Identification of Residues Essential for the Activity and Substrate Affinity of l-Carnitine Dehydrogenase

Recently, two l-carnitine dehydrogenases from soil isolates Rhizobium sp. (Rs-CDH) and Xanthomonas translucens (Xt-CDH) have demonstrated to exhibit mutually differing affinities toward l-carnitine. To identify residues important for affinity to the substrate, we compared the primary structure of Xt-CDH and Rs-CDH with the recognized 3D structure of 3-hydroxyacyl-CoA dehydrogenase (PDB code: 1F0Y). Then, six residues of Xt-CDH (Phe143, Gly188, Ile190, Ala191, Gly223, and Ala224) and the corresponding residues of Rs-CDH (Tyr140, Ala185, Val187, Gly188, Ser220, and Phe221) were selected for further mutagenesis. The residues of Xt-CDH were replaced with that of Rs-CDH at the corresponding position and vice versa. All Rs-CDH mutants exhibited slight effects on substrate affinity, except for the double mutants Rs-V187I/G188A, which was devoid of enzyme activity. All Xt-CDH mutants showed different K _m values. Xt-F143Y caused a higher increase in the K _m value. Furthermore, the kinetic parameters of 10 mutants at Xt-F143 and Rs-Y140 were investigated. All Rs-Y140 mutants, except aromatic residues (Phe, Trp), produced proteins that were almost entirely devoid of enzyme activity and with disrupted affinity to l-carnitine. All Xt-F143 variants showed a marked reduction (P ≤ 0.05) in enzyme activity. Overall, our results suggest that the aromatic rings of Tyr140 in Rs-CDH and Phe143 of Xt-CDH are essential for substrate recognition.     

Milk Clotting Enzyme from Microorganisms

The purification of the milk clotting enzyme from Mucor pusillus Lindt could be achieved by column chromatography on Amberlite IRC-50 by raising pH from 3.5 to 4.5 and about 70% of activity was recovered after this treatment. After the treatment through the column of DEAE-Sephadex A-25, the trace cellulase activity could be eliminated.

The homogeneity of the purified preparation was proved by ultracentrifugal analysis and electrophoretic patterns at various pH values.

Isoelectric point of this enzyme is considered to lie between pH 3.5 and 3.8.

The enzyme activity was inhibited by Hg⁺⁺ or Fe⁺⁺⁺.

Trypsinogenkinase activity was not contained in this enzyme.

The antiserum against the milk clotting enzyme from Mucor pusillus reacted with the purified and crude enzyme preparations in precipitin test and inhibited their enzyme activities, but did not react with other enzymes such as rennin, pepsin, acid proteases from Aspergillus saitoi and Aspergillus oryzae, or the culture filtrates of some strains of Mucor and Rhizopus.

The antigen-antibody reaction was so specific that it might be possible with this antibody to identify this enzyme and also the strain itself.

Normal sera from some mammals inhibited this enzyme activity too, but the degree was less than that with rennin.     

Mechanism of Antimycin A Resistance in Bacillus subtilis

The site of action of antimycin A is known to lie between cytochrome b and c in the respiratory chain of mammalian cells. But in general, bacteria, even those which have cytochromes similar to those of mammalian cells such as Bacillus subtilis, are naturally resistant to this antibiotic.

The mechanism of this natural resistance is studied using a strain of B. subtilis. Succinoxidase activity of the intact cells of this bacterium showed very low sensitivity to the antibiotic, but on disruption of the cells, the sensitivity increased 7.5 times. Moreover, the activity of the intact cells could be sensitized by treatment with cationic detergent. In addition to the permeability barrier suggested by the above results, it was found that the electron transport system of this bacterium contained antimycin A insensitive by-path.     

Characterization of Pseudomonas aeruginosa Bacteriophage 95

A new strain of bacteriophage, phage 95, specific to Pseudomonas aeruginosa and Ps. schuylkilliensis was isolated. It has been shown that the phage induces a lytic enzyme which hydrolyzes peptide bond between l-alanine and d-glutamic acid in the peptidoglycan of cell wall.

Here the characteristics of phage 95 are described. It possesses a hexagonal head of 650Å, a contractile tail of 1150Å and spike with fibers. Its DNA has a GC content of 48%, a density of 1.700 g · cm^?3 and Tm of 87.7°C. Photoreactivation was observed. Latent period is 20 min and burst size is 50. The phage is stable between pH 5 and 7.5, and unstable above 55°C. Culture condition and 300 liter scale cultivation are also presented.     

Studies on the Autolysis of Aspergillus oryzae

Mode of action of crystalline nuclease O obtained from autolyzed Aspergillus oryzae on RNA and synthetic homopolymers was examined. Crystalline nuclease O had no strict base specificity, although the velocity of hydrolysis was poly A > poly U > RNA > poly C. This enzyme did not degrade poly G. Digestion of high molecular weight RNA with an excess of this enzyme produced mono-, di- and trinucleotides with 5′-terminal phosphate. The amount of mono-, di- and trinucleotides was, respectively, 13.6, 70.0 and 16.4% of total degradation products. All the four bases were detected in mononucleotide fraction and 3′-terminals and 5′-terminals of oligonucleotides.     

A Method of Fractionation of Bacterial Cytoplasmic Membrane

A study was made of thermogravimetric analyses of microcrystalline cellulose, (Avicell), over a temperature range from 240°C to 300°C under air and nitrogen by means of a thermal balance. For comparative purpose, cellobiose and glucose were also used. The volatilization rate of cellulose was related to the amount of pyrolytic residue and accelerated owing to the oxidation in the presence of atmospheric oxygen. The apparent activation energies of pyrolysis were obtained from weight loss data.

Quantities of carbonyl and carboxyl groups in pyrocellulose increased linearly against degradation stages of cellulose irrespective of pyrolytic temperatures and times.     

Milk Clotting Enzyme from Microorganisms

Out of some 800 strains of microorganisms, a potent fungus for milk clotting enzyme was isolated from soil during the course of screening tests and was identified as one of strains of Mucor pusillus Lindt. Satisfactory results were obtained in cheese making experiments with this enzyme which could be produced effectively by solid culture on wheat bran at 30°C for about 70 hrs.

The balance between milk clotting activity and proteolytic activity of this enzyme resembled very much to that of rennet.

Microbial rennet from Mucor pusillus F-27 was obtained with high productivity by solid culture followed by water extraction. The enzyme could be precipitated by salting out with ammonium sulfate and also by mixing with various water-miscible organic solvents such as ethanol, methanol or acetone.

This enzyme is one of acid proteases having its optimal pH for milk casein digestion around 3.5. The ratio of milk clotting activity to proteolytic activity of this enzyme resembled that of calf rennet than those of other proteases of fungal origin. This was more heat stable and more resistant against pH changes than animal rennet. Apparent activity of milk clotting was more affected by Ca ion concentration in milk than that of calf rennet.

The liberation of 12% TCA soluble nitrogen from casein fraction was a little less specific than that of calf rennet. The optimal temperature for milk clotting lay around 56°C.

Electrophoretic patterns of α-peak of casein treated with this enzyme showed the weak proteolysis which resembled that with rennet.     

Effect of salt on the activity of Streptomyces prolyl aminopeptidase

A salt-tolerant prolyl aminopeptidase from Streptomyces aureofaciens TH-3 (TH-3PAP) was purified from a culture supernatant. The gene encoding TH-3PAP was cloned and sequenced. The primary structure of TH-3PAP showed 65% identity with that of PAP from Streptomyces lividans (SLPAP) and possessed a conserved catalytic motif, GxSxGG, which is conserved in the alpha/beta hydrolase fold family. The characterization of the recombinants TH-3PAP and SLPAP indicated a difference: in 4.0 M NaCl, TH-3PAP showed enzyme activity, whereas SLPAP was inactive. Next, we constructed chimeras between TH-3PAP and SLPAP using an in vivo DNA shuffling system and a sandwich chimera (sc-PAP), whose region from 63 to 78 amino acids of TH-3PAP was substituted with that of SLPAP. Comparison of the biochemical properties between TH-3PAP and the salt-sensitive sc-PAP suggested that the fine tuning of the N-terminal conformation of TH-3PAP by hydrophobic interaction is important for the salt tolerance mechanism of the enzyme.