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. 2022 Oct;69(5):2069-2080.
doi: 10.1002/bab.2268. Epub 2021 Oct 19.

Thermostable trypsin-like protease by Penicillium roqueforti secreted in cocoa shell fermentation: Production optimization, characterization, and application in milk clotting

Laísa Santana Nogueira  1 , Iasnaia Maria de Carvalho Tavares  1 , Nívio Batista Santana  1 , Sibelli Passini Barbosa Ferrão  1 , Jabson Meneses Teixeira  2 , Floriatan Santos Costa  3 , Tatielle Pereira Silva  4 , Hugo Juarez Vieira Pereira  4 , Muhammad Irfan  5 , Muhammad Bilal  6 , Julieta Rangel de Oliveira  7 , Marcelo Franco  7
Affiliations

Thermostable trypsin-like protease by Penicillium roqueforti secreted in cocoa shell fermentation: Production optimization, characterization, and application in milk clotting

Laísa Santana Nogueira et al. Biotechnol Appl Biochem. 2022 Oct.

Abstract

The increased demand for cheese and the limited availability of calf rennet justifies the search for milk-clotting enzymes from alternative sources. Trypsin-like protease by Penicillium roqueforti was produced by solid-state fermentation using cocoa shell waste as substrate. The production of a crude enzyme extract that is rich in this enzyme was optimized using a Doehlert-type multivariate experimental design. The biochemical characterization showed that the enzyme has excellent activity and stability at alkaline pH (10-12) and an optimum temperature of 80°C, being stable at temperatures above 60°C. Enzymatic activity was maximized in the presence of Na+ (192%), Co2+ (187%), methanol (153%), ethanol (141%), and hexane (128%). Considering the biochemical characteristics obtained and the milk coagulation activity, trypsin-like protease can be applied in the food industry, such as in milk clotting and in the fabrication of cheeses.

Keywords: Doehlert design; biochemical characterization; milk clotting; trypsin-like protease.

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