[0135] Preparation method of polypeptide
[0136] Polypeptides suitable for use in the methods of the present disclosure (e.g., polypeptides having decolorizing enzyme activity, including polypeptides having pyropheophytinase activity) can be obtained by culturing in a suitable fermentation medium under conditions that allow expression of the polypeptide as disclosed herein host cells and produce the polypeptide. Those skilled in the art understand how to perform methods of producing polypeptides as disclosed herein depending on the host cells used, such as the pH, temperature and composition of the fermentation medium. Host cells can be cultured in shake flasks or in fermentors having a volume of 0.5 liter or 1 liter or greater to 10 cubic meters to 100 cubic meters or greater. Culturing can be performed aerobically or anaerobically according to the needs of the host cells. If the host cell is a Pseudomonas (eg, Pseudomonas fluorescens), culturing of the host cell is performed under aerobic conditions.
[0137] Advantageously, a polypeptide as disclosed herein is recovered or isolated from the fermentation medium, for example by centrifugation or filtration known to those skilled in the art. Recovery of a polypeptide having pyropheophytinase activity may also include destruction of the cell in which the polypeptide is produced. Disruption of cells can be performed using glass beads and/or sonication known to those skilled in the art. Method for treating oil comprising chlorophyll derivatives with a polypeptide having decolorizing enzyme activity
[0138] In one embodiment, the methods of the present disclosure further comprise treating an oil comprising a chlorophyll derivative such as pyropheophytin with a polypeptide having depigmentase activity as disclosed herein or with a composition comprising a polypeptide disclosed above . In one embodiment, a polypeptide as disclosed herein has pyropheophytinase activity. In another embodiment, the polypeptide has pheophytinase activity. In another embodiment, the polypeptide has pyropheophytinase activity and pheophytinase activity. In another embodiment, the polypeptide has pyropheophytinase activity, pheophytinase activity, and chlorophyllase activity.
[0139] As discussed herein, a polypeptide having pyropheophytinase activity is capable of hydrolyzing pyropheophytin to pyropheophytin, a polypeptide having pheophytinase activity is capable of hydrolyzing pheopheophytin to pheophorbide, And the polypeptide with chlorophyllase activity can hydrolyze chlorophyll into dephytochlorophyll. Thus, in one embodiment, decolorizing enzyme treatment reduces the level of one or more chlorophyll substrates in the oil. In various embodiments, the chlorophyll substrate can be chlorophyll, pheophytin, and/or pyropheophytin. For example, treatment with a decolorizing enzyme can reduce the total concentration of chlorophyll substrates in the oil by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%. The decrease in the overall concentration of the chlorophyll substrate may be a result of the conversion of pyropheophytin to pyropheophytin, the conversion of pheophytin to pheophorbide and/or the conversion of chlorophyll to physochlorophyll.
[0140] In another embodiment, the chlorophyll substrate in the oil comprises pyropheophytin, and at least a portion of the pyropheophytin is converted to pyropheophytin by decolorizing enzyme treatment. For example, treatment with a decolorizing enzyme can reduce the total concentration of pyropheophytin in the oil by at least 5%, at least 10% by weight, compared to the total concentration (by weight) of pyropheophytin present in the oil prior to decolorizing enzyme treatment. %, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%. The decrease in the total concentration of pyropheophytin may be the result of the conversion of pyropheophytin to pyropheophytin. In another embodiment, the chlorophyll substrate in the oil comprises pheophorbide, and at least a portion of the pheophorbide is converted to pheophorbide as a result of the treatment. For example, treatment with a decolorizing enzyme can reduce the total concentration of pheophytin in the oil by at least 5%, at least 10%, by weight, compared to the total concentration of pheophytin present in the oil prior to decolorizing enzyme treatment (by weight). At least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%. The decrease in the total concentration of pheophorbide may be the result of the conversion of pheophorbide to pheophorbide. In such embodiments, the polypeptide exhibits pheophytinase activity.
[0141] In another embodiment, the chlorophyll substrate in the oil comprises chlorophyll, and at least a portion of the chlorophyll is converted to chlorophyll by decolorizing enzyme treatment. For example, treatment with a decolorizing enzyme can reduce the total concentration of chlorophyll in the oil by at least 5%, at least 10%, at least 20%, by weight, compared to the total concentration (by weight) of chlorophyll present in the oil before the decolorizing enzyme treatment. At least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%. The decrease in the total concentration of chlorophyll may be a result of the conversion of chlorophyll to chlorophyll. In such embodiments, the polypeptide exhibits chlorophyllase activity.
