Enzymatic Synthesis and Characterization of MLM-type Structured Lipid Using Grapeseed Oil and Capric Acid.

The objectives were to optimize the reaction conditions for C10:0 incorporation into grapeseed (GS) oil, characterize the structured lipid (SL) product, and study the changes in antioxidant activity of the SL. Taguchi method was used to optimize C10:0 incorporation by combining parameters in a total of 9 experiments. Lipozyme ® RM IM (Rhizomucor miehei immobilized lipase) and Lipozyme ® 435 (Candida antarctica recombinant immobilized lipase) were used as biocatalysts for the acidolysis reactions. C10:0 incorporation and triacylglycerol (TAG) species of the SL were analyzed to determine optimal conditions and enzyme type that gave higher incorporation. The optimal conditions were the same for both enzymes as follows: substrate molar ratio 1:3 (GS oil: C10:0), enzyme load 5% (w/w) of substrates, temperature 65℃, and time 12 h. HPLC analysis of SL gave MLM-type TAG species of 11.51±0.11 mol% and 12.68±0.34 mol% for Lipozyme ® RM IM and Lipozyme ® 435, respectively. GC analysis indicated that C10:0 incorporated at the sn-1,3 positions of the SL were 46.03±0.55 mol% and 47.28±1.22 mol%, respectively, for Lipozyme ® RM IM and Lipozyme ® 435. However, the total C10:0 incorporated into TAG species with Lipozyme ® RM IM was significantly higher (60.08±0.04 mol%) compared to 50.78±0.44 mol% for Lipozyme ® 435. Scaled-up (300 g) acidolysis reaction and characterization were done on SL synthesized using Lipozyme ® RM IM. SL reaction product was purified using short path distillation and fully characterized in terms of lipid classes, tocopherol, thermal behavior, and oxidative stability. The yield of purified scaled-up SL after short path distillation (SPD) was 72.96 wt%. The antioxidant in SL was reduced after SPD due to loss of tocopherols. This MLM-type-SL synthesized within 12 h using Lipozyme ® RM IM had a high content of C10:0 and may have functional and health benefits.

A Whole-Process Visible Strategy for the Preparation of Rhizomucor miehei Lipase with Escherichia coli Secretion Expression System and the Immobilization.

BACKGROUND: Rhizomucor miehei (RM) lipase is a regioselective lipase widely used in food, pharmaceutical and biofuel industries. However, the high cost and low purity of the commercial RM lipase limit its industrial applications. Therefore, it is necessary to develop cost-effective strategies for large-scale preparation of this lipase. The present study explored the high-level expression of RM lipase using superfolder green fluorescent protein (sfGFP)-mediated Escherichia coli secretion system. RESULTS: The sfGFP(-15) mutant was fused to the C-terminus of RM lipase to mediate its secretion expression. The yield of the fusion protein reached approximately 5.1 g/L with high-density fermentation in 5-L fermentors. Unlike conventional secretion expression methods, only a small portion of the target protein was secreted into the cell culture while majority of the fusion protein was still remained in the cytoplasm. However, in contrast to intracellular expression, the target protein in the cytoplasm could be transported efficiently to the supernatant through a simple washing step with equal volume of phosphate saline (PBS), without causing cell disruption. Hence, the approach facilitated the downstream purification step of the recombinant RM lipase. Moreover, contamination or decline of the engineered strain and degradation or deactivation of the target enzyme can be detected efficiently because they exhibited bright green fluorescence. Next, the target protein was immobilized with anion-exchange and macropore resins. Diethylaminoethyl sepharose (DEAE), a weak-basic anion-exchange resin, exhibited the highest bind capacity but inhibited the activity of RM lipase dramatically. On the contrary, RM lipase fixed with macropore resin D101 demonstrated the highest specific activity. Although immobilization with D101 didn't improve the activity of the enzyme, the thermostability of the immobilized enzyme elevated significantly. The immobilized RM lipase retained approximately 90% of its activity after 3-h incubation at 80 °C. Therefore, D101 was chosen as the supporting material of the target protein. CONCLUSION: The present study established a highly efficient strategy for large-scale preparation of RM lipase. This innovative technique not only provides high-purity RM lipase at a low cost but also has great potential as a platform for the preparation of lipases in the future.

Deciphering the immobilization of lipases on hydrophobic wrinkled silica nanoparticles.

In this work, the adsorption of Candida antarctica B (CALB) and Rhizomucor miehei (RML) lipases into hydrophobic wrinkled silica nanoparticles (WSNs) is investigated. WSNs are hydrophobized by chemical vapor deposition. Both proteins are homogeneously distributed inside the pores of the nanoparticles, as confirmed by Transmission Electron Microscopy and Energy Dispersive X-ray measurements. The maximum enzyme load of CALB is twice that obtained for RML. Fourier Transform Infrared Spectroscopy confirms the preservation of the enzyme secondary structure after immobilization for both enzymes. Adsorption isotherms fit to a Langmuir model, resulting in a binding constant (KL) for RML 4.5-fold higher than that for CALB, indicating stronger binding for the former. Kinetic analysis reveals a positive correlation between enzyme load and RML activity unlike CALB where activity decreases along the enzyme load increases. Immobilization allows for enhancing the thermal stability of both lipases. Finally, CALB outperforms RML in the hydrolysis of ethyl-3-hydroxybutyrate. However, immobilized CALB yielded 20 % less 3-HBA than free lipase, while immobilized RML increases 3-fold the 3-HBA yield when compared with the free enzyme. The improved performance of immobilized RML can be explained due to the interfacial hyperactivation undergone by this lipase when immobilized on the superhydrophobic surface of WSNs.

Lipase in-situ immobilized in covalent organic framework: Enzymatic properties and application in the preparation of 1, 3-dioleoyl-2-palmitoylglycerol.

In this study, the critical importance of designing an appropriate immobilized carrier and method for free lipase to ensure exceptional biological catalytic activity and stability was emphasized. Covalent organic frameworks (COF-1) were synthesized as a novel porous carrier with an azine structure (-CN-NC-) through the condensation of hydrazine hydrate and benzene-1,3,5-tricarbaldehyde at room temperature. Simultaneously, Rhizomucor miehei lipase (RML) was immobilized within the COF-1 carrier using an in-situ aqueous phase method. Characterization of the carrier and RML@COF-1 and evaluation of the lipase properties of RML and RML@COF-1 through p-Nitrophenyl palmitate hydrolysis were conducted. Additionally, application in the synthesis of 1, 3-dioleoyl-2-palmitoylglycerol (OPO) was explored. The results showed that RML@COF-1 exhibited a high enzymatic loading of 285.4 mg/g. Under 60℃ conditions, the activity of RML@COF-1 was 2.31 times higher than that of free RML, and RML@COF-1 retained 77.25% of its original activity after 10 cycles of repeated use, indicating its excellent thermal stability and repeatability. Under the optimal conditions (10%, 1:8 PPP/OA, 45℃, 5 h), the yield of OPO reached 47.35%, showcasing the promising application prospects of the novel immobilized enzyme synthesized via in-situ aqueous phase synthesis for OPO preparation.

Investigation of Structural Features of Two Related Lipases and the Impact on Fatty Acid Specificity in Vegetable Fats.

One of the indispensable applications of lipases in modification of oils and fats is the possibility to tailor the fatty acid content of triacylglycerols (TAGs), to meet specific requirements from various applications in food, nutrition, and cosmetic industries. Oleic acid (C18:1) and stearic acid (C18:0) are two common long fatty acids in the side chain of triglycerides in plant fats and oils that have similar chemical composition and structures, except for an unsaturated bond between C9 and C10 in oleic acid. Two lipases from Rhizomucor miehei (RML) and Rhizopus oryzae (ROL), show activity in reactions involving oleate and stearate, and share high sequence and structural identity. In this research, the preference for one of these two similar fatty acid side chains was investigated for the two lipases and was related to the respective enzyme structure. From transesterification reactions with 1:1 (molar ratio) mixed ethyl stearate (ES) and ethyl oleate (EO), both RML and ROL showed a higher activity towards EO than ES, but RML showed around 10% higher preference for ES compared with ROL. In silico results showed that stearate has a less stable interaction with the substrate binding crevice in both RML and ROL and higher tendency to freely move out of the substrate binding region, compared with oleate whose structure is more rigid due to the existence of the double bond. However, Trp88 from RML which is an Ala at the identical position in ROL shows a significant stabilization effect in the substrate interaction in RML, especially with stearate as a ligand.

Structural insights into the substrate recognition and catalytic mechanism of a fungal glycoside hydrolase family 81 ß-1,3-glucanase.

ß-1,3-Glucan constitutes a prominent cell wall component being responsible for rigidity and strength of the cell wall structure in filamentous fungi. Glycoside hydrolase (GH) family 81 endo-ß-1,3-glucanases which can cleave the long chain of ß-1,3-glucans play a major role in fungal cell wall remodeling. Here, we reported the complex structures of a fungal GH family 81 endo-ß-1,3-glucanase from Rhizomucor miehei (RmLam81A), revealing the triple-helical ß-glucan recognition and hydrolysis patterns. In the crystals, three structured oligosaccharide ligands simultaneously interact with one enzyme molecular via seven glucose residues, and the spatial arrangement of ligands to RmLam81A was almost identical to that of ß-1,3-glucan triple-helical structure. RmLam81A performed an inverting catalysis mechanism with Asp475 and Glu557 severing as the general acid and base catalyst, respectively. Furthermore, two hydrophobic patches involving Tyr93, Tyr106, Ile108, Phe619 and Tyr628 alongside the ligand-binding site possibly formed parts of the binding site. A ligand-binding motif, ß31-ß32, consisting of two key residues (Lys622 and Asp624), involved the recognition of a triple-helical ß-glucan. Our results provided a structural basis for the unique ß-1,3-glucan recognition pattern and catalytic mechanism of fungal GH family 81 endo-ß-1,3-glucanases, which may be helpful in further understanding the diverse physiological functions of ß-1,3-glucanases.

[Molecular modification and highly efficient expression of L-asparaginase from Rhizomucor miehei].

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.

High level expression of a xyloglucanase from Rhizomucor miehei in Pichia pastoris for production of xyloglucan oligosaccharides and its application in yoghurt.

The xyloglucanase gene (RmXEG12A) from Rhizomucor miehei CAU432 was successfully expressed in Pichia pastoris. The highest xyloglucanase activity of 25,700 U mL-1 was secreted using high cell density fermentation. RmXEG12A was optimally active at pH 7.0 and 65 °C, respectively. The xyloglucanase exhibited the highest specific activity towards xyloglucan (7915.5 U mg-1). RmXEG12A was subjected to hydrolyze tamarind powder to produce xyloglucan oligosaccharides with the degree of polymerization (DP) 7-9. The hydrolysis ratio of xyloglucan in tamarind powder was 89.8%. Moreover, xyloglucan oligosaccharides (2.0%, w/w) improved the water holding capacity (WHC) of yoghurt by 1.1-fold and promoted the growth of Lactobacillus bulgaricus and Streptococcus thermophiles by 2.3 and 1.6-fold, respectively. Therefore, a suitable xyloglucanase for tamarind powder hydrolysis was expressed in P. pastoris at high level and xyloglucan oligosaccharides improved the quality of yoghurt.

Crystal structure of a chitinase (RmChiA) from the thermophilic fungus Rhizomucor miehei with a real active site tunnel.

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.

Application of Rhizomucor miehei lipase-displaying Pichia pastoris whole cell for biodiesel production using agro-industrial residuals as substrate.

This work aimed the application of a new biocatalyst for biodiesel production from residual agro-industrial fatty acids. A recombinant Pichia pastoris displaying lipase from Rhizomucor miehei (RML) on the cell surface, using the PIR-1 anchor system, were prepared using glycerol as the carbon source. The biocatalyst, named RML-PIR1 showed optimum temperature of 45 °C (74.0 U/L). The stability tests resulted in t1/2 of 3.49 and 2.15 h at 40 and 45 °C, respectively. RML-PIR1 was applied in esterification reactions using industrial co-products as substrates, palm fatty acid distillate (PFAD) and soybean fatty acid distillate (SFAD). The highest productivity was observed for SFAD after 48 h presenting 79.1% of conversion using only 10% of biocatalyst and free-solvent system. This is about ca. eight times higher than commercial free RML in the same conditions. The stabilizing agents study revealed that the treatment using glutaraldehyde (GA) and poly(ethylene glycol) (PEG) enabled increased stability and reuse of biocatalyst. It was observed by SEM analysis that the treatment modified the cell morphology. RML-PIR1-GA presented 87.9% of the initial activity after 6 reuses, whilst the activity of unmodified RML-PIR decreased by 40% after the first use. These results were superior to those obtained in the literature, making this new biocatalyst promising for biotechnological applications, such as the production of biofuels on a large scale.

Recombinant Protein Production and Purification Using Eukaryotic Cell Factories.

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.

Effects of Solvents on the Glycerolysis Performance of the SBA-15 Supported Lipases.

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.

Crude glycerol impurities improve Rhizomucor miehei lipase production by Pichia pastoris.

Crude glycerol, a by-product of biodiesel production, was employed as the carbon source to produce lipase using Pichia pastoris. Under identical fermentation conditions, cell growth and lipase activity were improved using crude glycerol instead of pure glycerol. The impacts of crude glycerol impurities (methyl ester, grease, glycerol, methanol, and metal ions Na+, Ca2+, and Fe3+) on lipase production were investigated. Impurities accelerated P. pastoris entering the stationary phase. Na+, Ca2+, and grease in waste crude glycerol were the main factors influencing higher lipase activity. Through response surface optimization of Ca2+, Na+, and grease concentrations, lipase activity reached 1437 U/mL (15,977 U/mg), which was 2.5 times that of the control. This study highlights the economical and highly efficient valorization of crude glycerol, demonstrating its possible utilization as a carbon source to produce lipase by P. pastoris without pretreatment.

Efficient improvement of surface displayed lipase from Rhizomucor miehei in PichiaPink™ protease-deficient system.

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.

Rhizomucor miehei lipase-catalysed synthesis of cocoa butter equivalent from palm mid-fraction and stearic acid: Characteristics and feasibility as cocoa butter alternative.

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.

Molecular modification and highly efficient expression of L-asparaginase from Rhizomucor miehei / 生物工程学报

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.

Soluble enzyme cross-linking via multi-component reactions: a new generation of cross-linked enzymes.

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.

Use of polyethylenimine to produce immobilized lipase multilayers biocatalysts with very high volumetric activity using octyl-agarose beads: Avoiding enzyme release during multilayer production.

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.

One-pot cascade synthesis of benzopyrans and dihydropyrano[c]chromenes catalyzed by lipase TLIM.

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.

Improved production of recombinant Rhizomucor miehei lipase by coexpressing protein folding chaperones in Pichia pastoris, which triggered ER stress.

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%.