Separation and Purification of Hydroxylamine Oxidase from Agrobacterium tumefaciens LAD9

Abstract

Abstract: The metabolic pathway of heterotrophic nitrification-aerobic denitrification bacteria was directly determined by the actions of their hydroxylamine oxidase（HAO）. Isolating high-purity HAO from this kind of bacteria has become particularly important to explain the mechanism of nitrogen removal. In this study, the separation and purification technic of HAO from a novel heterotrophic nitrification-aerobic denitrification strain Agrobacterium tumefaciens LAD9 was established. Electrophoretic purity of HAO could be sucessfully purified through DEAE Sepharose CL-6B ion-exchange chromatography and Sephacryl S-100 gel filtration from its periplasm. The final purification fold was 5.79 and the yield was 39.71%. SDS-PAGE eclectrophoresis results revealed that the molecular weight of HAO in the strain LAD9 was 18.8 kD. Studies on enzymatic properties showed that the purified enzyme could oxidize hadroxylamine to nitrite and its activity could be enhanced by the addition of Fe2+.

Key words: Nitrogen removal, Hydroxylamine oxidase, Purification, Agrobacterium tumefaciens, LAD9 Heterotrophic nitrification-aerobic denitrification

Chen Qian, Ma Tao, Wang Ting. Separation and Purification of Hydroxylamine Oxidase from Agrobacterium tumefaciens LAD9[J]. Biotechnology Bulletin, 2014, 0(7): 69-73.

share this article

0
/ / Recommend

Add to citation manager EndNote|Ris|BibTeX

URL: https://biotech.aiijournal.com/EN/

https://biotech.aiijournal.com/EN/Y2014/V0/I7/69

References

[1] Bouchez T, Patureau D, Delgenes JP, Moletta R. Successful bacterial incorporation into activated sludge flocs using alginate [J]. Bioresource Tech, 2009, 100（2）：1031-1032.
[2] Yao YC, Zhang QL, Liu Y, Liu ZP. Simultaneous removal of organic matter and nitrogen by aheterotrophic nitrifying-aerobic denitrifying bacterial strain in a membrane bioreactor [J]. Bioresource Tech, 2013, 143：83-87.
[2] Anderson IC, Poth M, Homstead J, Burdige D. A comparison of NO and N²O production by the autotrophic nitrifier Nitrosomonas europaea and the heterotrophic nitrifier Alcaligenes-faecalis [J]. Appl Environ Microbiol, 1993, 59（11）：3525-3533.
[3] Robertson LA, Kuenen JG, Kleijntjens R. Aerobic denitrification and heterotrophic nitrification by Thiosphaera pantotropha [J]. Antonie Van Leeuwenhoek Journal of Microbiology, 1985, 51（4）：445-445.
[4] Joo HS, Hirai M, Shoda M. Improvement in ammonium removal efficiency in wastewater treatment by mixed culture of Alcaligenes faecalis no. 4 and L1 [J]. J Biosci Bioeng, 2007, 103（1）：66-73.
[5] Kim JK, Park KJ, Cho KS, et al. Aerobic nitrification-denitrification by heterotrophic Bacillus strains [J]. Bioresource Tech, 2005, 96（17）：1897-1906.
[6] Khardenavis AA, Kapley A, Purohit HJ. Simultaneous nitrification and denitrification by diverse Diaphorobacter sp. [J]. Appl Microbiol Biotechnol, 2007, 77（2）：403-409.
[7] Chen PZ, Li J, Li QX, et al. Simultaneous heterotrophic nitrification and aerobic denitrification by bacterium Rhodococcus sp. CPZ24 [J]. Bioresource Tech, 2012, 116：266-270.
[8] Yao S, Ni JR, Ma T, Li C. Heterotrophic nitrification and aerobic denitrification at low temperature by a newly isolated bacterium, Acinetobacter sp. HA2 [J]. Bioresource Tech, 2013, 139：80-86.
[9] Richardson DJ, Wehrfritz JM, Keech A, et al. The diversity of redox proteins involved in bacterial heterotrophic nitrification and aerobic denitrification [J]. Biochem Soc Trans, 1998, 26（3）：401-408.
[10] 黄珏, 何义亮, 赵彬, 等. Providencia rettgeri strain HNR菌株羟胺氧化酶超声波法提取的研究[J].生物技术通报, 2008（S1）：384-388.
[11] Chen Q, Ni JR. Heterotrophic nitrification-aerobic denitrification by novel isolated bacteria [J]. J Ind Microbiol Biot, 2011, 38（9）：1305-1310.
[12] Chen Q, Ni JR. Ammonium removal by Agrobacterium sp. LAD9 capable of heterotrophic nitrification-aerobic denitrification [J]. J BIOSCI BIOENG, 2012, 113（5）：619-623.
［13] Wehrfritz JM, Reilly A, Spiro S, Richardson DJ. Purification of hydroxylamine oxidase from Thiosphaera pantotropha. Identification of electron accepters that couple heterotrophic nitrification to aerobic denitrification [J]. Febs Lett, 1993, 335（2）：246-250.
[14] Shaw JG, Kelly DJ. Binding-protein dependent transport of C4-dicarboxylates in Rhodobacter capsulatus [J]. Arch Microbiol, 1991, 155（5）：466-472.
[15] Otte S, Schalk J, Kuenen JG, Jetten MSM. Hydroxylamine oxidation and subsequent nitrous oxide production by the heterotrophic ammonia oxidizer Alcaligenes faecalis [J]. Appl Microbiol Biot, 1999, 51（2）：255-261.
[16] 赵祖国, 孔庆鑫, 王景峰, 等.一种羟胺氧化酶基因序列的PCR扩增及其生物信息学分析[J].应用与环境生物学报, 2009, 15（1）：100-105.
[17] 杨航, 黄钧, 刘博.异养硝化-好氧反硝化菌Paracoccus pantotrophus ATCC35512的研究进展[J].应用与环境生物学报, 2008, 14（4）：585-592.
[18] Moir JWB, Wehrfritz JM, Spiro S, Richardson DJ. The biochemical characterization of a novel non-haem-iron hydroxylamine oxidase from Paracoccus denitrificans GB17 [J]. Biochem J, 1996, 319（3）：823-827.
[19] 于大禹, 张琳颖, 高波.异养硝化-好氧反硝化菌异养硝化性能的影响因素[J].化工进展, 2012（12）：2797-2800.
[20] 宋琴, 许雷.异养硝化作用酶学研究进展[J].生物技术通报, 2008（5）：60-66.
[21] Robertson LA, Kuenen JG. Combined heterotrophic nitrification and aerobic denitrification in Thiosphaera pantotropha and other bacteria [J]. Antonie Van Leeuwenhoek, 1990, 57（5）：139-152.

Related Articles 15

[1] LI Wen-shuo, WANG Lin-song, DU Gui-cai, GUO Qun-qun, ZHANG Ting-ting, YANG Hong LI Rong-gui. Gene Cloning of an Aldehyde Dehydrogenase from Bursaphelenchus xylophilus and Biochemical Characterization [J]. Biotechnology Bulletin, 2022, 38(9): 207-214.

[2] QIN Xue-jing, WANG Yu-han, CAO Yi-bo, ZHANG Ling-yun. Prokaryotic Expression and Preparation of Polyclonal Antibody of PwHAP5 Gene in Picea wilsonii [J]. Biotechnology Bulletin, 2022, 38(8): 142-149.

[3] GAO Yu-xuan, JIN Jing-chen, XU Li-shan, GAO Ya-Juan, ZHANG Wen-tian, LI Chen-chen, ZHANG Guo-wei, JIN Yong-sheng. Isolation of Halophilic Heterotrophic Nitrification-aerobic Denitrification Bacterium Bacillus megatherium N07 and Its Denitrification Characteristics [J]. Biotechnology Bulletin, 2022, 38(7): 247-257.

[4] YI Fang, LAI Peng-cheng, ZHENG Xi-ao, HU Shuai, GAO Yan-li. Research on the Preparation and Purification of Kod DNA Polymerase [J]. Biotechnology Bulletin, 2022, 38(5): 183-190.

[5] WANG Xiao-qin, HUANG Yin-ping, WANG Wei-qian, WU Ping, QUAN Shu. Expression and Purification of the MLL3_SET Protein with a Site-directed Mutation of an Unnatural Amino Acid [J]. Biotechnology Bulletin, 2022, 38(3): 194-202.

[6] ZHANG Feng-wen, ZHOU Li-ya, DONG Chao, SHI Yan-mao. Purification of Antioxidant Peptides from Natto Supernatant and Study on Its Activity [J]. Biotechnology Bulletin, 2022, 38(2): 158-165.

[7] CHEN Duo, LIU Yong-zhe. Prokaryotic Expression，Purification and Crystallization of N-terminal Domain of Nucleocapsid Protein in SARS-CoV-2 [J]. Biotechnology Bulletin, 2022, 38(12): 149-155.

[8] CAO Ru-fei, LI Ze-xuan, XU Huan, ZHANG Sha, ZHANG Min-min, DAI Feng, DUAN Xiao-lei. Expression,Purification,and Crystallization of Pif1 Helicase from Bacteroides fragilis [J]. Biotechnology Bulletin, 2021, 37(9): 180-190.

[9] TIAN Jia-hui, FENG Jia-li, LU Jun-hua, MAO Lin-jing, HU Zhu-ran, WANG Ying, CHU Jie. Isolation,Purification and Characterization of Laccase LacT-1 from Cerrena unicolor [J]. Biotechnology Bulletin, 2021, 37(8): 186-194.

[10] ZHANG Xi-xi, ZHANG Yi-qing, LI Yu-lin, HAN Xiao, WANG Guo-qiang, WANG Xiao-jun, WANG Xu-dong, WANG Yun-long. Prokaryotic Expression,Purification and Application of N Protein C-terminal Recombinant Protein in Novel Coronavirus（SARS-CoV-2） [J]. Biotechnology Bulletin, 2021, 37(5): 92-97.

[11] WANG Zhi-xin, LU Lei-zhen, ZHOU Jing-bo, FENG Cheng-ling, JIA Zi-wei, NING Ya-wei, JIA Ying-min. Advance in the Research of the Antifungal Peptides [J]. Biotechnology Bulletin, 2021, 37(3): 206-218.

[12] TANG Lu, DONG Li-ping, YIN Mo-li, LIU Lei, DONG Yuan, WANG Hui-yan. Preparation and Identification of a Novel FGF20 Monoclonal Antibody [J]. Biotechnology Bulletin, 2021, 37(10): 179-185.

[13] MENG Li, DU Cai-ping. Eukaryotic Expression,Purification and Activity Identification of Rat His-Akt1 Recombinant Protein [J]. Biotechnology Bulletin, 2020, 36(12): 98-103.

[14] ZHOU Yang, WANG Tao-tao, YAN Dan-dan, WANG Ying-ying, SHI Qin-xuan, SUN Li-hui, LIN Feng. Advances in Biotechnological Application of Elastin-like Polypeptides as Functional Nanomaterials [J]. Biotechnology Bulletin, 2020, 36(11): 198-208.

[15] DUAN Jia-qi, LI Bei-bei, REN Jie, QIU De-wen, LI Guang-yue. Research on a Preparation Process of Deoxynivalenol [J]. Biotechnology Bulletin, 2020, 36(10): 256-262.

Recommended Articles

Metrics

Viewed

Full text

Abstract

Comments

Abstract

References

Related Articles

Recommended Articles

Metrics

Comments

TOP

Separation and Purification of Hydroxylamine Oxidase from Agrobacterium tumefaciens LAD9

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	LI Wen-shuo, WANG Lin-song, DU Gui-cai, GUO Qun-qun, ZHANG Ting-ting, YANG Hong LI Rong-gui. Gene Cloning of an Aldehyde Dehydrogenase from Bursaphelenchus xylophilus and Biochemical Characterization [J]. Biotechnology Bulletin, 2022, 38(9): 207-214.
[2]	QIN Xue-jing, WANG Yu-han, CAO Yi-bo, ZHANG Ling-yun. Prokaryotic Expression and Preparation of Polyclonal Antibody of PwHAP5 Gene in Picea wilsonii [J]. Biotechnology Bulletin, 2022, 38(8): 142-149.
[3]	GAO Yu-xuan, JIN Jing-chen, XU Li-shan, GAO Ya-Juan, ZHANG Wen-tian, LI Chen-chen, ZHANG Guo-wei, JIN Yong-sheng. Isolation of Halophilic Heterotrophic Nitrification-aerobic Denitrification Bacterium Bacillus megatherium N07 and Its Denitrification Characteristics [J]. Biotechnology Bulletin, 2022, 38(7): 247-257.
[4]	YI Fang, LAI Peng-cheng, ZHENG Xi-ao, HU Shuai, GAO Yan-li. Research on the Preparation and Purification of Kod DNA Polymerase [J]. Biotechnology Bulletin, 2022, 38(5): 183-190.
[5]	WANG Xiao-qin, HUANG Yin-ping, WANG Wei-qian, WU Ping, QUAN Shu. Expression and Purification of the MLL3_SET Protein with a Site-directed Mutation of an Unnatural Amino Acid [J]. Biotechnology Bulletin, 2022, 38(3): 194-202.
[6]	ZHANG Feng-wen, ZHOU Li-ya, DONG Chao, SHI Yan-mao. Purification of Antioxidant Peptides from Natto Supernatant and Study on Its Activity [J]. Biotechnology Bulletin, 2022, 38(2): 158-165.
[7]	CHEN Duo, LIU Yong-zhe. Prokaryotic Expression，Purification and Crystallization of N-terminal Domain of Nucleocapsid Protein in SARS-CoV-2 [J]. Biotechnology Bulletin, 2022, 38(12): 149-155.
[8]	CAO Ru-fei, LI Ze-xuan, XU Huan, ZHANG Sha, ZHANG Min-min, DAI Feng, DUAN Xiao-lei. Expression,Purification,and Crystallization of Pif1 Helicase from Bacteroides fragilis [J]. Biotechnology Bulletin, 2021, 37(9): 180-190.
[9]	TIAN Jia-hui, FENG Jia-li, LU Jun-hua, MAO Lin-jing, HU Zhu-ran, WANG Ying, CHU Jie. Isolation,Purification and Characterization of Laccase LacT-1 from Cerrena unicolor [J]. Biotechnology Bulletin, 2021, 37(8): 186-194.
[10]	ZHANG Xi-xi, ZHANG Yi-qing, LI Yu-lin, HAN Xiao, WANG Guo-qiang, WANG Xiao-jun, WANG Xu-dong, WANG Yun-long. Prokaryotic Expression,Purification and Application of N Protein C-terminal Recombinant Protein in Novel Coronavirus（SARS-CoV-2） [J]. Biotechnology Bulletin, 2021, 37(5): 92-97.
[11]	WANG Zhi-xin, LU Lei-zhen, ZHOU Jing-bo, FENG Cheng-ling, JIA Zi-wei, NING Ya-wei, JIA Ying-min. Advance in the Research of the Antifungal Peptides [J]. Biotechnology Bulletin, 2021, 37(3): 206-218.
[12]	TANG Lu, DONG Li-ping, YIN Mo-li, LIU Lei, DONG Yuan, WANG Hui-yan. Preparation and Identification of a Novel FGF20 Monoclonal Antibody [J]. Biotechnology Bulletin, 2021, 37(10): 179-185.
[13]	MENG Li, DU Cai-ping. Eukaryotic Expression,Purification and Activity Identification of Rat His-Akt1 Recombinant Protein [J]. Biotechnology Bulletin, 2020, 36(12): 98-103.
[14]	ZHOU Yang, WANG Tao-tao, YAN Dan-dan, WANG Ying-ying, SHI Qin-xuan, SUN Li-hui, LIN Feng. Advances in Biotechnological Application of Elastin-like Polypeptides as Functional Nanomaterials [J]. Biotechnology Bulletin, 2020, 36(11): 198-208.
[15]	DUAN Jia-qi, LI Bei-bei, REN Jie, QIU De-wen, LI Guang-yue. Research on a Preparation Process of Deoxynivalenol [J]. Biotechnology Bulletin, 2020, 36(10): 256-262.