Brault Guillaume, Shareck François, Hurtubise Yves, Lépine François, Doucet Nicolas
INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada.
Innu-Science Canada, Inc., Local 119, Trois-Rivières, Québec, Canada.
PLoS One. 2014 Mar 26;9(3):e91872. doi: 10.1371/journal.pone.0091872. eCollection 2014.
Short-chain aliphatic esters are small volatile molecules that produce fruity and pleasant aromas and flavors. Most of these esters are artificially produced or extracted from natural sources at high cost. It is, however, possible to 'naturally' produce these molecules using biocatalysts such as lipases and esterases. A gene coding for a newly uncovered lipase was isolated from a previous metagenomic study and cloned into E. coli BL21 (DE3) for overexpression using the pET16b plasmid. Using this recombinant strain as a whole-cell biocatalyst, short chain esters were efficiently synthesized by transesterification and esterification reactions in organic media. The recombinant lipase (LipIAF5-2) showed good affinity toward glyceryl trioctanoate and the highest conversion yields were obtained for the transesterification of glyceryl triacetate with methanol. Using a simple cetyl-trimethylammonium bromide pretreatment increased the synthetic activity by a six-fold factor and the whole-cell biocatalyst showed the highest activity at 40°C with a relatively high water content of 10% (w/w). The whole-cell biocatalyst showed excellent tolerance to alcohol and short-chain fatty acid denaturation. Substrate affinity was equally effective with all primary alcohols tested as acyl acceptors, with a slight preference for methanol. The best transesterification conversion of 50 mmol glyceryl triacetate into isoamyl acetate (banana fragrance) provided near 100% yield after 24 hours using 10% biocatalyst loading (w/w) in a fluidized bed reactor, allowing recycling of the biocatalyst up to five times. These results show promising potential for an industrial approach aimed at the biosynthesis of short-chain esters, namely for natural flavor and fragrance production in micro-aqueous media.
短链脂肪族酯是具有果香和宜人香气及风味的挥发性小分子。这些酯类大多是人工合成或从天然来源中高成本提取的。然而，利用脂肪酶和酯酶等生物催化剂“天然”生产这些分子是可行的。从之前的宏基因组研究中分离出一个编码新发现脂肪酶的基因，并使用pET16b质粒将其克隆到大肠杆菌BL21(DE3)中进行过表达。以该重组菌株作为全细胞生物催化剂，在有机介质中通过酯交换和酯化反应高效合成了短链酯。重组脂肪酶（LipIAF5-2）对三辛酸甘油酯具有良好的亲和力，乙酸甘油酯与甲醇的酯交换反应获得了最高的转化率。使用简单的十六烷基三甲基溴化铵预处理可使合成活性提高6倍，全细胞生物催化剂在40°C和相对较高的10%（w/w）含水量下表现出最高活性。全细胞生物催化剂对酒精和短链脂肪酸变性表现出优异的耐受性。作为酰基受体测试的所有伯醇的底物亲和力同样有效，对甲醇略有偏好。在流化床反应器中使用10%（w/w）的生物催化剂负载量，50 mmol乙酸甘油酯转化为乙酸异戊酯（香蕉香味）的最佳酯交换转化率在24小时后接近100%，生物催化剂可循环使用多达五次。这些结果表明，一种旨在生物合成短链酯的工业方法具有广阔的前景，即在微水介质中生产天然香料和香精。
