Highly thermostable xylanase purified from Rhizomucor miehei NRL 3169

A thermostable xylanase was purified and characterized from the thermophilic fungus Rhizomucor miehei (Cooney & Emerson) Schipper. The enzyme was purified to homogeneity by ammonium sulfate precipitation, sephadex G-100 gel filtration and diethylaminoethyl cellulose anion exchange chromatography with a 29.1-fold. The enzyme was highly active within a range of pH from 5.0 to 6.5. The optimum temperature of the purified enzyme was 75°C. The enzyme showed high thermal stability at 70°C and 75°C and the half-life of the xylanase at 90°C was 30 min. Km and Vmax values at 50°C of the purified enzyme were 0.055 mg/ml and 113.5 μmol min?1 mg?1 respectively. The enzyme was activated by Ca2+, Cu2+, K+ and Na+. On the other hand, Ag2+, Hg2+, Ba2+, and Zn2+ inhibited the enzyme. The molecular weight of the xylanase was estimated to be 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The present study is among the first works to examine and describe a secreted highly thermostable endoxylanase from the Rhizomucor miehei fungus. This enzyme displays a number of biochemical properties that make it a potentially strong candidate for industrial and commercial application in pulp bleaching.     

Heat shock response in the thermophilic fungus Rhizomucor miehei

Changes in the composition of the membrane lipids and cytosol carbohydrates of the thermophilic fungus Rhizomucor miehei in response to heat shock were studied. Under optimal conditions (41–43°C), high trehalose content (8–11%) was found at all stages of growth of submerged culture. Heat shock (51–53°C) for 1 h did not result in enhanced trehalose synthesis, while increase in shock duration to 3 h resulted in a significant increase in trehalose content. The share of sterols and phosphatidic acids in the membrane lipids increased, while the share of phosphatidylcholines and phosphatidylethanolamines decreased. These processes resulted in increased content of non-bilayer lipids, while the unsaturation degree of the fatty acids of the major phospholipids did not decrease. Comparison of resistance to lethal heat shock in the control and experimental variants of R. miehei revealed that this thermophilic fungus exhibited no acquired heat resistance.     

Production of fungal rennet byMucor miehei using solid state fermentation

Summary Investigations have been carried out on the production of fungal rennet using a thermophilic strain ofMucor miehei under solid state fermentation conditions. A high milk clotting enzyme activity (58000 Soxhlet units/g) was achieved when optimum conditions were used. Further, a high ratio of 6.6:1 between milk clotting and proteolytic activities for this enzyme was obtained. Cheese prepared using this enzyme was also found to be acceptable in organoleptic quality. Large scale production of the enzyme in trays using the optimum conditions gave milk-clotting enzyme activities comparable to those in flask experiments.     

Optimisation of n-octyl oleate enzymatic synthesis over Rhizomucor miehei lipase

Octyl oleate is a useful organic compound with several applications in cosmetic, lubricant and pharmaceutical industry. At first, the enzymatic synthesis of n-octyl oleate by direct lipase-catalysed esterification of oleic acid and 1-octanol was investigated in a stirred batch reactor in solvent-free system. A systematic screening and optimisation of the reaction parameters were performed to gain insight into the kinetics mechanism. Particularly, enzyme concentration, reaction temperature, stirrer speed, water content, substrates concentration and molar ratio were optimised with respect to the final product concentration and reaction rate. The kinetics mechanism of the reaction was investigated. Finally, a comparison of the experimental results obtained in a solvent free-system with those using two different solvents, supercritical carbon dioxide (SC-CO₂) and n-hexane, was proposed. It resulted that in SC-CO₂ higher concentration of the desired product was attained, requiring lower enzyme concentrations to achieve comparable conversion of free fatty acid into fatty acid ester.     

Optimization of Synthesis of Ethyl Isovalerate Using Rhizomucor miehei Lipase

Immobilized lipase from Rhizomucor miehei (Lipozyme IM-20) was used to catalyze the esterification reaction between isovaleric acid and ethanol to synthesize ethyl isovalerate in n-hexane. Response surface methodology based on a four-variable, five-level, central composite rotatable design was employed to optimize four important reaction variables—enzyme/substrate (E/S) ratio, substrate concentration, incubation time, and temperature—affecting the synthesis of ethyl isovalerate. The optimum conditions predicted for achieving maximum ester yield (500 mM) are as follows: E/S ratio, 48.41 g/mol; substrate concentration, 1 M; reaction time, 60 h; and temperature, 60°C. The predicted value matched well with the experimentally obtained value of 487 mM.     

Synthesis of ethyl isovalerate using Rhizomucor miehei lipase: optimization

Immobilized lipase from Rhizomucor miehei (Lipozyme IM-20) was used to catalyze the esterification reaction between isovaleric acid and ethanol to synthesize ethyl isovalerate in n-hexane. Response surface methodology based on five-level four-variable central composite rotatable design was employed to optimize four important reaction variables such as enzyme/substrate E/S ratio, substrate concentration, incubation time, and temperature affecting the synthesis of ethyl isovalerate. The optimum conditions predicted for achieving maximum ester yield (500 mM) are as follows: E/S ratio, 48.41 g/mol; substrate concentration, 1 M; reaction time, 60 h; temperature, 60 degrees C. The predicted value matched well with experimentally obtained value of 487 mM.     

Enzymatic synthesis of isoamyl acetate using immobilized lipase from Rhizomucor miehei

The effects of important reaction parameters for enhancing isoamyl acetate formation through lipase-catalyzed esterification of isoamyl alcohol were investigated in this study. Increase in substrate (acid) concentration led to decrease in conversions. A critical enzyme concentration of 3 g l(-1) was detected for a substrate concentration of 0.06 M (each of alcohol and acid). Solvents with partition coefficient higher than 1000 (log P>3.0) supported enzyme activity to give high conversions. Acetic acid at higher concentrations could not be esterified easily probably owing to its role in lowering the microaqueous pH of the enzyme. Extraneous water/buffer addition decreased the isoamyl acetate yields slightly ( approximately 10%) at 0.005-0.01% v/v of the reaction mixture and drastically (>40%) at above 0.01% v/v. Buffer saturation of the organic solvent employed improved esterification (upto two-fold), particularly at moderately higher substrate concentrations (>0.18 M). Employing acetic anhydride instead of acetic acid resulted in a two-fold increase in the yields (at 0.25 M substrate). Use of excess nucleophile (alcohol) concentration by increasing the alcohol/acid molar ratio resulted in higher conversions in shorter duration (upto eight-fold even at 1.5 M acetic acid). Yields above 80% were achieved with substrate concentrations as high as 1.5 M and more than 150 g l(-1) isoamyl acetate concentrations were obtained employing a relatively low enzyme concentration of 10 g l(-1). The operational stability of lipase was also observed to be reasonably high enabling ten reuses of the biocatalyst.     

Structure-Guided Modification of Rhizomucor miehei Lipase for Production of Structured Lipids

To improve the performance of yeast surface-displayed Rhizomucor miehei lipase (RML) in the production of human milk fat substitute (HMFS), we mutated amino acids in the lipase substrate-binding pocket based on protein hydrophobicity, to improve esterification activity. Five mutants: Asn87Ile, Asn87Ile/Asp91Val, His108Leu/Lys109Ile, Asp256Ile/His257Leu, and His108Leu/Lys109Ile/Asp256Ile/His257Leu were obtained and their hydrolytic and esterification activities were assayed. Using Discovery Studio 3.1 to build models and calculate the binding energy between lipase and substrates, compared to wild-type, the mutant Asp256Ile/His257Leu was found to have significantly lower energy when oleic acid (3.97 KJ/mol decrease) and tripalmitin (7.55 KJ/mol decrease) were substrates. This result was in accordance with the esterification activity of Asp256Ile/His257Leu (2.37-fold of wild-type). The four mutants were also evaluated for the production of HMFS in organic solvent and in a solvent-free system. Asp256Ile/His257Leu had an oleic acid incorporation of 28.27% for catalyzing tripalmitin and oleic acid, and 53.18% for the reaction of palm oil with oleic acid. The efficiency of Asp256Ile/His257Leu was 1.82-fold and 1.65-fold that of the wild-type enzyme for the two reactions. The oleic acid incorporation of Asp256Ile/His257Leu was similar to commercial Lipozyme RM IM for palm oil acidolysis with oleic acid. Yeast surface-displayed RML mutant Asp256Ile/His257Leu is a potential, economically feasible catalyst for the production of structured lipids.     

Computational analysis of chain flexibility and fluctuations in Rhizomucor miehei lipase.

We have performed molecular dynamics simulation of Rhizomucor miehei lipase (Rml) with explicit water molecules present. The simulation was carried out in periodic boundary conditions and conducted for 1. 2 ns in order to determine the concerted protein dynamics and to examine how well the essential motions are preserved along the trajectory. Protein motions are extracted by means of the essential dynamics analysis method for different lengths of the trajectory. Motions described by eigenvector 1 converge after approximately 200 ps and only small changes are observed with increasing simulation time. Protein dynamics along eigenvectors with larger indices, however, change with simulation time and generally, with increasing eigenvector index, longer simulation times are required for observing similar protein motions (along a particular eigenvector). Several regions in the protein show relatively large fluctuations and in particular motions in the active site lid and the segments Thr57-Asn63 and the active site hinge region Pro101-Gly104 are seen along several eigenvectors. These motions are generally associated with glycine residues, while no direct correlations are observed between these fluctuations and the positioning of prolines in the protein structure. The partial opening/closing of the lid is an example of induced fit mechanisms seen in other enzymes and could be a general mechanism for the activation of Rml.     

Characterization of two novel family 12 xyloglucanases from the thermophilic Rhizomucor miehei

Two novel glycoside hydrolase (GH) family 12 xyloglucanase genes (designated RmXEG12A and RmXEG12B) were cloned from the thermophilic fungus Rhizomucor miehei. Both genes contained open reading frames of 729 bp encoding 242 amino acids. Their deduced amino acid sequences shared 68 % identity with each other and less than 60 % with other xyloglucanases. The two genes, without the sequences for the signal peptides, were cloned and successfully expressed in Escherichia coli as active xyloglucanases, designated RmXEG12A and RmXEG12B, with similar molecular masses—25.6 and 25.9 kDa, respectively. RmXEG12A showed optimal activity at pH?6.5 and 65 °C, RmXEG12B at pH?5.0 and 60 °C. Both recombinant xyloglucanases displayed very high specific activities, 6,681.4 and 3,092.2 U?mg^?1, respectively, toward tamarind xyloglucan, but no activity toward carboxymethylcellulose, Avicel, or p-nitrophenyl derivatives. The main products of tamarind xyloglucan hydrolysis by the two xyloglucanases were XXXG, XXLG/XLXG, and XLLG (where G is an unsubstituted β-d-Glc residue, X is a xylosylated β-d-Glc residue, and L is a β-d-Glc residue substituted by xylosyl-galactose).     

Enzymatic synthesis of isoamyl butyrate using immobilized Rhizomucor miehei lipase in non-aqueous media

Isoamyl butyrate, an important fruity flavor ester, was synthesized using Rhizomucor miehei lipase immobilized on a weak anion exchange resin (Lipozyme IM-20) by the esterification of isoamyl alcohol and butyric acid. The effects of various reaction parameters such as substrate and enzyme concentrations, substrate molar ratio, temperature and incubation time have been investigated. Yields above 90% were obtained with substrate concentrations as high as 2.0 M. No evidence of enzyme inhibition by butyric acid was present up to 1.0 M concentration. Acid inhibition and, to a small extent, alcohol inhibition were evident above 1.0 M substrate concentration. Conversions reached a saturation value by the end of 24–48 h of incubation due to the accumulation of the water of reaction. The equilibrium was successfully pushed forward towards esterification by removing the accumulated water using a molecular sieve.Journal of Industrial Microbiology & Biotechnology (2000) 25, 147–154. Received 09 February 2000/ Accepted in revised form 24 June 2000     

Immobilization of Rhizomucor miehei lipase onto montmorillonite K-10 and polyvinyl alcohol gel

Abstract

Immobilization of enzymes from different sources on various supports in designed systems increases enzymes’ stability by protecting the active site of it from undesired effect of reaction environment. Also, immobilization decreases the cost of separation and facilities the reuse of the enzymes. Therefore, the design of new immobilization enzyme preparations has been an inevitable area of modern biotechnology. Herein, Rhizomucor miehei lipase (RML) was immobilized on montmorillonite K-10 (MMT-RML) by adsorption and in polyvinyl alcohol (PVA-RML) by entrapment to obtain a more stable and active lipase preparation. The free and immobilized lipase preparations were characterized for p-nitrophenyl palmitate hydrolysis. The apparent Michaelis–Menten (K_mapp) constant was almost the same for the free RML and PVA-RML, whereas the corresponding value was 17.7-fold lower for MMT-RML. PVA-RML and MMT-RML have shown a 1.1 and 23.8 folds higher catalytic efficiency, respectively, than that of the free RML. The half-lives of PVA-RML and MMT-RML were found to be 7.4 and 3.4 times longer than the free RML at 35?°C, respectively. PVA-RML and MMT-RML maintained 65% and 87% of their initial activities after four reuses. These results showed that the catalytic performance of RML has improved significantly by immobilization.     

Enhanced Activity of Rhizomucor miehei Lipase by Deglycosylation of Its Propeptide in Pichia pastoris

Many studies have demonstrated that the properties of enzymes expressed in eukaryotes can be affected by the position and extent of glycosylation on enzyme. In this study, two potential glycosylation sites (the 8th and the 58th asparagine) were identified and the effect of propeptide glycosylation on Rhizomucor miehei lipase (RML) expressed in Pichia pastoris was investigated. To better understand the effect of glycosylation on the activity of RML, three mutants (M1, generated by N8A; M2, generated by N58A; and M3, generated by N8A and N58A) were designed to generate deglycosylated enzymes. The results showed that deglycosylated RML exhibited a twofold higher activity compared to the wild type. However, it was also found that glycosylation on the propeptide was important for the removal of the propeptide by Kex2 protease and secretion of the enzyme. Thus, our study provided a further understanding into the role of glycosylation on enzyme function.     

Enhanced activity of Rhizomucor miehei lipase by directed evolution with simultaneous evolution of the propeptide

Propeptides are short sequences that facilitate the folding of their associated proteins. The present study found that the propeptide of Rhizomucor miehei lipase (RML) was not proteolytically removed in Escherichia coli. Moreover, RML was not expressed if the propeptide was removed artificially during the cloning process in E. coli. This behavior in E. coli permitted the application of directed evolution to full-length RML, which included both propeptide and catalytic domain, to explore the role played by the propeptide in governing enzyme activity. The catalytic rate constant, k (cat), of the most active mutant RML protein (Q5) was increased from 10.63?±?0.80 to 71.44?±?3.20?min(-1) after four rounds of screening. Sequence analysis of the mutant displayed three mutations in the propeptide (L57V, S65A, and V67A) and two mutations in the functional region (I111T and S168P). This result showed that improved activity was obtained with essential involvement by mutations in the propeptide, meaning that the majority of mutants with enhanced activity had simultaneous mutations in propeptide and catalytic domains. This observation leads to the hypothesis that directed evolution has simultaneous and synergistic effects on both functional and propeptide domains that arise from the role played by the propeptide in the folding and maturation of the enzyme. We suggest that directed evolution of full-length proteins including their propeptides is a strategy with general validity for extending the range of conformations available to proteins, leading to the enhancement of the catalytic rates of the enzymes.     

Enzymatic hydrolysis of racemic ibuprofen esters using Rhizomucor miehei lipase immobilized on different supports

This research describes the immobilization of Rhizomucor miehei lipase (RML) and chemically aminated RML (NH₂-RML) on different supports including octyl-sepharose (octyl-RML), activated sepharose with cyanogen bromide (CNBr-RML and CNBr-NH₂-RML), glyoxyl sepharose (Gx-RML and Gx-NH₂-RML) and glyoxyl sepharose dithiothreitol (Gx-DTT-RML and Gx-DTT-NH₂-RML). The highest immobilization yield was achieved for octyl-RML (>98%) followed by CNBr-RML (88%). Octyl-RML had the most specific activity (13.6) among all derivatives. The other preparations had moderate activities likely because of chemical reaction during covalent attachment of the enzyme. The catalytic behavior of lipase immobilized in hydrolysis reactions was investigated using methyl, ethyl, propyl, butyl and isobutyl-ibuprofen esters and the influence of the alkyl chain and the alcoholic residue of the ester were studied. Butyl ester was the most interesting ester for carrying out hydrolysis. The highest enantioselectivity of enzyme (E = 8.8) was obtained with isooctane/sodium phosphate buffer pH 7.0 at temperature of 40 °C. Increasing temperature from 40 to 50 °C caused decreasing in enantioselectivities and conversions. Also esterification of ibuprofen was carried out in solvent systems containing isooctane and two ionic liquids (ILs); [BMIM][PF₆] and [BMIM][BF₄]. Poor conversions and enantioselectivities were observed during esterification in all solvents.     

Theoretical investigation of the dynamics of the active site lid in Rhizomucor miehei lipase.

Interfacial activation of Rhizomucor miehei lipase is accompanied by a hinge-type motion of a single helix (residues 83-94) that acts as a lid over the active site. Activation of the enzyme involves the displacement of the lid to expose the active site, suggesting that the dynamics of the lid could be of mechanistic and kinetic importance. To investigate possible activation pathways and to elucidate the effect of a hydrophobic environment (as would be provided by a lipid membrane) on the lid opening, we have applied molecular dynamics and Brownian dynamics techniques. Our results indicate that the lipase activation is enhanced in a hydrophobic environment. In nonpolar low-dielectric surroundings, the lid opens in approximately 100 ns in the BD simulations. In polar high-dielectric (aqueous) surroundings, the lid does not always open up in simulations of up to 900 ns duration, but it does exhibit some gating motion, suggesting that the enzyme molecule may exist in a partially active form before the catalytic reaction. The activation is controlled by the charged residues ARG86 and ASP91. In the inactive conformation, ASP91 experiences repulsive forces and pushes the lid toward the open conformation. Upon activation ARG86 approaches ASP61, and in the active conformation, these residues form a salt bridge that stabilizes the open conformation.     

High-throughput screening of B factor saturation mutated Rhizomucor miehei lipase thermostability based on synthetic reaction

Conventional lipase screening methods are mostly based on hydrolytic activity, which may not always be the best method to assess the enzyme activity, especially for evaluating synthetic activity. Here we developed a high throughput and visual method to screen clones with high synthetic activity and used it to assess lipases thermostability. All mutants' lipase synthetic activity were identified through esterification of caprylic acid and ethanol with methyl red as the pH indicator adding in the substrates on according to the color change halo around the colony on culture plates since synthetic reaction was often accompanied with a rise in pH. After two rounds operation with the pH indicator screening method, we obtained a double mutant Asn120Lys/Lys131Phe from the Rhizomucor miehei lipase saturation mutated library based on amino acid residue B factors. The mutant's initial synthetic activity was a little higher than wild type and its thermostability in synthetic reaction was enhanced, which remained 63.1% residual activity after being heated at 70°C for 5h comparing to 51.0% of wild type. The double mutant with the two residue replacements balanced well between stability and activity. Yeast surface display technology and the pH indicator method, combined with colony screening were shown to facilitate high-throughput screening for lipase synthetic activity.     

Modelling on isoamyl isovalerate synthesis from Rhizomucor miehei lipasein organic media: optimization studies

Immobilized lipase from Rhizomucor miehei (Lipozyme IM-20) was employed in the esterification of isovaleric acid and isoamyl alcohol to synthesize isoamyl isovalerate in n-heptane. Response surface methodology (RSM) based on a five-level, five-variable central composite rotatable design (CCRD) was used to evaluate the effects of important variables: enzyme concentration (20–40% w/w of acid), acid concentration (0.2–1.0 M), incubation period (24–120 h), alcohol concentration (0.25–1.25 M) and temperature (30–70 °C) on the esterification yield of isoamyl isovalerate. Extent of conversion was found to be excellent at all acid and alcohol concentrations employed in the range of 0.2–1.25 M, even at low enzyme concentration (20% w/w). The optimum conditions arrived at are as follows: 35% (w/w) enzyme concentration, 1.0 M acid concentration, 1.25 M alcohol concentration and 120 h incubation period, at 35 °C. Under these conditions, the predicted value was 680 mM ester matched very well with an experimental value of 678 mM.     

Highly enantioselective esterification of racemic ibuprofen in a packed bed reactor using immobilised Rhizomucor miehei lipase

A systematic study of the enantioselective resolution of ibuprofen by commercial Rhizomucor miehei lipase (Lipozyme(R) IM20) has been carried out using isooctane as solvent and butanol as esterificating agent. The main variables controlling the process (temperature, ibuprofen concentration, ratio butanol:ibuprofen) have been studied using an orthogonal full factorial experimental design, in which the selected objective function was enantioselectivity. This strategy has resulted in a polynomial function that describes the process. By optimizing this function, optimal conditions for carrying out the esterification of racemic ibuprofen have been determined. Under these conditions, enantiomeric excess and total conversion values were 93.8% and 49.9%, respectively, and the enantioselectivity was 113 after 112 h of reaction. These conditions have been considered in the design of a continuous reactor to scale up the process. The esterification of ibuprofen was properly described by pseudo first-order kinetics. Thus, a packed bed reactor operating as a plug-flow reactor (PFR) is the most appropriate in terms of minimizing the residence time compared with a continuous stirred tank reactor (CSTR) to achieve the same final conversion. This reactor shows a similar behavior in terms of enantioselectivity, enantiomeric excess, and conversion when compared with batch reactors. A residence-time distribution (RTD) shows that the flow model is essentially a plug flow with a slight nonsymmetrical axial dispersion (Peclet number = 43), which was also corroborated by the model of CSTR in series. The stability of the system (up to 100 h) and the possibility of reutilization of the enzyme (up to four times) lead to consider this reactor as a suitable configuration for scale up of the process.     

The crystal and molecular structure of the Rhizomucor miehei triacylglyceride lipase at 1.9 A resolution.

The crystal and molecular structure of a triacylglyceride lipase (EC 3.1.1.3) from the fungus Rhizomucor miehei was analyzed using X-ray single crystal diffraction data to 1.9 A resolution. The structure was refined to an R-factor of 0.169 for all available data. The details of the molecular architecture and the crystal structure of the enzyme are described. A single polypeptide chain of 269 residues is folded into a rather unusual singly wound beta-sheet domain with predominantly parallel strands, connected by a variety of hairpins, loops and helical segments. All the loops are right-handed, creating an uncommon situation in which the central sheet is asymmetric in that all the connecting fragments are located on one side of the sheet. A single N-terminal alpha-helix provides the support for the other, distal, side of the sheet. Three disulfide bonds (residues 29-268, 40-43, 235-244) stabilize the molecule. There are four cis peptide bonds, all of which precede proline residues. In all, 230 ordered water molecules have been identified; 12 of them have a distinct internal character. The catalytic center of the enzyme is made up of a constellation of three residues (His257, Asp203 and Ser144) similar in structure and function to the analogous (but not homologous) triad found in both of the known families of serine proteinases. The fourth residue in this system equivalent to Thr/Ser in proteinases), hydrogen bonded to Asp, is Tyr260. The catalytic site is concealed under a short amphipatic helix (residues 85 to 91), which acts as "lid", opening the active site when the enzyme is adsorbed at the oil-water interface. In the native enzyme the "lid" is held in place by hydrophobic interactions.