| 摘要: |
| 使用酶法对苎麻[Boehmeria nivea (L.) Gaudich]进行低温脱胶具有节省能耗和环境友好的优点，然而目前少有相关报道。基于此，本研究从土壤中筛选果胶酸裂解酶（Pel，pectate lyase）产生菌，并通过基因克隆表达获得该菌的重组Pel，测定重组Pel的酶学性质后将其应用于苎麻的低温脱胶中。结果表明，从土壤中获得一株短小芽孢杆菌（Bacillus pumilus）B.15-1，该菌株在液体培养时可产酶0.12 U/mL。将其Pel基因克隆并在大肠杆菌中表达，用Ni+-亲和层析纯化获得分子量为42.56 kDa的重组酶。该酶的最适作用pH为8.5，在pH 8.5和30 ℃孵育24 h后酶活力无损失；最适作用温度为55 ℃，在30 ℃较稳定。rBpumPel-Ec可在30 ℃对苎麻进行单独脱胶，当用酶量为25 U/mL时得苎麻失重率为18.65±0.65%。rBpumPel-Ec亦能通过与NaOH联用的方式提升碱法脱胶的效果，当将25 U/mL rBpumPel-Ec与0.5% NaOH联用时，所得苎麻失重率（30.20±0.65%）高于单独使用0.5% NaOH时所得失重率（23.54±0.43%）。本研究结果说明rBpumPel-Ec在苎麻的低温脱胶方面具有潜在应用价值。 |
| 关键词: 果胶酸裂解酶 基因克隆表达 酶学性质 短小芽孢杆菌 苎麻脱胶 清洁生产 |
| DOI： |
| 投稿时间：2025-01-24修订日期：2025-03-11 |
| 基金项目:中央引导地方科技发展专项（桂科ZY23055011），广西重点研发计划（桂科AB24010349），国家自然科学基金（32360560），广西科学院基本业务经费项目（2021YBJ703） |
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| Functional Identification of a Pectate Lyase from Bacillus pumilus and It’s Application in Low-Temperature Ramie Degumming |
|
ZHOU Jin1, LIU Sijia1, QIN Yan2, WANG Qingyan2, XIAN Liang2, LIANG Xinquan1
|
| (1.College of Light Industry and Food Engineering,Guangxi University,Nanning,Guangxi;2.National Key Laboratory of Non-food Biomass Energy Technology,NationalEngineeringResearchCenterforNon-FoodBiorefinery,Guangxi Academy of Sciences,Nanning,Guangxi) |
| Abstract: |
| Degumming of ramie using enzyme method at low temperature is cost-reduced and environment friendly, but there is seldom reports on low temperature degumming. To resolve this problem, screening of pectate lyase producing strain, obtaining recombinant enzyme by gene cloning and expression in Escherichia coli, determination of enzyme characteristics and application of enzyme in low-temperature ramie degumming were conducted. The result indicated that the pectate lyase producing bacterium strain B.15-1 (0.12 U/mL pectate lyase activity by liquid cultivation) was identified as Bacillus pumilus by analysis of the 16S rRNA coding sequence. The putative pectate lyase coding gene was cloned and expressed in Escherichia coli, and the recombinant protein with an apparent molecular weight of 42.56 kDa was one-step purified using Ni+- affinity chromatography. The optimum pH of the purified enzyme was 8.5, and the enzyme was stable at pH 8.5 after incubation at 30 ℃ for 24 h. The optimum temperature of the purified enzyme was 55 ℃, and the enzyme was stable at 30 ℃. rBpumPel-Ec could applied in the tow-temperature degumming of ramie by obtaining a weight loss value of 18.65±0.65% with the enzyme dose of 25 U/mL. rBpumPel-Ec could increased the degumming effect by combination with NaOH. The combination of 25 U/mL rBpumPel-Ec with 0.5% NaOH could obtain a weight loss value of 30.20±0.65%, which was higher than that of 0.5% NaOH was used only (23.54±0.43%). The result of this study indicated the potential of rBpumPel-Ec in low-temperature degumming. |
| Key words: pectate lyase gene cloning and expression enzyme characteristics Bacillus pumilus ramie deguming clean production |
