Search

Nowadays, breast milk is considered as the ideal food for infants owing to the most common oleic acid-palmitic acid-oleic acid (OA-PA-OA) fatty acid distribution of the human milk fat (HMF). This study reports the synthesis of 1,3-dioleoyl-2-palmotoylglycerol (OPO)-rich human milk fat substitutes in a two-step enzymatic acidolysis reaction with Rhizomucor miehei lipase (RML) immobilized on magnetic multi-walled carbon nanotubes(mMWCNTs). The immobilized RML (RML-mMWCNTs) showed better thermal and pH stability, convenient recovery and reusability than the free soluble form. Under optimized reaction conditions (1:8 tripalmitin (PPP)/OA, 10%wt. enzyme, 50 °C, 5 h), PA content at the sn-2 position and OA incorporation at the sn-1,3 positions reached 93.46% and 59.54%, respectively. Comparison tests have also showed that RML-mMWCNTs has better catalytic activity and reusability than the commercial lipase Lipozyme RM IM. The results suggest that RML-mMWCNTs is a promising biocatalyst for the synthesis of OPO-rich TAGs with potential use in infant formulas.

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.

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 from Rhizomucor miehei at 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.

The aim of this work was to produce diacylglycerols (DAG) and monoacylglycerols (MAG) with a high content of polyunsaturated fatty acids (PUFA). Rhizomucor miehei lipase mediated-hydrolysis of sardine oil was conducted at several water activities. The system was mechanistically modeled to predict the time evolution of the concentration of triacylglycerols, DAG, MAG and free fatty acids (FFA) and the concentration of saturated, mono- and polyunsaturated fatty acids. The release of the first fatty acid from the triacylglycerol was independent on the unsaturation degree. Contrary, the hydrolysis of the second one was highly affected by the degree of unsaturation, PUFA being the fatty acids that showed the highest resistance to hydrolysis. MAG percentage was maximum (7 mol%) at lower water activities, while DAG content was favored at higher water activities (35 mol%), achieving a 2-fold concentration of DHA.

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.

The development of sensitive, easy and reliable methods for the determination of Rhizomucor pusillus rennin (MPR) activity, in free and in immobilized form, along with the elucidation of the mechanism of action, represent challenges for the widespread use of the enzyme in industrial cheese production. These could be accomplished by using highly specific and sensitive substrates, as well as direct assay methods. We designed and synthesized novel substrates based on Fluorescence Resonance Energy Transfer (FRET) for the MPR by employing computational simulation techniques and peptide synthesis in liquid phase. Three FRET-substrates (Abz-GFY-pNA, Abz-SFY-pNA and Abz-GFI-pNA) were found active, while the Abz-GFY-pNA showed the highest reliability, sensitivity and specificity among them. Subsequently, a novel mechanism of MPR action was elucidated, with the development of novel methods for assaying activity in free and immobilized form, which both may contribute in the wider use of rennin in cheese production and other biotechnological applications.

Lipases B from Candida antarctica (CALB), Rhizomucor miehei (RML) and Thermomyces lanuginosus (TLL) were immobilized on octadecyl methacylate (OM) or octadecyl methacrylate (OMC) beads. Their specific activity and regioselectivity were studied in the synthesis of conjugated linoleic acid (CLA) partial glycerides, which presented nutraceutical properties. TLL derivatives were poor catalysts. Novozym® 435 was much better than Lipozyme® RM IM. RML activity (a GRAS enzyme) was modulated via immobilization. After only 3 h, OM−RML gave the highest CLA conversion (54% at 40 °C with 1:3 M ratio of glycerol to CLA). OM-RML reduced by a factor of 3.12 and 1.16 the activation energy of the reaction with Lipozyme® RM IM and Novozym® 435, respectively. The new GRAS preparation OM-RML brings forth an optimal regioselective preparation of sn -1 mono and sn -1,3 diacylglycerols rich in CLA, with a ratio of sn -1,3/ sn -1,2 regioisomers of 21.8, compared to 2.3 for Novozym® 435.

A novel aspartic protease gene (RmproA) was cloned from the thermophilic fungus Rhizomucor miehei CAU432 and expressed in Pichia pastoris. The RmproA was successfully expressed in P. pastoris as an active extracellular protease. High protease activity of 3480.4 U/mL was obtained by high cell-density fermentation. The protease was purified by the two step protocols to homogeneity. The molecular mass of the RmproA was estimated to be 52.4 kDa by SDS-PAGE and 50.6 kDa by gel filtration. The purified enzyme was optimally active at pH 5.5 and 55 °C, respectively. The enzyme exhibited a broad range of substrate specificity. RmproA-treated pork muscle showed lower shear force than papain-treated sample at a relative low concentration, suggesting its effectiveness on meat tenderization. Moreover, turtle hydrolysis by RmproA resulted in a large amount of small peptides, which exhibited high ACE-inhibitory activity. Thus, RmproA may be a potential candidate for several industrial applications.

A gene (RmGBE) encoding a glycogen branching enzyme from Rhizomucor miehei was cloned into the pET28a (+) vector and expressed in Escherichia coli, and biochemically analysed. RmGBE had an open reading frame of 2097bp encoding 698 amino acid residues. The purified enzyme was a monomer of 78.1kDa. RmGBE was optimally active at 25°C and pH 7.5. It displayed excellent cold adaptation over a low temperature range of 10-30°C, retaining over 85% of its relative activity. RmGBE showed the highest specificity to amylose, about ten times higher than to amylopectin. Addition of RmGBE to wheat bread resulted in a 26% increase in specific volume and a 38% decrease in crumb firmness in comparison with the control. Besides, the retrogradation of bread was significantly retarded along with the enzyme reaction. These properties make RmGBE highly useful in the food and starch industries.

The synthesis of docosahexaenoyl triacylglycerides at low temperature (e.g., 50°C) using biocatalysts of 6 commercial lipases adsorbed on hydrophobic supports was studied. In general, the triacylglyceride yields were very low with the exceptions of those produced with the enzymes from Candida antarctica fraction B, CALB (82%), and those produced with the enzyme from Pseudomonas fluorescens, PFL (57%). The reactions were performed under vacuum to remove the released ethanol. The yields varied widely when different derivatives of CALB were used, and they were higher when CALB adsorbed on hydrophobic supports was used (82%). One interesting by-product (18% of sn-2 monoacylglyceride of DHA) remained at the end of the synthetic process. CALB adsorbed on Sepabeads exhibited better activity and stability than did the commercial derivative Novozym 435. The best CALB biocatalyst preserved 90% of the activity after 30 days under the reaction conditions.

A novel β-glucosidase gene, designated RmBglu3B , was cloned from the thermophilic fungus, Rhizomucor miehei CAU432. Its 2196-bp open reading frame encoded 731 amino acids. Its deduced amino-acid sequence showed highest identity (66%) with a glycoside hydrolase family 3 β-glucosidase from R. miehei NRRL5382. RmBglu3B was successfully expressed in Escherichia coli . The recombinant enzyme was purified to homogeneity with 18.2-fold purification and 59% recovery yield. Molecular masses of 76.5 kDa, by SDS–PAGE, and 66.4 kDa, by gel filtration, suggested that it is a monomer. Optimal pH and temperature of the purified enzyme were 5.0 and 50 °C, respectively. RmBglu3B exhibited a broad range of substrate specificity, catalyzing the cleavage of β-1,2, β-1,3, β-1,4 and β-1,6 linkages, in various oligosaccharides, to liberate glucose. RmBglu3B also showed relatively high activity (19.1 U/mg) toward laminaran and transglycosylation activity, enabling gentiobiose production. This enzyme is a potential candidate for several industrial applications.