The alkaline pectate lyase PEL168 of Bacillus subtilis heterologously expressed in Pichia pastoris is more stable and efficient for degumming ramie fiber.
The alkaline pectate lyase PEL168 of Bacillus subtilis heterologously expressed in Pichia pastoris is more stable and efficient for degumming ramie fiber.
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DOI：
10.1186/1472-6750-13-26
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发表时间：
2013-03-19
影响因子：
3.5
通讯作者：
Ma Y
中科院分区：
文献类型：
--
作者：
Zhang C;Yao J;Zhou C;Mao L;Zhang G;Ma Y
苎麻常规高温碱处理脱胶工艺对环境污染严重。果胶裂解酶可用于脱胶过程中脱除苎麻中的果胶，减少环境污染，降低能耗。枯草芽孢杆菌果胶裂解酶PEL168已被鉴定并解析其蛋白质结构。然而，在脱胶过程中，芽孢杆菌不适合作为果胶裂解酶的宿主，因为大多数芽孢杆菌都是内源产生纤维素酶，对纤维有害。巴斯德毕赤酵母不表达内源性纤维素酶，具有较高的分泌能力，是理想的表达宿主。关于果胶裂解酶PEL168在巴斯德毕赤酵母中的异源表达及其在苎麻生物脱胶中的工业化应用尚未见报道。本研究在巴斯德毕赤酵母中表达了Pel168基因。在巴斯德毕赤酵母(PEL168P)中表达的重组蛋白PEL168在SDS-PAGE上显示48.6 kDa和51.4 kDa两条带，而在大肠杆菌(PEL168E)中表达的酶与预测的46 kDa相同。脱糖消化表明PEL168P是糖基化的。两种PEL168的最适反应温度为50℃，最适pH为9.5。在60℃预孵育20min后，PEL168E完全失去活性，而PEL168P保留了26%的剩余活性。PEL168P的比活力为1320U/mg，Km值为0.09 mg/ml，Vmax为18.13Mol/μ。K+、Li+、Ni2+和Sr2+对PEL168P的活性几乎没有抑制作用，而Ca~(2+)对酶的活性有38%的促进作用。PEL168P在脱胶过程中能有效脱除苎麻中的果胶。通过进一步的密码子优化，PEL168酶在巴斯德毕赤酵母中的表达提高了1.5倍。果胶裂解酶PEL168具有良好的蛋白质结构，可在巴斯德毕赤酵母中异源表达。经密码子优化后，PEL168在巴斯德毕赤酵母中的表达水平显著提高。因此，PEL168是进一步优化和工程化生物脱胶的理想候选菌。
The conventional degumming process of ramie with alkaline treatment at high temperature causes severe environmental pollution. Pectate lyases can be used to remove pectin from ramie in a degumming process with reduced environmental pollution and energy consumption. Pectate lyase PEL168 from Bacillus subtilis has been previously characterized and the protein structure was resolved. However, Bacillus is not a suitable host for pectate lyases during the degumming process since most Bacillus produce cellulases endogenously with a detrimental effect to the fiber. Pichia pastoris, which does not express endogenous cellulases and has high secretion capability, will be an ideal host for the expression. No previous work was reported concerning the heterologous expression of pectate lyase PEL168 in P. pastoris with an aim for industrial application in ramie bio-degumming. The gene pel168 was expressed in P. pastoris in this study. The recombinant protein PEL168 in P. pastoris (PEL168P) showed two bands of 48.6 kDa and 51.4 kDa on SDS-PAGE whereas the enzyme expressed in E. coli (PEL168E) was the same as predicted with a band of 46 kDa. Deglycosylation digestion suggested that PEL168P was glycosylated. The optimum reaction temperature of the two PEL168s was 50°C, and the optimum pH 9.5. After preincubation at 60°C for 20 min, PEL168E completely lost its activity, whereas PEL168P kept 26% of the residual activity. PEL168P had a specific activity of 1320 U/mg with a Km of 0.09 mg/ml and a Vmax of 18.13 μmol/min. K+, Li+, Ni2+ and Sr2+ showed little or no inhibitory effect on PEL168P activity, and Ca2+ enhanced enzyme activity by 38%. PEL168P can remove the pectin from ramie effectively in a degumming process. A 1.5 fold increase of PEL168 enzyme expression in P. pastoris was achieved by further codon optimization. Pectate lyase PEL168 with an available protein structure can be heterologously expressed in P. pastoris. The characterized recombinant PEL168P can be used to remove pectin from ramie efficiently and the expression level of PEL168 in P. pastoris was increased markedly by codon optimization. Therefore, PEL168 is an ideal candidate for further optimization and engineering for bio-degumming.
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影响因子：
1.8
作者：
Zhang, Gui-Min;Hu, Yong;Zhang, Xian-En
通讯作者：
Zhang, Xian-En
影响因子：
4.8
作者：
Solbak, AI;Richardson, TH;Kerovuo, J
通讯作者：
Kerovuo, J
影响因子：
2.7
作者：
Gao, Zhaowei;Li, Zhuofu;Zhang, Wei
通讯作者：
Zhang, Wei
DOI：
10.1016/s0304-4165(99)00017-3
发表时间：
1999-04-19
影响因子：
3
作者：
Kobayashi, T;Hatada, Y;Ito, S
通讯作者：
Ito, S
影响因子：
3.5
作者：
NASSER, W;AWADE, AC;ROBERTBAUDOUY, J
通讯作者：
ROBERTBAUDOUY, J