Aspergillus oryzae. Applied and Environmental Microbiology, May 2006, p , Vol. 72, No. 5

Transcription

1 Aspergillus oryzae Applied and Environmental Microbiology, May 2006, p , Vol. 72, No. 5 Speaker: Chia-wen Chen Advisor: Huang, C.-T 2007/10/30 1 Aspergillus oryzae Kingdom Phylum Subphylum Class Order Family Genus Species Fungi Ascomycota Pezizomycotina Eurotiomycetes Eurotiales Trichocomaceae Aspergillus A. oryzae 2 Rawlings, N.D., Morton, F.R. & Barrett, A.J. (2006) MEROPS: the peptidase database. Nucleic Acids Res 34, D270-D272.

2 Aspergillus oryzae GRAS (generally regarded as safe) A large amounts of enzymes (mainly hydrolytic enzymes ) A wide range (glucoamylse a, alanyl dipeptidyl peptidase ) Commercial production (often extracellular) 3 4

3 Solid-State Culture Nature environment Often used in filamentous fungi Low cost Hard to manipulate physical and chemical properties Purity low Purify?? Food Low price Submerged Culture Artificial environment Can be used in plant and animal cell High cost Easy to manipulate physical and chemical properties Purity high Purify Pharmaceutical products High price 5 Submerged Culture Solid-State Culture pilot solid-state bioreactor 6

4 glab (glucoamylase B) pepa (alanyl dipeptidyl peptidase) Glycolytic pathway & TCA cycle 7 Hydrolytic enzymes A solid state-specific expression marker Expression of glab was also induced by starch or malto-oligosacharides in surface cultures (Hata et al. 1998) 8

5 Hydrolysis of X-Ala, His-Ser, and Ser-Tyr dipeptides at a neutral ph optimum The encoding gene can t be expressed in submerged culture Can be expressed when cultured on steamed rice N-glycosylated. (Kitano et al. 2002) 9 Realize the mechanism causing the difference between 2 culture systems through transcriptional analysis proteomic analysis ESTs (Expressed Sequence Tags) 10

6 ESTs are generated by sequencing cdna Usually 200 to 500 nucleotides long 11 Database Tools BLAST, FASTA, Entrez, GenBank 12

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9 17 A comparative proteome analysis Protein secretion profiles Compare the difference between solid-state and submerged culture 18

10 Incubate 40 hours Yield 2-D gel electrophoresis Hydrophobic Chromatography Classifying proteins SDS-PAGE MALDI-TOF MS Peptide mass fingerprinting Northern analysis 19 Dry mycelium weight N- acetylglucosamine Protein concentration Bradford protein assay 3 independent experiments 20

11 The single unit of chitin Chitin is the major component of fungi s cell wall g 100 ml Solid-State fermentation: Submerged fermentation=4-6.4 fold (protein secreted/gram of dry mycelium) 22

12 Protein +Genome Proteome Nature 405, (15 June 2000) 23 Bakhtiar, R. et al. Mutagenesis : ; doi: /mutage/

13 25 mh + hv mh* mh* + M m - +[M+H]

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15 29 α-amylase α-amylase α-amylase α-amylase α-amylase α-amylase α-amylase α-amylase IPG:ph % SDS polyacrylamide gel 300 μg CBB stained Oryzin spot Only appears in solid-state culture 30

16 α-amylase α-amylase α-amylase α-amylase α-amylase α-amylase α-amylase α-amylase Culture conditions Wheat bran proteins degraded and vanished at 24 h Age of the culture Spots diffused-heterogeneity of proteins due to glycosylation 31 Reason: N-linked oligosaccharides can be difficult to see by MALDI-TOF MS Make peptides above 3500 kda Deglycosylation of proteins before 2-D PAGE protein insoluble A reasonable choice 32

17 Cleave the β-aspartylglucosamine bond of asparagine-linked oligosaccharides Trypsin Cleaves Exclusively C-terminal to Arginine and Lysine Residues

18 Solid-state culture Submerged culture Cell wall-bound protein in submerged culture Incubate 40 hours 10-20% SDS polyacrylamide gel CBB stained In-gel deglycosylated with PNGase F trypsin digestion Scores >60 IPG:ph Over 50% of visible proteins are α-amylases and their proteolytic products 36

19 Glucoamylase A, Xylanase G2. 37 α-amylase and β-glucosidase 38

20 Hydrophobic Chromatography A 280nm α-amylase activity 39 40

21 Solid state Submerged state Both conditions Be trapped in cell wall 41 Quantitative control Northern analysis 42

22 Low external osmotic pressure in submerged culture a static cell wall protein can be trapped Aspergillus kawachii can secrete α-amylase and β-glucosidase the help of extracellular polysacharrides 43 Submerged and Solid-State Culture produce different proteins. Solid-State Culture usually produce more proteins We can divide proteins into 4 types. 44

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