Enzymatic production lipase immobilization

A membrane cell recycle reactor with continuous ethanol extraction by dibutyl phthalate increased the productivity fourfold with increased conversion of glucose from 45 to 91%.249 The ethanol was then removed from the dibutyl phthalate with water. It would be better to do this second step with a membrane. In another process, microencapsulated yeast converted glucose to ethanol, which was removed by an oleic acid phase containing a lipase that formed ethyl oleate.250 This could be used as biodiesel fuel. Continuous ultrafiltration has been used to separate the propionic acid produced from glycerol by a Propionibacterium.251 Whey proteins have been hydrolyzed enzymatically and continuously in an ultrafiltration reactor, with improved yields, productivity, and elimination of peptide coproducts.252 Continuous hydrolysis of a starch slurry has been carried out with a-amylase immobilized in a hollow fiber reactor.253 Oils have been hydrolyzed by a lipase immobilized on an aromatic polyamide ultrafiltration membrane with continuous separation of one product through the membrane to shift the equilibrium toward the desired products.254 Such a process could supplant the current energy-intensive industrial one that takes 3-24 h at 150-260X. Lipases have also been used to prepare esters. A lipase-surfactant complex in hexane was used to prepare a wax ester found in whale oil, by the esterification of 1 hexadecanol with palmitic acid in a membrane reactor.255 After 1 h, the yield was 96%. The current industrial process runs at 250°C for up to 20 h. 

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. 

Optically active amines are important intermediates and chiral auxiliaries in the technical synthesis of agrochemicals and pharmaceuticals. BASF, one of the world s leading producers of chiral amines, developed a process based on the enzymatic resolution of racemic amines 49 with Burkholderia plantarii lipase immobilized on polyacrylate (Scheme 16) [75,76]. Methoxyacetic acid estars are particularly well suited for the stereospecific enzymatic differentiation, giving both the free amine (S)-49 and the acylated product R)-50 in high ee. The reaction stops at 50% conversion and the selectivity factor was calculated to be as high as 500. A plug-flow or batch reactor can be used for the enzymatic reaction and the residence time is in the range of 5-7 h. The more important amine (R)-49 can be liberated with the aid of base and is subsequently purified by distil-... 

An enzymatic production process for Diltiazem (54), a coronary vasodilator and calcium channel blocker, was started in 1993 by Tanabe Seiyaku, Japan [7, 77]. The epoxide (2i, 3S)-52 is a key intermediate in this synthesis (Scheme 17) and can be produced via asymmetric hydrolysis of rac-52 catalyzed by Serratia marescens lipase immobilized on spongy layers. The whole process takes place in a polyacrylonitrile hollow fiber membrane reactor and produces (2i, 3S)-52 in yields of 40-45%. The hydrolyzed product (2S,3i )-53 is not stable under the prevailing reaction conditions and decarboxylates to aldehyde 55, a strong enzyme deactivator. The aldehyde needs therefore to be removed, which is achieved by continuous filtration of its bisulfite adduct 56. Using this enzymatic process it was possible to bring down the number of required steps en route to 54 from nine to five. This process is also carried out by other companies (e.g., DSM) with a worldwide annual production of 1001. 

Chirazyme L2-C2 (CAL-B) proved to be a very useful enzyme for the development of an acylation process for the large-scale production of vitamin A (retinol, 91) at Roche (Scheme 27) [90,91]. In the plant process of vitamin A, intermediate 88 is partially acylated and then subjected to acid-catalyzed dehydration and isomerization to yield the vitamin A ester 90 via acetate 89. Contrary to the chemical acylation, an enzymatic approach allowed for a highly selective monoacylation of 88, and Chirazyme L2-C2 showed a very high conversion rate at 30% (w/w) substrate concentration. A first continuous process on the laboratory scale was set up with a 15 ml fixed-bed reactor containing 5.0-8.0 g of immobilized biocatalyst 4.9 kg of 89 was synthesized within 100 days in 99% yield and with 97% selectivity for the primary hydroxyl group. The laboratory process was implemented in a miniplant (120 g of biocatalyst), which could convert 1.4 kg of 88 into 1.6 kg 89 per day. After 74 days the conversion efficiency was still 99.4%. Further development of this transformation led to a modified process, which uses Thermomyces lanuginosus lipase immobilized on Accurel MPlOOl for the continuous production of 89 [92]. 

A combination of different enzymes has been shown to increase yield in the enzymatic production of EAAE. Eor example, Tiirkan and Kalay (2006) used three different immobilized lipases from RML, TLL and Candida. They found lipases from RML and TLL catalyzed the first step (TAG to DAG) of transesterification faster while hpase from Candida antarctica catalyzed the second (DAG to MAG) and third (MAG to FAAE and glycerol) steps faster. 

The first industrial plant for enzymatic production of biodiesel was built in China in 2006, with a capacity of 20,000 tons/year. Tert-butanol was selected as the reaction medium, and immobilized lipases like Lipozymes TL IM and Novozyms 435 were both used in this plant as enzymatic catalysts (Zhao et al., 2015). 

Chemo-enzymatic epoxidation of unsaturated fatty acids with aqueous H2O2 has been conducted with considerable success and here we have a remarkable situation that undesirable ring opening of the epoxide does not occur. Excellent activity and stability has been realized with Novozym 435, a Candida antartica lipase B immobilized on polyacryl. This enzyme is readily separable, can be used several times without loss of activity, and has a turnover of more than 2,00,000 moles of products per mole of catalyst (Bierman et al., 2000). 

Monoglyceride (MG) is one of the most important emulsifiers in food and pharmaceutical industries [280], MG is industrially produced by trans-esterification of fats and oils at high temperature with alkaline catalyst. The synthesis of MG by hydrolysis or glycerolysis of triglyceride (TG) with immobilized lipase attracted attention recently, because it has mild reaction conditions and avoids formation of side products. Silica and celite are often used as immobilization carriers [281], But the immobilized lipase particles are difficult to reuse due to adsorption of glycerol on this carriers [282], PVA/chitosan composite membrane reactor can be used for enzymatic processing of fats and oils. The immobilized activity of lipase was 2.64 IU/cm2 with a recovery of 24%. The membrane reactor was used in a two-phase system reaction to synthesize monoglyceride (MG) by hydrolysis of palm oil, which was reused for at least nine batches with yield of 32-50%. 

The final intriguing use of CLAs is in the immobilization of enzymes in the soapy shell in order to carry out an enzymatic reaction. Thus the hydrolysis of -nitrophenyl acetate to / -nitrophenol has been demonstrated by immobilizing a lipase into the shell of a CLA. The CLAs were then pumped through a cross-fiow membrane, where they were separated and recycled, with the product appearing in the permeate [70]. 

The asymmetric hydrolysis of (exo,exo)-7-oxabicyclo[2.2.1]heptane-2,3-dimethanol, diacetate ester (37) to the corresponding chiral monoacetate ester (38) (Fig. 12B) has been demonstrated with lipases [61]. Lipase PS-30 from P. cepacia was most effective in asymmetric hydrolysis to obtain the desired enantiomer of monoacetate ester. The reaction yield of 75 M% and e.e. of >99% were obtained when the reaction was conducted in a biphasic system with 10% toluene at 5 g/liter of the substrate. Lipase PS-30 was immobilized on Accurel PP and the immobilized enzyme was reused (5 cycles) without loss of enzyme activity, productivity, or e.e. of product (38). The reaction process was scaled up to 80 liters (400 g of substrate) and monoacetate ester (38) was isolated in 80 M% yield with 99.3% e.e. The product was isolated in 99.5% chemical purity. The chiral monoacetate ester (38) was oxidized to its corresponding aldehyde and subsequently hydrolyzed to give chiral lactol (33) (Fig. 12B). The chiral lactol (33) obtained by this enzymatic process was used in chemoenzymatic synthesis of thromboxane A2 antagonist (35). 

Lipase PS-30 was immobilized on Accurel PP and the immobilized enzyme was reused five times without any loss of activity or productivity in the resolution process to prepare A-(+)-(43). The enzymatic process was scaled up to a 640-liter preparative batch using immobilized lipase PS-30 at 4 g/liter racemic substrate (43) in toluene as a solvent. From the reaction mixture, i -(+)-(43) was isolated in 35 M% overall yield with 98.5% e.e. and 99.5% chemical purity. The undesired, S -(-)-acetatc (46) produced by this process was enzymatically hydrolyzed by lipase PS-30 in a biphasic system to prepare the corresponding S -(-)-alcohol (43). Thus both enantiomers of alcohol (43) were produced by the enzymatic process. 

An enzymatic process cannot be adopted for industrial production of BDF, if immobilized lipase cannot be used for long period. Studies on the stability of immobilized C. antarctica lipase revealed that it was the most stable when the reaction was conducted with 5 to 8 mol MeOH for total FAs in acid oil prepared from soapstock (Watanabe et al., 2007). The phenomenon, that there is the optimum region of MeOH concentration, may be explained as follows ... 

Compound 25 (Fig. 18.9), a prodrug of 9-P-D-arabinofuranosyl guanine (26), was developed for the potential treatment of leukemia. Compound 24 is poorly soluble in water and its synthesis by conventional techniques is difficult. An enzymatic demethoxylation process was developed using adenosine deaminase (Mahmoudian et al., 1999, 2001). Compound 25 was enzymatically prepared from 6-methoxyguanine (27) and ara-uracil (28) using uridine phosphorylase and purine nucleotide phosphorylase. Each protein was cloned and overexpressed in independent Escherichia coli strains. Fermentation conditions were optimized for production of both enzymes and a co-immobilized enzyme preparation was used in the biotransformation process at 200 g/L substrate input. Enzyme was recovered at the end of the reaction by filtration and reused in several cycles. A more water soluble 5 -acetate ester of compound 26 was subsequently prepared by an enzymatic acylation process using immobilized Candida antarctica lipase in 1,4-dioxane (100 g/L substrate) with vinyl acetate as the acyl donor (Krenitsky et al., 1992). 

The reaction is catalyzed by a variety of both acids and bases but simple bases such as NaOH and KOH are generally used for the industrial production of biodiesel [200, 201]. The vegetable oil feedstock, usually soybean or rapeseed oil, needs to be free of water (<0.05%) and fatty acids (<0.5%) in order to avoid catalyst consumption. This presents a possible opportunity for the application of enzymatic transesterification. For example, lipases such as Candida antarctica B lipase have been shown to be effective catalysts for the methanolysis of triglycerides. When the immobilized form, Novozyme 435, was used it could be recycled 50 times without loss of activity [201, 202]. The presence of free fatty acids in the triglyceride did not affect the enzymes performance. The methanolysis of triglycerides catalyzed by Novozyme 435 has also been successfully performed in scC02 as solvent [203]. 

SCFs offer a nonaqueous environment which can be desirable for enzymatic catalysis of lipophilic substrates. The lipophilic substance cholesterol is 2 to 3 orders of magnitude more soluble in CX>2-cosolvent blends than in waterQ). In CO2 based blends, it may be oxidized to cholest-4en-3one, a precursor for pharmaceutical production using an immobilized enzyim(22). The enzyme polyphenol oxidase has been found to be catalytically active in supercritical CO2 and fluoroform (22). The purpose of using a SCF is that it is miscible with one of the reactants-oxygen. Lipase may be used to catalyze the hydrolysis and interesterification of triglycerides in supercritical OO2 without severe loss of activity(24). These reactions could be integrated with SCF separations for product recovery. 

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