Identification and in silico structural and functional analysis of a trypsin-like protease from shrimp Macrobrachium carcinus.

Macrobrachium carcinus (Linnaeus, 1758) is a species of freshwater shrimp widely distributed from Florida southwards to southern Brazil, including southeast of Mexico. In the present work, we identified a putative trypsin-like protease cDNA fragment of 736 nucleotides from M. carcinus hepatopancreas tissue by the 3'RACE technique and compared the deduced amino acid sequence to other trypsin-related proteases to describe its structure and function relationship. The bioinformatics analyses showed that the deduced amino acid sequence likely corresponds to a trypsin-like protease closely related to brachyurins, which comprise a subset of serine proteases with collagenolytic activity found in crabs and other crustacea. The M. carcinus trypsin-like protease sequence showed a global sequence identity of 94% with an unpublished trypsin from Macrobrachium rosenbergii (GenBank accession no. AMQ98968), and only 57% with Penaeus vannamei trypsin (GenBank accession no. CAA60129). A detailed analysis of the amino acid sequence revealed specific differences with crustacean trypsins, such as the sequence motif at the beginning of the mature protein, activation mechanism of the corresponding zymogen, amino acid residues of the catalytic triad and residues responsible for substrate specificity.

Optimization of five environmental factors to increase beta-propeller phytase production in Pichia pastoris and impact on the physiological response of the host.

Recently, we engineered Pichia pastoris Mut(s) strains to produce several beta-propeller phytases, one from Bacillus subtilis and the others designed by a structure-guided consensus approach. Furthermore, we demonstrated the ability of P. pastoris to produce and secrete these phytases in an active form in shake-flask cultures. In the present work, we used a design of experiments strategy (Simplex optimization method) to optimize five environmental factors that define the culture conditions in the induction step to increase beta-propeller phytase production in P. pastoris bioreactor cultures. With the optimization process, up to 347,682 U (82,814 U/L or 6.4 g/L culture medium) of phytase at 68 h of induction was achieved. In addition, the impact of the optimization process on the physiological response of the host was evaluated. The results indicate that the increase in extracellular phytase production through the optimization process was correlated with an increase in metabolic activity of P. pastoris, shown by an increase in oxygen demand and methanol consumption, that increase the specific growth rate. The increase in extracellular phytase production also occurred with a decrease in extracellular protease activity. Moreover, the optimized culture conditions increased the recombinant protein secretion by up to 88%, along with the extracellular phytase production efficiency per cell.

Shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris bioreactor cultures.

Recently, we engineered a Pichia pastoris Mut(+) strain to produce and secrete recombinant Litopenaeus vannamei trypsinogen. Despite the observed toxicity of the recombinant shrimp trypsinogen to the P. pastoris cell host, when high density cell cultures in shake flasks with alanine in the induction medium were used recombinant shrimp trypsinogen could be produced. To further improve the product yield, in this work, we evaluated L. vannamei trypsinogen production in P. pastoris using a bioreactor and two recombinant P. pastoris strains with different methanol utilization (Mut) phenotypes. The effect of pH and temperature during the induction step on the trypsinogen production was also evaluated. The results indicate that temperature, pH, and Mut phenotypes influence the production of the recombinant protein, with almost no observed effect on cell growth. All cultures with the Mut(+) strain had significant operational difficulties, such as in lowering the induction temperature, maintaining dissolved oxygen (DO) above 20%, and maintaining the methanol concentration at a constant value, and showed a decrease in metabolic activity due to trypsinogen toxicity to the cell host. In the culture with the Mut(s) strain, however, the temperature, methanol concentration, and DO could be more easily controlled, the temperature could be easily decreased, and the trypsinogen caused the lowest toxicity to the host cells. After 96 h of Mut(s) strain induction (pH 6 and 25°C), about 250 mg/L recombinant trypsinogen was detected in the culture medium.