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Isolation of a rhamnogalacturonan lyase expressed during ripening of the Chilean strawberry fruit and its biochemical characterization.
Plant Physiology and Biochemistry ( IF 6.1 ) Pub Date : 2019-11-29 , DOI: 10.1016/j.plaphy.2019.11.041 Angela Méndez-Yañez 1 , Makarena González 1 , Cristian Carrasco-Orellana 1 , Raúl Herrera 1 , María A Moya-León 1
Plant Physiology and Biochemistry ( IF 6.1 ) Pub Date : 2019-11-29 , DOI: 10.1016/j.plaphy.2019.11.041 Angela Méndez-Yañez 1 , Makarena González 1 , Cristian Carrasco-Orellana 1 , Raúl Herrera 1 , María A Moya-León 1
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Fragaria chiloensis (L.) Mill. fruit has exotic organoleptic properties however commercialization is a challenge due to its fast and intensive softening. Texture modifications associated to ripening are related to cell wall metabolism. Main cell wall polysaccharides metabolized in F. chiloensis fruit are pectins, being rhamnogalacturonan I (RG-I) an abundant pectin domain in strawberry. Several enzymes belonging to the fruit molecular machinery have been described to act on different cell wall polysaccharides in F. chiloensis, but none acting on the main chain of RG-I until now. A gene sequence coding for a rhamnogalacturonan endolyase (RG-lyase) (EC 4.2.2.23) was isolated from F. chiloensis. The FchRGL1 sequence belongs to Polysaccharide Lyase family 4 and contains the three functional domains of RG-lyases: RGL4 domain, fibronectin type III and the carbohydrate binding module. In addition, it contains key amino acid residues for activity and Ca2+ coordination. qRT-PCR analyses indicate that FchRGL1 transcripts increase in fruit throughout ripening. RG-lyase activity evidences a remarkable increase as the fruit ripens. The heterologous expression of FchRGL1 in Pichia pastoris provided an active protein that allows its biochemical characterization. RG-lyase activity is optimum at pH 5.0, 25-30 °C and 2 mM Ca2+. A KM of 0.086 mg mL-1 was determined for potato RG-I, and the enzyme undergoes inhibition at high substrate concentration. The enzyme is also able to degrade the mucilage of germinating A. thaliana's seeds. Finally, the properties of FchRGL1 and its expression pattern are congruent with a crucial role in cell wall re-organization during softening of F. chiloensis fruit.
中文翻译：
智利草莓果实成熟过程中表达的鼠李糖半乳糖醛酸裂合酶的分离及其生化特性。
草莓草莓 水果具有奇异的感官特性，但是由于其快速而强烈的软化，商业化是一个挑战。与成熟相关的质地修饰与细胞壁代谢有关。在F. chiloensis果实中代谢的主要细胞壁多糖是果胶，是鼠李糖半乳糖醛酸聚糖I（RG-I），是草莓中丰富的果胶结构域。已经描述了几种属于果实分子机制的酶，它们作用于Chiloensis中的不同细胞壁多糖，但到目前为止，还没有一种作用于RG-1的主链。从沙门氏菌中分离出编码鼠李糖半乳糖醛酸聚糖内切酶（RG-裂解酶）（EC 4.2.2.23）的基因序列。FchRGL1序列属于多糖裂解酶家族4，包含RG裂解酶的三个功能域：RGL4域，III型纤连蛋白和碳水化合物结合模块。此外，它还包含关键的氨基酸残基，可实现活性和Ca2 +配位。qRT-PCR分析表明，在整个成熟过程中，FchRGL1转录本在果实中都会增加。随着果实成熟，RG-裂合酶活性显着增加。FchRGL1在巴斯德毕赤酵母中的异源表达提供了一种活性蛋白，可对其进行生化鉴定。RG裂解酶活性在pH 5.0、25-30°C和2 mM Ca2 +时最佳。马铃薯RG-1的KM确定为0.086 mg mL-1，并且该酶在高底物浓度下受到抑制。该酶还能够降解发芽拟南芥种子的黏液。最后，FchRGL1的特性及其表达模式与F软化过程中细胞壁重组的关键作用是一致的。
更新日期：2019-11-30
中文翻译：
智利草莓果实成熟过程中表达的鼠李糖半乳糖醛酸裂合酶的分离及其生化特性。
草莓草莓 水果具有奇异的感官特性，但是由于其快速而强烈的软化，商业化是一个挑战。与成熟相关的质地修饰与细胞壁代谢有关。在F. chiloensis果实中代谢的主要细胞壁多糖是果胶，是鼠李糖半乳糖醛酸聚糖I（RG-I），是草莓中丰富的果胶结构域。已经描述了几种属于果实分子机制的酶，它们作用于Chiloensis中的不同细胞壁多糖，但到目前为止，还没有一种作用于RG-1的主链。从沙门氏菌中分离出编码鼠李糖半乳糖醛酸聚糖内切酶（RG-裂解酶）（EC 4.2.2.23）的基因序列。FchRGL1序列属于多糖裂解酶家族4，包含RG裂解酶的三个功能域：RGL4域，III型纤连蛋白和碳水化合物结合模块。此外，它还包含关键的氨基酸残基，可实现活性和Ca2 +配位。qRT-PCR分析表明，在整个成熟过程中，FchRGL1转录本在果实中都会增加。随着果实成熟，RG-裂合酶活性显着增加。FchRGL1在巴斯德毕赤酵母中的异源表达提供了一种活性蛋白，可对其进行生化鉴定。RG裂解酶活性在pH 5.0、25-30°C和2 mM Ca2 +时最佳。马铃薯RG-1的KM确定为0.086 mg mL-1，并且该酶在高底物浓度下受到抑制。该酶还能够降解发芽拟南芥种子的黏液。最后，FchRGL1的特性及其表达模式与F软化过程中细胞壁重组的关键作用是一致的。
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