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Expression of glaB was also induced by starch ormalto-oligosacharides in surface cultures(Hata et al. 1998)
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Hydrolysis of X-Ala, His-Ser, and Ser-Tyrdipeptides at a neutral pH optimumThe encoding gene can’t be expressed insubmerged cultureCan be expressed when cultured onsteamed riceN-glycosylated.
(Kitano et al. 2002)
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Realize the mechanism causing the differencebetween 2 culture systems
Culture conditionsAge of the cultureSpots diffused-heterogeneity of proteins due to glycosylation
Wheat bran proteins degraded and vanished at 24 h
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Reason: N-linked oligosaccharides can bedifficult to see by MALDI-TOF MSMake peptides above 3500 kDaDeglycosylation of proteins before 2-D PAGE→protein insolubleA reasonable choice
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Cleave the β-aspartylglucosamine bond ofasparagine-linked oligosaccharides
Trypsin Cleaves Exclusively C-terminal toArginine and Lysine Residues
http://gandalf.umd.edu/BCHM461/Exam2/Ex2_sol.pdf
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Solid-state culture Submerged culture
Cell wall-bound proteinin submerged culture
Incubate 40 hours10-20% SDS polyacrylamide gelCBB stainedIn-gel deglycosylated with PNGase F → trypsin digestionScores >60IPG:ph4-7
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Over 50% of visible proteins areα-amylases and their proteolytic products
37Glucoamylase A, Xylanase G2…….
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α-amylase and β-glucosidase
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Hydrophobic ChromatographyA280nm
α-amylase activity
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Solid state
Submerged state
Both conditions
Be trapped in cell wall
42Northern analysis
Quantitative control
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Low external osmotic pressure in submergedculture→ a static cell wall→ protein can be trappedAspergillus kawachii can secrete α-amylaseand β-glucosidase → the help of extracellularpolysacharrides
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Submerged and Solid-StateCulture produce different