Biocatalysts. Hydrolases Production

Abstract

The need to reduce waste generation and the use of toxic materials for waste disposal has led to agrowing interest in the development of sustainable bioprocesses using enzymes. Hydrolases can beapplied in different fields such as biofuels, pharmaceutical, detergent, oleochemical, organic synthesis and food industries. Many hydrolases such as, lipases, proteases and glycosyl hydrolases are extracellular, and to make possible its use economically, it is necessary to optimize yields through optimization of culture conditions and/or genetic manipulation to increase the microbial enzyme activities.The objective of this work was to optimize hydrolases production, and the design of bioprocessscale-up. The culture medium to obtain high enzyme activities by different strains belongingto Firmicutes phylum was optimized by means of statistical-based designs, followed by a partialpurification to assess the enzyme preparations properties.A Box Behnken design-based statistical analysis indicated that the variables that most influencedthe lipase and protease production were peptone and sucrose concentration. Increments on lipaseproduction up to 33 U/g at 5 g/L of sucrose were observed, while further increments on the carbonsource concentration reduced the enzyme production. The optimized medium (sucrose, 5g/L; peptone, 7.5 g/L; CaCl2, 0.05 M) produced 35.1 U/mL and 4.60 U/ml for lipase and protease respectively. Both, protease and lipase, were used to enrich refined fish oils in polyunsaturated fatty acids(ω-3 PUFA); as a result, an increment of 1.2-fold of DHA (docosahexaenoic acid) content was obtained.The examination of the culture media components influencing bacterial growth and endoglucanaseproduction demonstrated that mono and disaccharides are useful substrates for enzyme production.Moreover, their combination with carboxymethyl-cellulose (CMC) showed a synergistic effect onenzyme production. Consequently, by using a peptone-based medium amended with sucrose andCMC, a cellulolytic cocktail was efficiently produced in a 1-L stirred tank reactor on batch operation mode, reaching a maximum of 3.12 IU/mL. It is important to point out that the use of simplesugars favors both operative culture conditions and downstream processing.Finally, raw and alkali-pretreated sugarcane bagasse were the carbon sources that better promotedxylanase production. The xylanases obtained were further utilized for: (i) a cocktail formulationsupplementing commercial enzymes that improved the glucose releasement (~38 %) from sugarcane pretreated material for second generation ethanol production. (ii) generation of arabinoxylooligosaccharides, potential prebiotics, from the pretreated sugarcane bagasse and wheat bran.Scaling-up these bioprocesses to evaluate the technical feasibility and the commercial viability isnow being performed in a pilot plant with a 150 L Fermentor. Scale parameters were selected foreach of the enzymes.