Enzymes for Research, Diagnostic and Industrial Use

Neutral Protease

Official Full Name
Neutral Protease
Neutral protease (Dispase) is a non-mammalian animal origin free (AOF) metallo, neutral protease, purified by methods developed at Worthington. Its mild proteolytic action makes the enzyme especially suitable for the preparation of primary cells and secondary (subcultivation) cell culture, since it is gentle on cell membranes. This protease is also used as a secondary enzyme in cell isolation and tissue dissociation applications, commonly used with collagense.
Bacillolysin; EC; Bacillus metalloendopeptidase; Bacillus subtilis neutral proteinase; anilozyme P 10; Bacillus metalloproteinase; Bacillus neutral proteinase; megateriopeptidase; Neutral Protease (Dispase)

EC No.
CAS No.9080-56-2
CAS No.9001-92-7
SourceBacillus polymy...
Related Reading

Neutral protease refers to a class of proteases that can act catalysis in a neutral, weakly acidic, or weakly alkaline environment. Its optimal pH is between 6.0 and 7.5, and can catalyze the hydrolysis of peptide bonds of proteins, releasing amino acids or peptides. Neutral protease has advantages of quick react rate, no industrial pollution, and wide adaptability of reaction conditions.


Neutral proteases can be divided into four major categories depending on the catalytic mechanism and the functional group of the active site: serine protease, aspartic protease, cysteine protease, and metalloprotease.

Serine protease: serine proteases typically catalyze the hydrolysis of substrate proteins in two steps. During the hydrolysis process, an intermediate dimer of the covalent enzyme-peptide complex is formed followed with the loss of amino acids and the broken of peptide chains. The acyl group is then removed, and the peptide bond is hydrolyzed by the nucleophilic attack of the intermediate by water. Most of the serine proteases form a typical triple structure of Ser-His-Asp, and a small amount of serine serves as the catalytic basis.

Aspartic acid protease: the activity site of aspartic acid protease is dependent on the aspartic acid residue. The aspartic acid residues in the activity site of most of the pepsin family are located in the Asp-Thr-Gly-Xaa motif, which is located at the bilobate amino and carboxyl ends. Xaa can be replaced by Ser or Thr, the side chain is linked to Asp by hydrogen bonding.

Cysteine protease: catalytic mechanisms of cysteine protease are similar to those of serine protease. The study of the catalytic mechanism of several cysteine proteases from different evolutionary sources shows extremely obvious similarities. Papain is considered to be the prototype for the catalytic mechanism of cysteine proteases. The difference of the catalytic mechanism is mainly due to the different conformations of active sites: serine proteases contain a Ser-His-Asp catalytic triad, while there is an acid group and an acyl mercaptan intermediate formed during the hydrolysis of cysteine proteases.

Metalloproteinase: metalloproteinase has a different catalytic mechanism compared with the proteases above. This class of enzymes depends on the presence of divalent metal cations and can be inactivated by dialysis or metal chelates. X-ray crystallography studies have shown that most metalloproteases form a site for metal binding in the enzyme structure during crystal formation. The metal cation is usually Zn2+, and also may be other metal cations, such as Mg2+ and Cu2+. The metal ion at the active site of the enzyme can be chelated by a chelating agent such as EDTA or OP, so that the enzyme loses its partial or full activity. This process is usually reversible and the enzyme activity can recover by re-adding metal ions.


Due to the special role in metabolism, neutral proteases are widely present in animals, plants and microorganisms. Compared with other sources, microbial protease has unparalleled advantages due to its culture conditions, production cost control, and preparation scale. Neutral proteases can be divided into bacterial neutral proteases, fungal neutral proteases, and viral neutral proteases according to their sources.

Bacterial neutral protease: the most commonly used neutral proteases in the market is the bacterial neutral proteases, especially produced by Bacillus, such as Bacillus subtilis and Bacillus licheniformis. The enzyme activity of bacterial neutral protease mostly depends on divalent cations, such as Mg2+, Zn2+, and Ca2+. Bacterial protease has strong hydrolysis ability, quick react rate, the hydrolyzed product has less bitterness, so it has been widely used in the food industry.

Fungal neutral protease: many fungi can produce neutral proteases such as Aspergillus oryzae, Rhizopus, and Mucor. Moreover, the catalytic pH of the fungal protease is wide (usually 4 to 11). Aspergillus oryzae can produce acidic proteases, neutral proteases, and alkaline proteases. The production of fungal proteases is mainly through solid-state fermentation. Their activity of protease is mainly dependent on divalent cations which can be affected by metal chelates. In general, the react rate and stability of fungal proteases are relatively lower than bacterial proteases.

Viral neutral protease: research of viral proteases has largely focused on the medical field. Aspartic proteases, serine proteases, and cysteine proteases have been included in viral proteases. All virus-derived proteases are endonucleases, and their activity does not require the participation of metal ions. Due to its special role in medicine, more and more researches have been carried out on the three-dimensional structure, translation, expression mechanism, and function mechanism of viral proteases.


Applications in the food industry: neutral proteases are widely used in the food industry to improve food quality, stability, and solubility. Neutral proteases produced by Aspergillus oryzae can change the properties of gluten in flour by hydrolysis, and bacterial-derived neutral proteases can be used to increase the extension and toughness of the dough. Neutral proteases can also be applied to beverage clarification. Since the leachate of black tea is digested with neutral protease, the ultrafiltration flux changes significantly due to its high soluble protein content. Neutral proteases can also be used for meat tenderization, mainly because neutral proteases can dissolve myofibrils and elastin. Neutral protease also play an important role in the processing of soy products. In the case of bean paste, for example, the neutral protease produced by the fungus during fermentation makes the protein in the soybean material fully hydrolyzed. While providing necessary growth factors for microorganisms, it also promotes the production of many beneficial amino acids, effectively increasing the nutrients of products.

Applications in the leather industry: protease is mainly used to selectively hydrolyze non-pectin components in leather and remove non-fibrin, such as albumin and globulin. The use of proteases in the leather industry can reduce the soaking time. The choice of neutral protease preparation depends on the specificity of the matrix protein, and the amount of enzyme depends on the softness and hardness of the treated leather. The application of neutral protease in the processing of leather products not only helps to reduce environmental pollution but also saves energy.

Applications in cosmetics: neutral proteases are widely used in the production and development of cosmetics. Neutral proteases used in cosmetics are mostly derived from microorganisms and plants. The addition of neutral protease to toothpaste helps to remove tartar, especially Bacillus subtilis neutral protease. Neutral proteases are added to the cream to dissolve the dandruff, make the skin soft, promote skin metabolism, increase skin absorption of drugs, and reduce the resistance of pathogenic bacteria in the stratum corneum. At the same time, the microbial source of neutral protease is more heat-resistant and can remove the aging keratinocytes, enhance the removal effect, and prevent the formation of acne.

Applications in the pharmaceutical industry: in the pharmaceutical industry, proteases are used to produce pharmaceuticals, such as wound debridement ointment, burn scar softening ointment, anti-inflammatory swelling and sputum drugs. Neutral protease has a wide variety of diversity and substrate specificity, making it an obvious advantage in the development of highly effective therapeutic agents.

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