Structure-guided modification of Rhizomucor miehei lipase for production of structured lipids.
Structure-guided modification of Rhizomucor miehei lipase for production of structured lipids.
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DOI：
10.1371/journal.pone.0067892
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Han SY
中科院分区：
文献类型：
--
作者：
Zhang JH;Jiang YY;Lin Y;Sun YF;Zheng SP;Han SY
为了提高酵母表面显示的米黑根瘤菌脂肪酶（RML）在生产人乳脂肪代用品（HMFS）中的性能，我们基于蛋白质疏水性对脂肪酶底物结合袋中的氨基酸进行了突变，以提高其酯化活性。获得了5个突变体Asn87Ile、Asn87Ile/Asp91Val、His108Leu/Lys109Ile、Asp256Ile/His257Leu和His108Leu/Lys109Ile/Asp256Ile/His257Leu，并测定了它们的水解和酯化活性。利用Discovery Studio 3.1建立模型并计算脂肪酶与底物之间的结合能，与野生型相比，突变体Asp256Ile/His257Leu在油酸（降低3.97 KJ/mol）和三棕榈素（降低7.55 KJ/mol）为底物时能量显著降低。该结果与Asp256Ile/His257Leu的酯化活性一致（是野生型的2.37倍）。对这四个突变体在有机溶剂和无溶剂体系中生产HMFS进行了评估。Asp256Ile/His257Leu催化三棕榈素与油酸的油酸掺入率为28.27%，催化棕榈油与油酸的油酸掺入率为53.18%。Asp256Ile/His257Leu酶的效率分别是野生型酶的1.82倍和1.65倍。Asp256Ile/His257Leu的油酸掺入与商用脂酶RM IM的油酸水解棕榈油相似。酵母表面显示的RML突变体Asp256Ile/His257Leu是一种潜在的、经济上可行的结构性脂质生产催化剂。
To improve the performance of yeast surface-displayed Rhizomucor miehei lipase (RML) in the production of human milk fat substitute (HMFS), we mutated amino acids in the lipase substrate-binding pocket based on protein hydrophobicity, to improve esterification activity. Five mutants: Asn87Ile, Asn87Ile/Asp91Val, His108Leu/Lys109Ile, Asp256Ile/His257Leu, and His108Leu/Lys109Ile/Asp256Ile/His257Leu were obtained and their hydrolytic and esterification activities were assayed. Using Discovery Studio 3.1 to build models and calculate the binding energy between lipase and substrates, compared to wild-type, the mutant Asp256Ile/His257Leu was found to have significantly lower energy when oleic acid (3.97 KJ/mol decrease) and tripalmitin (7.55 KJ/mol decrease) were substrates. This result was in accordance with the esterification activity of Asp256Ile/His257Leu (2.37-fold of wild-type). The four mutants were also evaluated for the production of HMFS in organic solvent and in a solvent-free system. Asp256Ile/His257Leu had an oleic acid incorporation of 28.27% for catalyzing tripalmitin and oleic acid, and 53.18% for the reaction of palm oil with oleic acid. The efficiency of Asp256Ile/His257Leu was 1.82-fold and 1.65-fold that of the wild-type enzyme for the two reactions. The oleic acid incorporation of Asp256Ile/His257Leu was similar to commercial Lipozyme RM IM for palm oil acidolysis with oleic acid. Yeast surface-displayed RML mutant Asp256Ile/His257Leu is a potential, economically feasible catalyst for the production of structured lipids.
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影响因子：
2.7
作者：
Kourist, Robert;Brundiek, Henrike;Bornscheuer, Uwe T.
通讯作者：
Bornscheuer, Uwe T.
影响因子：
3.5
作者：
OTERO, C;RUA, ML;ROBLEDO, L
通讯作者：
ROBLEDO, L
影响因子：
7.7
作者：
Lutz, Stefan
通讯作者：
Lutz, Stefan
影响因子：
2.9
作者：
KOBORI, H;SATO, M;OSUMI, M
通讯作者：
OSUMI, M
提高变形杆菌脂肪酶 LipK107 的催化活性。
影响因子：
--
作者：
Gao, Bei;Xu, Tao;Wei, Dongzhi
通讯作者：
Wei, Dongzhi