Изучение in silico структурных особенностей липаз из различных продуцентов как потенциальных агентов для иммобилизации на заряженных и гидрофобных носителях

Липаза – гидролитический фермент, получаемый из многих организмов, таких как животные, растения, грибы и бактерии. Она осуществляет расщепление триглицеридов до моноглицеридов и жирных кислот, при этом обладает широкой субстратной специфичностью. Липазы применяются в пищевой промышленности и других сферах человеческой деятельности. Характерная особенность многих липаз – явление поверхностной активации, обуславливающее свойственную им зависимость скорости каталитической реакции от концентрации и агрегатного состояния субстрата. Доказано, что функционирование ферментов зависит от их структурных особенностей. Внутренние полости, туннели и поры являются неотъемлемыми компонентами нативной конформации белка. Они играют важную роль в транспорте субстрата, кофакторов и ионов к активному и регуляторным центрам фермента. Кроме того, их конфигурация может влиять на термостабильность энзимов, в связи с чем изучение вышеперечисленных структур является необходимым для понимания механизмов функционирования биокатализаторов. Также важным элементом структуры ферментов являются скопления заряженных и гидрофобных аминокислотных остатков на их поверхности. Данное свойство необходимо учитывать при планировании путей адсорбционной иммобилизации биокатализаторов на различных носителях для использования в промышленности и медицине. В работе исследованы состав, локализация и конфигурация внутренних полостей, туннелей, пор, а также поверхностных скоплений заряженных и гидрофобных аминокислотных остатков в молекулах липаз из Rhizopus niveus (PDB ID: 1LGY), Rhizomucor miehei (PDB ID: 3TGL), Burkholderia cepacia (PDB ID: 1OIL), панкреатических липаз Homo sapiens (PDB ID: 1N8S) и Equus caballus (PDB ID: 1HPL). Для расчета параметров и визуализации данных структур использовались программы MOLE и Maestro. Показано наличие соответственно 2, 1, 5, 5 и 2 туннелей и отсутствие пор для данных ферментов; а также наличие по 6 внутренних полостей для липаз из R. niveus и E. caballus, 5 внутренних полостей для молекулы из R. miehei и по 2 внутренних полости для энзимов из B. cepacia и H. sapiens. Приведены аминокислотный состав и профили туннелей изучаемых липаз. Выявлено, что данные структуры не сообщаются друг с другом общими пустотами. Установлено преобладание гидрофобных аминокислотных остатков в большинстве туннелей данных ферментов. Изучены структура, локализация и состав скоплений заряженных и гидрофобных аминокислотных остатков на поверхностях макромолекул. Выявлено возможное влияние расположения данных скоплений на связывание липаз с носителем при их адсорбционной иммобилизации.

Lipase-Catalyzed Chemoselective Ester Hydrolysis of Biomimetically Coupled Aryls for the Synthesis of Unsymmetric Biphenyl Esters

Lipases are among the most frequently used biocatalysts in organic synthesis, allowing numerous environmentally friendly and inexpensive chemical transformations. Here, we present a biomimetic strategy based on iron(III)-catalyzed oxidative coupling and selective ester monohydrolysis using lipases for the synthesis of unsymmetric biphenyl-based esters under mild conditions. The diverse class of biphenyl esters is of pharmaceutical and technical relevance. We explored the potency of a series of nine different lipases of bacterial, fungal, and mammalian origin on their catalytic activities to cleave biphenyl esters, and optimized the reaction conditions, in terms of reaction time, temperature, pH, organic solvent, and water–organic solvent ratios, to improve the chemoselectivity, and hence control the ratio of unsymmetric versus symmetric products. Elevated temperature and increased DMSO content led to an almost exclusive monohydrolysis by the four lipases Candida rugosa lipase (CRL), Mucor miehei lipase (MML), Rhizopus niveus lipase (RNL), and Pseudomonas fluorescens lipase (PFL). The study was complemented by in silico binding predictions to rationalize the observed differences in efficacies of the lipases to convert biphenyl esters. The optimized reaction conditions were transferred to the preparative scale with high yields, underlining the potential of the presented biomimetic approach as an alternative strategy to the commonly used transition metal-based strategies for the synthesis of diverse biphenyl esters.

Spectroscopic studies on the comparative refolding of guanidinium hydrochloride denatured hen egg-white lysozyme and Rhizopus niveus lipase assisted by cationic single-chain/gemini surfactants via artificial chaperone protocol

Referred to as second generation surfactants, the gemini surfactants have shown promise in various potential areas of surfactant application.

Correction: Spectroscopic studies on the comparative refolding of guanidinium hydrochloride denatured hen egg-white lysozyme and Rhizopus niveus lipase assisted by cationic single-chain/gemini surfactants via artificial chaperone protocol

Correction for ‘Spectroscopic studies on the comparative refolding of guanidinium hydrochloride denatured hen egg-white lysozyme and Rhizopus niveus lipase assisted by cationic single-chain/gemini surfactants via artificial chaperone protocol’ by Nuzhat Gull et al., RSC Adv., 2017, 7, 28452–28460.

Isolasi fragmen gen LIPASE dari kapang Absidia corymbifera, Rhizopus oryzae dan Rhizopus oligosporus Isolation of LIPASE gene fragment from Absidia corymbifera, Rhizopus oryzae and Rhizopus oligosporus fungi

Diversification of oil palm products, suchas healthy oil, needs lipase sustainability as abiocatalist. Many attempts have beendeveloped to produce lipase, includingintensive exploration and screening of severalspecies of molds. Genetic engineering for overexpression of LIPASE gene in the selectedmold is considered to be the potentialapproach for efficient production of thisenzyme. This research was aimed to isolate theLIPASE gene fragment of Indonesianindigenous fungi, namely Absidia corymbifera,Rhizopus oryzae and R. oligosporus by meansof RT-PCR (Reverse Transcriptase PolymeraseChain Reaction) technique using heterologousprimers. The result showed that a cDNAfragment of 462 bp has been amplified andisolated from the three fungi with differentconcentration. The highest quantity was foundfrom A. corymbifera. The RT-PCR productsisolated from A. corymbifera was cloned,sequenced and analyzed for its homology to thesequence of LIPASE gene from other species.BLAST analysis showed that the DNA sequenceof the cloned RT-PCR product derived fromA. corymbifera was highly homologous withLIPASE gene from Rhizopus niveus.AbstraksDiversifikasi produk kelapa sawit, sepertiminyak sehat (healthy oil) memerlukanketersediaan lipase sebagai biokatalis. Berbagaiupaya untuk produksi lipase telah dikembang-kan, termasuk eksplorasi dan skrining terhadapbeberapa spesies kapang secara intensif.Rekayasa genetika untuk mengoverekspresi-kan gen LIPASE pada kapang hasil skriningtersebut dipandang merupakan satu pendekatanpotensial untuk produksi enzim ini secaraefisien. Penelitian ini bertujuan untukmengisolasi fragmen gen LIPASE dari tigakapang indigenous Indonesia, yaituA. corymbifera, R. oryzae dan R. oligosporus,menggunakan teknik RT-PCR (ReverseTranscriptase Polymerase Chain Reaction).Hasil penelitian menunjukkan bahwa fragmencDNA sepanjang 462 bp dari ketiga kapangtelah diisolasi, masing-masing dengankuantitas yang berbeda. Hasil tertinggidiperoleh dari kapang A. corymbifera. ProdukRT-PCR dari A. corymbifera diklon, disekuenkemudian dianalisis homologinya dengansekuen gen LIPASE dari spesies lain. AnalisisBLAST menunjukkan bahwa sekuen DNA dariproduk RT-PCR terklon yang berasal dariA. corymbifera memiliki homologi tinggidengan gen LIPASE dari Rhizopus niveus.

Isolasi fragmen gen LIPASE dari kapang Absidia corymbifera, Rhizopus oryzae dan Rhizopus oligosporus Isolation of LIPASE gene fragment from Absidia corymbifera, Rhizopus oryzae and Rhizopus oligosporus fungi

Diversification of oil palm products, suchas healthy oil, needs lipase sustainability as abiocatalist. Many attempts have beendeveloped to produce lipase, includingintensive exploration and screening of severalspecies of molds. Genetic engineering for overexpression of LIPASE gene in the selectedmold is considered to be the potentialapproach for efficient production of thisenzyme. This research was aimed to isolate theLIPASE gene fragment of Indonesianindigenous fungi, namely Absidia corymbifera,Rhizopus oryzae and R. oligosporus by meansof RT-PCR (Reverse Transcriptase PolymeraseChain Reaction) technique using heterologousprimers. The result showed that a cDNAfragment of 462 bp has been amplified andisolated from the three fungi with differentconcentration. The highest quantity was foundfrom A. corymbifera. The RT-PCR productsisolated from A. corymbifera was cloned,sequenced and analyzed for its homology to thesequence of LIPASE gene from other species.BLAST analysis showed that the DNA sequenceof the cloned RT-PCR product derived fromA. corymbifera was highly homologous withLIPASE gene from Rhizopus niveus.AbstraksDiversifikasi produk kelapa sawit, sepertiminyak sehat (healthy oil) memerlukanketersediaan lipase sebagai biokatalis. Berbagaiupaya untuk produksi lipase telah dikembang-kan, termasuk eksplorasi dan skrining terhadapbeberapa spesies kapang secara intensif.Rekayasa genetika untuk mengoverekspresi-kan gen LIPASE pada kapang hasil skriningtersebut dipandang merupakan satu pendekatanpotensial untuk produksi enzim ini secaraefisien. Penelitian ini bertujuan untukmengisolasi fragmen gen LIPASE dari tigakapang indigenous Indonesia, yaituA. corymbifera, R. oryzae dan R. oligosporus,menggunakan teknik RT-PCR (ReverseTranscriptase Polymerase Chain Reaction).Hasil penelitian menunjukkan bahwa fragmencDNA sepanjang 462 bp dari ketiga kapangtelah diisolasi, masing-masing dengankuantitas yang berbeda. Hasil tertinggidiperoleh dari kapang A. corymbifera. ProdukRT-PCR dari A. corymbifera diklon, disekuenkemudian dianalisis homologinya dengansekuen gen LIPASE dari spesies lain. AnalisisBLAST menunjukkan bahwa sekuen DNA dariproduk RT-PCR terklon yang berasal dariA. corymbifera memiliki homologi tinggidengan gen LIPASE dari Rhizopus niveus.

Catálise Enzimática: Transesterificação do Óleo de Soja e Esterificação do Ácido Oleico via Lipases

Resumo: O presente trabalho descreve o estudo da catálise enzimática em reações de transesterificação do óleo de soja e esterificação do ácido oleico. Foram estudadas as seguintes enzimas: Candida antarctica, Candida rugosa, Candida cylindracea, Hog pancreas, Porcine pancreas, Rhizopus niveus e Pseudomonas fluorescens. Todas as reações foram realizadas com 5,0% do biocatalisador e os rendimentos das reações foram determinados por CG-FID e os resultados expressos em percentagem (%) de conversão de ésteres. Tanto em reações de transesterificação quanto esterificação a lipase de maior eficiência foi a de Candida antarctica resultando em um rendimento na esterificação de 96,5% e na transesterificação o óleo de soja 84,1%. Todas as reações foram analisadas por CG-FID e RMN.Palavras-chave: Biodiesel, biocatálise, bioenergia.