Microbially Derived Enzyme Preparations

One of the first applications of the microbial hydrolysis of epoxides for the synthesis of a bioactive compound is based on the resolution of a 2,3-disub-stituted oxirane having a cis-configuration (Scheme 14). Thus, by using an enzyme preparation derived from Pseudomonas sp., the (91 ,10S)-enantiomer was hydrolyzed in a frans-specific fashion (i.e. via inversion of configuration at C-10) yielding the 9R,10R)-threo-diol. The remaining (9S,101 )-epoxide was converted into (-1-)-dispar lure, the sex pheromone of the gypsy moth in >95% ee [101]. 

Enzyme Preparations used in food processing are derived from animal, plant, or microbial sources (see Classification, below). They may consist of whole cells, parts of cells, or cell-free extracts of the source used, and they may contain one active component or, more commonly, a mixture of several, as well as food-grade diluents, preservatives, antioxidants, and other substances consistent with good manufacturing practices. 

Enzyme preparations are produced in accordance with good manufacturing practices. Regardless of the source of derivation, they should cause no increase in the total microbial count in the treated food over the level accepted for the respective food. 

Application and Principle This procedure is to be applied to enzyme preparations derived from either animal or microbial sources. 

D-p-Hydroxyphenylglycine and its derivatives are important as side-chain precursors for semisynthetic penicillins and cepharosporines. Yamada and coworkers of our laboratory found that these amino acids can be efficiently prepared from the corresponding 5-monosubstituted hydantoins using the microbial enzyme D-hydantoinase [4]. 

Paclitaxel and related compounds have also been found in various Taxus species in addition to the Pacific yew, occurring in roots, stems, wood, and needles as well as bark. Yew extracts contain a complex mixture of taxanes, with paclitaxel usually constituting less than 20% of the total taxanes. Isolation of paclitaxel from these mixtures is a difficult purification problem and contributed to the slow development of this compound as a drug. The most valuable material in this mixture for semisynthesis is 10-deacetylbaccatin-III. Microbial strains were isolated from soil samples containing C-13 deacylase and C-lO-deacetylase enzyme activities that are able to convert mixtures of taxanes to 10-deacetylbaccatin-III, thereby increasing the amount and ease of isolation of this precursor for semisynthesis (Scheme 17.14). Treatment of ethanol extracts, prepared either from whole plants of a variety of renewable yew cultivars or from material derived from the bark of... 

Ansamycins. Synthesis of 25-deacetoxy-25-epi-hydroxy rifenycin S (RF S) was reported. The additional hydroxyl function introduced by the transformation did not have the expected effect on antibacterial activity, although the proper conformation for interaction with the target enzyme was proved. RF derivatives containing 3-amidino and 4-aminoimidazolo [4,S-c] moieties were prepared and some showed good antinycobacterial activity in mice. LM 427, a spiropiperidyl RF, was particularly effective in mouse tubercular infection models. Microbial transformation of RF B to RF O and RF S, a intermediate in the thesis of rifempicin,... 

Oxidations - Enzymes which functionalize inactivated carbon are often difficult to obtain and handle. Many examples exist of oxidations using microbial fermentations, e.g. for steroids, and recently in olefin oxidation.Such preparative transformations have not been achieved with purified enzymes and are unlikely to be amenable to large-scale in vitro approaches because of instability and complexity of the enzyme systems. Klibanov and coworkers, however, have developed systems with horseradish peroxidase and xanthine oxidase for oxidation of aromatic alcohols and amines, for use in syntheses and waste water treatment. Hydroxyphenyl compounds can be oxidized to dihydroxy derivatives. L-DOPA has been made from L-tyrosine in this manner.Cyclohexanone was oxidized to t-caprolactone with a bacterial oxygenase. 

Pancreatic preparations have been widely used as digestive aids, because they contain proteases, amylase and lipase. They have been prescribed for patients who have pancreatic disorders or after removal of the pancreas. The various activities present in the pancreatic preparations can be duplicated by in vitro methods from blends of microbial enzymes derived from Bacillus subtilis, Aspergillus flavus-oryzae and Aspergillus niger. Cellulase derived from Aspergillus niger is often added to the microbial preparation. The pancreatic preparations still hold the major share of the market, but this could be a useful application for the right combination of microbial enzymes. 

There are considerable differences in the hydrolysis rates of different amino acids. If the rate is too low for practical purposes, then the chloroacetyl derivatives of the racemates can be applied as substrates instead of the acetyl derivatives. Of course, it is often worthwhile to recover the unchanged D-acylamino acid and hydrolyze it with aqueous acid to produce the D-enanthiomer of the amino acid. The unnatural D isomers are frequently used as building components in studies of structure-activity relationships, in the preparation of hormone analogs resistant to the action of proteolytic enzymes and in the synthesis of microbial peptides. 

The native Pasteurella and the recombinant Escherichia co//-derived preparations of the various microbial Pasteurella GAG synthases rapidly form long polymer chains in vitro (2). The sugar transfer specificity of the native-sequence enzymes is exquisite and only the authentic sugars are incorporated into polymer products. For example, the native synthase enzymes do not utilize significantly the C4 epimer precursors in comparison to the natural UDP-sugars. 

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