The neutral protease is the earliest proteinase used in industrial production. Most microbial neutral proteases are metalloenzymes, with Zn2+ in the enzyme protein. They are the most unstable enzymes in microbial proteinases, and easily autodigest, even causing significant decreases in relative molecular weight during low-temperature freeze-drying. The representative neutral proteases are thermolysin produced by Thermus thermophilus and neutral protease produced by Bacillus subtilis.
Most microbial neutral proteases are metalloenzymes, with one zinc atom in one molecule of enzyme protein. The molecular weight is 35,000-40,000, and the isoelectric point is pH 8-9. They are the most unstable enzymes in microbial proteinases, and easily autodigest, even causing significant decreases in molecular weight during low-temperature freeze-drying.
The representative neutral proteases are thermolysin produced by Thermus thermophilus and neutral protease produced by Bacillus subtilis. These enzymes are stable at pH 6-7, but rapidly inactivated beyond this range. When casein is used as a substrate, the optimal pH for protease from Bacillus subtilis is 7-8, for thermolysin is 7-9, and for protease from Aspergillus is pH 6.5-7.5.
The zinc ion in the active center of the enzyme plays a bridging role between the enzyme and the substrate. Some enzymes contain several calcium atoms in their molecules. Calcium has a stabilizing effect on the conformation. If the zinc in the active center is removed by EDTA, the enzyme can be obtained with different activities by replacing it with other metals. This method can be used to improve the activity of proteinases and prepare highly active proteinase crystals. It is believed that the active center of the high-activity enzyme contains calcium, cobalt, copper, manganese, nickel, and zinc atoms. If these atoms are replaced with barium, cadmium, iron, sodium, or lead, the resulting proteinases are inactive. The calcium type enzyme obtained by crystallization has an activity three times higher than that of the ordinary enzyme. When manganese replaces the zinc in the active center of the neutral protease from Bacillus stearothermophilus, the enzyme activity is increased by 56%, and the heat resistance is also improved. The amino acid composition of many neutral proteases has been determined. Neutral proteases from Bacillus subtilis do not contain disulfide bonds composed of cysteine and may not contain methionine. The order of amino acids is still unclear. In the neutral protease enzyme of Bacillus stearothermophilus, 2-3 tyrosine residues and 1 histidine residue in the enzyme molecule, together with the zinc atom, are essential for its activity, and 1 tryptophan residue and several calcium atoms play an important role in maintaining the conformation of the enzyme. In the neutral protease of Clostridium sporogenes, in addition to zinc, the enzyme activity also requires serine residues. The amino acids in the active center of the heat-resistant neutral protease thermolysin and neutral protease A from Bacillus subtilis are the same.
The minimum synthetic substrate of a neutral protease is Z-Gty-X-NH:, where X must be a hydrophobic amino acid. If the carboxyl group cleavable by cutting the peptide bond is provided by alanine, serine, threonine, or histidine residue, the hydrolysis activity is enhanced. Neutral proteases have almost no effect on the synthetic substrates of alkaline proteases. Hydroxyapatite can be used to adsorb neutral proteases and separate them from alkaline proteases. Since neutral proteases preferentially cleave peptide bonds composed of leucine or phenylalanine, the precise determination can be carried out using the substrate of phenylalanyl-leucine acylamides with furan-acrylic glycine as a ligand by spectrophotometry.
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