Naslov
Isolation of fumarase from baker's yeast

Autori
Džaja, Ana ; Nemeth, Gergely ; Findrik, Zvjezdana ; Vasić-Rački, Đurđa

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Applied Biocatalysis, 6th meeting of students and university professors, Book of Abstracts / Vasić-Rački, Đurđa ; Vrsalović Presečki, Ana - Zagreb : Pasanec d.o.o., 2010, 12-12

ISBN
978-553-6470-50-1

Skup
Applied Biocatalysis, 6th meeting of students and university professors

Mjesto i datum
Zagreb, Hrvatska, 09.06.2010

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
fumarase; isolation; purification

Sažetak
Fumarase is a lyase that catalyzes the hydration of fumaric acid to L-malic acid. The enzyme can be found in various sources [1, 2, 3], like for instance baker’s yeast [3]. In this work fumarase was isolated from commercially available baker’s yeast. The purification procedure was done in several steps, and after each step protein concentration and fumarase activity were measured. The first step of enzyme isolation was the cell disruption. For this purpose glass spheres of different dimensions were used, mixed with yeast and buffer on homogenizer and after a certain time, the mixture was centrifuged and the supernatant used for fumarase activity testing. It was found that bigger glass spheres were better, since they gave higher fumarase activity and higher protein concentration. It was also found that by increasing the homogenization time, higher fumarase activity can be achieved, but only up to 50 minutes. After that fumarase activity drops, probably due to local heating which destroys the protein. After the cells are disrupted, the proteins were precipitated with ammonium sulphate. The literature data show that fumarase precipitates when there is 40 - 65 % of ammonium sulphate saturation concentration, and therefore this fraction was used for further purification. Salt was removed from the protein solution in the next purification step – gel filtration on Sephadex G-50. The results show good separation of salt and protein peak. The fraction containing proteins was used for further purification by gel filtration on Sephadex G-100. Different peaks were obtained by using this method, which indicate the presence of proteins with different molecular masses. The fractions containing fumarase activity were used in further measurements. The obtained enzyme was kinetically characterized on fumaric and L-malic acid as a substrate. Enzyme kinetic data were estimated from these measurements. The enzyme purity was also estimated by measuring the extinction coefficient at 280 nm and compared to the literature data. [1] Massey V., Biochem J, 1952, 51(4): 490-494. [2] Mizobata T., Fujioka T., Yamasaki F., Hidaka M., Nagai J., Kawata Y., Arch Biochem Biophys, 1998, 355(1): 49-55. [2] Keruchenko J.S., Keruchenko I.D., Gladilin K.L., Zaitsev V.N., Chirgadze N.Y., Biochim Biophys Acta, 1992, 1122: 85-92.

Izvorni jezik
Engleski

Znanstvena područja
Biotehnologija

POVEZANOST RADA