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!!