Eastern Regional Research Center, Agricultural Research Service , U.S. Department of Agriculture, Wyndmoor, Pennsylvania 19038, United States.
J Agric Food Chem. 2013 Dec 26;61(51):12711-9. doi: 10.1021/jf403914u. Epub 2013 Dec 13.
Despite the longstanding importance of the thermally tolerant pectin methylesterase (TT-PME) activity in citrus juice processing and product quality, the unequivocal identification of the protein and its corresponding gene has remained elusive. TT-PME was purified from sweet orange [ Citrus sinensis (L.) Osbeck] finisher pulp (8.0 mg/1.3 kg tissue) with an improved purification scheme that provided 20-fold increased enzyme yield over previous results. Structural characterization of electrophoretically pure TT-PME by MALDI-TOF MS determined molecular masses of approximately 47900 and 53000 Da for two principal glycoisoforms. De novo sequences generated from tryptic peptides by MALDI-TOF/TOF MS matched multiple anonymous Citrus EST cDNA accessions. The complete tt-pme cDNA (1710 base pair) was cloned from a fruit mRNA library using RT- and RLM-RACE PCR. Citrus TT-PME is a novel isoform that showed higher sequence identity with the multiply glycosylated kiwifruit PME than to previously described Citrus thermally labile PME isoforms.
尽管耐热果胶甲酯酶（TT-PME）活性在柑橘汁加工和产品质量方面具有长期的重要性，但该蛋白质及其相应基因的确切鉴定仍然难以捉摸。TT-PME 从甜橙[Citrus sinensis（L.）Osbeck]精制浆（8.0 mg/1.3 kg 组织）中被纯化出来，采用改进的纯化方案，与之前的结果相比，酶产量提高了 20 倍。通过 MALDI-TOF MS 对电泳纯 TT-PME 的结构特征进行分析，确定了两种主要糖基化同工型的分子量约为 47900 和 53000 Da。通过 MALDI-TOF/TOF MS 从胰蛋白酶肽产生的从头序列与多个匿名柑橘 EST cDNA 克隆相匹配。使用 RT-PCR 和 RLM-RACE PCR 从果实 mRNA 文库中克隆出完整的 tt-pme cDNA（1710 个碱基对）。柑橘 TT-PME 是一种新型同工型，与多糖基化猕猴桃 PME 的序列同一性高于先前描述的柑橘热不稳定 PME 同工型。
