1. 30/09/2010Microbial Transformations (NRM)1 30/09/2010Microbial Transformations (NRM)2 Bioconversions or Microbial Transformation The ability of microbes to chemically modify a wide variety of organic compounds. The substrate may be metabolized or take place without energy gain (cometabolism). Bioconversion is often preferable: 1. Substrate specificity : only one specific reaction step is normally catalyzed by an enzyme 2. Sitespecificity (Regiospecificity): only one type functional group may be affected 3. Stereospecificity: only one specific enantiomer is converted. The reaction product is normally optically active. 4. Reaction conditions: enzymatic reactions do not cause destructionof sensitive substrates, due to the mild condiotions of conversion.

2. 30/09/2010Microbial Transformations (NRM)3 Types of Bioconversion Reactions 1. Oxidations 2. Reductions 3. Hydrolytic Reactions 4. Condensations Procedures for Biotransformation 1. Culture (spores, growing cultures, resting cells, cell-free enzyme extracts, immobilized cells), enzymes or immobilized enzymes 2. Suitable medium 3. Conversion time is related to the type of reaction, the substrate concentration, and the microorganism used 4. Applications of bioconversions: industrial, more cost effective processes, optimized for specific proceses. 30/09/2010Microbial Transformations (NRM)4 Transformation of Antibiotics The objective is the development of modified antibiotics with improved effectiveness, reduced toxicity, wider antimicrobial spectrum, improved oral absorption, decreased development of resistance, or lower allergic effects. Types of antibiotic transformation: 1. Indirect transformation. The addition of inhibitors or modified precursors to the medium may result in the synthsis of altered antibiotics via controlled biosynthesis. E.g. Streptomyces parvulus produces two new actinomycins which proline is replaced by this proline analog. 2. Direct transformation. Acylation reactions, deacylation reactions, phosphorylation, hydrolysis reaction, and hydroxylation. E.g. Hydrolysis reactions  splitting of the lactone ring of  -lactam antibiotics (penicillins & cephalosporins) by  -lactamase, leading to inactivation of the antibiotics.

3. 30/09/2010Microbial Transformations (NRM)5 The photo (courtesy of Merck & Co., Inc.) shows how the growth of bacteria on the agar in a culture dish has been inhibited by the three circular colonies of the fungus Penicillium notatum. The antibiotic penicillin, diffusing outward from the colonies, is responsible for this effect. Today, penicillin is made from cultures of Penicillium chrysogenum that has been specially adapted for high yields. http://users.rcn.com/jkimball.ma.ultranet/Biol ogyPages/A/Antibiotics.html The beta-lactams get their name from the characteristic ring structure — shown here in blue — that they all share. (The green arrow shows the bond that is broken by the beta-lactamases that are synthesized by many penicillin-resistant bacteria.) 30/09/2010Microbial Transformations (NRM)6 Transformation of Pesticides Microbial transformation is of interest not for the production of new active agents, but for the greatest possible detoxification of the environment. Removal of pesticides can be accomplished through various mechanisms: 1. Pesticides as carbon sources. E.g. The herbicide Dalapon, a chlorinated fatty acid, which is converted by Arthrobacter sp. into pyruvate after oxidative dehalogenation. Dalapon

4. 30/09/2010Microbial Transformations (NRM)7 Dalapon Status:BSI IUPAC:2,2-dichloropropionic acidCAS:2,2-dichloropropanoic acid Reg. No.:75-99-0 Formula:C 3 H 4 Cl 2 O 2 Activity:herbicides (halogenated aliphatic herbicides) Notes:There is no ISO common name for this substance; the name “dalapon” is approved by the British Standards Institution and the Weed Science Society of America. When this substance is used as an ester or a salt, its identity should be stated, for example dalapon-calcium [53606-78-3], dalapon-magnesium [29110-22-3], dalapon-sodium [127-20-8]. The 2-(2,4,5-trichlorophenoxy)ethyl ester has its own ISO common name, erbon. 30/09/2010Microbial Transformations (NRM)8 2. Cometabolism. Microorganisms do not obtain energy from the transformation reaction and require another substrate for growth. Dehalogenation or oxidative dehalogenation reactions which make pesticide molecules accessible for further breakdown. E.g. Reductive dehalogenation DDT (dichlorodiphenyl trichloroethane)  TDE by Aerobacter aerogenes Dehydrohalogenation DDT  DDE by Trichoderma viride Synonyms for DDT: 1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane; Chlorophenothane Synonyms for TDE: 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethane; Tetrachlorodiphenylethane; Rhothane; Synonyms for p,p'-DDE:1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene; p,p'- dichlorodiphenyldichloroethylene Bioassays of technical-grade DDT, TDE, and p,p'-DDE for possible carcinogenicity were conducted using Osborne-Mendel rats and B6C3F1 mice http://ntp.niehs.nih.gov/index.cfm?objectid=0704C8A4-F52A-BE78-5C81C16C5C1F14C9

5. 30/09/2010Microbial Transformations (NRM)9 DDT has an extremely low volatility and may be the least soluble chemical known, which makes it extremely persistent in soils and aquatic sediments. It has relatively low acute mammalian toxicity and is toxic to a wide range of insects. It kills insects by affecting the transmission of nerve impulses, probably by influencing the delicate balance of sodium and potassium within the neuron. A comparison of a normal Peregrine falcon eggshell and one thinned byexposure to the pesticide DDT. (©Galen Rowell/Corbis. Reproduced by permission.) http://www.pollutionissues.com/Co-Ea/DDT-Dichlorodiphenyl-Trichloroethane.html 30/09/2010Microbial Transformations (NRM)10 3. Conjugate formation. Linkage of pesticides or decomposition products with naturally compounds such as amino acids or carbohydrates results only in a temporary detoxfication. E.g. Conversion of a dithiocarbamate fungicide by E. coli, S. cerevisiae, A. niger, and Hansenula anomale. Thiocarbamates are mainly used in agriculture as insect- icides, herbicides, and fungicides. Additional uses are as biocides for industrial or other commercial applications, and in household products. Some are used for vector control in public health. http://www.intox.org/data bank/documents/chemica l/thiocarb/ehc76.htm

6. 30/09/2010Microbial Transformations (NRM)11 Biotransformation of L-ascorbic acid (Vitamin C) D-Glukosa  D-sorbitolL-sorbose  2-keto-L- sorbitol dehydrogenase Acetobacter suboxydans A. xylinum gulonic acid  sodium salt/2-keto-L-gulonic acid  L-ascorbic acid