Response Surface Methodology Modelling of an Aqueous Two-Phase System for Purification of Protease from Penicillium candidum (PCA 1/TT031) under Solid State Fermentation and Its Biochemical Characterization.

Amaal Alhelli,Mohd Abdul Manap,Hamed Mirhosseini,Zahra Shad,Nameer Mohammed,Eilaf Suliman,Anis Meor Hussin,Abdulkarim Mohammed

doi:10.3390/ijms17111872

Abstract

Penicillium candidum (PCA 1/TT031) synthesizes different types of extracellular proteases. The objective of this study is to optimize polyethylene glycol (PEG)/citrate based on an aqueous two-phase system (ATPS) and Response Surface Methodology (RSM) to purify protease from Penicillium candidum (PCA 1/TT031). The effects of different PEG molecular weights (1500–10,000 g/mol), PEG concentration (9%–20%), concentrations of NaCl (0%–10%) and the citrate buffer (8%–16%) on protease were also studied. The best protease purification could be achieved under the conditions of 9.0% (w/w) PEG 8000, 5.2% NaCl, and 15.9% sodium citrate concentration, which resulted in a one-sided protease partitioning for the bottom phase with a partition coefficient of 0.2, a 6.8-fold protease purification factor, and a yield of 93%. The response surface models displayed a significant (p ≤ 0.05) response which was fit for the variables that were studied as well as a high coefficient of determination (R2). Similarly, the predicted and observed values displayed no significant (p > 0.05) differences. In addition, our enzyme characterization study revealed that Penicillium candidum (PCA 1/TT031) produced a slight neutral protease with a molecular weight between 100 and 140 kDa. The optimal activity of the purified enzyme occurred at a pH of 6.0 and at a temperature of 50 °C. The stability between different pH and temperature ranges along with the effect of chemical metal ions and inhibitors were also studied. Our results reveal that the purified enzyme could be used in the dairy industry such as in accelerated cheese ripening.

Highlights

Microbial proteases (EC 3.4) are considered as the most important group of industrial enzymes with a great number of industrial and biotechnological applications accounting for about 40% of the total enzyme sales and 60% of the total enzyme market worldwide [1]
As it can be seen from the results, the response variables displayed significant (p ≤ 0.05) values for the response surface models and a high R2 value that was noted which was in the range of 0.94–0.98
We studied the effects of four independent variables, i.e., citrate concentration of buffer (8%–16% (w/w), X3), polyethylene glycol (PEG) concentration (9%–20% (w/w), X2), PEG molecular mass (1500–10,000 (g/mol), X1), and NaCl concentration (0%–10% (w/w), X4) and their effect was observed on the purification factor (Y2), the partition coefficient (Y1), and total yield (Y3) for the purified enzyme from the fungus, P. candidum (PCA 1/TT031)

Summary

Introduction

Microbial proteases (EC 3.4) are considered as the most important group of industrial enzymes with a great number of industrial and biotechnological applications accounting for about 40% of the total enzyme sales and 60% of the total enzyme market worldwide [1]. P. camemberti produce two extracellular proteinases [5]: a neutral proteinase and an acid proteinase [6,7,8,9]. P. camemberti synthesis exopeptidases [10]: an exocellular acid carboxypeptidase [11,12], a neutral carboxypeptidase which is mycelial bound [12], and an exocellular aminopeptidase owing to two components [8,13,14,15]. Neutral proteases are significant in the dairy industry since they influence a specific function in hydrolyzing hydrophobic amino acid bonds at a neutral pH, so they reduce the bitterness of food protein hydrolysates [16]

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