| Agaricus bisporus was nutritious and delicious. However, Agaricus bisporus was easy to occur enzymatic browning during processing and storage, seriously affecting the product appearance, flavor, nutrition, processing performance and then resulting in great economic losses. Polyphenol oxidase (PPO) was the main enzyme which was responsible for enzymatic browning. In addition, in recent years it was found that PPO could catalyze enzymatic cross-linking of proteins, which can improve the functional properties of proteins. Therefore, Agaricus bisporus PPO deserves further research.The research work was composed of four parts, including purification and biochemical characterisation of PPO from Agaricus bisporus, effect of Agaricus bisporus PPO on cross-linking of milk proteins, cloning of new polyphenol oxidase cDNAs from Agaricus bisporus, cloning and expression of mature forms of PPO3 and PPO4 cDNAs in prokaryotic expression system. The main methods, results and conclusions were as follows:1 Agaricus bisporus PPO was purified by ammonium sulfate fractional precipitation, DEAE-Sepharose Fast Flow ion-exchange chromatography and phenyl Sepharose CL-4B chromatography. The molecular weight of the purified PPO was about 43 kDa by SDS-PAGE, and the N-terminal sequence was identified as Ala-Thr-Asn-Ser-Gly-Thr-Leu-Ile-Ile-Phe by Edman degradation.2 The optimum temperature and pH for the PPO activity was 20℃and pH 6.5-7.0. PPO was thermal labile. When the temperature was up to 60℃, the PPO activity was completely lost. Moreover, PPO activity was completely inactivated by Al3+, while Cu2+ and Zn2+ showed hearvy inhibition on the PPO activity. The Km and Vmax values of this PPO for catechol were 0.67 mmol/L and 3,333 U/ml min-1, respectively.3 The optical parameters of cross linking of caseins with Agaricus bisporus PPO were as follows:(1) PPO activity was 600 U/ml, (2) pH value was 7, (3) cross linking performed at 30℃for 6 h. The optical parameters of cross linking of whey proteins were that, (1) pH value was 3-5, (2) PPO activity was 600 U/ml, (3) cross linking performed at 40℃for 4 h. Following the cross linking with PPO, the emulsifying activity index, emulsifying stability index, foam capacity and foam stability of the milk proteins were evaluated to be modified. In addition, the antigenicity ofβ-casein,α-lactalbumin andβ-lactoglobulin was decreased obviously after cross linking by PPO, respectively.4 Two new conserved regions of PPO cDNAs from Agaricus bisporus were amplified using degenerate primers, which were designed corresponding to well conserved regions of several fungi PPOs. Then two new PPO full length cDNAs (PPO3 and PPO4) were obtained by RACE-PCR. PPO3 was 1,844 bp in length with an open reading frame of 1,731 bp, while that of PPO4 was 2,042 bp with an open reading frame of 1,836 bp. PPO3 and PPO4, with 52% identity at the nucleic acid level, encoded a 596-amino acid protein and 611-amino acid protein respectively. They were deposited at the Genbank database under the accession numbers GQ354801 and GQ354802, respectively.5 Biotic softwares and network servers were used to analysis the amino acids, phylogenetic tree, secondary and tertiary structures of PPO3 and PPO4. The results indicated that PPO3 and PPO4 were the new encolded genes for Agaricus bisporus PPO.6 By analysising the cleavage of C-terminal region, we knew that the mature form of PPO3 was 1,173 bp in length encoded a total of 391 amino acids with a molecular weight of 45.3 kDa, and the mature form of PPO4 was 1,134 bp in length encoded a total of 378 amino acids with a molecular weight of 43.2 kDa. Mature forms of PPO3 and PPO4 were both expressed in E. coli BL21 (DE3) RIPL using pGEX-4T-1 vector and the expressed proteins were purified. The molecular weight of the reconbinant proteins were 71 kDa and 69 kDa, respectively. Both of them could be probed by the sheep anti-Agaricus bisporus PPO antibody.
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