Mucor miehei

The ability of lipases to synthesize the amide bond has been shown (26). Mucor miehei lipase (NOVO tipozyme) has been used in the reaction of laurylamine and oleic acid at 60°C. Water was shown to inhibit the synthesis of this Ai-lauryloleamide (27). 

Mucor miehei, 84 Multiblock copolymers, 7-8, 26 Multichip modules, dielectrics for, 270-271 Mylar, 21... 

Conduritols and inositols are cyclic polyalcohols with significant biological activity. The presence of four stereogenic centers in the stmcture of conduritols allows the existence of 10 stereoisomers. Enzymatic methods have been reported for the resolution of racemic mixtures or the desymmetrization of meso-conduritols. For example, Mucor miehei lipase (MML) showed enantiomeric discrimination between all-(R) and all-(S) stereoisomers ofconduritol E tetraacetate (Figure 6.52). Alcoholysis resulted in the removal of the four acetyl groups ofthe all-(R) enantiomer whereas the all-(S) enantiomer was recovered [141]. 

Ester formation catalyzed by lipase (Mucor miehei) in conjunction with hydrogenation catalyzed by a rhodium complex Sol-gel immobilization of both catalysts... 

Immobilized Mucor miehei lipase (lipase MM) induced the polycondensation of adipic acid and 1,4-butanediol in ether solvents [26]. A horizontal two-chamber reactor was employed to facilitate the use of the molecular sieves. A low disper-sity polyester with DP = 20 was obtained by two-stage polymerization. 

The p-acetoxybenzyloxycarbonyl (AcOZ) group can be removed efficiently by a lipase from Mucor miehei and an acetyl esterase from the flavedo of oranges under exceptionally mild conditions (pH 5-6).110,11 121 Acetyl esterase discriminated between acetyl and longer acyl side chains (Scheme 8). 

A similar enzyme-catalyzed stereoselective synthesis of enantiomers of propanolamines has been recently reported30. Addition of a lipozyme from the fungus Mucor miehei to the epoxide ( )-8 in toluene and then a slightly more than one half molar equivalent of 2-propylamine gave a 29% conversion of ( )-8 to (S)-9 with an ee of 90%. For some benzene ring-substituted epoxides, both the percent conversion of the epoxide and the ee of product are slightly higher30. 

Lipase from Mucor miehei Kl buffer, NaSH, 20% MeOH, 30 °C, pH 5, 65%... 

Mucor miehei lipase (ITGL) [59] Seri 44 Leul45 Ser82 Ser82 OG ... 

An elegant method to suppress the undesired spontaneous hydrolysis of a 5(47/)-oxazolone in aqueous media uses a lipase-catalyzed alcoholysis reaction. Of particular importance is the synthesis of /crt-leucine, a non-proteinogenic a-amino acid that has found widespread use both as a chiral auxiliary and as a component of potentially therapeutic pseudopeptides. Racemic 4-/ert-butyl-2-phenyl-5(47/)-oxazolone 238 was submitted to Mucor miehei catalyzed alcoholysis using butanol as a nucleophile. Addition of a catalytic amount of triethylamine promoted in situ racemization. In this way, the enantiomericaUy pure butyl ester of (5)-A-benzoyl-/ert-leucine 239 was obtained in excellent yield (Scheme 7.75). 

The stability of the ester surfactants against enzymatic hydrolysis by two different microbial Upases, Mucor miehei lipase (MML) and Candida antarc-tica lipase B (CALB) added separately to the surfactant solutions, was also investigated, see Fig. 5 [19]. It is obvious that hydrolysis of the unsubstituted surfactant is much faster with both CALB and MML than that of the substituted surfactants, i.e., increased steric hindrance near the ester bond leads to decreased hydrolysis rate. Since the specificity of the enzyme against its substrate is determined by the structure of the active site, it can be concluded, as expected, that the straight chain surfactant most easily fits into the active site of both enzymes. 

Fig. 5 Hydrolysis of the esters surfactants in the presence of lipase at 20 °C. Left ( ) Linear ester-I- MML, (0) linear ester + CALB, and (A) methyl substituted ester-t CALB. Right (A) Methyl substituted ester + MML, ( ) ethyl substituted ester + MML, ( ) dimethyl substituted ester + MML, ( ) ethyl substituted ester + CALB, and (o) dimethyl substituted ester + CALB. MML and CALB stand for Mucor miehei and Candida antarctica B, respectively. (Redrawn from [19])...

The activity of three ester spHtting enzymes, Candida antarctica lipase B (CALB), Mucor miehei lipase (MML) and esterase, towards the carbonate surfactant was studied. While CALB and esterase were found to catalyze the hydrolysis of the carbonate bond, MML showed no activity. 

The chemical stability of the amide bond is high. When the surfactant containing an amide bond was subjected to 1 M sodium hydroxide during five days at room temperature, only 5% of the amide surfactant was cleaved. The corresponding experiment performed in 1 M HCl resulted in no hydrolysis. The amide bond was, however, found to be slowly hydrolyzed when lipase from Candida antarctica or peptidase was used as catalyst. Amidase and lipase from Mucor miehei was found to be ineffective. Despite the very high chemical stability, the amide surfactant biodegrades by a similar path in the... 

Esterification between oleic acid and oleyl alcohol, catalyzed by the Mucor miehei immobihzed hpase in a batch-stirred tank reactor with supercritical carbon dioxide as solvent produced higher reaction rates at supercritical conditions than in the solvent-free system (Knez et al., 1995). 

Lipases are serine hydrolases that catalyse the hydrolysis of lipids to fatty acids and glycerol [2]. In contrast to esterases, they work at the lipid-water interface and show only little activity in aqueous solutions. Studies of the X-ray structures of human lipase [3,4] and Mucor miehei lipase [5,6] revealed a change in conformation at the lipid-water interface, which explains the increase of activity. 

Lipolysed milk fat was one of the first flavours produced with the help of enzymes. The original process was based on the controlled lipase-catalysed hydrolysis of cream [18]. For instance, Mucor miehei lipase possesses a high selectivity towards flavour-active short-chain fatty acids. Additionally, lipases that prefer long-chain fatty acids or lipases without particular preferences can be found. The free fatty acids produced can be isolated by steam distillation and further purified. Thus, it is possible to obtain pure short-chain fatty acids like butanoic, hexanoic, octanoic and decanoic acid. 

The biotechnological production of flavour compounds is particularly focused on esters and lactones. Lipase from Mucor miehei is the most widely studied fungal lipase [30-35]. Esters of acids from acetic acid to hexanoic acid and alcohols from methanol to hexanol, geraniol and citronellol have been synthesised using lipases from Mucor miehei, Aspergillus sp., Candida rugosa, Rhizopus arrhizus and Trichosporum fermentans [32-37]. 

Methylbutanoates and methylbutyl esters are essential flavour compounds in fruit flavours they can be produced biotechnologically as mentioned before. Chowdary et al. [33] have described the production of a fruit-like flavour isoamyl isovalerate by direct esterification of isoamyl alcohol and isovaleric acid in hexane with the help of Mucor miehei lipase immobilised on a weak anion-exchange resin. 

Large-scale synthesis of (Z)-3-hexenyl acetate in hexane with lipase, (Z)-3-hexenol and acetic acid was described by several authors [40-42]. (Z)-3-Hex-enyl acetate has a fruity odour and shows a significant green note flavour. It can be produced using lipase from Candida antarctica immobilised on an acrylic resin [40, 41] or using immobilised lipase from Mucor miehei [42]. The conversion was reported to be about 90%. 

The biotechnological synthesis of lactones has reached a high standard. Besides microbial production, lactones can also be enzymatically produced. For instance, a lipase-catalysed intramolecular transesterification of 4-hydroxy-carboxylic esters leads enantioselectively (ee>80%) to (S)-y-lactones the chain length may vary from C5 to Cl 1 [13]. y-Butyrolactone can be produced in that way with lipase from Mucor miehei [30]. 

Finally, Sanfilippo and coworkers describe the enzymatic kinetic resolution of atropisomeric ( )-3,3 -bis(hydroxymethyl)-2,2 -bipyridine N,N-dioxide by enantioselective esterifcation in an unusual medium of 2-propanol/vinyl acetate (20 80) [139]. Lipase from Mucor miehei (immobilized lipase preparation, Lipozyme ) was found to give good enantioselectivity with an (aS)-enantiopreference in the axial recognition and allowed efficient preparation of both enantioforms with > 98%. Despite the fact that the propanol reacts with the acyl donor, this did not diminish its positive effects on the solubility of the bipyridyl substrate. 

Figure 6.8 Regioselective deacylation of the polyacetilated flavonoid quercetin (28a) by action of different lipases (CalB, lipase from Candida antarctica Mml, lipase from Mucor miehei f-BME, ferf-butyl methyl ether)...

Nicolosi and coworkers have intensively investigated the exploitation of lipases for the selective deprotection of bioactive compounds [90]. For instance, as shown in Figure 6.8, the alternative use of the lipases from Candida antarctica (CalB) and Mucor miehei (Mml) enabled the preparation of different derivatives of the flavonoid quercetin (28) [91]. Similar results, this time exploiting the lipase from Pseudomonas cepacia, were obtained with the polyacetylated catechin 29 [92]. 

Similarly, the esterification of geraniol and citronellol is carried out in hexane and catalyzed by the immobilized lipase Mucor miehei. The geraniol and citronellol esters are extensively used for the fragrance industry, where the use of lipases benefits mild conditions and low impurity side products [14, 15]. 

We collaborated with Professor Palligamai Vasudevan of the Chemical Engineering Department of the University of New Hampshire on a study of immobilization of lipases on CoFoam. Immobilization was performed at the Hydrophilix facility in Portland, ME. Approximately 2 g lipase (from porcine pancreas and Mucor miehei) were stirred into 500 ml deionized water. The enzyme solution was emulsified with an equal volume of a methylene diisocyanate (MDI)-based hydrophilic polyurethane... 

TIL Thermomyces lanuginosus lipase, RdL Rhizopus delemar lipase, RnL Rhizopus niveus lipase, MmE Mucor miehei esterase, PsL Pseudomonas sp. lipase, MmL Mucor miehei lipase, RoL Rhizopus orvzae lipase, CaLA Candida antarctica lipase A, CaLB Candida antarctica lipase B, PLE Pig liver esterase, EP Enteropeptidase, PKA Porcine kidney acylase, CE Cholesterol esterase Figure 8.1 (S)-Selective enzyme hits from hydrolase screening. ... 

Prins, J. and Nielsen, T. K. 1970. Microbial rennet. Mucor miehei. Process Biochem. 5, 34-35. 

Ramet, J. P. and Weber, F. 1981. Cheesemaking properties of a thermolabile milkclotting enzyme form Mucor miehei. Lait 61, 458-464. 

Sternberg, M. Z. 1971. Crystalline milk clotting protease from Mucor miehei and some of its properties. J. Dairy Sci 54, 159-167. 

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