|本期目录/Table of Contents|

[1]费忠,李恒,龚劲松,等.基于宏基因组学壳聚糖酶挖掘研究[J].应用与环境生物学报,2014,20(04):597-601.[doi:10.3724/SP.J.1145.2014.01039]
 FEI Zhong,LI Heng,GONG Jinsong,et al.Studies of chitosanase mining based on metagenomic technology[J].Chinese Journal of Applied & Environmental Biology,2014,20(04):597-601.[doi:10.3724/SP.J.1145.2014.01039]
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基于宏基因组学壳聚糖酶挖掘研究()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
20卷
期数:
2014年04期
页码:
597-601
栏目:
研究论文
出版日期:
2014-08-25

文章信息/Info

Title:
Studies of chitosanase mining based on metagenomic technology
作者:
费忠 李恒 龚劲松 杨涛 许正宏 史劲松
江南大学药学院 无锡 214122
Author(s):
FEI Zhong LI Heng GONG Jinsong YANG Tao XU Zhenghong SHI Jinsong
School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, China
关键词:
宏基因组文库Fosmid壳聚糖酶壳寡糖大肠杆菌
Keywords:
metagenomic library Fosmid chitosanase oligochitosan Escherichia coli
分类号:
Q939.97 : Q78
DOI:
10.3724/SP.J.1145.2014.01039
文献标志码:
A
摘要:
壳聚糖酶(EC3.2.1.132)可有效地将壳聚糖降解为活性良好和应用前景广泛的低分子壳聚糖或壳寡糖,但目前存在专一性强的壳聚糖酶发酵水平低、种类单一、应用性能较差等问题. 本研究从虾蟹堆积场淤泥采样,采用宏基因组文库方法,从其Fosmid文库中筛选出壳聚糖酶基因ChiE. 测序表明,ChiE由1 362 bp组成,编码453个氨基酸,预测分子量(Mr)为42 × 103左右. 将ChiE与pET-28a (+)载体连接,转化大肠杆菌Escherichia coli Rosetta-gami (DE3)构建了壳聚糖酶重组菌. 采用离子交换柱(DEAE Sepharose FF)和凝胶色谱柱(SuperdexTM 75)纯化目的蛋白. 酶学性质结果显示,该酶最适作用温度70 ℃,最适pH 6.0,Li+、Sr2+、K+等离子对其具有一定激活作用,Fe3+、Pb2+、Fe2+等离子则具有不同程度的抑制. 在最适作用条件下,该酶比酶活为2 158 U/mg. 本研究表明,重组菌E. coli Rosetta-gami (DE3)/ChiE具有培养简便、酶发酵产量大等优点,结果可为利用基因工程菌生产壳聚糖酶奠定基础. 图5 表2 参17
Abstract:
Chitosanase (EC3.2.1.132) can effectively degrade chitosan into low molecular chitosan or oligosaccharide, which plays an important role in a wide range area due to its biological activity. However, the application performance of chitosanase is limited by several problems including low fermentation level, lack of variety, and poor catalytic properties. Using chitosanase segregation agar containing chitosan as the medium components, we obtained chitosanase gene ChiE from shrimp activated sludge microbial metagenomic Fosmid library. The chitosanase gene ChiE, which was inserted into the expression vector pET-28a(+) and transformed into Escherichia coli Rosetta-gami (DE3), was shown by SDS-PAGE to consist of 1 362 bp, encode 453 amino acid residues, and have a molecular mass of 42 × 103. The recombinant chitosanase purified by DEAE Sepharose FF and SuperdexTM 75 was most active at 70 oC and pH 6.0. The activity of chitosanase was significantly enhanced by Li+, Sr2+, K+ and obviously inhibited by Fe3+, Pb2+, and Fe2+. The highest chitosanase activity was 2 158 U/mg with an optimal pH and temperature of 6.0 and 70 oC, respectively. This study demonstrated the advantages of simple cultivation and high fermentation throughput of E. coli Rosetta-gami (DE3)/ChiE, which should be helpful in producing chitosanase by genetically engineered strain.

参考文献/References:

1 Aam BB, Heggset EB, Norberg AL, Sorlie M, Varum KM, Eijsink VGH. Production of chitooligosaccharides and their potential applications in medicine [J]. Mar Drugs, 2010, 8: 1482-1517
2 乔莹, 白雪芳, 杜昱光. 壳寡糖医药保健功能的研究进展[J]. 中国生化药物杂志, 2008, 29 (3): 210-213 [Qiao Y, Bai XF, Du YG. Research advances of chitosan oligosaccharides on keeping healthy [J]. Chin J Biochem Pharm, 2008, 29 (3): 210-213]
3 Dahiya N, Tewari R, Hoondal GS. Biotechnological aspects of chitinolytic enzymes: a review [J]. Appl Microbiol Biotechnol, 2006, 71: 773-782
4 Hoell IA, Vaaje-Kolstada G, Eijsink VGH. Structure and function of enzymes acting on chitin and chitosan [J]. Biotechnol Genet Eng, 2010, 27: 331-366
5 Handelsman J, Rondon MR, Brady SF, Clardy L, Goodman RM. Molecular biological access to the chemistry of unknown soil microbes: a new frontien for natural products [J]. Chem Biol, 1998, 5: R245-249
6 Ammiraju JSS, Yu YS, Luo MZ, Kudrna D, Kim HR, Goicoechea JL, Katayose Y, Matsumoto T, Wu JZ, Sasaki T,Wing RA. Random sheared fosmid library as a new genomic tool to accelerate complete finishing of rice (Oryza sativa spp. Nipponbare) genome sequence: sequencing of gap-specific fosmid clones uncovers new euchromatic portions of the genome [J]. Theor Appl Genet, 2005, 111: 1596-1607
7 Hjort K, Bergstrom M, Adesina MF, Jansson JK, Smalla K, Sjoling S. Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen-suppressive soil [J]. FEMS Microbiol Ecol, 2010, 71: 197-207
8 Jeon JH, Kim JT, Kang SG, Lee JH, Kim SJ. Characterization and its potential application of two esterases derived from the arctic sediment metagenome [J]. Mar Biotechnol, 2009, 11: 307-316
9 Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar [J]. Anal Chem, 1959, 31: 426-428
10 Steele HL, Jaeger KE, Daniel R, Streit WR. Advances in recovery of novel biocatalysts from metagenomes [J]. J Mol Microbiol Biotechnol, 2009, 16: 25-37
11 Pechsrichuang P, Yoohat k, Yamabhai M. Production of recombinant Bacillus subtilis chitosanase, suitable for biosynthesis of chitosan-oligosaccharides [J]. Bioresour Technol, 2013, 127: 407-414
12 Saito A, Ooya T, Miyatsuchi D, Fuchigami H, Terakado K, Nakayama SY, Watanabe T.Nagata Y, Ando A. Molecular characterization and antifungal activity of a family 46 chitosanase from Amycolatopsis sp. CsO-2 [J]. FEMS Microbiol Lett, 2009, 293: 79-84
13 Johnsen MG, Hansen OC, Stougaard P. Isolation, characterization and heterologous expression of a novel chitosanase from Janthinobacterium sp. strain 4239 [J]. Microb Cell Fact, 2010, doi: 10.1186/1475-2859-9-5
14 Yoon HG, Kim HY, Lim YH, Kim HK, Shin DH Hong BS, Cho HY. Thermostable chitosanase from Bacillus sp. strain CK4: cloning and expression of the gene and characterization of the enzyme [J]. Appl Environ Microbiol, 2000, 66: 3727-3734
15 Jiang XY, Chen DC, Chen LH, Yang GN, Zou SM. Purification, characterization, and action mode of a chitosanase from Streptomyces roseolus induced by chitin [J]. Carbohydr Res, 2012, 355: 40-44
16 Wang SL, Chen SJ, Wang CL. Purification and characterization of chitinases and chitosanases from a new species strain Pseudomonas sp. TKU015 using shrimp shells as a substrate [J]. Carbohydr Res, 2008, 343: 1171-1179
17 Kabayashi T, Koido O, Deguchi S, Horikoshi K. Characterization of chitosanase of a deep biosphere Bacillus strain [J]. Biosci Biotechnol Biochem, 2011, 75: 669-673

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备注/Memo

备注/Memo:
“十二五”国家科技支撑计划课题(2012BAD33B06)、国家高技术研究发展计划项目(2012AA022204C)、国家自然科学基金项目(21206055)和江苏省自然科学基金项目(BK2012127)资助 Supported by the Sci-tech Pillar Project of the Twelfth Five-year Plan of China (2012BAD33B06), the National High-tech R&D Program of China (2012AA022204C), the National Natural Science Foundation of China (21206055) and the Natural Science Foundation of Jiangsu, China (BK2012127)
更新日期/Last Update: 2014-08-26