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Design of Experiments as Approach for Enhancing the Production of the Nicotinamide Cofactor by ��Saccharomyces Cerevisiae��



	
Najla BEN AKACHA a*, Rabaa BEN ASKERa and Mohamed GARGOURIb









a Laboratory of Natural Substances,
National Institute of Research and Physical and Chemical Analysis. (INRAP),
Biotechnop�le Sidi Thabet, 2020 Ariana, Tunisia.


b Biocatalysis and Industrial Enzymes Group, 
Laboratory of Microbial Ecology and Technology, 
National Institute of Applied Sciences and Technology. (INSAT) Tunis, University of Carthage Tunisia.












(*) HYPERLINK "mailto:benakachanajla@yahoo.fr"benakachanajla@yahoo.fr

Phone number: +216.71.537.666   +216.71.537.659       
(  Fax number�: 216.71.537.688    



Abstract
Nicotinamide adenine dinucleotide (NAD+) has crucial roles in metabolic and biosynthetic reactions. Although these cofactors have long been indispensable in analytical assays, their practical applications remain limited because of the high cost of these molecules. The aim of this work was to produce the oxidized form of cofactor efficiently and with higher concentrations. To make the production more reliable, simpler and cheaper, Saccharomyces cerevisiae was used as source of this cofactor. The production of NAD+ was improved by an optimization of the growth medium of yeast cells. The experimental program proposed on the basis of a full 23 factorial design demonstrated that the better production was achieved by the respective use of 60 g glucose/L and 30 g peptone/L in anaerobically medium. Signi�cant amounts of NAD+ (up to 232.2 mg NAD+ / 100g extract) were obtained in this case.  

Keywords: cofactor, factorial design, optimization, yeast
                Introduction
The yeast cell  Saccharomyces cerevisiae  is well-studied organism due to the good control of its molecular biology and fermentation techniques. These cells are therefore widely used in many branches of industry. Their physiology has been well recognized and their production has been applied at the industrial scale. Furthermore, the flexibility of yeast�s metabolism, which can be fermentative, respiratory or mixed respire-fermentative, makes it applicable in many biotechnological processes [1]. The baker�s yeast is also considered as an important natural source used to produce nicotinamide cofactor which constitutes a high added value molecule since it can find application in diverse domains. The pyridine nucleotides NAD(H) and NADP(H) play major roles in the formation of by-products. These cofactors offer the flexibility of being a single probe for the studies of many systems. While the importance of utilizing these cofactors for expansive and wide-ranging studies is evident, stabilizing the synthesized cofactors for long term storage has posed a great challenge and has prevented extensive use of nicotinamide cofactors as probes. �
In the case of �Saccharomyces cerevisiae�, redox cofactors (NAD+/NADH) participate in more than 300 different biochemical reactions associated with the enzymatic oxidation/reduction of a specific substrate [2,3]. Special attention has been focused on the intracellular concentration of nicotinamide nucleotide because of its special role as compound through which the energy released during the catabolic processes is trapped and transferred to anabolic reactions. 
The energy and redox potential required to support the growth of the microorganism are supplied mainly by the consumption of carbohydrates. So, the medium (the carbon source) as well as the aeration conditions used for the growth of �Saccharomyces cerevisiae� affect the intracellular concentration ratio of the reduced and oxidized nicotinamide nucleotide. The intracellular concentration of NADH is in general higher in glucose than in galactose grown cells [4]. During the growth of yeast on sugars, energy produced by glycolysis is directed either to the reduction of NAD+. Heux et al. [5] have demonstrated that during the batch fermentation under controlled microaeration conditions, expression of NADH oxidase under the control of a yeast promoter, leads to large decreases in the intracellular NADH concentration and NADH/NAD+ ratio. This increased NADH consumption caused a large redistribution of metabolic fluxes.
During the growth of �Saccharomyces cerevisiae�, biosynthesis of proteins, nucleic acids, lipids and excretion of metabolites result on the production of a surplus of cytosolic NADH [6]. During fully respiratory metabolism, �Saccharomyces cerevisiae� couples the oxidation of cytosolic NADH directly to the mitochondrial electron transport chain via the NADH dehydrogenase and the glycerol 3-P shuttles [7,8].
Commercial cofactor production is of great interest because of the increasing demand for these molecules. The use of commercial form is however no longer practical or cost effective. The aim of the present work was to optimize the production of cofactor from low cost matrix Saccharomyces cerevisiae. It was necessary to find an optimized growth medium which allows to produce high amounts of NAD+. The production was optimized by using 23 full factorial design.
Material and Methods
                     Media and culture conditions
�Saccharomyces cerevisiae� was cultured anaerobically at 32�C (Rotation speed of 120 rpm in 250-mL  flasks). The pH was controlled at pH 5.5. Various growth media have been prepared : YEPD (10 g yeast extract/L, 10 or 30 g peptone/L, 30 or 50 or 60 g D-glucose /L); YEP gly (10 g yeast extract/L, 10 g peptone/L, 1.2 g ammonium sulfate/L, 1 g potassium phosphate/L, 20 g glycerol/L); YEP eth (10 g yeast extract/L, 10 g peptone/L, 1.2 g ammonium sulfate/L, 1 g potassium phosphate/L, 2% ethanol). Cell growth was determined by measuring the turbidity of the culture at OD 600 using UV-Visible spectrophotometer.
Extraction of cofactor
The NAD+ was extracted during the growth cycle of yeast cells. The extraction of oxidized form of nicotinamide nucleotide was achieved as the method described by Zhang et al.,9 with some modifications. 1 mL of yeast suspension was sampled from different growth medium into a tube containing 125 �L of HCl buffer 0.4 M. The HCl extraction destroyed the reduced form (NADH) [9]. Triton X100 (0.5%) was then added and the mixture was made and left on ice for 10 min. NAD+ was then extracted at 50 �%C for 10 min. The neutralization of obtained extract (pH 7.2-7.4) was carried out by using KOH solution (2 M). The samples were centrifuged at 12000 rpm, 4�%C for 10 min. The NAD+ extract was then lyophilized and the concentration was determined spectrophotometrically at 260 nm. The molar extinction coefficient of NAD+ experimentally determined was 1650 M-1cm-1. The extracted cofactor was also used to test the alcohol-dehydrogenase activity.
Improvement of NAD+ production: 23 Factorial design
The improvement of NAD+ production was achieved with a full 23 factorial design after an appropriate choice of the most important variables implied in the production of cofactor by yeast cells. The production was optimized through varying the concentration of two principal elements of the growth medium of Saccharomyces cerevisiae cells. Application of this method requires the adequate selection of factors and levels. Three parameters were studied (glucose concentration, peptone concentration and the presence of oxygen), and their effects on a response were investigated. Each factor was present as two levels, + and -, which were used to represent the low and high levels of the factors, respectively (Table 1). Selection of factors was based on the composition and on the aeration mode of the culture medium that have a significant influence on the production of cofactor NAD+, and selection of levels was carried out based on results obtained in preliminary studies. Eight runs representing treatment combinations are shown in the design matrix as in Table 2. The culture was carried out at 32�C under an agitation of 120 rpm and the pH was adjusted to 5.5. 
   	Results and Discussion
              Production of NAD+ during the growth cycle of �Saccharomyces cerevisiae�
Maintaining the cellular redox balance is a basic requirement for living cells to sustain metabolism and for growth. The intracellular redox state is to a large extent dependant on the intracellular concentrate on ratios of the two pyridine nucleotide systems NADH/NAD+ and NADPH/NADP+ [10,11]. We were focused on this work on the extraction of oxidized form of cofactor (NAD+) from yeast cells cultivated on various growth media. The extraction of cofactor was performed through the growth cycle. Fig. 1 shows the concentration of NAD+ produced after 32 h of culture. It was demonstrated that the best cofactor production was obtained after 24h of culture for all media tested. YEPD (50 g glucose/L) medium allowed to reach a concentration of NAD+ which was 2.64 and 2.18 times higher than the production obtained by respective use of YEPgly and YEPeth media. Thus, YEPD was used to conduct further experiments.
              Optimization of NAD+ production
Experimental design
Oxidized cofactors were quantified in extracts from cells cultivated on various growth media. The cultures were conducted at 32�C and at pH5.5. This pH has been chosen in order to favor the production of oxidized form of nicotinamide. It has been well established that oxidized cofactors are most stable at lower pH (e.g. 5.5-6.0) and reduced cofactors at higher pH (e.g.9.0-9.2) [12]. A 23 experimental design was performed. It consists of eight runs that allowed the study of three parameters (see design matrix in Table 2). The values of each parameter at levels � and + are given in Table 1. The response was determined by calculation of the NAD+ amount produced by yeast cells cultivated in YEPD medium. This production was calculated for each treatment combination in the design.
All experiments were conducted in duplicates, and results were obtained as the average of the experiments under the same conditions (Table 2). According to the obtained contrast coefficients (Table 2), it may be concluded that all factors: X1 (glucose concentration), X2 (peptone concentration) and X3 (aeration mode) have a comparable significant effect on the production of NAD+. The factor X1, when combined with the other factors, its effect on the response was accentuated. This is demonstrated by the study of interactions, which shows that interaction 12, interaction 13, and the interaction of third order 123 have significant contrast coefficients. The important amounts of production (e.g. 232.2 mg NAD+/100g extract) were obtained in anaerobic condition where glucose and peptone concentrations were of 60 and 30 g/L, respectively. These conditions (run 4) allowed to reach a production which was 2.39 times higher when compared to run 7, in which only the peptone concentration was kept and the other parameters were changed. 
Effect of glucose and oxygen on production of NAD+
Oxidized cofactor was quantified in extracts of cells from cultures grown on YEPD and YEPeth media. We can conclude from Table 3 that the presence of glucose affected beneficially the NAD+ production since the cofactor production was higher when this later was extracted from yeast cells grown on YEPD (50 g glucose/L) medium. In this case, the production was 2.31-fold higher if compared to YEPeth medium. The enrichment of growth medium with glucose has a favorable effect on the production. This can be confirmed by Table 2, where the most important production yields were obtained in runs in which high level of glucose concentration was used. This means that the oxidative metabolism of sugar was higher in glucose grown cells.
Table 3 shows also that, for all media tested, the amounts of NAD+ were more important where cultures were conducted under anaerobic conditions. These amounts were 1.09 and 1.47 fold higher in anaerobic extracts if compared to aerobic extracts when YEPD (50g glucose/L) and YEPeth media have been respectively used (Table 3). These results allow to highlight the effect of oxygen on the production of NAD+ and allow to confirm that in the case of fermentative matabolism of Saccharomyces cerevisiae, the krebs cycle, is not activated and the nicotinamide cofactor will be mainly present under the oxidized form. 
           The yeast cells grown under anaerobic condition in YEPD medium containing glucose as source of carbon, allowed to produce the higher amounts of NAD+. This was discussed in previous work of Nissen et al., [6] who have demonstrated that in fermentative metabolism, a part of glucose is not converted to pyruvate, but to glycerol via the glycerol 3P-dehydrogenase. The synthesis of glycerol allows to re-oxidize the excess of NADH produced during the biomass development. The glycerol represents the marker of redox status of yeast [13].  
Conclusion
              The production of NAD+ by using cells of �Saccharomyces cerevisiae�, was studied in this work. The results have demonstrated that the oxidized form of cofactor was highly influenced by the presence of glucose as source of carbon and by the fermentative metabolism of Saccharomyces cerevisiae. The desired level of cofactor purification depends on the ultimate application. Industrial oxidoreductases enzymes produced in bulk as well as their cofactors require little downstream processing, and hence are relatively crude preparations.�The use of NAD+ extracts isolated from yeast is therefore very interesting due to their reduced cost.









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List of figures

Figure 1.
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