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Effect of culture temperature alteration of Aspergillus niger A-25 on its production of xylanase
Abstracts / Journal of Biotechnology 136S (2008) S290–S344
peratures were collected and analyzed by qRT-PCR to determine the transcriptional levels of three operons for all eight necessary genes of the validamycin A biosynthesis (Yu et al., 2005; Bai et al., 2006). It was found that remarkable difference of the gene transcription at different fermentation temperatures contributed to the distinct production performance. Study related to the biosynthetic pathway at an enzyme level was also conducted and will be discussed in this paper. Acknowledgement We appreciate the financial support from the National High Technology R&D Program (863 Program project #2006AA10A202) of the Ministry of Science & Technology of China (MOST), and the Shanghai Leading Academic Discipline Project (project #B203).
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conditions showed that there emerged 3 active bands (XynI, XynII, XynIII) in the first condition while only 2 (XynI, XynII) emerged in the latter. Since when detected after 84 h in the latter condition all 3 zymogram bands emerged, the reasonable explanation would be that temperature alteration performance promoted the XynIII to be synthesized earlier or to be synthesized more than that of constant temperature performance. The real-time PCR of xynIII gene demonstrated that at 48 h the transcriptional level of xynIII gene in temperature alteration performance was high as 4.5-fold as that in constant temperature performance. These results provided biochemical support for carrying out culture temperature alteration, a simple technique during fungal fermentation process in order to raise enzyme production level at a low cost. Keywords: Xylanase; Aspergillus niger; Temperature alteration; Real time PCR
References References Bai, L.Q., Li, L., Xu, H., Minagawa, K., Yu, Y., Zhang, Y., Zhou, X., Deng, Z.X., 2006. Functional analysis of the validamycin biosynthetic gene cluster and engineered production of validoxylamine A. Chem. Biol. 13 (4), 387–397. Iwasa, T., Yamamoto, H., Shibata, M., 1970. Studies on validamycins, new antibiotics. I. Streptomyces hygroscopicus var. limoneus nov. var., validamycin-producing organism. J. Antibiot. (Tokyo) 23, 595–602. Shen, Y.C., 1996. 25 years of research and development of jinggangmycin. Plant Protect. (Chin.) 22 (4), 44–45. Yu, Y., Bai, L.Q., Minagawa, K., Jian, X., Li, L., Li, J., Chen, S., Deng, Z.X., 2005. Gene cluster responsible for validamycin biosynthesis in Streptomyces hygroscopicus subsp. jinggangensis 5008. Appl. Environ. Microb. 71 (9), 5066–5076.
doi:10.1016/j.jbiotec.2008.07.1922 V1-P-053 Effect of culture temperature alteration of Aspergillus niger A-25 on its production of xylanase Hongge Chen ∗ , Xinyu Liu, Xiaojuan Lu, Liangwei Liu, Xincheng Jia College of Life Sciences, Henan Agricultural University, Zhengzhou 450002, PR China E-mail address: [email protected] (H. Chen). Xylanases have been the focus of much attention due to their extensive applications in the pulp and paper, food industries, animal feed, and bioconversion of agricultural wastes to useful materials (Coughlan and Hazlewood, 1993). At present, filamentous fungi are the most common industrial sources of xylanases because of their high productivity (Tanaka et al., 2005). The strain Aspergillus niger A-25 has a good ability to produce xylanases, and to further improve its enzyme level, the culture temperature alteration was carried out during fermentation process of A-25 and its effect on xylanase production was investigated.As shown by measurement of mycelium weight and xylanase activity, A-25 had an optimal growth temperature of 33 ◦ C and an optimal xylanase production temperature of 29 ◦ C with the yield of 610 IU/ml. The two temperatures were obviously inconsistent, so different alterations of culture temperature were performed during A-25 xylanase fermentation. The results showed that operating culture temperature 33 ◦ C for early 48 h then down to 29 ◦ C afterwards could not only shorten the culture time to reach peak value of enzyme production, but also remarkably enhance the enzyme yield to 721 IU/ml as compared with the fermentation under constant 29 ◦ C.The further analyses revealed that temperature alteration performance increased the total mycelium biomass, as well as xylanase productivity per unit weight of mycelium compared with the constant temperature condition. Detection of xylanase zymogram of culture supernatant after 48 h and 52 h cultivation under above two
Coughlan, M.P., Hazlewood, G.P., 1993. Beta-1,4-D-xylan-degrading enzyme systems: biochemistry, molecular biology and applications. Biotechnol. Appl. Biochem. 17, 259–289. Tanaka, H., Nakamura, T., Hayashi, S., Ohta, K., 2005. Purification and properties of an extracellular endo-1,4-beta-xylanase from Penicillium citrinum and characterization of the encoding gene. J. Biosci. Bioeng. 100, 623–630.
doi:10.1016/j.jbiotec.2008.07.1923 V1-P-055 Optimization of medium and process parameters for the production of inulinase from Kluyveromyces marxianus Y1 Wenjie Yuan ∗ , Fengwu Bai Department of Bioscience and Bioengineering, Dalian University of Technology, Dalian 116023, China E-mail address: [email protected] (W. Yuan). Abroad attention has been focused on the utilization of Jerusasalem artichoke as a carbohydrate raw material to produce fuel ethanol. A yeast strain of Kluyveromyces marxianus Y1 was selected for ethanol production using Jerusasalem artichoke by means of simultaneous saccharification and fermentation. The optimum condition for extra cellular inulinase production by K. marxianus Y1 was: inulin 40, yeast extract 4, peptone 4, urea 1, corn stream 10 (g/L). Fermentation medium pH 5.0, cultivation temperature 38 ◦ C, agitation rate of 150 rpm were optimal for enzyme production (54 U/ml) with a fermentation time of 120 h at shake flask level. K+ , Fe2+ , Mg2+ can increased the activity of inulinase, Ca2+ had little effect on inulinase, but Mn2+ inhibit it. The optimum reaction parameter of the inulinase was 55 ◦ C, pH 5.5. This enzyme also showed a good pH and temperature adaptability and stability. References Bernardo, O., Silva-Santisteban, Y’epez, Maugeri Filho, Francisco, 2005. Agitation, aeration and shear stress as key factors in inulinase production by Kluyveromyces marxianus. Enzyme Microb. Technol. 36, 717–724. Gill, Prabhjot Kaur, Sharma, Arun Dev, Harchand, Rajesh Kumari, 2003. Effect of media supplements and culture conditions on inulinase production by an actinomycete strain. Bioresour. Technol. 87, 359–362. Passador-Gurgel, G.C., Furlan, S.A., Meller, J.K., 1996. Application of a microtitre reader system to the screening of inulinase nulinase-producing yeasts. Microbiol. Biotechnol. (45), 158–161. Singh, R.S., Sooch, Balwinder Singh, Puri, Munish, 2007. Optimization of medium and process parameters for the production of inulinase from a newly isolated Kluyveromyces marxianus YS-1. Bioresour. Technol. 98, 2518–2525.
peratures were collected and analyzed by qRT-PCR to determine the transcriptional levels of three operons for all eight necessary genes of the validamycin A biosynthesis (Yu et al., 2005; Bai et al., 2006). It was found that remarkable difference of the gene transcription at different fermentation temperatures contributed to the distinct production performance. Study related to the biosynthetic pathway at an enzyme level was also conducted and will be discussed in this paper. Acknowledgement We appreciate the financial support from the National High Technology R&D Program (863 Program project #2006AA10A202) of the Ministry of Science & Technology of China (MOST), and the Shanghai Leading Academic Discipline Project (project #B203).
S317
conditions showed that there emerged 3 active bands (XynI, XynII, XynIII) in the first condition while only 2 (XynI, XynII) emerged in the latter. Since when detected after 84 h in the latter condition all 3 zymogram bands emerged, the reasonable explanation would be that temperature alteration performance promoted the XynIII to be synthesized earlier or to be synthesized more than that of constant temperature performance. The real-time PCR of xynIII gene demonstrated that at 48 h the transcriptional level of xynIII gene in temperature alteration performance was high as 4.5-fold as that in constant temperature performance. These results provided biochemical support for carrying out culture temperature alteration, a simple technique during fungal fermentation process in order to raise enzyme production level at a low cost. Keywords: Xylanase; Aspergillus niger; Temperature alteration; Real time PCR
References References Bai, L.Q., Li, L., Xu, H., Minagawa, K., Yu, Y., Zhang, Y., Zhou, X., Deng, Z.X., 2006. Functional analysis of the validamycin biosynthetic gene cluster and engineered production of validoxylamine A. Chem. Biol. 13 (4), 387–397. Iwasa, T., Yamamoto, H., Shibata, M., 1970. Studies on validamycins, new antibiotics. I. Streptomyces hygroscopicus var. limoneus nov. var., validamycin-producing organism. J. Antibiot. (Tokyo) 23, 595–602. Shen, Y.C., 1996. 25 years of research and development of jinggangmycin. Plant Protect. (Chin.) 22 (4), 44–45. Yu, Y., Bai, L.Q., Minagawa, K., Jian, X., Li, L., Li, J., Chen, S., Deng, Z.X., 2005. Gene cluster responsible for validamycin biosynthesis in Streptomyces hygroscopicus subsp. jinggangensis 5008. Appl. Environ. Microb. 71 (9), 5066–5076.
doi:10.1016/j.jbiotec.2008.07.1922 V1-P-053 Effect of culture temperature alteration of Aspergillus niger A-25 on its production of xylanase Hongge Chen ∗ , Xinyu Liu, Xiaojuan Lu, Liangwei Liu, Xincheng Jia College of Life Sciences, Henan Agricultural University, Zhengzhou 450002, PR China E-mail address: [email protected] (H. Chen). Xylanases have been the focus of much attention due to their extensive applications in the pulp and paper, food industries, animal feed, and bioconversion of agricultural wastes to useful materials (Coughlan and Hazlewood, 1993). At present, filamentous fungi are the most common industrial sources of xylanases because of their high productivity (Tanaka et al., 2005). The strain Aspergillus niger A-25 has a good ability to produce xylanases, and to further improve its enzyme level, the culture temperature alteration was carried out during fermentation process of A-25 and its effect on xylanase production was investigated.As shown by measurement of mycelium weight and xylanase activity, A-25 had an optimal growth temperature of 33 ◦ C and an optimal xylanase production temperature of 29 ◦ C with the yield of 610 IU/ml. The two temperatures were obviously inconsistent, so different alterations of culture temperature were performed during A-25 xylanase fermentation. The results showed that operating culture temperature 33 ◦ C for early 48 h then down to 29 ◦ C afterwards could not only shorten the culture time to reach peak value of enzyme production, but also remarkably enhance the enzyme yield to 721 IU/ml as compared with the fermentation under constant 29 ◦ C.The further analyses revealed that temperature alteration performance increased the total mycelium biomass, as well as xylanase productivity per unit weight of mycelium compared with the constant temperature condition. Detection of xylanase zymogram of culture supernatant after 48 h and 52 h cultivation under above two
Coughlan, M.P., Hazlewood, G.P., 1993. Beta-1,4-D-xylan-degrading enzyme systems: biochemistry, molecular biology and applications. Biotechnol. Appl. Biochem. 17, 259–289. Tanaka, H., Nakamura, T., Hayashi, S., Ohta, K., 2005. Purification and properties of an extracellular endo-1,4-beta-xylanase from Penicillium citrinum and characterization of the encoding gene. J. Biosci. Bioeng. 100, 623–630.
doi:10.1016/j.jbiotec.2008.07.1923 V1-P-055 Optimization of medium and process parameters for the production of inulinase from Kluyveromyces marxianus Y1 Wenjie Yuan ∗ , Fengwu Bai Department of Bioscience and Bioengineering, Dalian University of Technology, Dalian 116023, China E-mail address: [email protected] (W. Yuan). Abroad attention has been focused on the utilization of Jerusasalem artichoke as a carbohydrate raw material to produce fuel ethanol. A yeast strain of Kluyveromyces marxianus Y1 was selected for ethanol production using Jerusasalem artichoke by means of simultaneous saccharification and fermentation. The optimum condition for extra cellular inulinase production by K. marxianus Y1 was: inulin 40, yeast extract 4, peptone 4, urea 1, corn stream 10 (g/L). Fermentation medium pH 5.0, cultivation temperature 38 ◦ C, agitation rate of 150 rpm were optimal for enzyme production (54 U/ml) with a fermentation time of 120 h at shake flask level. K+ , Fe2+ , Mg2+ can increased the activity of inulinase, Ca2+ had little effect on inulinase, but Mn2+ inhibit it. The optimum reaction parameter of the inulinase was 55 ◦ C, pH 5.5. This enzyme also showed a good pH and temperature adaptability and stability. References Bernardo, O., Silva-Santisteban, Y’epez, Maugeri Filho, Francisco, 2005. Agitation, aeration and shear stress as key factors in inulinase production by Kluyveromyces marxianus. Enzyme Microb. Technol. 36, 717–724. Gill, Prabhjot Kaur, Sharma, Arun Dev, Harchand, Rajesh Kumari, 2003. Effect of media supplements and culture conditions on inulinase production by an actinomycete strain. Bioresour. Technol. 87, 359–362. Passador-Gurgel, G.C., Furlan, S.A., Meller, J.K., 1996. Application of a microtitre reader system to the screening of inulinase nulinase-producing yeasts. Microbiol. Biotechnol. (45), 158–161. Singh, R.S., Sooch, Balwinder Singh, Puri, Munish, 2007. Optimization of medium and process parameters for the production of inulinase from a newly isolated Kluyveromyces marxianus YS-1. Bioresour. Technol. 98, 2518–2525.