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Experimental design of medium optimization for invertase production by Pichia sp.

ABSTRACT: The culture medium requirement for invertase production by Pichia sp. was optimized and identified by initial screening method of Plackett-Burman. Furthermore, optimum concentrations of medium components, which were selected by in initial screening by Plackett-Burman, were determined by the Box-Behnken and its representative three-factor response-surface method. The regression models showed significantly high R (2) values of 97% for invertase activities, indicating that they are appropriate for predicting relationships between yeast extract, peptone and sucrose concentration with invertase production. According to the model the optimal concentrations of sucrose, yeast extract and peptone were 40, 5 and 4 g/ml, respectively. These predicted conditions were verified by validation experiments. In the optimized medium Pichia sp. produced invertase with activity of 38.71 U/ml, which is 4 times higher than that produced in original medium. Thus, this statistical approach enabled rapid identification and integration of key medium parameters for Pichia sp. BCCS M1, resulted the high invertase production.

SUBMITTER: Ghasemi Y

PROVIDER: S-EPMC3907660 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Experimental design of medium optimization for invertase production by Pichia sp.

Ghasemi Younes Y Mohkam Milad M Ghasemian Abdollah A Rasoul-Amini Sara S

Journal of food science and technology 20110817 2

The culture medium requirement for invertase production by Pichia sp. was optimized and identified by initial screening method of Plackett-Burman. Furthermore, optimum concentrations of medium components, which were selected by in initial screening by Plackett-Burman, were determined by the Box-Behnken and its representative three-factor response-surface method. The regression models showed significantly high R (2) values of 97% for invertase activities, indicating that they are appropriate for ...[more]

PMID: 24493883

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Project description:Margatoxin (MgTx) is a high-affinity blocker of voltage-gated potassium (Kv) channels. It inhibits Kv1.1–Kv1.3 ion channels in picomolar concentrations. This toxin is widely used to study physiological function of Kv ion channels in various cell types, including immune cells. Isolation of native MgTx in large quantities from scorpion venom is not affordable. Chemical synthesis and recombinant production in Escherichia coli need in vitro oxidative refolding for proper disulfide bond formation, resulting in a very low yield of peptide production. The Pichia pastoris expression system offers an economical approach to overcome all these limitations and gives a higher yield of correctly refolded recombinant peptides. In this study, improved heterologous expression of recombinant MgTx (rMgTx) in P. pastoris was obtained by using preferential codons, selecting the hyper-resistant clone against Zeocin, and optimizing the culturing conditions. About 36 ± 4 mg/L of >98% pure His-tagged rMgTx (TrMgTx) was produced, which is a threefold higher yield than has been previously reported. Proteolytic digestion of TrMgTx with factor Xa generated untagged rMgTx (UrMgTx). Both TrMgTx and UrMgTx blocked the Kv1.2 and Kv1.3 currents (patch-clamp) (Kd for Kv1.2 were 64 and 14 pM, and for Kv1.3, 86 and 50 pM, respectively) with comparable potency to the native MgTx. The analysis of the binding kinetics showed that TrMgTx had a lower association rate than UrMgTx for both Kv1.2 and Kv1.3. The dissociation rate of both the analogues was the same for Kv1.3. However, in the case of Kv1.2, TrMgTx showed a much higher dissociation rate with full recovery of the block than UrMgTx. Moreover, in a biological functional assay, both peptides significantly downregulated the expression of early activation markers IL2R and CD40L in activated CD4+ TEM lymphocytes whose activation was Kv1.3 dependent. In conclusion, the authors report that the Pichia expression system is a powerful method to produce disulfide-rich peptides, the overexpression of which could be enhanced noticeably through optimization strategies, making it more cost-effective. Since the presence of the His-tag on rMgTx only mildly altered the block equilibrium and binding kinetics, recombinant toxins could be used in ion channel research without removing the tag and could thus reduce the cost and time demand for toxin production.

Project description:BackgroundPretreatments are one of the main bottlenecks for the lignocellulose conversion process and the search for cheaper and effective pretreatment methodologies for each biomass is a complex but fundamental task. Here, we used a 2ν5-1 fractional factorial design (FFD) to optimize five pretreatment variables: milling time, temperature, double treatment, chemical concentration, and pretreatment time in acid-alkali (EA) and acid-organosolv (EO) pretreatments, applied to elephant grass leaves.ResultsFFD allowed optimization of the pretreatment conditions using a reduced number of experiments and allowed the identification of secondary interactions between the factors. FFD showed that the temperature can be kept at its lower level and that the first acid step can be eliminated in both pretreatments, without significant losses to enzymatic hydrolysis. EA resulted in the highest release of reducing sugars (maximum of 205 mg/g substrate in comparison to 152 mg/g in EO and 40 mg/g in the untreated sample), using the following conditions in the alkali step: [NaOH] = 4.5% w/v; 85 °C and 100 min after ball milling the sample. The factors statistically significant (P < 0.05) in EA pretreatment were NaOH concentration, which contributes to improved hydrolysis by lignin and silica removal, and the milling time, which has a mechanical effect. For EO samples, the statistically significant factors to improved hydrolysis were ethanol and catalyst concentrations, which are both correlated to higher cellulose amounts in the pretreated substrates. The catalyst is also correlated to lignin removal. The detailed characterization of the main hemicellulosic sugars in the solids after pretreatments revealed their distinct recalcitrance: glucose was typically more recalcitrant than xylose and arabinose, which could be almost completely removed under specific pretreatments. In EA samples, the removal of hemicellulose derivatives was very dependent on the acid step, especially arabinose removal.ConclusionThe results presented herewith contribute to the development of more efficient and viable pretreatments to produce cellulosic ethanol from grass biomasses, saving time, costs and energy. They also facilitate the design of enzymatic cocktails and a more appropriate use of the sugars contained in the pretreatment liquors, by establishing the key recalcitrant polymers in the solids resulting from each processing step.

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Experimental design of medium optimization for invertase production by Pichia sp.

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Experimental design of medium optimization for invertase production by Pichia sp.

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