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1.
Sheng Wu Gong Cheng Xue Bao ; 37(9): 3242-3252, 2021 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-34622632

RESUMO

L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.


Assuntos
Asparaginase , Rhizomucor , Asparaginase/biossíntese , Asparaginase/genética , Bacillus subtilis/genética , Microbiologia Industrial , Engenharia de Proteínas , Rhizomucor/enzimologia , Alinhamento de Sequência
2.
Biochim Biophys Acta Proteins Proteom ; 1869(11): 140709, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34358705

RESUMO

A chitinase gene (RmChiA) encoding 445 amino acid (aa) residues from a fungus Rhizomucor miehei was cloned and overexpressed in Escherichia coli. Two kinds of RmChiA crystal forms, with space groups P32 2 1 and P1, were obtained by sitting-drop vapor diffusion and the structures were determined by X-ray diffraction. The overall structure of RmChiA monomer, which is the first structure of bacterial-type chitinases from nonpathogenic fungi, adopts a canonical triosephosphate isomerase (TIM) barrel fold with two protruding chitinase insertion domains. RmChiA exhibited a unique NxDxE catalytical motif and a real active site tunnel structure, which are firstly found in GH family 18 chitinases. The motif had high structural homolog with the typical DxDxE motif in other GH family 18 chitinases. The tunnel is formed by two unusual long loops, containing 15 aa and 45 aa respectively, linked by a disulfide bond across the substrate-binding cleft. Mutation experiments found that opening the roof of tunnel structure increased the hydrolysis efficiency of RmChiA, but the thermostability of the mutants decreased. Moreover, the tunnel structure endowed RmChiA with the exo-chitinase character.


Assuntos
Domínio Catalítico , Quitinases/química , Proteínas Fúngicas/química , Rhizomucor/enzimologia , Quitinases/genética , Quitinases/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hidrólise , Mutação
3.
Methods Mol Biol ; 2290: 215-228, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009593

RESUMO

Cloning proteins enables their production and characterization for further studies. This requires inserting the gene of the studied protein to be inserted in a vector, which then will be transformed to the host cell used as "factory." Consequently, the "biomass" of host cells will be produced using bioreactors. Here we describe the production of Rhizomucor miehei lipase (RML) by cloning the corresponding genes in the yeast Pichia pastoris. This enzyme is used as a biocatalyst for biofuel production. The successfully produced recombinant proteins are then purified using ion exchange chromatography.


Assuntos
Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Rhizomucor/química , Cromatografia por Troca Iônica/métodos , Clonagem Molecular/métodos , Células Eucarióticas/metabolismo , Expressão Gênica/genética , Lipase/metabolismo , Pichia/genética , Rhizomucor/enzimologia , Rhizomucor/genética
4.
J Oleo Sci ; 70(3): 385-395, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33658468

RESUMO

In this study, Candida antarctica lipase B (CALB), Rhizomucor miehei lipase (RML) and Lecitase® Ultra (LU) were immobilized onto the mesoporous silica SBA-15. The glycerolysis performance of the obtained supported lipases (lipase@SBA-15) in solvent systems was carefully investigated. LU@SBA-15 exhibited good glycerolysis performance in solvent-free system, with diacylglycerols (DAG) content and triacylglycerols (TAG) conversion at 52.4 and 98.6% respectively obtained after 12 h reaction at 60°C. CALB@SBA-15 showed good glycerolysis activity in tert-pentanol and tert-butanol systems, with TAG conversion over 90% obtained. In addition, the present CALB@SBA-15 exhibited selectivity for monoacylglycerols (MAG) production, with glycerol to TAG molar ratio increased to 3:1, MAG content over 80% and TAG conversion over 99% could be obtained from both tert-pentanol and tert-butanol systems. However, RML@SBA-15 showed low glycerolysis activity neither in solvent nor in solvent-free systems. The present results favor the practical enzymatic design for MAG and DAG production.


Assuntos
Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Glicerol/química , Lipase/química , Dióxido de Silício/química , Solventes , Butanóis/química , Diglicerídeos/química , Monoglicerídeos/química , Pentanóis/química , Rhizomucor/enzimologia , Fatores de Tempo , Triglicerídeos/química
5.
Food Chem ; 343: 128407, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33129620

RESUMO

In this study, cocoa butter equivalents (CBEs) were prepared through enzymatic interesterification of palm mid-fraction (PMF) with stearic acid (SA). The reaction process parameters were experimented and the performance of the product was analysed. PMF and stearic acid (at a mass ratio of 1:2) were catalysed by 80 g kg-1 enzyme loading of Lipozyme RM IM fromRhizomucor mieheiat 60 °C for 120 min. The yield of the CBE product was more than 92%, and the CBE resembled cocoa butter (CB) in terms of its triacylglycerol composition. The hardness of the CBE product was higher than that of CB at different storage temperatures, but this difference was not obvious at 25 °C. The polymorphic structures and SFC curve of the CBE were similar to those of the CB. In addition, the CBE could be mixed with CB in any ratio without an obvious eutectic phenomena. Up to 40% CBE could be added to CB without significantly affecting the thermodynamic properties of CB. Thus, replacing CB with the CBE product is feasible.


Assuntos
Gorduras na Dieta , Lipase/química , Óleo de Palmeira/química , Rhizomucor/enzimologia , Ácidos Esteáricos/química , Catálise , Cristalização , Esterificação , Estudos de Viabilidade , Lipase/metabolismo , Termodinâmica , Triglicerídeos/química
6.
Protein Expr Purif ; 180: 105804, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33276128

RESUMO

Lipase from Rhizomucor miehei (RML) is a promising biocatalyst used in food industry, fine chemicals, and biodiesel production. Yeast surface display allows direct application of lipase in form of whole-cell biocatalyst, avoiding purification and immobilization process, but the protease of the host cell may affect the activity of displayed lipase. Herein, we used the protease-deficient Pichia pastoris, PichiaPink™ as host to display RML efficiently. RML gene, GCW21 gene and α-factor gene were co-cloned into plasmid pPink LC/HC and transformed into protease-deficient P. pastoris. After inducution expression for 96 h, the lipase activity of displayed RML reached 121.72 U/g in proteinase-A-deficient P. pastoris harboring high-copy plasmid, which exhibited 46.7% higher than recombinant P. pastoris without protease defect. Displayed RML occurred the maximum activity at pH 8.0 and 45 °C and the optimal substrate was p-nitrophenyl octanoate. Metal ions Li+, Na+, K+, and Mg2+ of 1-10 mM had activation towards displayed RML. Displayed RML was effectively improved in PichiaPink™ protease-deficient system, which may promote the further research and development for the industrial application of RML.


Assuntos
Técnicas de Visualização da Superfície Celular , Proteínas Fúngicas/biossíntese , Lipase/biossíntese , Rhizomucor/genética , Saccharomycetales , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Lipase/química , Lipase/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Rhizomucor/enzimologia , Saccharomycetales/genética , Saccharomycetales/metabolismo
7.
Chem Commun (Camb) ; 56(67): 9683-9686, 2020 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-32696765

RESUMO

By using a isocyanide-based multi-component reaction for the immobilization of the soluble forms of Rhizomucor miehei lipase (RML) and Thermomyces lanuginosa lipase (TLL), the first step of enzyme aggregation or crystallization in the traditional methods of cross-linking was bypassed. High immobilization yields and specific activities were achieved for both lipases.


Assuntos
Lipase/metabolismo , Cristalização , Cianetos/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Eurotiales/enzimologia , Lipase/química , Tamanho da Partícula , Rhizomucor/enzimologia
8.
Bioorg Chem ; 99: 103888, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32388204

RESUMO

Lipase TLIM was reported to be an efficient, commercially available and reusable catalyst for the Knoevenagel-Michael cascade reactions of aldehydes, malononitrile/ethyl cyanoacetate and 4-hydroxycoumarin/1, 3-cyclohexanedione/dimedone in aqueous DMSO. This methodology presents many superiorities such as simple procedure, mild reaction conditions, commercially available and reusable catalyst, high substrate applicability, the ability to be scaled up, and good to excellent yields.


Assuntos
Benzopiranos/metabolismo , Lipase/metabolismo , Benzopiranos/química , Biocatálise , Estrutura Molecular , Rhizomucor/enzimologia
9.
Enzyme Microb Technol ; 137: 109535, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32423679

RESUMO

A strategy to obtain biocatalysts formed by three enzyme layers has been designed using lipases A and B from Candida antarctica (CALA and CALB), the lipases from Rhizomucor miehei (RML) and Thermomyces lanuginosus (TLL), and the artificial chimeric phospholipase Lecitase Ultra (LEU). The enzymes were initially immobilized via interfacial activation on octyl-agarose beads, treated with polyethylenimine (PEI) and a new enzyme layer was immobilized on the octyl-enzyme-PEI composite by ion exchange, producing octyl-enzyme-PEI-enzyme biocatalysts. Except when using LEU, when the two-layer biocatalysts, a large percentage of the PEI-immobilized enzyme was released when a new batch of PEI was added. This was prevented by glutaraldehyde crosslinking. The enzyme modifications produced more active preparations in some cases while in other cases, the effect of the modifications was negative for enzyme activity. These effects of the enzymes modifications were also different when the enzyme was immobilized by interfacial activation or by ion exchange. In all cases, the 3-layer biocatalysts were more active than the single- or bi-layer biocatalysts with some of the assayed substrates. However, as the substrate diffusion problems increased when new enzyme layers were added, even a decrease in enzyme activity with some substrates was found after increasing the number of enzyme layers.


Assuntos
Biocatálise , Enzimas Imobilizadas/metabolismo , Lipase/metabolismo , Polietilenoimina/metabolismo , Sefarose/metabolismo , Candida/enzimologia , Estabilidade Enzimática , Proteínas Fúngicas/metabolismo , Glutaral/metabolismo , Cinética , Rhizomucor/enzimologia
10.
Bioengineered ; 11(1): 375-385, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32175802

RESUMO

Rhizomucor miehei lipase (RML) is a biocatalyst that widely used in laboratory and industrial. Previously, RML with a 70-amino acid propeptide (pRML) was cloned and expressed in P. pastoris. Recombinant strains with (strain containing 4-copy prml) and without ER stress (strain containing 2-copy prml) were obtained. However, the effective expression of pRML in P. pastoris by coexpressing ER-related elements in pRML-produced strain with or without ER stress has not been reported to date. In this study, an efficient way to produce functional pRML was explored in P. pastoris. The coexpression of protein folding chaperones, including PDI and ERO1, in different strains with or without ER stress, was investigated. PDI overexpression only increased pRML production in 4-copy strain from 705 U/mL to 1430 U/mL because it alleviated the protein folded stress, increased the protein concentration from 0.56  mg/mL to 0.65 mg/mL, and improved enzyme-specific activity from 1238 U/mg to 2186 U/mg. However, PDI coexpression could not improve pRML production in the 2-copy strain because it increased protein folded stress, while ERO1 coexpression in the two strains all had a negative effect on pRML expression. We also investigated the effect of the propeptide on the substrate specificity and the condition for pRML enzyme powder preparation. Results showed that the relative activity exceeded 80% when the substrates C8-C10 were detected at 35°C and pH 6, and C8-C12 at 45°C and pH 8. The optimal enzyme powder preparation pH was 7, and the maximum recovery rate for pRML was 73.19%.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Lipase/metabolismo , Pichia/enzimologia , Rhizomucor/enzimologia , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Regulação Fúngica da Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Dobramento de Proteína
11.
Molecules ; 25(3)2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-32012738

RESUMO

Functional properties of each enzyme strictly depend on immobilization protocol used for linking enzyme and carrier. Different strategies were applied to prepare the immobilized derivatives of Rhizomucor miehei lipase (RML) and chemically aminated RML (NH2-RML). Both RML and NH2-RML forms were covalently immobilized on glyoxyl sepharose (Gx-RML and Gx-NH2-RML), glyoxyl sepharose dithiothreitol (Gx-DTT-RML and Gx-DTT-NH2-RML), activated sepharose with cyanogen bromide (CNBr-RML and CNBr-NH2-RML) and heterofunctional epoxy support partially modified with iminodiacetic acid (epoxy-IDA-RML and epoxy-IDA-NH2-RML). Immobilization varied from 11% up to 88% yields producing specific activities ranging from 0.5 up to 1.9 UI/mg. Great improvement in thermal stability for Gx-DTT-NH2-RML and epoxy-IDA-NH2-RML derivatives was obtained by retaining 49% and 37% of their initial activities at 70 °C, respectively. The regioselectivity of each derivative was also examined in hydrolysis of fish oil at three different conditions. All the derivatives were selective between cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in favor of EPA. The highest selectivity (32.9 folds) was observed for epoxy-IDA-NH2-RML derivative in the hydrolysis reaction performed at pH 5 and 4 °C. Recyclability study showed good capability of the immobilized biocatalysts to be used repeatedly, retaining 50-91% of their initial activities after five cycles of the reaction.


Assuntos
Enzimas Imobilizadas/química , Óleos de Peixe/química , Lipase/química , Rhizomucor/enzimologia , Catálise , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Solventes/química , Temperatura
12.
Appl Biochem Biotechnol ; 191(3): 1294-1314, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32096059

RESUMO

DHA-rich medium- and long-chain triacylglycerols (MLCT) were produced by lipase-catalyzed acidolysis of microbial oil from Schizochytrium sp. with medium-chain fatty acids (MCFA). Four commercial lipases, i.e., NS40086, Novozym 435, Lipozyme RM IM, and Lipozyme TL IM were screened based on their activity and fatty acid specificity. The selected conditions for MLCT synthesis were Lipozyme RM IM as catalyst, reaction time 6 h, lipase load 8 wt%, substrate molar ratio (MCFA/microbial oil) 3:1, and temperature 55 °C. Under the selected conditions, the lipase could be reused successively for 17 cycles without significant loss of lipase activity. The obtained product contained 27.53% MCFA, 95.29% at sn-1,3 positions, and 44.70% DHA, 69.77% at sn-2 position. Fifty-nine types of triacylglycerols (TAG) were identified, in which 35 types of TAG contained MCFA, the content accounting for 55.35%. This product enriched with DHA at sn-2 position and MCFA at sn-1,3 positions can improve its digestion and absorption under an infant's digestive system, and thus has potential to be used in infant formula to increase the bioavailability of DHA.


Assuntos
Ácidos Docosa-Hexaenoicos/química , Ácidos Graxos/química , Lipase/metabolismo , Estramenópilas/metabolismo , Triglicerídeos/biossíntese , Aspergillus oryzae/enzimologia , Basidiomycota/enzimologia , Catálise , Esterificação , Microbiologia Industrial , Rhizomucor/enzimologia , Espectrometria de Massas em Tandem , Temperatura
13.
Food Chem ; 305: 125447, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31499289

RESUMO

A novel α-amylase gene (RmAmyA) from Rhizomucor miehei was cloned and expressed in Pichia pastoris. RmAmyA showed 70% amino acid identity with the α-amylase from Rhizomucor pusillus. A high α-amylase activity of 29,794.2 U/mL was found through high cell density fermentation. The molecular mass of RmAmyA was determined to be 49.9 kDa via SDS-PAGE. RmAmyA was optimally active at 75 °C and pH 6.0, and it did not require Ca2+ to improve its activity. It exhibited broad substrate specificity towards amylose, amylopectin, soluble starch, pullulan, and cyclodextrins. High level of maltose (54%, w/w) was produced after liquefied starch was hydrolysed with RmAmyA for 16 h. Moreover, the addition of RmAmyA into Chinese steamed bread resulted in 7.7% increment in the specific volume, and 17.2% and 11.5% reduction in the chewiness and hardness, respectively. These results indicate that RmAmyA might be a potential candidate for applications in the food industry.


Assuntos
Maltose/metabolismo , Rhizomucor/enzimologia , alfa-Amilases/metabolismo , Pão/análise , Indústria Alimentícia , Concentração de Íons de Hidrogênio , Hidrólise , Pichia/metabolismo , Amido/metabolismo , Especificidade por Substrato , Temperatura , alfa-Amilases/química , alfa-Amilases/genética
14.
Int J Biol Macromol ; 145: 856-864, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31655153

RESUMO

This paper shows the step by step coimmobilization of up to five different enzymes following two different orders in the coimmobilization to alter the effect of substrate diffusion limitations. The enzymes were the lipases A and B from Candida antarctica, the lipases from Rhizomocur miehei and, Themomyces lanuginosus and the phospholipase Lecitase Ultra. The utilized strategy was a layer by layer immobilization, coating the immobilized enzymes with polyethylenimine followed by the crosslinking of the enzyme and PEI with glutaraldehyde to prevent enzyme release, and them adding a new lipase layer. The use of previously inactivated biocatalysts (using diethyl p-nitrophenylphosphate) permitted to visualize the immobilization of each enzyme layer, which was later confirmed by SDS-PAGE. This also confirmed the successful and complete covalent crosslinking of the glutaraldehyde treated enzyme layers. Activity of the combibiocatalysts was followed using diverse substrates. The protocol was successful and permitted to immobilize in an ordered way the 5 different enzymes in a down-up distribution.


Assuntos
Enzimas Imobilizadas/metabolismo , Lipase/metabolismo , Candida/enzimologia , Candida/metabolismo , Estabilidade Enzimática/fisiologia , Eurotiales/enzimologia , Eurotiales/metabolismo , Proteínas Fúngicas/metabolismo , Glutaral/metabolismo , Fosfolipases/metabolismo , Polietilenoimina/metabolismo , Rhizomucor/enzimologia , Rhizomucor/metabolismo
15.
J Biotechnol ; 306: 16-23, 2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31520680

RESUMO

Rhizomucor miehei lipase (RML), a GRAS catalyst with wide applications, was overexpressed in Yarrowia lipolytica, also a GRAS unconventional yeast, via a combined strategy, optimization for promoter, gene dosage and fermentation process. The lipase activity of the recombinant strain was first increased from 19.5 to 26.9 U/mL via codon optimization of rml gene. Subsequently, a method was developed for constructing hybrid promoters harboring different copy number of upstream activation sequences fragment (UAS1B), and the recombinant strain Po1g/hp12d-rml 25# reached 38.9 U/mL. On this basis, expression vectors with different optimized rml gene copy numbers were constructed and introduced into Y. lipolytica Po1g. The recombinant strain Po1g/hp12d-2rml 14# carrying 12 copies of UAS1B in the upstream of pLEUmin and 2 copies of rml gene obtained the highest lipase activity of 59.6 U/mL. Moreover, in optimized shaking flask culture parameters: 5% (m/v) of d-Sorbitol, 2% (v/v) inoculation density, initial pH 7.0, and 30 mL initial culture medium, the RML activity of Po1g/hp12d-2rml 14# further reached 157 U/mL after 84-h of incubation at 28 ℃. Overall, RML activity was enhanced about 8-fold compared with the initial recombinant strain via the combined strategy, which provides a consolidated basis for the large-scale production of RML in Y. lipolytica to match urgent demand of the market.


Assuntos
Proteínas Fúngicas/genética , Lipase/genética , Rhizomucor/enzimologia , Yarrowia/genética , Meios de Cultura , Fermentação , Proteínas Fúngicas/metabolismo , Dosagem de Genes , Expressão Gênica , Vetores Genéticos , Lipase/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhizomucor/genética , Yarrowia/metabolismo
16.
Biomolecules ; 9(8)2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31404957

RESUMO

Isomaltulose is mainly produced from sucrose by microbial fermentation, when the utilization of sucrose contributes a high production cost. To achieve a low-cost isomaltulose production, soy molasses was introduced as an alternative substrate. Firstly, α-galactosidase gene from Rhizomucor miehei was expressed in Yarrowia lipolytica, which then showed a galactosidase activity of 121.6 U/mL. Under the effects of the recombinant α-galactosidase, most of the raffinose-family oligosaccharides in soy molasses were hydrolyzed into sucrose. Then the soy molasses hydrolysate with high sucrose content (22.04%, w/w) was supplemented into the medium, with an isomaltulose production of 209.4 g/L, and the yield of 0.95 g/g. Finally, by virtue of the bioremoval process using Pichia stipitis, sugar byproducts in broth were transformed into ethanol at the end of fermentation, thus resulting in high isomaltulose purity (97.8%). The bioprocess employed in this study provides a novel strategy for low-cost and efficient isomaltulose production from soybean molasses.


Assuntos
Etanol/metabolismo , Fermentação , Isomaltose/análogos & derivados , Melaço , Soja/classificação , alfa-Galactosidase/metabolismo , Etanol/química , Concentração de Íons de Hidrogênio , Hidrólise , Isomaltose/química , Isomaltose/metabolismo , Rhizomucor/enzimologia , Temperatura
17.
Food Funct ; 10(6): 3142-3149, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31157352

RESUMO

In this work, Candida antarctica lipase A was applied to selectively remove saturated fatty acids from palm oil to prepare palm oil acylglycerol concentrate (POAC), where palmitic acid decreased from 40.0 to 28.7% and oleic acid increased from 40.0 to 50.5% after 3 h of hydrolysis. Lipozyme RMIM from Rhizomucor miehei was then used to incorporate either alpha linolenic acid (ALA) or eicosapentaenoic acid (EPA) into the resulting POAC. Optimum omega-3 incorporation was achieved when POAC to omega-3 ratio was 6 : 3, reaction temperature was 40 °C and reaction time was 18 h. Under these conditions, the ALA content in the separated ALA incorporated structured lipid (POAC-ALA) was 27.1%, and the EPA content in the EPA incorporated structured lipids (POAC-EPA) was 30.9%. The formed structured lipids had lower levels of saturated fatty acids, and significantly lower melting points, in both cases below 8 °C. The enzymatic process developed produces new structured lipids, with lower saturated fat and higher omega-3, with potential as a healthy palm oil derived lipid ingredient.


Assuntos
Ácidos Graxos Ômega-3/química , Proteínas Fúngicas/química , Lipase/química , Óleo de Palmeira/química , Biocatálise , Candida/enzimologia , Ácido Oleico/química , Ácido Palmítico/química , Rhizomucor/enzimologia
18.
J Biosci Bioeng ; 128(4): 416-423, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31130335

RESUMO

ß-Mannosidase (EC 3.2.1.25) is an exoglycosidase specific for the hydrolysis of terminal ß-1,4-glycosidic linkage in mannan which can be applied in the food manufacture, animal feed, bioethanol making and coffee extraction industries. A novel ß-mannosidase gene (Lrman5A) from Lichtheimia ramosa was synthesized and recombinantly expressed in Pichia pastoris X33. Lrman5A encodes 444 amino acids with a calculated molecular mass of 51.0 kDa which shares the highest identity (73%) with the ß-mannosidase from Rhizomucor miehei. Purified recombinant Lrman5A showed the maximal activity at pH 6.0 and 65°C, had broad-range pH stability (retaining >65% activity after incubation at pH 3.0-8.5 at 37°C for 24 h), and was highly thermostable (retaining >80% activity after incubation at 65°C for 10 min). The specific activity, and Km of Lrman5A was 17.5 U/mg and 1.377 mM, respectively. Lrman5A and GH5 ß-mannanase displayed significant synergistic effects on the degradation of locust bean gum (LBG) and released more mannose (up to 2.89 folds) by simultaneous or sequential addition. Due to its hydrolytic properties, Lrman5A may have potential applications in the area of bioenergy, feed and food processing.


Assuntos
Galactanos/metabolismo , Mananas/metabolismo , Mucorales/enzimologia , Gomas Vegetais/metabolismo , beta-Manosidase/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Mucorales/genética , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhizomucor/enzimologia , Rhizomucor/genética , beta-Manosidase/genética
19.
Bioorg Med Chem Lett ; 29(10): 1236-1240, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30898405

RESUMO

Lipase RMIM was firstly used as a promiscuous biocatalyst to catalyze the Knoevenagel-Michael cascade reactions of 4-hydroxycoumarin with aromatic, heterocyclic or aliphatic aldehydes to synthesize dicoumarol derivatives in water. Results showed that the adopted methodology could offer many advantages, such as mild reaction conditions, pure aqueous reaction system, wide substrate applicability, recyclable catalyst, excellent yields (81-98%), operational simplicity, and environmentally friendly reactions.


Assuntos
Dicumarol/síntese química , Lipase/química , Rhizomucor/enzimologia , Aldeídos/química , Catálise , Dicumarol/análogos & derivados , Química Verde/métodos , Estrutura Molecular , Temperatura , Fatores de Tempo , Água/química
20.
Food Chem ; 271: 739-746, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30236739

RESUMO

In this study, mesoporous silica SBA-15 was modified by organic functional groups through silanization. Series of organosilane compounds were grafted onto the SBA-15, and the obtained functionalized carriers were then used to immobilize the lipase from Rhizomucor miehei (RML). The enzymatic properties of the obtained immobilized RML samples were evaluated, and the catalytic efficiencies in glycerolysis of triacylglycerols (TAG) reaction were studied. Compared with the parent SBA-15 immobilized RML, the organic modification gave a maximum improvement of enzymatic activity from 200.00 to 13211.11 U/g; in addition, TAG conversion and diacylglycerols (DAG) content increased from 21.28 to 84.24% and 15.45 to 59.03% respectively. The organic modification also decreased the sensitivity of immobilized RML in extreme pH values and increased their thermostability.


Assuntos
Enzimas Imobilizadas/química , Lipase/química , Rhizomucor/enzimologia , Diglicerídeos , Dióxido de Silício
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