Hiroyasu Ogino

Enhancement of the Organic Solvent-stability of the LST-03 Lipase by Directed Evolution

LST‐03 lipase from an organic solvent‐tolerant Pseudomonas aeruginosa LST‐03 has high stability and activity in the presence of various organic solvents. In this research, enhancement of organic solvent‐stability of LST‐03 lipase was attempted by directed evolution. The structural gene of the LST‐03 lipase was amplified by the error prone‐PCR method. Organic solvent‐stability of the mutated lipases was assayed by formation of a clear zone of agar which contained dimethyl sulfoxide (DMSO) and tri‐n‐butyrin and which overlaid a plate medium. And the organic solvent‐stability was also confirmed by measuring the half‐life of activity in the presence of DMSO. Four mutated enzymes were selected on the basis of their high organic solvent‐stability in the presence of DMSO. The organic solvent‐stabilities of mutated LST‐03 lipase in the presence of various organic solvents were measured and their mutated amino acid residues were identified. The half‐lives of the LST‐03‐R65 lipase in the presence of cyclohexane and n‐decane were about 9 to 11‐fold longer than those of the wild‐type lipase, respectively. Some substituted amino acid residues of mutated LST‐03 lipases have been located at the surface of the enzyme molecules, while some other amino acid residues have been changed from neutral to basic residues.

Amino acid residues involved in organic solvent-stability of the LST-03 lipase

LST-03 lipase from Pseudomonas aeruginosa LST-03 is highly active and stable in the presence of various organic solvents. To further characterize and improve the organic solvent-stability of the LST-03 lipase, residues that potentially provide this stability were identified and mutated to other amino acids in an effort to increase the organic solvent-stability of the protein. S155L, G157R, S164K, S194R, and D209N mutations were found to improve the organic solvent-stability of the wild-type LST-03 lipase. Such mutations were found to induce structural changes, including the formation of a salt bridge, hydrogen bonds, lead to an improved packing of the hydrophobic core, and pI shift of side chain. These changes increased the stability of the protein, thereby improving the organic solvent-stability of the wild-type LST-03 lipase. In addition, a single mutation was found to stabilize the lipase by single or multiple factors.

Stabilities and Conformational Transitions of Various Proteases in the Presence of an Organic Solvent

The half‐life of the activity of the PST‐01 protease that was secreted by organic solvent‐tolerant Pseudomonas aeruginosa PST‐01 was very long in the presence of methanol as compared to that in the absence of methanol. The conformational transitions of the PST‐01 protease, α‐chymotrypsin, thermolysin, and subtilisin in the presence and absence of methanol were monitored by measuring the CD spectra. The conformational stabilities of the PST‐01 protease and subtilisin in the presence of methanol were higher than those in the absence of methanol. This resulted in high stability of these proteases in the presence of methanol. Furthermore, it was suggested that the organic solvent stabilities of enzymes were closely related to the secondary structure by monitoring the conformational transitions of polyamino acids, which form the particular conformations, in the presence and absence of methanol.

Peptide Synthesis of Aspartame Precursor Using Organic‐Solvent‐Stable PST‐01 Protease in Monophasic Aqueous‐Organic Solvent Systems

The PST‐01 protease is a metalloprotease that has zinc ion at the active center and is very stable in the presence of water‐soluble organic solvents. The reaction rates and the equilibrium yields of the aspartame precursor N‐carbobenzoxy‐l‐aspartyl‐l‐phenylalanine methyl ester (Cbz‐Asp‐Phe‐OMe) synthesis from N‐carbobenzoxy‐l‐aspartic acid (Cbz‐Asp) and l‐phenylalanine methyl ester (Phe‐OMe) in the presence of water‐soluble organic solvents were investigated under various conditions. Higher reaction rate and yield of Cbz‐Asp‐Phe‐OMe were attained by the PST‐01 protease when 30 mM Cbz‐Asp and 500 mM Phe‐OMe were used. The maximum reaction rate was obtained pH 8.0 and 37 °C. In the presence of dimethylsulfoxide (DMSO), glycerol, methanol, and ethylene glycol, higher reaction rates were obtained. The equilibrium yield was the highest in the presence of DMSO. The equilibrium yield of Cbz‐Asp‐Phe‐OMe using the PST‐01 protease attained 83% in the presence of 50% (v/v) DMSO.

Enhancement of the aspartame precursor synthetic activity of an organic solvent-stable protease

The PST-01 protease is highly stable and catalyzes the synthesis of the aspartame precursor with high reaction yields in the presence of organic solvents. However, the synthesis rate using the PST-01 protease was slower than that observed when thermolysin was used. Structural comparison of both enzymes showed particular amino acid differences near the active center. These few residue differences in the PST-01 protease were mutated to match those amino acid types found in thermolysin. The mutated PST-01 proteases at the 114th residue from tyrosine to phenylalanine showed enhancement of synthetic activity. This activity was found to be similar to thermolysin. In addition, mutating the residue in the PST-01 protease with arginine and serine showed more improvement of the activity. The mutant PST-01 protease should be more useful than thermolysin for the synthesis of the aspartame precursor, because this enzyme has higher stability and activity in the presence of organic solvents. The results show the potential of organic solvent-stable enzymes as industrial catalysts.

Characterization of Recombinant Glyoxylate Reductase from Thermophile Thermus thermophilus HB27

A glyoxylate reductase gene from the thermophilic bacterium Thermus thermophilus HB27 (TthGR) was cloned and expressed in Escherichia coli cells. The recombinant enzyme was highly purified to homogeneity and characterized. The purified TthGR showed thermostability up to 70 °C. In contrast, the maximum reaction condition was relatively mild (45 °C and pH 6.7). Although the kcat values against co‐enzyme NADH and NADPH were similar, the Km value against co‐enzyme NADH was ∼18 times higher than that against NADPH. TthGR prefers NADPH rather than NADH as an electron donor. These results indicate that a phosphate group of a co‐enzyme affects the binding affinity rather than the reaction efficiency, and TthGR demands appropriate amount of phosphate for a high activity. Furthermore, it was found that the half‐lives of TthGR in the presence of 25% dimethyl sulfoxide and diethylene glycol were significantly longer than that in the absence of an organic solvent.