的新型几丁质酶, 能在酸性和高温环境中发挥作用, 这些特性赋予了 T. lanuginosus 几丁质酶在几丁质的生物转化及其它生物技术中极大的应用优势关键词 : 几丁质酶, 疏绵状嗜热丝孢菌, 嗜热真菌, 货架稳定性 Production and Stability of a Thermostable Chitinase GUO Run-Fang 1* SHI Xiao-Qin 2 LI Duo-Chuan 3 WEI Qing 1 JIA Ying-Min 1 (1. Department of Bioengineering, Hebei Agricultural University, Baoding ) (2. Medical College of Hebei University, Baoding ) (3. Department of Environmental Biology, Shandong Agricultural University, Tai an ) Abstract: An extracellular chitinase from Thermomyces lanuginosus SY2 was produced when SY2 was grown in mineral liquid medium containing colloidal chitin as the only carbon source. The chitinase was stable at 50 for 1h, and the half-life time of the enzyme at 65 was 25 min. when it was kept at room temperature for 12 week, the chitinase activity was retained about 45% activity. The ph stability was kept in the range , and the enzyme was able to obtain 70% of the full activity, when the enzyme was incubated in the buffer (ph 2.5). The chitinase activity was enhanced obviously by metal ion Ca 2+, but inhibited under the high content chemical reagents. The results suggested that the chitinase from Thermomyces lanuginosus SY2 was a novel enzyme due to its high activity and stability under the high temperature and acid environment, which make it applicable for biotransformation and other biotechnological purposes. Keywords: Chitinase, Thermomyces lanuginosus, Thermophilic fungi, Shelf-life stability,, [1], [2,3] (EC ), N- 基金项目 :(No D) * 通讯作者 : : runfangg@163.com 收稿日期 : ; 接受日期 :

2 482 微生物学通报 2008, Vol.35, No.4, [4],,,, Thermococcus chitonophagus Thermococcus kodakaraensis KOD1 Bacillus BG-11 StreptomycesOPC520 [5-7],, ph,, Thermomyces lanuginosus SY2,, ph, 1 材料和方法 1.1 菌株来源及鉴定 (Thermomyces lanuginosus) SY2 Cooney & Emerson [8], [8, 9] 1.2 培养基 K 2 HPO mg, KH 2 PO mg, KCl 200 mg, MgSO mg, 4 g, 750 ml, 250 ml, 25 g, 1 g ( ), kpa 30 min 1.3 酶活性和蛋白含量测定 DNS [10], (U) 1 μmol Bradford [11], 1.4 产酶条件研究初始氮源对产酶的影响 : SY2 (0.1 ), (120 r/min, 50 ) 6 d, NaNO 3 NH 4 NO 3 (NH 4 ) 2 SO 初始底物浓度对产酶的影响 : SY2 (0.5%~2.5%), (120 r/min, 50 ) 6 d, 初始 ph 值对产酶的影响 : SY2 ph, (120 r/min, 50 ) 6 d, 产酶的时间谱 :, 2,, 13,, Time-course 1.5 酶的提取及纯化 SY2 7 d, r/min,, 85 DEAE-Sepharose Phenyl-Sepharose,, 1.6 几丁质酶稳定性研究几丁质酶的温度 ph 值稳定性 : ( ) 10min 20 min 30 min 40 min 50 min 60 min, 0, 50, ph 50 1 h, ph 5.0,, ph 金属离子和化学变性剂对酶活性的影响 :, ( 50 mmol/l),, 100, 几丁质酶的货架稳定性 : [7] KCl(15, W/V), (25 ~30 ) (0 ~4 ), 12,,

: 483 2 结果与分析 2.1 疏绵状嗜热丝孢菌 SY2 菌株分离鉴定 PDA,,,,, 1.

2 几丁质酶产生的最佳培养条件, [4], [12, 13 ],, 0.1 NH 4 NO 3, NaNO 3, (NH 4 ) 2 SO 4, 图 1 疏绵状嗜热丝孢菌显微特征 Fig.

3 : 结果与分析 2.1 疏绵状嗜热丝孢菌 SY2 菌株分离鉴定 PDA,,,,, 1.5 μm~ 4 μm,, 10 μm~15 μm,,,,, 6 μm~10 μm,,,,, ;, ( 1), 疏绵状嗜热丝孢菌 (Thermomyces lanuginosus) 2.2 几丁质酶产生的最佳培养条件, [4], [12, 13 ],, 0.1 NH 4 NO 3, NaNO 3, (NH 4 ) 2 SO 4, 图 1 疏绵状嗜热丝孢菌显微特征 Fig. 1 Micro-features of Thermomyces lanuginosus NH 4 NO 3, 2,, 1.5,,,, 1.5 ph,, Fig. 2 图 2 T. lanuginosus 胞外几丁质酶的产生 Production of extracellular chitinase from T. lanuginosus ph NH 4 NO ph 5.5 T. lanuginosus SY2 4 d,, 2.8 U/mL;, 几丁质酶的最适作用温度 ph 值及其稳定性 [1],

4 484 微生物学通报 2008, Vol.35, No.4, Sutrisno [14] 50 ~60 55,, 55 ; 60, 60 min, 85; min; min, 24% ( 3) Aspergillus fumigatus 45, 55 1 h, 30 [12] 5 ~15, ph, ph 4.5 ph 3.0~9.0 Thermomyces lanuginosus, ph 2.5, 70, 图 3 T. lanuginosus 几丁质酶的 ph 稳定性 (a) 和热稳定性 (b) Fig. 3 ph (a) and temperature (b) stabilities of T. lanuginosus chitinase 2.4 不同金属离子和变性剂对酶活性的影响 1 Ca 2+ Ba 2+ Na + K +, Ca 2+, 170 ; Ag + Hg 2+ Fe 2+ Cu 2+, EDTA, 1 mmol/l, 44.28%, 10 mmol/l, EDTA,, 2.5 几丁质酶的货架期 4 15 (W/V) KCl 4 8, 100, 8, ; 4,, 12, 25, 表 1 金属离子和化学试剂对几丁质酶活性的影响 Table 1 Effects of metal ions and chemical reagents on T. lanuginosus chitinase Metal ions and reagents Concentration Relative activity (%) H 2 O 50 mmol/l 100 Ca mmol/l 170 Mg mmol/l 70.9 Ba mmol/l Cu mmol/l 4.97 Zn mmol/l 39.5 Fe mmol/l 15.5 Mn mmol/l 50.9 Ag + 50 mmol/l 0 Hg mmol/l 1.32 Na + 50 mmol/l K + 50 mmol/l Urea 0.5 mol/l Urea 3.0 mol/l SDS 0.1%(W/V) SDS 1% (W/V) EDTA 1.0 mmol/l EDTA 10 mmol/l PMSF 1.0 mmol/l PMSF 10 mmol/l HS 1.0 mmol/l HS 10 mmol/l 37.61

5 : 485,,, 参考文献图 4 T. lanuginosus 几丁质酶的货架稳定性 Fig. 4 Shelf-life stability of T. lanuginosus chitinase,, 8 60, 12 45,,,,, Bhushan Bacillus BG-11, 3 讨论,,,,, T. lanuginosus SY2, 0.1 NH 4 NO ph 5.5, 50 4 d,, 2.8 U/mL,, Thermomyces lanuginosus SY2,, ph ph , 60 ; 8, 15, ph 3.0~9.0,, T. lanuginosus [1] Haki GD, Rakshit SK. Developments in industrially important thermostable enzymes: a review. Bioresource Technology, 2003, 89(1): [2] Nwe N, Stevens W. Production of fungal chitosan by solid substrate fermentation followed by enzymatic extraction. Biotechnol Lett, 2002, 24(2): [3] Suntornsuk W, Pochanakanich P, Suntornsuk L. Fungal chitosan production on food processing by-products. Proc Biochem, 2002, 37(7): [4] Patil RS, Ghormade V, Deshpande MV. Chitinolytic enzymes: an exploration. Enzyme Microb Technol, 2000, 26(7): [5] Andronopoulou E, Vorgias CE. Purification and characterization of a new hyperthermostable, allosamidin insensitive and denaturation resistant chitinase from the hyperthermophilic archaeon Thermococcus chitonophagus. Extremophiles, 2003, 7(1): [6] Takeshi T, Toshiaki F, Tadayuki I. Different cleavage specificites of the dual catalytic domain in chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Journal of Biological Chemistry, 2001, 276(38): [7] Bhushan B. Production and characterization of a thermostable chitinase from a new alkalophilic Bacillus sp. BG-11. J Appl Microbiol, 2000, 88(5): [8] Cooney DG, Emerson R. Thermophilic Fungi: An account of their biology, activities and classification. San Francisco: W H Freeman &Co, 1964, pp [9] Tsiklinsky P. Thermomyces lanuginosus. Ann Inst Pasteur Paris, 1899, 13(4): [10] Miller GL. Use of dinitrosalicyclic acid regent for determination of reducing sugar. Anal Chem, 1959, 31(3): [11] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the priciple of proteins-dye binding. Analytical Biochemistry, 1976, 72(1): [12] Xia GQ, Jin CS, Zhou J, et al. A novel chitinase having a unique mode of action from Aspergillus fumigatus YJ-407. Eur J Biochem, 2001, 268(14): [13] Awani, NN, Kapadnis BP, Das AD, et al. Purification and charcterization of a thermophilic and acidophilic chitinase from Microbispora sp. V2. J Appl Microbiol, 2002, 93(6): [14] Sutrisno A, Ueda M, Abe Y, et al. A chitinase with high activity toward partially N-acetylated chitosan from a new, moderately thermophilic, chitin-degrading bacterium Ralstonia sp. A-471. Appl Microbiol Biotechnol, 2004, 63(4):

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Microsoft Word - 20120619-188_editing 固定时间 - 离子选择性电极浓度直读法快速测定腐竹中的硼砂魏姜勉 1 高向阳 1 * 张娜 (1 河南农业大学食品科学技术学院, 郑州, 450002, 中国 ; 2 郑州科技学院, 郑州,450064, 中国 ) 2 摘要 : 为建立一种快速测定食品中硼砂的新方

微生物学通报 APR 20, 2008, 35(4): 481~485 Microbiology 2008 by Institute of Microbiology, CAS 研究报告 一种耐热几丁质酶的产生及其稳定性研究 郭润芳 1* 史小琴 2 李多川 3 魏青

微生物学通报 APR 20, 2008, 35(4): 481~485 Microbiology 2008 by Institute of Microbiology, CAS 研究报告一种耐热几丁质酶的产生及其稳定性研究郭润芳 1* 史小琴 2 李多川 3 魏青