Proteases (proteinases or peptidases) are a class of hydrolases that cleave peptide chains in proteins. Endopeptidases are a type of protease that hydrolyze the internal peptide bonds of proteins, forming shorter peptides; exopeptidases hydrolyze the terminal peptide bonds from the C-terminal or N-terminal, forming free amino acids. Microbial proteases are a popular instrument in many industrial applications. In this review, the classification, detection, identification, and sources of microbial proteases are systematically introduced, as well as their applications in food, detergents, waste treatment, and biotechnology processes in the industry fields. In addition, recent studies on techniques used to express heterologous microbial proteases are summarized to describe the process of studying proteases. Finally, future developmental trends for microbial proteases are discussed.

β-Alanine is the only naturally occurring β-type amino acid in nature, and it is also one of the very promising three-carbon platform compounds that can be applied in cosmetics and food additives and as a precursor in the chemical, pharmaceutical and material fields, with very broad market prospects. β-Alanine can be synthesized through chemical and biological methods. The chemical synthesis method is relatively well developed, but the reaction conditions are extreme, requiring high temperature and pressure and strongly acidic and alkaline conditions; moreover, there are many byproducts that require high energy consumption. Biological methods have the advantages of product specificity, mild conditions, and simple processes, making them more promising production methods for β-alanine. This paper provides a systematic review of the chemical and biological synthesis pathways, synthesis mechanisms, key synthetic enzymes and factors influencing β-alanine, with a view to providing a reference for the development of a highly efficient and green production process for β-alanine and its industrialization, as well as providing a basis for further innovations in the synthesis of β-alanine.

A new serine carboxypeptidase gene, capA, was identified in Aspergillus niger CBS 513.88 by reading genomic information and performing sequence alignment, and the gene was cloned and expressed in Pichia pastoris GS115. In a shake flask, the enzyme activity of the recombinant strain GS115 (pPIC9K-capA) reached 209.3 U mg⁻¹. The optimal temperature and pH for enzyme activity were determined to be 45 °C and 6.0, respectively. After incubation at 40–50 °C or at pH 4.0–8.0 for 1 h, the enzyme retained more than 80% or 60% of its initial activity. The presence of 1–10 mmol L⁻¹ Mg²⁺ enhanced the activity of CapA, whereas 1–10 mmol L⁻¹ Cu²⁺, Fe²⁺, or Co²⁺, 10 mmol L⁻¹ Mn²⁺, or 1–10 mmol L⁻¹ phenylmethylsulfonyl fluoride (PMSF) significantly inhibited its activity. CapA had a broad substrate specificity and preferred the hydrophobic amino acids Leu and Lys at the C terminus of proteins, and N-benzyloxycarbonyl-l-phenylalanyl-l-leucine (Cbz-Phe-Leu) was the optimal substrate, for which CapA exhibited K_m 0.063 mmol L⁻¹ and k_cat/K_m 186.35 mmol L⁻¹ s⁻¹. The good thermostability, pH stability and hydrolysis characteristics of CapA provide a solid foundation for application in the food and biotechnology fields.

A predicted metalloproteinase gene, HypZn, was cloned from Aspergillus niger CGMCC 3.7193 and expressed in Pichia pastoris GS115, and the physicochemical characteristics of recombinant HypZn were investigated after separation and purification. The results showed that the specific activity of the purified HypZn reached 1859.2 U/mg, and the optimum temperature and pH value of HypZn were 35°C and 7.0, respectively. HypZn remained stable both at 40°C and at pH values between 5.0 and 8.0. The preferred substrate of HypZn was soybean protein isolates, and the K_m and V_max values were 21.5 μmol/mL and 4926.6 μmol/(mL∙min), respectively. HypZn was activated by Co²⁺ and Zn²⁺ and inhibited by Cu²⁺ and Fe²⁺. The degree of soybean protein isolate hydrolysis reached 14.7%, and the hydrolysates were of uniform molecular weight. HypZn could tolerate 5000 mM NaCl and completely lost its activity after 30 min at 50°C. The enzymological characterizations indicated that HypZn has great application potential in the food industry, especially in fermented food processing.

A new serine carboxypeptidase gene, capA, was identified in Aspergillus niger CBS 513.88 by reading genomic information and performing sequence alignment, and then, the gene was cloned and expressed in Pichia pastoris. In a shake-flask, the enzyme activity of the recombinant strain GS115 (pPIC9K-capA) reached 109.7 U/mL. The optimum temperature and pH for enzyme activity were determined to be 45°C and 6.0, respectively. After incubation at 40°C-50°C or at pH 4.0–8.0 for 1 h, the enzyme retained more than 80% or 60% of its initial activity. Mg²⁺ enhanced the activity of CapA, whereas Cu²⁺, Fe²⁺, Co²⁺ and PMSF inhibited its activity. Among the six substrates tested, five could be hydrolyzed by CapA. CapA had a broad substrate specificity and preferred the hydrophobic amino acids Leu and Lys in the C-terminus of proteins, and CBZ-Phe-Leu was the preferred substrate for CapA. The good thermostability, pH stability and hydrolysis characteristics provide a solid foundation for the application of CapA in the food and biotechnology fields.