Analysis of the Barley Grain Protease Spectrum
Angela Bell, Peter C. Morris & James H. Bryce
International Centre For Brewing and Distilling,
School of Life Sciences,
Heriot – Watt University, Edinburgh, UK
Goals of the Project
• To characterise the protease activity in malted & germinating barley grains
• To identify individual proteases using
proteomic techniques
• To investigate the significance of specific proteases in the malting process
Overview
• Malting = Germination under controlled conditions and up to a specific point in germination process
• Malting stopped at point of grain modification
• Proteases = essential components of modification process
Proteases
• Very important biologically• Four mechanistic classes:
– Serine– Cysteine– Metallo– Aspartate
• Classes can be differentiated between on the basis of specific inhibitors
Barley Grain Proteases
• Cysteine proteases = the most active protease class, followed by aspartate, serine then the metalloproteases
• Cysteine already well characterised:EP A & EP B purified, analysed & sequenced in 1980’s (Koehler, S., Ho, Tuan – Hua, D (1988), Davy, A., et al (1998))
• Other classes not so well studied
Project So Far. . . .
I. Developed a reproducible & sensitive protease assay
II. Carried out preliminary protease activity studies using crude extracts of 4 - day Oxbridge malt, with & without class specific inhibitors
III. Carried out physiological studies on protease activated starch degrading enzymes
IV. Worked on method optimisation for protease purification
Crude Extract Studies – 4 Day Oxbridge Malt
Proteases During Germination
Divalent Cations
• Mn2+ & Mg2+
• Fe2+
Physiological Studies
• Three major starch degrading enzymes putatively influenced by proteolytic activity:
Limit Dextrinaseα – Amylaseβ – Amylase• Assay enzymes in the presence of class
specific protease inhibitors & by germinating grains in the presence of different endoprotease inhibitors
Limit Dextrinase
• Limit dextrinase = a key starch degrading enzyme present in germinating barley grains
• Present in an inactive form bound to a proteinaceous inhibitor molecule
• Proposed to be activated by cysteine protease mediated breakdown of inhibitory complex and also reducing conditions
Limit Dextrinase – 4 Day Malt
α - Amylase• Is synthesised during germination & is one of
the few enzymes present in the barley grain during germination that can initiate native starch hydrolysis
• Due to the presence of α – amylase inhibitors in the grain, inhibitor degradation is required for full activity
• Inhibitory complexes thought to be broken down by protease activity
α – Amylase, Four Day Malt: Inhibitors at Assay Stage
α – Amylase & Germination
Western Blotting
Serine & Aspartate Proteases are +ve regulators of the amount α – amylase present during grain germination
2 3 4 2 3 4 2 3 4 2 3 4
72 KDa55 KDa36 KDa
Day of Germination
Serine and Aspartate Proteases Inhibited
Only Aspartate Proteases Inhibited
Only Serine Proteases Inhibited
Control
α – Amylase & GA3
β - Amylase
• Is synthesised during grain development & stored in an inactive form bound to a proteinaceous inhibitor
• Putatively activated during grain germination by protease activity & / or reducing conditions
• Been suggested that β – amylase degraded by serine class protease activity
β – Amylase 4 Day Malt• Extraction Stage:
• Assay Stage:
β – Amylase During Germination
0
1
2
3
4
5
6
7
8
Day 1 Day 2 Day 3 Day 4 Day 5
Mea
n U
nits
Bet
a - A
myl
ase
/ g F
lour
Control
5 mM PMSF
Free β - Amylase
Conclusions• limit dextrinase is indeed activated by cysteine class
proteases and reducing conditions, and may require presence of ion2+ / metalloproteases for activity
• α – amylase may be activated by aspartate and serine class proteases
• both aspartate and serine class proteases are important for the amount of α – amylase present in grains during germination in a process involving GA3
• β – amylase is degraded by serine class proteases and requires divalent cations for activity
Current & Future Prospects
• Protease purification: many issues!- Sample stability- “dirty” samples- Too many proteins for positive identification• The future = Investigations into the roles of
aspartate and serine proteases in the gibberellic acid induced expression of alpha - amylase
• Thank you to:
• Maltsters Association of Great Britain and Lindisfarne Trust for funding my project
• All in Peter Morris’s Lab, Heriot – Watt University, Edinburgh
Thank you!!