Aspergillus Oryzae

Applied and Environmental Microbiology, May 2006, p. 3448-3457, Vol. 72, No. 5

Speaker: Chia-wen Chen Advisor: Huang, C.-T

2007/10/30 1

z Aspergillus oryzae Kingdom Fungi Phylum Ascomycota Subphylum Pezizomycotina Class Eurotiomycetes Order Eurotiales Family Trichocomaceae Genus Aspergillus Species A. oryzae

Rawlings, N.D., Morton, F.R. & Barrett, A.J. (2006) MEROPS: the peptidase database. Nucleic Acids Res 34, D270-D272. z 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)

4 Solid-State Culture Submerged Culture Nature environment Artificial environment

Often used in filamentous fungi Can be used in plant and animal cell Low cost High cost Hard to manipulate physical and Easy to manipulate physical and chemical properties chemical properties Purity low Purity high Purify ?? Purify ☆ Food Pharmaceutical products Low price High price

Submerged Culture Solid-State Culture

pilot solid-state bioreactor

http://www.vesuite.org/index.php 6 http://www.unitus.it/dipartimenti/dabac/progetti/ssbioreactors/solidstatebioreactor.htm z glaB (glucoamylase B) z pepA (alanyl dipeptidyl peptidase) z Glycolytic pathway & TCA cycle

z Hydrolytic enzymes z A solid state-specific expression marker z Expression of glaB was also induced by starch or malto-oligosacharides in surface cultures (Hata et al. 1998)

8 z Hydrolysis of X-Ala, His-Ser, and Ser-Tyr dipeptides at a neutral pH optimum z The encoding gene can’t be expressed in submerged culture z Can be expressed when cultured on steamed rice z 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 z ESTs are generated by sequencing cDNA 11 z Usually 200 to 500 nucleotides long http://www.ncbi.nlm.nih.gov/About/primer/est.html

z Database http://www.ncbi.nlm.nih.gov/projects/dbEST z Tools BLAST, FASTA, Entrez, GenBank

12 13

14 15 http://www.nrib.go.jp/ken/EST/db/index.html

16 http://www.nrib.go.jp/ken/EST/db/blast.html 17 http://www.bio.nite.go.jp/dogan/Top

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

18 Incubate 40 hours

Yield

MALDI-TOF MS 2-D gel Hydrophobic SDS-PAGE electrophoresis Chromatography Peptide mass fingerprinting Classifying proteins

Northern analysis 19

z Dry mycelium weight → N- acetylglucosamine z Protein concentration → Bradford protein assay z 3 independent experiments

20 z The single unit of chitin z Chitin is the major component of fungi’s cell wall

http://www.ocean.udel.edu/horseshoecrab/Research/chitin.html 21

10g 100 mL

Solid-State fermentation: Submerged fermentation=4-6.4 fold (protein secreted/gram of dry mycelium) 22 Protein +Genome Æ Proteome

23 Nature 405, 837-846(15 June 2000)

24 Bakhtiar, R. et al. Mutagenesis 2000 15:415-430; doi:10.1093/mutage/15.5.415 25 http://www.psrc.usm.edu/mauritz/maldi.html

mH + hv → mH* - + mH* + M →m +［M+H］ 26 http://www.psrc.usm.edu/mauritz/maldi.html http://qbab.aber.ac.uk/roy/mss/tofspec.htm 27

28 http://www.expasy.org/ 29 http://www.expasy.org/tools/aldente/

α-amylase α-amylase α-amylase α-amylase

z IPG:ph4-7 Oryzin spot z 10-20% SDS polyacrylamide gel Only appears in solid-state culture z 300 μg 30 z CBB stained α-amylase α-amylase α-amylase α-amylase

α-amylase α-amylase α-amylase α-amylase

z Culture conditions Wheat bran proteins degraded and vanished at 24 h z Age of the culture

z Spots diffused-heterogeneity of proteins due to glycosylation 31

z Reason: N-linked oligosaccharides can be difficult to see by MALDI-TOF MS z Make peptides above 3500 kDa z Deglycosylation of proteins before 2-D PAGE →protein insoluble z A reasonable choice

32 z Cleave the β-aspartylglucosamine bond of asparagine-linked oligosaccharides

http://www.rcsb.org/pdb/explore.do?structureId=1PNG 33 http://www.glycoforum.gr.jp/science/word/glycoprotein/GPA00E.html

z Trypsin Cleaves Exclusively C-terminal to Arginine and Lysine Residues

http://gandalf.umd.edu/BCHM461/Exam2/Ex2_sol.pdf 34 http://www.rcsb.org/pdb/explore.do?structureId=2PTN Solid-state culture Submerged culture

Cell wall-bound protein in submerged culture

z Incubate 40 hours z 10-20% SDS polyacrylamide gel z CBB stained z In-gel deglycosylated with PNGase F → trypsin digestion z Scores ＞60 35 z IPG:ph4-7

Over 50% of visible proteins are α-amylases and their proteolytic products 36 Glucoamylase A, Xylanase G2……. 37

α-amylase and β-glucosidase

38 z Hydrophobic Chromatography z A280nm z α-amylase activity

40 Solid state

Submerged state

Both conditions

Be trapped in cell wall

Quantitative control

Northern analysis 42 z Low external osmotic pressure in submerged culture → a static cell wall → protein can be trapped z Aspergillus kawachii can secrete α-amylase and β-glucosidase → the help of extracellular polysacharrides

Submerged and Solid-State Culture produce different proteins.

Solid-State Culture usually produce more proteins

We can divide proteins into 4 types.

44 45