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Gene cloning
of phenolic acid decarboxylase from Bacillus subtilis
and expression in top-fermenting yeast strain
Xiaohong Cao1*, Yunqian Cui1,2,
Xinghua Liao1, Guangtian Zhou2 and Leo
Thamm2
1College
of Food Engineering and Biotechnology, Tianjin University of
Science and Technology, Tianjin 300457, P. R. China.
2China-Germany
Brewing Technical Service Center, Shandong Institute of
Light Industry, Jinan 250353, P. R. China.
*Corresponding author. E-mail:
cuiyunqian@yahoo.com.cn or
caoxh@tust.edu.cn.
Tel: +86-22-60601428.
Abbreviations: 4VG,
Vinylguaiacol; 4VP, 4-vinylphenol; 4EG,
4-ethylguaiacol; 4EP, 4-ethylphenol; POF,
phenolic off-flavor; PADC, phenolic acid
decarboxylase gene; HPLC, high
performance liquid chromatography; YPD, yeast peptone
dextrose; LB, Luria Bertani; PCR, polymerase
chain reaction; PET, polyethylene terephthalate.
Accepted 28 July, 2010 |
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Phenolic acid decarboxylase (PADC) gene, encoding
phenolic acid decarboxylase, was cloned from Bacillus
subtilis and ligated with a shuttle vector YEp352 to
generate a novel plasmid YPADC. By analysis of sequencing
and the restriction endonuclease digestion, the validity of
construction was proved. Subsequently, the new vector was
successfully transformed into wild-type top-fermenting yeast
strain W303-1A; the mutant yeast strain W303+padc was
obtained, which was tested on the laboratory-scale mashing
and fermentation experiments. At the end of fermentation,
the results showed an obvious increase of 4-vinylguaiacol
content in top-fermented beers brewed with mutant yeasts.
The final 4-vinylguaiacol concentration obtained with
wild-type and mutant yeasts was 1.20 and 1.70mg/l,
respectively. Additionally, the level of esters produced by
the mutant strain was higher than that of the wild-type;
there were therefore a marked clove-like and ester aroma in
top-fermented beers brewed with the former. However, no
evident differences were found in brewing characteristic
between wild-type and mutant strains, especially the ability
of utilizing fermentable sugar and reducing diacetyl. Taken
together, these approaches indicated the possibility of
cloning PADC gene and enhancing the concentration of
4-vinylguaiacol in top-fermented beers.
Key words:
Clone, phenolic acid decarboxylase, top-fermenting yeast,
4-vinylguaiacol. |
