José L. Adrio

Microbial Enzymes: Tools for Biotechnological Processes

Microbial enzymes are of great importance in the development of industrial bioprocesses. Current applications are focused on many different markets including pulp and paper, leather, detergents and textiles, pharmaceuticals, chemical, food and beverages, biofuels, animal feed and personal care, among others. Today there is a need for new, improved or/and more versatile enzymes in order to develop more novel, sustainable and economically competitive production processes. Microbial diversity and modern molecular techniques, such as metagenomics and genomics, are being used to discover new microbial enzymes whose catalytic properties can be improved/modified by different strategies based on rational, semi-rational and random directed evolution. Most industrial enzymes are recombinant forms produced in bacteria and fungi.

Stimulatory effect of growth in the presence of alcohols on biotransformation of penicillin G into cephalosporin-type antibiotics by resting cells of Streptomyces clavuligerus NP1

Abstract Growth of Streptomyces clavuligerus NP1 in the presence of methanol or ethanol resulted in a marked increase in production of cephalosporin(s) from penicillin G by resting cells. The mycelium produced in alcohol-supplemented medium was fragmented and dispersed as compared with growth in control medium. HPLC analysis showed that at least two products were present in the biotransformation supernatant fluid after 1 h incubation. One of them has been identified as deacetoxycephalosporin G (DAOG).

Inactivation of deacetoxycephalosporin C synthase in extracts of Streptomyces clavuligerus during bioconversion of penicillin G to deacetoxycephalosporin G

By using cell-free extracts of Streptomyces clavuligerus containing deacetoxycephalosporin C synthase, we can convert penicillin G into deacetoxycephalosporin G. The degree of bioconversion was low (below 1% based on charged substrate) and decreased as substrate concentration was increased. Formation of product occurred during the first 2 h and then ceased. Addition of increasing levels of cofactors and/or substrate at the 2-h point did not result in more product. Preincubation of the crude extracts in the presence of ferrous ions plus either ascorbate or α-ketoglutarate led to complete loss of activity. Such inactivation could not be reversed by catalase, superoxide dismutase, mannitol, thiourea, dimethylsulfoxide, dithiothreitol, or β-mercaptoethanol.

Further studies on the bioconversion of penicillin G into deacetoxycephalosporin G by resting cells of Streptomyces clavuligerus NP-1

Resting cells of Streptomyces clavuligerus NP-1, which posses deacetoxy-cephalosporin C synthase activity, have been shown previously to perform oxidative ring expansion of penicillin G in the presence of iron, ascorbic acid, and α-ketoglutaric acid to form deacetoxycephalosporin G. Further studies on this bioconversion indicated that use of MOPS or HEPES buffer at pH 6.5 more than doubled the extent of the reaction observed with the previously used Tris-HCl at pH 7.4. Levels of bioconversion as high as 16.5% were achieved at low penicillin G concentrations. Previously, conversion yields were <1%.

Screening of Microorganisms for Enzymatic Biosynthesis of Nondigestible Oligosaccharides

Nondigestible oligosaccharides (NDOs), namely prebiotic oligosaccharides, are functional food ingredients that possess properties that are beneficial to the health of consumers. These include noncariogenicity, a low calorific value, and the ability to stimulate the growth of beneficial bacteria in the colon. Fructooligosaccharides (FOS) represent one of the major classes of oligosaccharides in terms of their production volume. They are manufactured by two different processes, which result in slightly different end products. First, FOS are produced by controlled hydrolysis of inulin. Secondly, FOS can be produced from sucrose by using the transfructosylating activity of the enzyme β-fructofuranosidase (EC 3.2.1.26) at high concentrations of the starting material. This chapter summarizes the methods used for the detection and characterization of enzymes of fungal origin with fructosyltransferase activity and the analytical methods utilized to identify the oligosaccharides produced.

Essential Role of Genetics in the Advancement of Biotechnology

Microorganisms are one of the greatest sources of metabolic and enzymatic diversity. In recent years, emerging recombinant DNA and genomic techniques have facilitated the development of new efficient expression systems, modification of biosynthetic pathways leading to new metabolites by metabolic engineering, and enhancement of catalytic properties of enzymes by directed evolution. Complete sequencing of industrially important microbial genomes is taking place very rapidly and there are already hundreds of genomes sequenced. Functional genomics and proteomics are major tools used in the search for new molecules and development of higher-producing strains.