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ThesisItem Open Access Identification and characterization of enzyme(s) causing browning of button mushroom, Agaricus bisporus (Lange) Sing.(Punjab Agricultural University, Ludhiana, 2020) Ravneet Kaur; Sodhi, H.S.Agaricus bisporus is the most acceptable mushroom in the world but being perishable it loses sheen as fresh produce. Present study has dealt with the enzymes related to browning of button mushroom, namely, tyrosinase (E.C 1.14.18.1), laccase (EC 1.10.3.2), peroxidase (EC 1.11.1.7), phenylalanine ammonia lyase (E.C. 4.3.1.5), lipoxygenase (EC 1.13.11.12) and catalase (EC 1.11.1.6). These were estimated in submerged state and the compost/casing (solid substrate). The enzyme activity of A. bisporus mycelium in broth was found to increase with time and maximum activity was that of peroxidase (7.41U/mg) and laccase (6.05U/mg). Mycelial run in compost indicated maximum specific activity of laccase enzyme (12.3 U/mg) while the activity of the other enzymes was significantly lower. During the pin head stage, there was an increase in the activity of tyrosinase, laccase, peroxidase, PAL, catalase, lipoxygenase enzymes as 17.67, 13.98, 14.67, 4.5, 2.33 and 17.75U/mg respectively. After harvesting during the first flush, tyrosinase activity was reduced up to 12.03 U/mg and remained at par during the second and third flush harvesting. A similar pattern was observed for laccase and PAL. Peroxidase activity showed a decline up to second flush and remained stable thereafter. Catalase activity reduced from the pin head stage to first flush and no significant change in the lipoxygenase activity was observed during reproductive phase of mushroom production. During the postharvest period, the activity of melanin synthesizing enzymes that is tyrosinase, laccase and peroxidase was found to increase with time during storage and maximum increase was that for tyrosinase leading to increase in browning index (BI) value. The activity of the lipoxygenase enzyme increased which led to increased electrolytic leakage and hence lipid peroxidation. During the storage period, there was a decrease in the activity of PAL while the activity of catalase was found to increase. Mushroom washing treatments (KMS, Citric acid and Salicylic acid) and fumigation (cinnamon oil and clove oil) along with packing treatments (polypropylene bags, carton trays with cling film cover and plastic trays with cling film cover) were studied up to 16d at 4 day intervals. The washing treatments using 0.2% KMS, 1% citric acid and 200µmole/L salicylic acid showed a significant decline in the browning associated enzymes and BI in all the packagings during 16d storage. In general mushrooms treated with 200µmole/L salicylic acid and packed in PT showed the best results with minimum BI value (23.09) and tyrosinase activity (27.72 U/mg) at 16th day of storage. Cinnamon oil and clove oil fumigation indicated a gradual decline in activity of melanogenic enzymes and BI with increasing oil concentration up to 40 ppm. Tyrosinase enzyme was purified and found active over pH range 6.4 to 7.2 showing 90% and 88% of the maximum activity at pH 7. The enzyme was stable at a temperature range 25-40°C with maximum activity at 35°C. The kinetic studies of enzyme showed that in case of catechol as substrate, the Km was found to be 0.71 mM with Vmax 2518 µmole/min/ml. In case of L-Dopa from Lineweaver-Burk plot, the Km value was found to be 0.87 mM with Vmax 1714 µmole/ml/min. The SDS-PAGE electrophoresis of the enzyme gave a single prominent band at 43 kDa. FTIR spectra of purified enzyme indicated a secondary structure that reflected the amide I and amide II bands. This study indicated that mushroom browning is a complex process triggered after the harvesting involving the enzymatic reactions. The study of the browning associated enzymes at the different stages of the cultivation and during the storage with different postharvest treatments showed that browning can be decelerated by the postharvest treatments which mainly act by restricting the tyrosinase and related enzymes.
