Lipase Rhizomucor miehei

A very attractive and efficient method for the synthesis of L-aminoacids via DKR has been reported by Turner et al. [41a,b]. They employed enzyme-catalyzed ring opening of 5(4H)-oxazolones in combination with a catalytic amount of Et3N. The relatively low pKa of the C-4 proton (8.9) of oxazolones facilitates racemization. Hydrolysis of the ester obtained through DKR, followed by debenzoylation, yields L-aminoacids in excellent enantiomeric excess (99.5%) (Figure 4.16). In their initial studies, they employed Rhizomucor miehei lipase (Lipozyme) as the biocatalyst [41]. More recently, they have obtained excellent results employing CALB [41bj. This method has also been employed by Bevinakatti [41c,d] and Sih [41e,fj. 

In a first experiment, the esterification of oleic acid with 1-butanol catalysed by a Rhizomucor miehei lipase was investigated (Scheme 4.3). Lipases usually function at the water/organic interface, which make them extremely suitable for use in the CCS. 

In this communication a study of the catalytic behavior of the immobilized Rhizomucor miehei lipase in the transesterification reaction to biodiesel production has been reported. The main drawbacks associated to the current biodiesel production by basic homogeneous catalysis could be overcome by using immobilized lipases. Immobilization by adsorption and entrapment have been used as methods to prepare the heterogeneous biocatalyst. Zeolites and related materials have been used as inorganic lipase supports. To promote the enzyme adsorption, the surface of the supports have been functionalized by synthesis procedures or by post-treatments. While, the enzyme entrapping procedure has been carried out by sol-gel method in order to obtain the biocatalyst protected by a mesoporous matrix and to reduce its leaching after several catalytic uses. 

Other microbial lipases have also been successfully used in anhydrous ionic liquids, e.g., from Alcaligenes sp. (AsL) [54, 58], CaLA, Rhizomucor miehei lipase (RmL), and Thermomyces lanuginosus lipase (TIL) [54]. The lipase from pig pancreas (porcine pancreas lipase, PPL), the only mammalian lipase that has been subjected to ionic liquids, catalyzed transesterificationin[BMIm][NTf2]butnotin[BMIm][PF6]... 

The annual production would be 550 t of oleyl oleate with market price 5 EURO/kg. As biocatalyst, an immobilized Rhizomucor miehei lipase - Lipozyme IM - product from NOVO Nordisk was used. 

Maruyama, K., Shimada, Y., Baba,T., Ooguri,T., Sugihara, A.,Tominaga, Y., and Mori-yama, S. 2000. Purification of ethyl docosahexaenoate through selective alcoholysis with immobilized Rhizomucor miehei lipase. J. Jpn. Oil Chem. Soc., 49, 793-799. 

Enzyme preparation. The enzyme preparation Lipozyme IM, which is a Rhizomucor miehei lipase, immobilized on a macroporous anion exchange resin, was kindly donated from NOVO Nordisk AS (Copenhagen, Denmark). 

Esters of fatty acids with monohydric alcohols find applications as emollients in cosmetics. They are prepared by acid- or base-catalyzed (trans)esterifications [200, 205]. As with biodiesel production, the use of enzymatic catalysis offers potential benefits but in the case of these specialty fatty acid esters there is a special advantage the products can be labelled as natural. Consequently, they command a higher price in personal care products where natural is an important customer-perceived advantage. Examples include the synthesis of isopropylmy-ristate by CaLB-catalyzed esterification [206] and n-hexyl laurate by Rhizomucor miehei lipase (Lipozyme IM-77)-catalyzed esterification [207] (see Fig. 8.38). 

Human milk lipids are unique in that they contain predominantly palmitate at the sn-2 position. To produce an infant formula whose lipids resemble those of mothers milk, Yang et al. (84) employed Rhizomucor miehei lipase to catalyze the acidolysis of lard, which is rich in 2-palmitoyl acylglycerols, with soybean fatty acids. As the enzyme had tight specificity for reaction at the sn- and sn-3 positions of the... 

Abbreviations GcL, Geotrichum candidum lipase hPL, human pancreatic lipase RmL, Rhizomucor miehei lipase hHL, human hepatic lipase hLPL, human lipoprotein lipase hLAL, human lysosomal acid lipase hGL, human gastric lipase BAL, bile salt-activated lipase HSL, hormone-sensitive lipase CLP, colipase AChE, Torpedo cal omica acetylcholinesterase cDNA, complementary deoxyribonucleic acid VLDL, very low-density lipoprotein IDL, intermediate-density lipoprotein HDL, high-density lipoprotein apoC-II, apolipoprotein C-II. 

Aucoin, M.G. Erhardt, F.A. Legge, R.L. Hyperactivation of Rhizomucor miehei lipase by hydrophobic xerogels. Biotechnol. Bioeng. 

The application of ionic liquids in lipase biocatalysis has not remained entirely restricted to CaLB, PcL or CrL. Other lipases have been used in ionic liquids for ester synthesis such as Candida antarctica lipase A (CaLA) [15,16], Thermomyces lanuginosus lipase [17] (TLL), Rhizomucor miehei lipase (PmL), Pseudomonas fluorescens lipase (PJL) [18], Pig pancreas lipase (PpL) [17] and Alcaligenes sp. lipase (A5 L) [16]. 

Rhizomucor miehei lipase immobilized on a macroporous anion exchange resin (lipozym IM 20) and Candida antarctica lipase SP 435 (immobilized on an unspecified carrier) were kindly donated by Novo Nordisk A/S. The a- and -methyl glucosides and galactosides were supplied by Sigma. 

The enzyme-catalyzed production of (R)-2-(2-aminobutyl)-3-chlorothio-phene ((R)-42) was a research project at Zeneca a few years ago [72]. Compound 42 was an intermediate in the preparation of adenosine derivatives and Scheme 13 outlines the relevant enzymatic step. Racemic alcohol rac-40 was treated with Rhizomucor miehei lipase immobilized on an ion-exchange resin (Lipozyme RM IM from Novozymes) in the presence of vinyl butyrate to yield the free (S)-alcohol 40 and the (R)-ester 41. Alcohol 40 was subsequently converted into the strategic intermediate 42, which found further use in the synthesis of antihypertension drugs. 

Rhizomucor miehei lipase vinyl butyrate, TBME, RT, 7 h... 

It has been observed that not all porons snpports are suitable for immobilization, owing to the limitations of pore size, which shonld be at least the same as that of the lipase. By studying the adsorption of Rhizomucor miehei lipase on a porous inorganic support, it was found that the activity depended on a pore size in the range of 100 nm. Beyond this value, the activity is independent suggesting that the internal diffusion restriction is insignificant (Bosley and Clayton, 1994). Similar observations have also been noted with the immobilization of Candida rugosa lipase on different hydrophobic carriers of various pore sizes (Al-Duri et al., 1995). 

Selectivity improvement is a critical requirement for industrial applications of lipase. This includes substrate selectivity, stereoselectivity, regioselectivity, and enantio-selectivity. Adsorption of Candida rugosa on celite was reported to enhance the stability of lipase and improve its enantioselectivity up to 3-fold (Ogino, 1970). Entrapment in cellulose acetate-Ti02 gel fiber improved the selectivity of Rhizomucor miehei lipase in the hydrolysis of 1,2-diacetoxypropane (Ikeda and Kurokawa, 2001). Also the enantioselectivity of pegylated P. cepacea lipase was increased 3-fold by... 

Direct esterification of ibuprofen was also carried out with immobilized Rhizomucor miehei lipase (Lipozyme IM20) and butanol using PER and CSTR... 

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