Low-cost media statistical design for laccase rPOXA 1B production in P. pastoris

Abstract

are multicopper oxidases of great importance in the industry due to their non-specificity and high oxidative potential. Laccases are useful to bleach synthetic dyes, oxidize phenolic compounds and degrade pesticides, among others. Hence, the objective of this work was to optimize low cost culture media for recombinant (rPOXA 1B) laccase production from Pleurotus ostreatus in Pichia pastoris. To this end, low cost nitrogen sources were studied, such as malt extract, isolated soy protein and milk serum. Following, two central composite designs (CCD) were performed. In CCD-1 different concentrations of glucose USP (0-13.35 gL -1 ), protein isolated soy protein (5-25 gL -1 ), malt extract (3.5-17.5 gL -1 ) and (NH 4 ) 2 SO 4 (1.3-6.5 gL -1 ) were evaluated. In CCD-2 only different concentrations of glucose USP (7.9-22 gL -1 ) and isolated soy protein (15.9-44.9 gL -1 ) were evaluated. CCD-2 results led to a One Factor Experimental design (OFED) to evaluate higher isolated soy protein (20-80 gL -1 ) concentrations. In all designs, (CCD-1, CCD-2 and OFED) CuSO 4 (0.16 gL -1 ) and chloramphenicol (0.1 gL -1 ) concentrations remained unchanged. For the OFED after sequential statistical optimization, an enzyme activity of 12,877.3 AE 481.2 UL À1 at 168 h was observed. rPOXA 1B activity increased 30.54 % in comparison with CCD-2 results. Final composition of optimized media was: 20 gL -1 glucose USP, 50 gL -1 isolated soy protein 90 % (w/w), 11.74 gL -1 malt extract, and 4.91 gL -1 (NH 4 ) 2 SO 4 . With this culture media, it was possible to reduce culture media costs by 89.84 % in comparison with improved culture media previously described by our group.

Methanol addition after glucose depletion improves rPOXA 1B production under the pGap in P. pastoris X33: breaking the habit

“…In this study, different culture media (A, B, C and D) resulting from statistical processes of improvement, optimization and cost reduction were used [5,6] to test the expression of the laccase POXA 1B from Pleurotus ostreatus expressed in P. pastoris under the control of the constitutive promoter pGap. Glucose was used as the primary carbon source, followed by the addition or not of methanol, after glucose depletion.…”

“…In this study, we conducted assays to demonstrate the positive effect of methanol supplementation after glucose depletion to produce rPOXA 1B laccase under the control of the pGap promoter in a P. pastoris culture. Different culture media previously devised by statistical designs were used [5,6]. As previously suggested, statistical designs are more efficient than the traditional one factor at time of optimization process [11].…”

“…Culture media A, B and C were based on previously published sequential statistical design and improved processes [5]. Culture media D was based on a sequential statistical design and improved process, where nitrogen and carbon sources were substituted to generate a low-cost culture media for rPOXA 1B production [6] (Table 1). All these media were assayed in triplicate for 0.5% methanol (v/v) addition [26,27], every 24 h, starting at 24 h. In general, samples were collected at 0, 12, 24 and every 24 h up to 168 h (for media A, B, C and D without methanol), and up to 192 h (for media A, B, C and D with methanol).…”

“…Culture media D (without methanol) had the following modifications in comparison to other media: glucose was substituted for a two-fold increase in glucose USP concentration (compared to media A, B and C without methanol). Additionally, peptone and yeast extract were both substituted by isolate soy protein [6], resulting in a media (C/N) 0 ratio of 6.7 ± 0.02 (Table 1). This modification represented a 7.0-, 6.4-and 6.5-fold increase in volumetric enzyme activity compared with culture A, B and C, respectively (Media D volumetric enzyme activity 8771.61 ± 218.79 U L −1 ).…”

A Brief History of Colour, the Environmental Impact of Synthetic Dyes and Removal by Using Laccases

“…Laccases have expressed in hosts as Saccharomyces cerevisiae [228], Yarrowia lipolytica [229], Pichia pastoris [44,204,[230][231][232], Kluyveromyces lactis [233] and even filamentous fungi such as Aspergillus niger [234], Aspergillus sojae [235], and Trichoderma reesei [236]. Even so, P. pastoris is the most successful expression host used in the production of recombinant proteins, mainly for biopharmaceuticals and industrial enzymes [41,42] and one of the most used for the expression of fungal laccases (Table 6).…”