Enzymes hydrolysis process

Olestra is prepared by a solvenfless transesterification process in which sucrose is treated with methyl ester of fatty acids in the presence of sodium methoxide between 100—180°C for 14 hours (68). The manufacturing process involves removal of the unreacted fatty acid esters by enzymic hydrolysis... 

Enzyme—Heat—Enzyme Process. The enzyme—heat—enzyme (EHE) process was the first industrial enzymatic Hquefaction procedure developed and utilizes a B. subtilis, also referred to as B. amjloliquefaciens, a-amylase for hydrolysis. The enzyme can be used at temperatures up to about 90°C before a significant loss in activity occurs. After an initial hydrolysis step a high temperature heat treatment step is needed to solubilize residual starch present as a fatty acid/amylose complex. The heat treatment inactivates the a-amylase, thus a second addition of enzyme is required to complete the reaction. 

An intriguing influence of a cosolvent immiscible with water on the enantioselec-tivity of the enzyme-catalyzed hydrolysis was observed. It was proven that enzyme enantioselectivity is directly correlated with the cosolvent hydrophobicity. In the best example, for ethyl ether as cosolvent, the reaction proceeded with E = 55, and the target compound was obtained in 33% yield with 92.7% ee. This finding may be of great practical importance, particularly in industrial processes [24], since it will enable better optimization of enzyme-catalyzed processes. It is clear that, in future, immobilized enzymes, as heterogeneous catalysts, wiU be widely used in most industrial transformations, especially in the preparation of pharmaceuticals [25]. 

The isomerism existing between the pairs of nucleotides was attributed to the different locations of the phosphoryl residues in the carbohydrate part of the parent nucleoside,49 63 since, for instance, the isomeric adenylic acids are both hydrolyzed by acids to adenine, and by alkalis or kidney phosphatase to adenosine. Neither is identical with adenosine 5-phosphate since they are not deaminated by adenylic-acid deaminase,68 60 and are both more labile to acids than is muscle adenylic acid. An alternative explanation of the isomerism was put forward by Doherty.61 He was able, by a process of transglycosidation, to convert adenylic acids a" and 6 to benzyl D-riboside phosphates which were then hydrogenated to optically inactive ribitol phosphates. He concluded from this that both isomers are 3-phosphates and that the isomerism is due to different configurations at the anomeric position. This evidence is, however, open to the same criticism detailed above in connection with the work of Levene and coworkers. Further work has amply justified the original conclusion regarding the nature of the isomerism, since it has been found that, in all four cases, a and 6 isomers give rise to the same nucleoside on enzymic hydrolysis.62 62 63 It was therefore evident that the isomeric nucleotides are 2- and 3-phosphates, since they are demonstrably different from the known 5-phosphates. The decision as to which of the pair is the 2- and which the 3-phosphate proved to be a difficult one. The problem is complicated by the fact that the a and b" nucleotides are readily interconvertible.64,64... 

To break up cellulose/hemicellulose, it is treated physically (milling), with heat, and hydrolyzed (sulfuric acid + enzymes). Also in this case, improved (bio)cata-lytic hydrolysis processes for cellulose/hemicellulose are needed. The sugar can then serve as feedstock for standard fermentation plants. 

There has also been extensive activity towards the replacement of the entire chemical route to 7-ADCA (Scheme 1.14) with a biocatalytic one. This is somewhat more complex than the above example, as the penicillin fermentation product requires ring expansion as well as side-chain hydrolysis in order to arrive at the desired nucleus. The penicillin nucleus can be converted to the cephalosporin nucleus using expandase enzymes, a process that occurs naturally during the biosynthesis of cephalosporin C by Acremonium chryso-genum and cephamycin C by Streptomyces clavuligems from isopenicUhn N (6-APA containing a 6-L-a-aminoadipoyl side chain). ... 

The main cyanogenic glycoside in laurel is prunasin, the P-o-glucoside of benzaldehyde cyanohydrin. The enzymic hydrolysis of prunasin may be visualized as an acid-catalysed process, first of all hydrolysing the acetal linkage to produce glucose and the cyanohydrin. Further hydrolysis results in reversal of cyanohydrin formation, giving HCN and benzaldehyde. 

When autohydrolysed aspen was treated with cellulase enzyme, poor results were obtained. Based on a ton of dry wood, only about 300 lb of fermentable sugars were obtained, and the fermentation was very inefficient with low yields of ethanol. These results compare poorly with those shown in Figure 5 when autohy-drolysed-caustic extracted aspen was treated with cellulase enzyme, and then fermented. It would appear from these results that the caustic extraction step for lignin removal would be necessary if the enzymatic hydrolysis process were to be adopted and advisable when acid hydrolysis is used. 

Second, the polymer needs to be broken down into small fragments. Microorganisms excrete extracellular enzymes that cleave the polymeric chains [4]. This enzymatic cleavage reaction, on the one hand, needs functional sites within the polymer backbone where the enzymes can catalyze the cleavage of chemical bonds. On the other hand the polymeric chain needs to be flexible enough that the chain can enter the catalytic site of the enzyme. In most cases, the chemical reaction catalyzed by the exo-enzymes is a hydrolysis process that converts the polymer chain into smaller oligomers and monomers [4]. 

This is one process in which the initial research was carried out in an academic lab. Whitesides and co-workers showed that resolution by stereoselective enzyme hydrolysis of racemic (+/-) glycidyl butanoate was possible (Figure 4.16). 

In another study, an antioxidant peptide has been isolated from hoki frame protein by gastrointestinal digestion (Kim et ah, 2007). A number of enzymes have been used for the hydrolysis process, and the isolated peptides showed strong antioxidant activities in various model systems. 

Enzyme-based processes for the resolution of chiral amines have been widely reported [2, 3] and are used in the manufacture of pharmaceuticals, for example, BASF s process for chiral benzylic amine intermediates. Scheme 13.1 [4]. The methods used are enantioselective hydrolysis of an amide and enantioselective synthesis of an amide, both of which are kinetic resolutions. For high optical purity products the processes depend upon a large difference in the catalyzed reaction rates of each enantiomer. 

In addition to the variations in the LHC composition that occur from species to species, each species has its extractives, which include resins and waxes. These constituents are capable of interfering with cellulose hydrolysis because of their hydrophobic nature. Tannins and other highly reactive materials are constituents of some woody species. When LHC is obtained from nonwoody (herbaceous) species, the range of interfering constituents increases greatly. Sugars, starches, dextran, carotenoids, and many isoprenoids are to be found. Operators of a cellulose hydrolysis process that uses municipal solid waste as its biomass resource may experience seasonal variations in composition and chance inclusion of crankcase oil and other products that inhibit enzymes or kill yeast. 

Figure 12. Relation of glucose selling price to delivered cellulose substrate costs for enzymatic hydrolysis process. Data for 250,000 t/yr plant 90% overall process efficiency, 50% enzyme recovery (reuse).

D. R. Dodds, Process for the preparation of chiral propanoic adds via enantioselective enzymic hydrolysis of their water-soluble esters, PCT Int. Appl. WO 8909765,1989. 

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