Aspartic Acid Protease from Botrytis cinerea Removes Haze-Forming Proteins during White Winemaking

The Problem: How can we prevent heat-induced hazes from forming during transportation and storage of white wine?

Existing methods
Bentonite removal of proteins
Loss of wine during process, change in taste Highly labor intensive

Enzymatic Hydrolysis digestion of proteins

Temperature required for digestion conflicting with normal wine making temperatures No viable proteases available yet

Our new process

Proteases secreted by Botrytis cinerea Primarily aspartic acid proteases,
Some others

Some evidence that these proteases negatively affect sparkling wine foam

Protein of interest BcAP8

Main liquid secretion of Botrytis cinerea fungus Several known mutants
None showed significant differences in function or main structure

A pair of antiparallel -sheets and an -helix

BcAP8 functionality
When combined with juice from grapes, successfully removed chitinases No effect on Thaumatin-like proteins Normal temperature and conditions

BcAP8 functionality
Little effect on Pathogenesis-related proteins Still caused a decrease in haze formed over time Potential for heat stabilization or reduction of need for bentonite

Methods & Experiments

Production of BcAP8 protein

Known sequence used to produce cDNA Expressed and optimized to produce in cells of Pichia pastoris Protein secreted in synthetic material, PNB1 Characterized by SDS-PAGE
Produced single band No further purification needed

Vector expression of protein

cDNA detailed sequence & corresponding amino acids

Samples for BcAP8 experiment with grape juice

200uL BcAP8 added to samples of Australian Semillon and Sauvignon Blanc juices 200uL pepsin added to identical samples as negative control Pepstatin A (in DMSO) was added to replicate preparations of samples as an inhibitor
Samples for preparation

Australian Semillon
Control Control w/ pepA BcAP8 BcAP8 w/ pepA Pepsin Pepsin w/ pepA Control Control w/ pepA

Sauvignon Blanc
BcAP8 BcAP8 w/ pepA Pepsin Pepsin w/ pepA

Experimental procedure for BcAP8 activity in grape juice

Half (100uL) of each sample stored at 22C for 21 days Accelerated experiment remaining sample was incubated at 40C for 18h Proteins extracted with MeOH/CHCl3 and characterized by SDS-PAGE
Stained with Brilliant Blue R-250

Results of grape juice experiments

BcAP8 top arrow Putative chitinase middle arrow Putative thaumatin-like protein bottom arrow

Results of grape juice experiments, contd.

Significant decrease in concentration of chitinase Little effect on TLP in all cases

Preparation of synthetic sample for BcAP8 activity in fermentation experiment

Zymaflore VL3 yeast in yeast peptone dextrose broth, incubated at 30C for 24h Culture added to synthetic juice with ammonium sulfate solution Incubated at 28C in orbital shaker overnight

Preparation of wine samples for BcAP8 activity in fermentation experiments

Sauvignon blanc and Semillion tested BcAP8 added to a sample of each of the wines on ice, incubated at 17C for 17h Yeast starter culture added, Diammonium phosphate added Monitored for enzymatic sugars over time

RP-HPLC quantitation of chitinase and thaumatin-like proteins in wines

Significant decrease in chitinase peaks Small decrease in TLP peaks Matches results from juice only tests

SDS-PAGE analysis of chitinase and thaumatin-like proteins in wines

Show same decrease as in RP-HPLC Absence of 35kDa peak in non-synthetic
Possible side effect of BcAP8 Present in synthetic

13kDa peak removed in BcAP8 samples, lipid transfer protein

Long term analysis by RP-HPLC for TLP and chitinase concentration

Samples stored at 4C for 1 year All samples showed difference between chitinase and TLP in BcAP8 vs. control Trend continued after one year

Heat stability and relation to hazeforming proteins in wine

One year samples tested for haze presence Small volumes were treated with Pepstatin to stop activity of BcAP8 All samples heated to 55C for 18h Measured on a 96-well plate at 540nm

Results of haze concentration tests

Little difference between pepstatin and no pepstatin
Little to no BcAP8 activity after one year

Massive difference in absorbance between BcAP8 and empty vector samples

Haze versus TLPs and chitinases

TLP concentration and haze concentration very disproportionate Chitinase concentrations show greater correlation Chitinase likely candidate for formation of haze Absence of 35kDa protein in BcAP8 wines could also have effect

Synthetic wine versus authentic wines

Much higher haze concentration in synthetic versus authentic More chitinase in control samples than others Authentic wines may have some other protein that contributes to stability

Benefits of BcAP8 over bentonite

Decreases concentration of haze forming proteins significantly Doesnt have to be removed from wine in any way Effective at normal winemaking temperatures and doesnt change taste of wine

Where do we go from here?

Future goals and experiments

Optimize production of BcAP8 to be able to produce on a larger scale
Study protein more closely

Optimize ratio of BcAP8 to wine to find most efficient Increase to a larger scale experiment
More wine, more BcAP8 Test on larger scale and compare results

References
Q334I5. The Protein Model Portal: Structural Biology Knowledgebase. http://www.proteinmodelportal.org/?pid=modelDetail&provider=MODBASE&tem plate=2y7lA&pmpuid=1000013120035&range_from=1&range_to=386&ref_ac=Q3 34I5&zid=async Van Sluyter, Steven C.; Warnock, Nicholas I.; Schmidt, Simon; Anderson, Peter; van Kan, Jan A. L.; Bacic, Antony; Waters, Elizabeth J. Aspartic Acid Protease from Botrytis cinerea Removes Haze-Forming Proteins during White Winemaking. Journal of Agricultural and Food Chemistry Article ASAP