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The present study aimed to discuss the applicability of a clean fermentation-organic acid method for various sources of shrimp waste (shrimp head and shell). Six major cultivated shrimp species in China, including Penaeus vannamei , Penaeus monodon , Fenneropenaeus chinensis , Penaeus japonicus , Macrobrachium rosenbergii , and Procambarus clarkii , were selected as the sources of shrimp waste. The numerical ranges of the corresponding characteristic biochemical indicators were as follows: moisture, 69.47%–79.44%; ash, 21.01%–39.11%; crude protein, 29.65%–46.04%; and crude lipid, 3.18%–9.64%; the data indicated that these types of shrimp waste were good sources for the extraction of chitin, protein and elemental calcium. The traditional chemical method and fermentation-organic acid method were used, and the results of processing the shrimp waste were compared. The results indicated that the purity of the chitin extracted by the fermentation-organic method was as good as the purity of the extracted by the chemical method. Moreover, chitin extracted by the fermentation-organic method had lower acetylation rate, crystallinity index and thermal stability compared with that of chitin extracted by the chemical method. These characteristics indicate that chitin extracted by the fermentation-organic method could be easily deacetylated or degraded leading to easier production of chitin derivatives, such as chitosan or chitooligosaccharides, from this type of chitin. Protein and calcium could be efficiently recycled during the chitin extraction process producing high nutrition protein raw material and high purity calcium citrate. These results suggest a cleaner method for the treatment of the shrimp waste generated from the major cultivated shrimp species in China. [Display omitted] • Chemical composition of six kinds of shrimp wastes were systematically analyzed. • Chitin extracted by new method has lower acetylation rate, crystallinity and thermal stability. • Protein and calcium could be recycled during chitin extraction process. [ABSTRACT FROM AUTHOR]

Thermal activity and stability are important characteristics for proteases applied in the detergent, pharmaceutical, food, and other green industries. With the intent to discover thermostable novel proteases, we constructed a fosmid metagenomic library from marine sediments in the East China Sea and isolated a clone endowed with high proteolytic activity from this library. Sequence analysis of the positive subclones allowed the identification of a coding region of 1254 bp related to protease activity. The unrooted phylogenetic tree and alignment results revealed that the sequence might be derived from Anaerolineaceae bacterium and encodes a new member of the peptidase S8A subfamily with the typical catalytic triad Asp 119 /His 150 /Ser 325 . The fusion protein, named pF1AL2, was expressed in Escherichia coli and showed a molecular weight of 35 kDa. pF1AL2 was active in the pH range of 5.0-11.0 with an optimal pH at 10.0 and had high stability under alkaline conditions, retaining more than 95% of its activity after 24 h at pH 11.0. The optimal temperature of pF1AL2 was 80 °C, and it retained nearly 80% of its activity after 6 h at 70 °C, showing great thermal activity and stability. In addition, the enzyme had great salt tolerance (the residual activity when kept in 3 M NaCl was 40%). Its thermal activity and stability, along with its halotolerance and pH-tolerance, indicate the high potential value of pF1AL2 in industrial applications. The exploitation of pF1AL2 could lay the foundation for the development and utilization of proteases with special features from marine resources by a metagenomic strategy. KEY POINTS: • A novel protease, pF1AL2, from marine sediments, was screened out by a metagenomic strategy. • The protease pF1AL2 analyzed in silico, cloned, and characterized. • pF1AL2 had an optimal temperature of 80 °C and retained nearly 80% of activity after 6 h at 70 °C. • pF1AL2 had great tolerance for high-temperature and acid, alkaline, and high salt environments.