A Study on Separation of Thermolabile and Thermostable Pectinesterase from Valencia Orange

Valencia 오렌지에서 내열성 및 비내열성 Pectinesterase 분리 정제

Pectinesterase(PE) has been definitively established as the causative agent for clarification of citrus juice and gelation of frozen concentrates. This enzyme is present in multiple forms in citrus fruit. Although representing a minor fraction of the total PE activity, thermostable pectinesterase(TSPE) accounts for the severe time/temperature processing treatment required to inactivate PE. This study was undertaken to separate and purify thermolabile pectinesterase(TLPE) and TSPE from the crude PE extracts of Valencia orange rag-pulp powder. The approach taken was to carry out the three kind of chromatographies of CM-Sephadex C-50, Phenyl Sepharose CL-4B and CM-Biogel A to the unheated crude PE or the heated crude PE. All of them used could increase the purity of PEs and, specially, Phenyl Sepharose CL-4B chromatography could separate crude PE as the mixture of PEs into two forms of TLPE and TSPE. The purified TLPE had specific activity of 1,005 units/mg, yield of 13.6% and purification of 35 fold, while the TSPE separated from the unheated crude PE showed specific activity of 3,115 units/mg, yield of 1.5% and purification of 100.5 fold, and another TSPE from the heated crude PE was found to be specific activity of 1,803 units/mg, yield of 15.4% and purification of 140 fold.

Valencia 오렌지로부터 추출한 펙틴에스테라제(pectinesterase, PE) 중에서 가열처리하지 않은 PE 조효소를 CM-Sephadex C-50, Phenyl Sepharose CL-4B와 CM-Biogel A의 chromatography를 차례로 수행하여 내열성이 약한 TLPE(thermolabile pectinesterase)와 내열성이 강한 TSPE(thermostable pectinesterase)를 분리 정제하였다. PE 중에서 가열처리한 PE 조효소에서는 위와 같은 정제과정으로 TSPE 만을 분리하였다. TLPE는 비활성 1,005 units/mg이고 35배로 정제된 것을 13.6% 회수할 수 있었으며, 반면에 가열하지 않고 분리한 TSPE는 비활성 3,115 units/mg이고 100.5배로 정제된 것을 1.5% 얻을 수 있었으나 가열하여 분리한 TSPE는 비활성 1,803 units/mg이고 가열처리한 조효소가 140배로 정제된 것을 15.4% 얻을 수 있었다.