果胶裂解酶是一类通过β-消除机制降解果胶的多糖裂解酶,对果胶的利用具有重要意义。该研究利用真核表达载体,克隆表达了来自曲霉属Aspergillus costaricensis的酸性果胶裂解酶PnlF,经过亲和层析和凝胶过滤层析对该酶的发酵液进行了分离纯化,并对其酶学性质和降解果胶的产物进行了系统性研究。该研究成功实现了PnlF在毕赤酵母中的分泌表达,粗酶活力达到526 U/mg。PnlF的最适反应温度为50 ℃,最适反应pH为5.0;其酶活力在20~30 ℃及pH 5~6内稳定。Ca2+和Mg2+可以大幅度提高PnlF的活力;而Ag+和Cu2+则会严重抑制其活力;此外SDS、TritonX-100、苯甲磺酰氟等化学试剂对该酶活性的抑制作用较强。
Pectin lyases are a class of polysaccharide lyases that degrade pectin through a β-elimination mechanism, and are of great significance to the utilization of pectin. In this study, the eukaryotic expression system was used to clone and express the pectin lyase PnIF from Aspergillus costaricensis. The broth of PnlF was isolated and purified by affinity chromatography and gel filtration chromatography, and its enzymatic properties as well as the degradation products from pectin through the PnlF activity were systematically investigated. The results showed that the secretory expression of PnIF in Pichia pastoris was successfully achieved, and the crude enzyme activity reached 526 U/mg. The optimum reaction temperature of PnlF was 50 °C, and the optimum reaction pH was 5.0. The enzyme activity of PnlF was stable at 20-30 ℃ and pH 5-6. Ca2+and Mg2+ can strongly enhanced the activity of PnlF, while Ag+ and Cu2+ can seriously inhibit its activity. In addition, chemical reagents such as SDS, TritionX-100 and PMSF have strong inhibitory effects on the enzyme activity.
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