Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Preparation and comparative characterization of immobilized Aspergillus oryzae expressing Fusarium heterosporum lipase for enzymatic biodiesel production

  • 480 Accesses

  • 39 Citations


In this paper, we provide the first report of utilizing recombinant fungal whole cells in enzymatic biodiesel production. Aspergillus oryzae, transformed with a heterologous lipase-encoding gene from Fusarium heterosporum, produced fully processed and active forms of recombinant F. heterosporum lipase (FHL). Cell immobilization within porous biomass support particles enabled the convenient usage of FHL-producing A. oryzae as a whole-cell biocatalyst for lipase-catalyzed methanolysis. The addition of 5% water to the reaction mixture was effective in both preventing the lipase inactivation by methanol and facilitating the acyl migration in partial glycerides, resulting in the final methyl ester content of 94% even in the tenth batch cycle. A comparative study showed that FHL-producing A. oryzae attained a higher final methyl ester content and higher lipase stability than Rhizopus oryzae, the previously developed whole-cell biocatalyst. Although both FHL and R. oryzae lipase exhibit 1,3-regiospecificity towards triglyceride, R. oryzae accumulated a much higher amount of sn−2 isomers of partial glycerides, whereas FHL-producing A. oryzae maintained a low level of the sn−2 isomers. This is probably because FHL efficiently facilitates the acyl migration from the sn−2 to the sn−1(3) position in partial glycerides. These findings indicate that the newly developed FHL-producing A. oryzae is an effective whole-cell biocatalyst for enzymatic biodiesel production.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. Abe K, Gomi K, Hasegawa F, Machida M (2006) Impact of Aspergillus oryzae genomics on industrial production of metabolites. Mycopathologia 162:143–153

  2. Atkinson B, Black GM, Lewis PJS, Pinches A (1979) Biological particles of given size, shape, and density for use in biological reactors. Biotechnol Bioeng 21:193–200

  3. Ban K, Kaieda M, Matsumoto T, Kondo A, Fukuda H (2001) Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized within biomass support particles. Biochem Eng J 8:39–43

  4. Ban K, Hama S, Nishizuka K, Kaieda M, Matsumoto T, Kondo A, Noda H, Fukuda H (2002) Repeated use of whole-cell biocatalysts immobilized within biomass support particles for biodiesel fuel production. J Mol Catal B: Enzymatic 17:157–165

  5. Christensen T, Woeldike H, Boel E, Mortensen SB, Hjortshoej K, Thim L, Hansen MT (1988) High-level expression of recombinant genes in Aspergillus oryzae. Biotechnology 6:1419–1422

  6. Fukuda H, Kondo A, Noda H (2001) Biodiesel fuel production by transesterification of oils. J Biosci Bioeng 92:405–416

  7. Gomi K, Iimura Y, Hara S (1987) Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans argB gene. Agric Biol Chem 51:2549–2555

  8. Hama S, Yamaji H, Kaieda M, Oda M, Kondo A, Fukuda H (2004) Effect of fatty acid membrane composition on whole-cell biocatalysts for biodiesel-fuel production. Biochem Eng J 21:155–160

  9. Hama S, Tamalampudi S, Fukumizu T, Miura K, Yamaji H, Kondo A, Fukuda H (2006) Lipase localization in Rhizopus oryzae cells immobilized within biomass support particles for use as whole-cell biocatalysts in biodiesel-fuel production. J Biosci Bioeng 101:328–333

  10. Hama S, Yamaji H, Fukumizu T, Numata T, Tamalampudi S, Kondo A, Noda H, Fukuda H (2007) Biodiesel-fuel production in a packed-bed reactor using lipase-producing Rhizopus oryzae cells immobilized within biomass support particles. Biochem Eng J 34:273–278

  11. Hama S, Tamalampudi S, Shindo N, Numata T, Yamaji H, Fukuda H, Kondo A (2008) Role of N-terminal 28-amino-acid region of Rhizopus oryzae lipase in directing proteins into secretory pathway of Aspergillus oryzae. Appl Microbiol Biotechnol 79:1009–1018

  12. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580

  13. Iso M, Chen B, Eguchi M, Kudo T, Shurestha S (2001) Production of biodiesel fuel from triglycerides and alcohol using immobilized lipase. J Mol Catal B: Enzymatic 16:53–58

  14. Kaieda M, Samukawa T, Matsumoto T, Ban K, Kondo A, Shimada Y, Noda H, Nomoto F, Ohtsuka K, Izumoto E, Fukuda H (1999) Biodiesel fuel production from plant oil catalyzed by Rhizopus oryzae lipase in a water-containing system without an organic solvent. J Biosci Bioeng 88:627–631

  15. Kaieda M, Samukawa T, Kondo A, Fukuda H (2001) Effect of methanol and water contents on production of biodiesel fuel from plant oil catalyzed by various lipases in a solvent-free system. J Biosci Bioeng 91:12–15

  16. Kaieda M, Nagayoshi M, Hama S, Kondo A, Fukuda H (2004) Enantioselective transesterification using immobilized Aspergillus oryzae overexpressing lipase. Appl Microbiol Biotechnol 65:301–305

  17. Kamini NR, Iefuji H (2001) Lipase catalyzed methanolysis of vegetable oils in aqueous medium by Cryptococcus spp. S-2. Process Biochem 37:405–410

  18. Koda A, Minetoki T, Ozeki K, Hirotsune M (2004) Translation efficiency mediated by the 5’ untranslated region greatly affects protein production in Aspergillus oryzae. Appl Microbiol Biotechnol 66:291–296

  19. Li W, Du W, Liu D (2007) Rhizopus oryzae IFO 4697 whole cell catalyzed methanolysis of crude and acidified rapeseed oils for biodiesel production in tert-butanol system. Process Biochem 42:1481–1485

  20. Minetoki T, Kumagai C, Gomi K, Kitamoto K, Takahashi K (1998) Improvement of promoter activity by the introduction of multiple copies of the conserved region III sequence, involved in the efficient expression of Aspergillus oryzae amylase-encoding gene. Appl Microbiol Biotechnol 50:459–467

  21. Nagao T, Shimada Y, Sugihara A, Tominaga Y (1996) Expression of lipase cDNA from Fusarium heterosporum by Saccharomyces cerevisiae: high-level production and purification. J Ferment Bioeng 81:488–492

  22. Nagao T, Shimada Y, Sugihara A, Murata A, Komemushi A, Tominaga Y (2001) Use of thermostable Fusarium heterosporum lipase for production of structured lipid containing oleic and palmitic acids in organic solvent-free system. J Am Oil Chem Soc 78:167–172

  23. Oda M, Kaieda M, Hama S, Yamaji H, Kondo A, Izumoto E, Fukuda H (2005) Facilitatory effect of immobilized lipase-producing Rhizopus oryzae cells on acyl migration in biodiesel-fuel production. Biochem Eng J 23:45–51

  24. Ozeki K, Kanda A, Hamachi M, Nunokawa Y (1996) Construction of a promoter probe vector autonomously maintained in Aspergillus and characterization of promoter regions derived from A. niger and A. oryzae genomes. Biosci Biotechnol Biochem 60:383–389

  25. Satou C, Kobayashi M, Takeda A, Ito T, Shimada K, Yamato S (2004) Simple determination of lipase-catalyzed cleavage products of triacylglycerol by gas chromatography. Bunseki Kagaku 53:1025–1029

  26. Shimada Y, Koga C, Sugihara A, Nagao T, Takada N, Tsunasawa S, Tominaga Y (1993) Purification and characterization of a novel solvent-tolerant lipase from Fusarium heterosporum. J Ferment Bioeng 75:349–352

  27. Shimada Y, Watanabe Y, Samukawa T, Sugihara A, Noda H, Fukuda H, Tominaga Y (1999) Conversion of vegetable oil to biodiesel using immobilized Candida antarctica lipase. J Am Oil Chem Soc 76:789–793

  28. Tamalampudi S, Talukder MMR, Hama S, Tanino T, Suzuki Y, Kondo A, Fukuda H (2007) Development of recombinant Aspergillus oryzae whole-cell biocatalyst expressing lipase-encoding gene from Candida antarctica. Appl Microbiol Biotechnol 75:387–395

  29. Unkles SE, Campbell EI, Ruiter-Jacobs YMJT, Broekhuijsen M, Macro JA, Carrez D, Contreas R, Hondel CAMJJ, Kinghorn JR (1989) The development of a homologous transformation system for Aspergillus oryzae based on the nitrate assimilation pathway: a convenient and general selection system for filamentous fungal transformation. Mol Gen Genet 218:99–104

Download references


This work was partly supported by the Japanese Ministry of the Environment for Technical Development of Measures to Prevent Global Warming (2007). Generous gifts of the fungal expression vector pNAN8142 and Aspergillus oryzae niaD300 from Ozeki (Hyogo, Japan) are gratefully acknowledged. We also thank Dr. Yuji Shimada (Osaka Municipal Technical Research Institute, Osaka, Japan) for providing pYGF2.

Author information

Correspondence to Akihiko Kondo.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hama, S., Tamalampudi, S., Suzuki, Y. et al. Preparation and comparative characterization of immobilized Aspergillus oryzae expressing Fusarium heterosporum lipase for enzymatic biodiesel production. Appl Microbiol Biotechnol 81, 637–645 (2008).

Download citation


  • Whole-cell biocatalyst
  • Biomass support particles
  • Filamentous fungi
  • Fusarium heterosporum lipase
  • Methanolysis