A library of artificial random proteins of 141 amino acid residues of which 95 are random and which includes the 20 kinds of amino adds was prepared. Out of the 25 identified random proteins, 5 were soluble in the cell lysate, indicating that about 20% of the random proteins expressed in £schericMa ¢oli are expected to be soluble. The soluble random proteins RP3-42 and RP3-45 and insoluble RP3-70 were purified. The solubility of the purified form is the same as that in the cell lysate.

The structural and catalytic properties of two soluble random proteins, RP3-42 and RP3-45, of 141 amino acid residues were investigated. Although no marked secondary structure was detected by CD spectrum, sedimentation equilibrium and small-angle X-ray scattering studies showed that they form an oligomeric structure and are as compact as the molten globule. The random proteins have low but distinct esterase activity; the values of the second-order rate constant for the hydrolysis of p-nitrophenol were 0.78 and 1.39 M 3I s 3I for RP3-42 and RP3-45, respectively. The differences in the properties of the random and the native proteins are discussed from the evolutionary point of view.z 1998 Federation of European Biochemical Societies.

Through selective cultivation with 6-aminohexanoate linear dimer, a by-product of nylon-6 manufacture, as the sole source of carbon and nitrogen, Pseudomonas aeruginosa PAO, which initially has no enzyme activity to degrade this xenobiotic compound, was successfully expanded in its metabolic ability. Two new enzyme activities, 6-aminohexanoate cyclic dimer hydrolase and 6-aminohexanoate dimer hydrolase, were detected in the adapted strains. Recent developments in the chemical industry have resulted in the production and distribution of various synthetic compounds. Nylon-6 is produced by ring cleavage polymerization of caprolactam, in which the monomeric unit, 6-aminohexanoate (Ahx), is combined by amide bonds with a degree of polymerization of Ͼ100, producing by-products of linear or cyclic oligomers of Ahx (called nylon oligomers). We have previously isolated two microorganisms, Flavobacterium sp. strain KI72 (7) and Pseudomonas sp. strain NK87 (6), that grow with the Ahx cyclic dimer (Acd) as the sole source of carbon and nitrogen. The degradation of xenobiotic compounds is highly dependent on specific enzymes, i.e., 6-aminohexanoate cyclic dimer hydrolase (enzyme I [EI]) (8), 6-aminohexanoate dimer hydrolase (EII) (9), and endo-type 6-aminohexanoate oligomer hydrolase (EIII) (11), and the responsible genes are encoded on plasmids (12, 13, 15). If a new metabolic ability could be directly evolved under laboratory conditions, it would be interesting from the standpoint of enzyme evolution and would also provide a good system to study the adaptation of microorganisms to xenobiotic compounds. In this study, we investigated the possibility of creating a new metabolic activity that would degrade the Ahx oligomer in a strain that is not inherently capable of such degradation. Pseudomonas aeruginosa PAO was clinically isolated in New Zealand and has been well studied biochemically and genetically as a standard strain of Pseudomonas (5). The wild-type PAO1 did not use Acd (Fig. 1) and the Ahx linear dimer (Ald) (data not shown); therefore, this strain was used to study whether microorganisms can acquire the ability to metabolize nylon oligomers experimentally. P. aeruginosa PAO1 was grown on M9 minimal medium (18) containing 2 g of glucose and 1 g of NH 4 Cl per liter (M9-Glu medium). Various dilutions of the culture broth were spread on M9-Ahx plates (M9 minimal plates containing 2 g of Ahx per liter as the sole carbon and nitrogen source). After 9 days of incubation at 30ЊC, hypergrowing colonies were obtained at a frequency of 10 Ϫ3. As a control experiment, the same culture broth was spread on an M9 plate containing no carbon source. However, no colonies were observed even after 9 days of incubation. One of the hypergrowing mutants (PAO5501) was purified on an M9-Ahx plate and was cultured on Ahx minimal medium (3 g of Ahx per liter containing salt mixture A [1 g of K 2 HPO 4 , 3 g of KH 2 PO 4 , 5 g of NaCl, 0.2 g of MgSO 4 ⅐ 7H 2 O, 3 mg of FeCl 3 per liter, pH 6.4]) for 3 days. The culture broth (10 9 cells per

Chromium is hazardous pollutant for ecosystem caused chromium especially in

This study was aimed to compare the ability of some yeast strains to consume sugars (sucrose, glucoseand fructose) and to convert them into ethanol during fermentation. The results of this comparison will be thebasis of considerations in choosing the right strain to be used as a mixed culture to increase the productionof ethanol from substrate containing a mixture of sucrose, glucose and fructose, such as juice of cane andsweet sorghum. The study was conducted using fermentation in substrate consisting of glucose, fructose,and sucrose separately, glucose-fructose mixture, and glucose-fructose-sucrose mixture using some yeaststrains: FNCC3012, OUT7009, OUT7027, OUT7055, OUT7080, OUT7096, OUT7903, OUT7913, and OUT7921.Following the fermentation, analysis of the produced ethanol and the remaining sugar was conducted. Theresults of study indicated that the strains with the highest substrate consumption were OUT7921, OUT7096,OUT7055, OUT7027, and OUT7913 for glucose, fructose, glucose-fructose mixture, sucrose, and glucosefructose-sucrose mixture, respectively. Strains that produced highest concentration ethanol were OUT7096 inglucose and sucrose substrates, OUT7921 in substrate of glucose-fructose mixture and sucrose, OUT7913 insubstrate of glucose-fructose-sucrose mixture. Upon consideration of each strain capacity, both in consumingsugar and producing ethanol, the recommended strains for use in mixed culture in bioethanol fermentationusing mixed substrate of glucose, fructose and sucrose are OUT7096, OUT7913, and OUT7921.