Summary
While Aspergillus strains are also being considered as potential hosts for production of extracellular heterologous proteins, the proteases produced by the host are highly problematic in that they typically modify and degrade the recombinant proteins. Culture-based approaches for minimization of protease activity in culture supernatants of Aspergillus niger NRRL-3 included reduction or elimination of peptide nitrogen in the medium, preferential use of a defined salts medium rather than a non-peptide nitrogen medium containing yeast-nitrogen base, supplementation of the medium with carboxymethylcellulose and cultivation at pH 6.5 rather than 7.5. In general, increased proteolytic activity was observed after maximum biomass was observed and biomass was declining suggesting the majority of protease activity was released by cell lysis. Carboxymethylcellulose shifted mycelial morphology from pelleted to filamentous. Mycelium lysis in the centre of pellets, with resultant release of intracellular proteases, would explain why filamentous cultures exhibited much lower proteolytic activity than pelleted cultures.
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References
Berka R.M., Hayenga K.J., Lawlis V.B. & Ward M., 2003. Aspartic proteinase deficient filamentous fungi. US Patent 6,509,171
M.P. Broekhuijsen I.E. Mattern R. Contreras J.R. Kinghorn C.A.M.J.J. vanden-Hondel (1993) ArticleTitleSecretion of heterologous proteins by Aspergillus niger: production of active human interleukin-6 in a protease deficient mutant by KEX2-like processing of a glucoamylase-hIL6 fusion protein Journal of Biotechnology 31 135–145 Occurrence Handle10.1016/0168-1656(93)90156-H Occurrence Handle1:CAS:528:DyaK2cXivFChtw%3D%3D
J.L. Brookman D.W. Denning (2000) ArticleTitleMolecular genetics in Aspergillus fumigatus Current Opinion in Microbiology 3 468–474 Occurrence Handle10.1016/S1369-5274(00)00124-7 Occurrence Handle1:CAS:528:DC%2BD3cXotVOitL4%3D
G.S. Byrne O.P. Ward (1989) ArticleTitleGrowth of Rhizopus arrhizus in fermentation media Journal of Industrial Microbiology 4 155–161 Occurrence Handle10.1007/BF01569800
Christensen, T. & Lehmbeck, J. 2000. Fungus wherein the areA, pepC and/or pepE genes have been inactivated. United States Patent 6,013,452
A. Conesa P.J. Punt N. Luijk C.A.M.J.J. Hondel Particlevan den (2001) ArticleTitleThe secretion pathway in filamentous fungi: a biotechnological view Fungal Genetics and Biology 33 155–171 Occurrence Handle10.1006/fgbi.2001.1276 Occurrence Handle1:CAS:528:DC%2BD3MXlslyjsL8%3D
D.O. Donnell L. Wang J. Xu D. Ridgway T. Gu M. Moo-Young (2001) ArticleTitleEnhanced heterologous protein production in Aspergillus niger through pH control of extracellular protease activity Biochemical Engineering Journal 8 187–193
M. Gyamerah G. Merichetti O. Adedayo J.M. Scharer M. Moo-Young (2002) ArticleTitleBioprocessing strategies for improving hen egg-white lysozyme (HEWL) production by recombinant Aspergillus niger (HEWLWT-13–16) Applied Microbiology and Biotechnology 60 403–407 Occurrence Handle1:CAS:528:DC%2BD38Xpt1Cjuro%3D
K. Iwashita (2002) ArticleTitleRecent studies of protein secretion by filamentous fungi: review Journal of Biosciences and Bioengineering 94 530–535 Occurrence Handle1:CAS:528:DC%2BD3sXhsVOnsLw%3D
D.A. MacKenzie J.A Spencer M.F. Gal-Coeffet D.B. Archer (1996) ArticleTitleEfficient production from Aspergillus niger of a heterologous protein and an individual protein domain, heavy isotope labeled for structure–function analysis Journal of Biotechnology 46 85–93 Occurrence Handle10.1016/0168-1656(95)00179-4 Occurrence Handle1:CAS:528:DyaK28XivFOht7c%3D
G.L. Miller (1959) ArticleTitleUse of dinitrosalicylic acid reagent for determination of reducing sugar Analytical Chemistry 31 426–428 Occurrence Handle1:CAS:528:DyaG1MXmtFKiuw%3D%3D
M. Morrin O.P. Ward (1989) ArticleTitleBiotransformation of progesterone into 11α-hydroxyprogesterone by different morphological forms of Rhizopus arrhizus Biotechnology Letters 11 319–324 Occurrence Handle10.1007/BF01024511 Occurrence Handle1:CAS:528:DyaL1MXkt1aisL4%3D
M. Morrin O.P. Ward (1990) ArticleTitleRelationdship between fungal growth, morphology and fumaric acid production by Rhizopus arrhizus Mycological Research 94 505–510 Occurrence Handle1:CAS:528:DyaK3MXktV2msQ%3D%3D Occurrence Handle10.1016/S0953-7562(10)80011-5
M. Papagianni M.M. Young (2002) ArticleTitleProtease secretion in glucoamylase producer Aspergillus niger cultures: fungal morphology and inoculum effects Process Biochemistry 37 1271–1278 Occurrence Handle10.1016/S0032-9592(02)00002-X Occurrence Handle1:CAS:528:DC%2BD38XhvVWhu74%3D
R. Radzio U. Kueck (1997) ArticleTitleSynthesis of biotechnologically relevant heterologous proteins in filamentous fungi Process Biochemistry 32 529–537 Occurrence Handle10.1016/S0032-9592(97)00004-6 Occurrence Handle1:CAS:528:DyaK2sXksVGlsr0%3D
I.N. Roberts D.J. Jeenes D.A. Mackenzie A.P. Wilkinson I.G. Summer D.B. Archer (1992) ArticleTitleHeterologous gene expression in A. niger: a glucoamylase–porcine pancreatic phospholipase A2 fusion protein is secreted and processed to yield mature enzyme Gene 122 155–161 Occurrence Handle10.1016/0378-1119(92)90043-O Occurrence Handle1:CAS:528:DyaK3sXnsVCksw%3D%3D
E. Schuster N. Dunn-Coleman J.C. Frisvad P.W.M. Dijck Particlevan (2002) ArticleTitleOn the safety of Aspergillus niger – a review Applied Microbiology and Biotechnology 59 426–435 Occurrence Handle1:CAS:528:DC%2BD38Xmslygsrc%3D
J.P.T.W. Hombergh Particlevan den P.J.I. Vondervoort Particlevan de L. Fraissinet J. Visser (1997) ArticleTitleAspergillus as a host for heterologous protein production: the problem of proteases Trends in Biotechnology 15 256–263
L. Wang D. Ridgway T. Gu M.M. Young (2003) ArticleTitleEffects of process parameters on heterologous protein production in Aspergillus niger fermentation Journal of Chemical Technology and Biotechnology 78 1259–1266 Occurrence Handle1:CAS:528:DC%2BD3sXpsV2hsrw%3D
Ward, O.P., Qin, W.M., Dhanjoon, J., Ye, J. & Singh, A. 2005 Physiology and biotechnology of Aspergillus. Advances in Applied Microbiology 58.
O.P. Ward (1989) Fermentation Biotechnology Open University Press Milton Keynes, UK
O.P. Ward (1991) Bioprocessing Open University Press Milton Keynes, UK
J. Xu L. Wang D. Ridgway T. Gu M. Moo-Young (2000) ArticleTitleIncreased heterologous protein production in Aspergillus niger fermentation through extracellular proteases inhibition by pelleted growth Biotechnology Progress 16 222–227 Occurrence Handle10.1021/bp000006s Occurrence Handle1:CAS:528:DC%2BD3cXhsVaktro%3D
V.B. Yadwad S. Wilson O.P. Ward (1996) ArticleTitleEffect of culture conditions and induction strategies on production of human interleukin-6 by a recombinant Aspergillus nidulans strain Mycological Research 100 356–360 Occurrence Handle1:CAS:528:DyaK28XislKhs7Y%3D Occurrence Handle10.1016/S0953-7562(96)80169-9
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Ahamed, A., Singh, A. & Ward, O.P. Culture-Based Strategies for Reduction of Protease Activity in Filtrates from Aspergillus niger NRRL-3. World J Microbiol Biotechnol 21, 1577–1583 (2005). https://doi.org/10.1007/s11274-005-8121-5
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DOI: https://doi.org/10.1007/s11274-005-8121-5