Research Overview ( 0304 )
Research Group: Research and Application of Microbial Enzymes
Research Area:
Our group is primarily engaged in enzyme technology, structure and function of enzymes. Our aim is enhancing enzyme activity, modifying enzyme properties through modern biotechnology (gene engineering, metabolic engineering, DNA shuffling, protoplast mutation, ion beam mutation etc); exploiting new enzymes; and enlarging fields of enzyme application.
Group Leader: Prof. Shijun Qian
1962-1967 Department of chemistry, Fudan University
1968- Institute of microbiology, Chinese Academy of Sciences
1980.10-1981.10 International Post-Graduate University Course in Microbiology, Osaka University, Japan
1988.3-1990.4 School of Medicine, University of Washington, USA
2000.10-2000.11 University of Cergy Pontoise, France
Research group Members:
Assistant Professor: Dr. Chao Yapeng, MS. Shi Jiaji, MS. Zhang Guoqing
Technician: Yang Jing
Graduate student: Sun Yan, Liu Weixiao, Liu Xiaoqiu, Yang Xiuqing, Song Liya, Xie Fuhong, Hu Meirong, Liu Shuzhen, Bao Hongbo, Zhang Chuanbao, Zhang Lianhui, Lu Jinghua, Tan Huifang
Background and Significance:
In the recent surge of interest in industrial biotechnology, considerable emphasis has been placed upon advance in enzymes, which is a key factor in bioconversion and biocatalysis.
The enzymes have been applied in every aspect of the national economy in China with great social and economic benefit.
Our research focuses on the exploitation, improvement and new application of enzymes related to textiles, food, medicine, glycotechnology and environment protection, etc. We have been studying laccase, cellulase, transglutaminase, heparinase, polychlorinated biphenyl- degrading enzymes, in an effort to expand new application, overcome low biocatalytic capabilities and high production cost, and improve adaptability in the reaction process of these enzymes.
Moreover, we also have been studying on structure, function and mechanism of enzymes.
Major Achievements:
During the past 4 years, we have got many funds supported by the government, Chinese Academy of Sciences and company etc. The research items are as following:
1. National Tenth Five years Plan Key Technologies R&D program of China: The Study and application of neutral-cellulase
2. Innovative program of Chinese Academy Sciences: The enzymes relateing to oligosaccharides and their applications
3. The National Natural Sciences Foundation of China: The relationship of level of DNA repair enzymes and telomerase with oesophagus cancer and lung cancer
4. National High Technology R&D program of China (863program): Construction of alkaline xylanase-secreting and laccase-secreting engineered strain with high activity and their application in paper processing
5. National High Technology R&D program of China (863 program): The exploitation and application of biodegradation enzyme gene resources
6. The National Key Basic Research Program of China (973 program): Methodology and catalytic mechanism of nitrile hydration and hydrolysis reaction
7. Innovative program of Chinese Academy of Sciences: The single ion beam microirradiation and its study of molecular and cellular effect
8. The National Natural Science Foundation of China: The analysis of fine structure of the heparin oligosaccharides with bioactivity
9. The National Major Natural Science Foundation of China: The study of chiralblock biosynthesis on chiral drug
10. Collaborative research project of Institute of microbiology, Chinese Academy of Sciences- P&G :Intelligent screening for new enzyme activity
11. Research project of Institute of microbiology: The new technology and new method of molecular enzyme engineering
12．A France-Chinese collaborative research project: Comparison of various transglutaminases to catalyze the sol-gel transition of protein
Major achievements:
- Neutral Cellulase
We got a high yield strain for producing neutral cellulase by screening, mutagenesis (low-energy ion bean, chemical and protoplast mutation), and optimization of culturing conditions. Now, we have finished pilot scale test in 20-ton fermenter, and got enzyme preparation by filter press, ultra-filter membrane concentration and spray drying, which filled in the gaps in the enzyme preparation in China. We made neutral cellulase to test in the textile industry and got good results.
- Laccase
Laccases have potential uses in environment protection, the textile industry, organic synthesis and biosensor etc.
We screened a White rot fungus strain producing Laccase and purified the enzyme. cDNA encoding a laccase was isolated from the white rot fungus by RT-PCR. It contained an open reading frame of 1157bp. The cDNA was cloned into the vector pGAPZA, and expressed in the Pichia pastoris. The activity of laccase secreted by the transformant was three times as high as that of the native strain. The optimal secreting time of laccase was significantly shorter than the native strain. Moreover, we treated laccase gene with ethyl methane-sulfonate to generate a mutant library. Then, the mutant genes were cloned into a vector to construct recombinants, which were transfometed to Pichia pastoris. We got a clone from 5000 clones by agar plate and 96-well plate screening, with a laccase activity 3 folds of the original strain
- Uracil N-Glycosylase
By RT-PCR, human Uracil N-Glycosylase (UNG) coding sequence is cloned onto pET-21(a), and expressed in E.coli after induction. Taking the recombinant plasmid, whose copy number of UNG is already known, as quantification standard, we examined the number of copies of UNG mRNA in the esophagus cancer biopsy samples by real-time quantitative RT-PCR, and determined that there is direct positive correlation between high-level expression of UNG and the presence of esophagus cancer.
- Heparinase
Heparinase is of great importance in the fine structural analysis of heparin /heparan sulfate as well as development of heparin oligosaccharides; moreover, heparinase itself showed pharmaceutical potentials.
Heparinase was produced by a novel species of Sphingobacterium. It was purified to apparent homogeneity by a combination of SP-Sepharose and Source 30S chromatographies. MALDI-TOF mass spectrum of the purified heparinase gave a molecular weight of 75KDa of the native enzyme. Peptide mass spectrum showed low homology with the database in the peptide bank. Inhibition of enzyme activity by N-acetylimidazole indicated that tyrosine residues were necessary for enzyme activity. The heparinase showed similar activity on both heparin and heparan sulfate except for the heparin from bovine lung.
Porcine intestinal mosca heparin was degraded into a series of oligosaccharides by the purified heparinase. Heparin disaccharides were successfully separated by HPLC. Conformation and charges of disulfated disaccharides were analyzed by computer models.
Electrospray ion-mass spectrometry ( ESI-MS ) was used to identify the structures of the disaccharides obtained by HPLC above. The mass spectrometry of peak I mainly gave the non-sulfated disaccharide with the mass of 379. Peak II and III indicated two major monosulfated disaccharides with different mass, and peak III occupied majority. Both of Peak IV and V showed the same mass. No tri-sulfate modified disaccharides were detected in the mixture of the heparin digest though they were abundant in the heparin structures. The results revealed that the heparinase may specifically cut the low sulfated sites of heparin.
Functional analysis of the heparin oligosaccharides revealed that it can bind with the cytokine-granulocyte-colony stimulating factor by capillary electrophoresis and mass spectrometry.
- Cloning and functional analysis of genes involved in polychlorinated biphenyls (PCBs) degradation by Rhodococcus sp. R04:
PCB is known as one of 12 persistent contaminants in the world, PCB biodegradation by microorganism is not only cheap and efficient, but also free of secondary pollution.
A novel PCBs degrading strain Rhodococcus pyridinovorans was isolated from oil field in northern China, and the degradation pathway of biphenyl was investigated. The studies on capability of degrading and dechlorination of PCBs revealed that R04 strain was a strong PCB degrader.
A plasmid library was constructed, and approximately 40 positive clones were obtained. The sequence of the positive clones indicated that its inserted fragment contained six ORFs. Based on the homology, these six ORFs are presumed to be bphB (dihydrodia dehydroxygenase) (nucleotide positions 581 to 1397), bphC (2,3-dihydroxybiphenyl dioxygenase) (1426 to 2313) bphA1 (large α- subunit) (3278 to 4627), bphA2 (small β-subunit) (4671 to 5198), bphA3 (ferredoxin) (5532 to 5921), and bphA4 (ferredoxin reductase) (6374 to 7612) in turn. By PCR amplification, a fragment contained possible bphD was obtained. The bphB, bphC, bphA1, bphA2, bphA3, bphA4 and bphD were organized in a gene cluster (Fig ).
To identify the products of bphB, bphC, bphA and bphD, the proteins were analyzed by SDS-PAGE. The interested proteins corresponding to size of 30, 32, 50, 20, 14, 48 and 30 KD were observed. Activities of bphA, bphB, bphC, and bphD were determined by HPLC or photometer, and when necessary, by GC-MS. The resulting assays indicated that bphA, bphB, bphC, and bphD were able to transform biphenyl 2,3-dihydro-dihydroxybiphenyl 2,3-dihydroxybiphenyl and 2-hydroxyl-6-oxo-6-pheylhexa-2,4-dienoic acid separately.
Fig. The organization of pbh gene clusters (bphB, bphC, bphA1, bphA2, bphA3, bphA4 and bphD) in R04 strain
- Nitrile hydratase
Nitrile hydratase has wide application in the synthesis of carboxylic acid and amide, as well as chiral bioconversion of nitrile compoundes.
The new culture medium and conditions of Rhodococcus sp. AJ270 was found, which would help us obtain doubled cell density; Nitrile hydratase was purified by sonication, ammonium sulfate fractionation, DEAE-Sephacel column chromatography, second DEAE-Sephacel column chromatography, Phenyl Sepharose column chromatography and Sephacryl S200 column chromatography. The molecular mass of α subunit and β subunit of the enzyme were 22957Da and 23493Da by mass spectrum. The enzyme could hydrate varieties of aliphatic nitriles and aromatic nitriles. Some substrates could be enantioselectively hydrated to the corresponding amides; Crystal of the enzyme was obtained by hanging-drop vapor-diffusion method at 4℃. After X-ray diffraction and data collection the phase calculation was carried out by molecular replacement.
7. Transglutaminase
Transglutimase has an applicable prospect in the food and textile industry.
The whole gene of transglutaminase was obtained from Streptomyces fraedia through genome walking. The whole gene was cloned into pGEM T-easy vector and transformed into BL21(DE3), the protein was expressed with a yield 500mg/L , but it existed in the form of inclusion body.
The Streptomyces was irradiated by ion beam. The energy and dosage of ion beam were 10kev and 30meq-50meq respectively. We investigated the transglutaminase gene of the mutants by polymerase chain reaction (PCR), nonradioisotopic single strand conformation polymorphism (SSCP)，and DNA sequencing. We have already got 21 strains with change in transglutaminase gene.
8. Other enzymes
Endo-type inulinase attacked inulin endo-wise producing inulo- oligosacchorides. An endoinulinase produced by Chaetomium sp. C34 was purified to electrophoretic homogeneity. Some of its properties were investigated.
Mn-Peroxidase is key-enzyme for lignin degradation. We screened a Trametes versicolor strain producing Mn-peroxidase and purified the enzyme.
Keratinase is used for feed industry and detergent. We screened a strain for degradation of human skin flake, then isolated and purified the keratinase.
Future Research Plan:
1. Exploitation and application of new enzymes:
Enzymes related to the textiles industry including cellulase, laccase, transglutaminase, catalase and pectinase will be exploited. Our aim is to produce two of these enzymes in pilot plant.
Enzymes related to bioconversion of biomass including laccase Mn-peroxidase and lignin peroxidase will be applied in test of conversion of biomass.
- Improvement of enzymes:
Transglutaminase, laccase and neutral proteinase will be improved by in vitro directed evolution and site directed mutagenesis to enhance enzyme activity and to change enzyme properties.
3. Study of structure and function of the Nitrile hydratase and the amidase.
Publications
1. Yinda Wang,Shijun Qian ,Guangzheng Meng and Shuzheng Zhang. Cloning and expressing of L-asperaginase gene in Escherichia. coli. Applied Biochemistry and Biotechnology.2001, 95(2):93-101.
2. Shi Jiaji, Cui Fumian, The immobilization of penicillin G acylase on γ-alumina by glutaraldehyde cross-linking, Chinese Journal of antibiotics, 2001，26（5）：334-336.
3. Shi Jiaji, Cui Fumian, Ge Meng, The epoxidation of cis-propenylphophonic acid to fosfomycin by pencillium sp., Acta Microbiology Sinica, 2001，4（3）：353-356．
4. Shi Jiaji, Cui Fumian, Selection of β-glucanase-producing trichoderma Kōningii T199 and its fermentation conditions, Acta Microbiology Sinica, 2001，41（6）：750-752.
5. Chao Yapeng, Qian Shijun, Fungal laccase and its application, Journal of Chinese biotechnology, 2001, 21(5): 23-29.
6. Liu Jianguo, Wang Jinfang, Li Gaoxiang, Culture conditions for creatininase formation by pseudomonas sp. K9510, Microbiology, 2001, 28(2): 7-11.
7. Zhu Xianjun, Liu Jianguo, Li Gaoxiang, Cloning and expression of urate oxidase and its application in serum uric acid analysis, Chinese Journal of Biotechnology, 2001, 17（1）：68-72.
8. Zhu Yuwen, Liu Jianguo, Li Gaoxiang, Cloning and expression of MGMT cDNA and analysis of the DNA repair activity of the recombinant protein, Chinese Journal of Biotechnology, 2001，17（4）：396-399.
9. Sun and S.Qian, Flux control analysis for biphenyl metabolism by Rhodococcus pyridinovorans R04. Biotechnology Letters,2002,24(18): 1525-1529.
10. Liu Shuzhen, Qian Shijun, Purification and properties of laccase from Basidiomycete, Acta Microbiology Sinica, 2003, 43(1): 73-78.
11. Bao Hongbo, Qian Shijun, Cloning of human uracil N-glycosylase and its detection in cancer tissues by quantitative RT-PCR, Chinese Joural of Biotechnolgy, 2003, 19(5): 561-565.
12. Sun Yan, Qian Shijun, Study on the degradation pathway of biphenyl by Rhodococcus pyridinovorans R04, Acta Microbiologica Sinica, 43(5): 653-658.
13. Weixiao Liu, Shijun Qian, Molecular cloning and characterization of a laccase gene from the Basidomycete Fome lignosus and expressionin Pichia postoris, Appl Microbiol. Biotech., 63（2）174-181，2003.
14. Chao Yapeng, Qian Shijun, Rapid purification characterization and substract specificity of Heparinase from a novel species of Sphingobacterium sp, J. Biochem., 134（3）365-371, 2003.
15. Qian Shijun, progress in study on enzyme preparation, Chinese Joural of Bioprocess Engineering, 1(1): 28-31, 2003.
16. Liu Xiaoqiu, Qian Shijun, Transglutaminase in medical research, Journal of Chinese biotechnology, 2003, 23(11)：32-36.
17. Bao Hongbo, Wang Jinfang, Qian Shijun, Advances in research of uracil N-glycosylase, Journal of Chinese biotechnology, 2003, 23（1）43-47.
18. Cui Dafu, Zhang Guoqing, Qian Shijun, et al. Detection of O6-methylguanine-DNA methyltransferase activity using surface plasmon resonance biosensor, Progress in Natural Science, 13(8): 874-876, 2003.
19. Wei xiao Liu, Yapeng Chao, Xiuqing Yang,Hong bo Bao ,Shijun Qian, Biodecolorization of azo,anthraqninonic and triphenylmethane dyes by white rot fungi and a Laccase- secreting engineered strain J.Ind Microbiol Biotechnol, 2004, 31: 127-132.
20. Xiuqing Yang, Yan Sun, Shijun Qian.Biodegradation of Seven polychlorinated biphenyls by a newly isolated aerobio bacterium (Rhdodcoccun Sp RO4). J.Ind Microbiol Biotechnol,2004, 31 : 415-420.
21. Yapeng Chao, Shaoxiang Xiong, Xiulan Cheng, and Shijun Qian, Mass Spectrometric Evidence of Heparin discchrides for the Catalytic Characterization of A Novel Endolytic Heparinase.Acta Biochimica et Biophysica Sinica, 2004, 36( 12 ):840-844.
22. Liu Weixiao, Chao Yapeng, Qian Shijun,Construction and Culture Conditions of Laccase-secreting Engineered Strain, Acta Microbiologica Sinica 2004,44(5):596-599.
23. Chao Yapeng, Xu Guanzhu, Cheng Xiulan, Qian Shijun, Preparation and characteristics of the immobilized endolytic heparinase, Acta Microbiologica Sinica, 2004,44(5):689- 691.
24. Liu Weixiao, Qian Shijun, Methods of the directed molecular evolution of enzyme in vitro, Microbiology, 2004, 31(2): 100- 104.
25. Liu Weixiao, Chao Yapeng, Qian Shijun, Construction of laccase-secreting engineering strain and decolorization of RBBR by purified laccase, Chinese Journal of bioprocess engineering, 2004, 2(1): 21- 24.
26. Sun Yan, Yang Xiuqing, Qian Shijun,Cloning and expression of poly chlorobiphenyl/biphenyl degrading gene from Rhodococcus Pyridinovorans, Chinese Environmental Sciences, 2004,24(6): 734-737.
27. Zhang Guoqing, Cui Fumian, Yang Xiuqing, Qian Shijun, Purification and Properties of endoinulinase from Chaetomium sp. ,Acta Microbiology Sinica, 2004, 44(6): 785-788.
28. Sun Yan, Qian Shijun, Characterization and identification of a biphenyl degrading strain, Microbiology, 2004,31(6):23- 26.
29. Aiye Liang ，Yapeng Chao（co-authored），Xiaojun Liu ，Yuguang Du ，Keyi Wang ，Shijun Qian and Bingcheng Lin ,Heparin oligosaccharides separation and interaction with Granulocyte- colony stimulating factor by capillary electrophoresis. Electrophoresis.(Accepted)
30. ZhangLian-hui Yang Xiu qing GeKeshan , Qian Shijun, Purification and Properties of Manganese Peroxidase from Tramates verscolor, Acta Microbiology Sinica,2005, ( in press )
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