ENGLISH ABSTRACT: Hen egg white lysozyme (HEWL; muramidase; EC 3:2:1:17) is an enzyme
present in high concentrations in chicken (Gallus gallus) egg whites. It hydrolyses
the link between N-acetylmuramic acid and N-acetylglucosamine in Gram positive
bacterial cell walls, resulting in cell death. It is thus active against lactic acid
bacteria (LAB), which may be present in grape juices and musts. These bacteria
are responsible for malolactic fermentation of wines although many species, particularly
of the genera Lactobacillus and Pediococcus, are considered spoilage organisms.
The growth of LAB is therefore closely monitored and controlled during
winemaking. The most common means of control is growth inhibition by chemical
treatment (usually with SO2). Lysozyme is a commonly used wine processing aid,
complementing the antimicrobial activity of SO2 . It allows for lower doses of SO2
to be used, thus improving the wholesomeness of wine. The OIV (Organisation
Internationale de la Vigne et du Vin) approved its use in quantities up to 500 mg per
liter of wine in 1997.
This study evaluated the effect of different secretion signals on the secretion of
lysozyme by the haploid auxotroph Saccharomyces cerevisiae strain FY23. Secretion
by an industrial strain (VIN13) transformed with a single copy of the HEWL gene with the MF-a secretion signal under the control of the PGK1 (phosphoglycerate
kinase 1) prompter and terminator was also evaluated. In the case of FY23 four
secretion signals were used, namely the native lysozyme signal and the S. cerevisiae
mating factor-a signal as well as mutants of these signals. These mutants incorporated
two additional arginines at the N-terminus of the signals immediately downstream
of the terminal methionine. The effect of these mutations was to increase the
positive charge of the secretion signal N-terminals. The secretion signal-lysozyme
fusions were placed under the regulation of the S. cerevisae PGK1 gene’s promoter
and terminator. The resulting expression cassettes were cloned into integrating vectors
YIpLac211 and pDMPOF1b and episomal vector pHVX2. These were used to
transform FY23 and VIN13.
FY23 as well as VIN13 transformants were evaluated in an artificial medium
designed to reflect the nutrient content of grape juice, with particular attention being
paid to assiminable nitrogen. Three hexose concentrations were tested in order to
determine the effect thereof on lysozyme secretion titer.
Lysozyme secreted under all tested growth conditions was found to be too low
for detection by either enzymatic assay or HPLC-FLD. For this reason secreted
lysozyme was isolated and concentrated 10x by means of cation-exchange. Subsequently,
lysozyme concentrations in the concentrates was determined by means of
the aforementioned techniques. SDS-PAGE analysis of lysozyme concentrates was
also performed.
No significant differences were found between native or MF-a secretion signals
and their mutated counterparts in terms of secretion titer or proteolytic maturation.
Lysozyme secreted with the MF-a signal was found to be misprocessed in all cases,
with both an authentically processed and a larger form, in which the secretion signal
was not completely removed, being present. Lysozyme secreted with the native
signal appeared to be correctly processed in all cases. Secretion titer from high
copy number episomal FY23 tranformants was similar to that of integrants containing
a single copy of the gene. Sugar concentration affected lysozyme production,
with higher quantities of the enzyme being secreted when higher initial sugar concentrations
were used. Lysozyme titers were extremely low (< 0:25 mg/L) with
all expression cassettes under all the tested conditions with both FY23 and VIN13.
In the case of the VIN13’s a lower final biomass was found for the secretor strain
tested in comparrison to the VIN13 wild-type.
AFRIKAANSE OPSOMMING: Hoendereierwitlisosiem (HEWL; muramidase, EG 3:2:1:17) is ´n ensiem teenwoordig
in hoë konsentrasies in hoender (Gallus gallus) eierwitte. Dit hidroliseer
die binding tussen N-asetielmuramiensuur en N-asetielglukosamien in Gram positiewe
bakteriese selwande, wat tot seldood lei. Dit is dus aktief teen melksuurbakterieë
(MSB), wat in druiwesap en mos teenwoordig kan wees. Hierdie bakterieë
is verantwoordelik vir appelmelksuurgisting van wyne, hoewel baie spesies, veral
van die genera Lactobacillus en Pediococcus, ook as bederforganismes beskou
word. Die groei van MSB word dus noukeurig tydens wynbereiding gemoniteer en
beheer. Die algemeenste wyse van beheer is groei-inhibisie deur chemiese behandeling
(gewoonlik SO2). Lisosiem is ´n algemeen gebruikte wyntoevoegingsmiddel
en vul die antimikrobiese aktiwiteit van SO2 aan. Met lisosiem kan ´n laer dosis
van SO2 gebruik word, wat lei tot ´n verbetering van die heilsaamheid van die wyn.
Die OIV (Organisasie Internationale de la Vigne et du Vin) het die gebruik daarvan
goedgekeur tot en met 500 mg per liter wyn vanaf 1997. Hierdie studie het
die effek van verskillende sekresieseine op die uitskeiding van lisosiem deur die haploïede ouksotrofe Saccharomyces cerevisiae stam, FY23, geëvalueer. Uitskeiding
deur ´n industriële stam (VIN13), wat getransformeer is met ´n enkelkopie van die
HEWL-gene met die MF-a sekresiesein onder die beheer van die PGK1 (Fosfogliseraat
kinase 1) promotor en termineerder, is ook geëvalueer. In die geval van FY23
is vier sekresieseine gebruik, naamlik die inheemse lisosiemsein, S. cerevisiae MF-
a sein, asook mutante van hierdie seine. Hierdie mutante het twee bykomende
arginienresidu’s by die N-terminus van die seine direk stroom-af van die terminale
metionien. Die effek van hierdie mutasies was om die positiewe lading van die
uitskeidingsein N-terminale te verhoog. Die gevolglike uitdrukkingskassette is in
die integrasievektor YIpLac211 en pDMPOF1b, en die episomale vektor pHVX2,
gekloneer. Dit is gebruik om VIN13 en FY23 te transformeer. FY23, sowel as
VIN13-transformante, is geëvalueer in ´n kunsmatige medium wat ontwerp is om
die voedingsinhoud van druiwesap te weerspieël, met besondere aandag aan assimileerbare
stikstof. Drie heksose konsentrasies is getoets om te bepaal wat die
uitwerking daarvan op die lisosiemsekresietiter is. Onder alle groeitoestande was
die isosiem wat uitgeskei is, te laag om deur ensimatiese toetse of HPLC-FLD
bepaal te word. Om hierdie rede is uitgeskeide lisosiem geïsoleer en 10x gekonsentreer
deur middel van katioon-uitruiling. Daarna is lisosiemkonsentrasies bepaal
deur middel van bogenoemde tegnieke. SDS-PAGE-ontleding van lisosiemkonsentraat
is ook uitgevoer. In terme van sekresietiter of proteolitiese maturasie, is geen
beduidende verskille gevind tussen inheemse of MF-a sekresieseine en hul gemuteerde
eweknieë nie. Lisosiem wat deur die MF-a sein uitgeskei is, is in alle gevalle
foutief geprosesseer, met ´n teenwoordigheid van beide die regte produk en ´n groter
produk, waarin die uitskeidingsein nie heeltemal verwyder word nie. Lisosiem wat
met die inheemse sein uitgeskei is, blyk in alle gevalle korrek verwerk te wees.
Sekresietiter van ´n aantal hoë-kopie episomale FY23-transformante was soortgelyk
aan dié van integrante met ´n enkelkopie van die geen. Suikerkonsentrasie beïnvloed
lisosiemproduksie, met ´n hoër hoeveelheid van die ensiem wat uitgeskei word wanneer
die aanvanklike suiker in hoër konsentrasies gebruik is. Lisosiemtiters was
baie laag (< 0:25 mg/L), met al die kassette onder al die getoetste toestande vir
beide FY23 en VIN13. In die geval van die VIN13’s, is ´n laer finale biomassa vir
die uitskeidingstam in vergelyking met die VIN13 wilde-tipe gevind.
