Neutral protease is an extremely stable Zn-metalloendopeptidase that is produced by Paenibacillus polymyxa. It is involved in the generation of beta-amylases and alpha-amylases from the large amylase precursor. Although related to trypsin, neutral protease is significantly less harmful to cells and can help prevent unwanted cell clumping without cell membrane damage after one hour incubations (Alvarez et al. 2006).

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 catalyse 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.


In the 1950s neutral protease was first crystallized and characterized from Bacillus subtilis and was thought to be an extracellular product (Fukumoto and Negoro 1951, Guntelburg 1954, Ottesen and Spector 1960).

Into the 1970s the proteases of Bacillus the rmoproteolyticus (Keay et al. 1970), Bacillus megaterium (Millet et al. 1969), Bacillus cereus (Feder et al. 1971), Streptomyces griseus (Nomoto et al. 1959), Aspergillus oryzae (Misaki et al. 1970), and Serratia (Miyata 1971) were preliminarily studied. Subsequently, Griffin and Fogarty investigated the proteolytic and starch degrading enzymes of Bacillus polymyxa (Griffin and Fogarty 1971 and 1973), and reported on the effects of different factors influencing the proteolytic activities.

Work done in the early 1980s highlighted the use of neutral protease as a means of separating epidermal sheets (Kitano and Okada 1983).

In 1993 Ash et al. (1991 and 1993) compared the 16S rRNA gene sequences of different Bacillusspecies and defined a new genus named Paenibacillus, to which the former Bacillus polymyxa now belongs to as Paenibacillus polymyxa.

Types of Neutral Proteinase

  • Soybean proteins
  • Wheat proteins
  • Pea Proteins
  • Rice Proteins
  • Sunflower Proteins
  • Animal proteins
  • Casein
  • Whey protein
  • Meat proteins
  • Egg albumin
  • Silk proteins


The neutral protease utilized in this study was the one expressed by B. subtilis SMSIOS cells harbouring plasmid pSM127. This plasmid contains a fragment of DNA coding for neutral protease isolated from B. subtilis BGSC 1A341 and cloned into the Bam HI site of PUB1 10. The enzyme was produced at 37°C in a 10-1 fermenter, as previously described. The growth medium, separated from the 222 cells by centrifugation, was concentrated by ultrafiltration and proteins were precipitated by addition of ammonium sulphate to 80% saturation. The precipitate was recovered by centrifugation, re-dissolved in 50 mm calcium acetate pH 5.0 and dialyzed against 25 mm Tris/HCl pH 8.0 containing 5 mm calcium acetate.


Polyacrylamide gel electrophoresis in the presence of SDS was carried out using a vertical slab gel apparatus. An exponential gradient of 15-24% along the direction of migration was used. The gels (1 mm thickness) were stained with Coomassie brilliant blue R-250. For amino acid analyses, protein samples were hydrolyzed in 6 M HC1 at 11 0 “C for 22 h in sealed evacuated tubes. The hydrolysis mixture contained 0.1 Y’n phenol as a scavenger for tyrosine destruction. Hydrolyzates were analyzed on a C. Erba (Milan) model 3A29 amino acid analyser using the singlecolumn methodology, according to the manufacturer’s instructions. Tryptophan in protein and protein fragment samples was analyzed qualitatively by recording fluorescence emission spectra of solutions in 6 M Gdn. HCI upon excitation at 295 nm and observing emission near 350 nm. Edman degradations were carried out manually using the phenylisothiocyanate reagent and the phenylthiohydantions of amino acids were identified by HPLC using a Biosil ODS55 CI8 column (Bio-Rad). Experimental details on other analytical methods have been reported previously.


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.

Neutral Proteinase (Neutral Protease) Market Geography Segment, Regional Supply:


  • North America
  • South America
  • Asia & Pacific
  • Europe
  • MEA (the Middle East and Africa)

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