Pectinase production from immobilized and free cells of Geotrichum candidum AA15 in galacturonic acid and sugars containing medium

2.1 Microorganism and inoculum preparation

The yeast strain, G. candidum AA15, was retrieved on Sabouraud’s Dextrose agar (SDA) plates and inoculum was prepared by transferring an isolated colony into 250 mL Erlenmeyer flask containing 50 mL SDB (Oxoid). The flask was incubated at 30 °C for 24 h. The density of the culture was adjusted to 1.0 by taking optical density (OD) at 600 nm using spectrophotometer. For pectinase production by using free cells, 5% inoculum was transferred to mineral salt medium (MSM). Mineral salt medium was prepared by dissolving 10 mL of 10X solution A (2% KH₂PO₄, 1.4% (NH₄)₂SO_4, 0.3% MgSO₄·7H₂O, 0.1% CaCl_2, 1% Peptone, 2% Tween 80) and 100 µL of 100X solution B (0.5% FeSO₄·7H₂O, 0.16% MnSO₄·H₂O, 0.14% ZnSO₄·7H₂O, 0.29% CoCl₂·6H₂O) in 98.9 mL of distilled water containing suitable substrate. Inoculum for immobilized cells was prepared using pretreated CB of 0.5 ± 0.1 cm³ size. After washing with tap water, the pieces of CB were boiled for 5 min and then dried at 45 °C for 24 h (Lakkana and Pattana, 2012). The alkali pretreatment to CB was followed by immobilization as reported elsewhere (Ejaz et al., 2018). Two pieces of immobilized CB were taken as inoculum.

2.2 Pectinase production in the presence of different sugars

Initially the effect of presence of sugars was investigated by transferring the inoculum of free and immobilized cells to the MSM containing 2% of different sugars including xylose, glucose and galactose and incubated at 28 °C for 72 h. After incubation, the media was centrifuged at 3500 rpm for 15 min and cell-free culture supernatant (CFCS) was assayed for pectinase by determining reducing sugars. The substrate, 0.5% pectin (Sigma-Aldrich) (in 50 mM sodium citrate buffer, pH 4.8) was mixed with CFCS and incubated at 37 °C in a water bath for 30 min. The reaction was quenched by the addition of Dinitrosalicylic acid and was boiled for 5 min. After chilling on ice bath, distilled water was added. OD₅₄₀ was measured against blank. The blank was prepared using heat inactivated CFCS. One international unit of pectinase was defined as the amount of enzyme that liberates 1 µmol of galacturonic acid per min under the standard assay conditions.

2.3 Determination of pectinase activity using pectin and pectin with galacturonic acid as substrate

Subsequent to the finding that galacturonic acid stimulated the production of pectinase, in another experiment, the free and immobilized cells were transferred separately to the flasks containing MSM supplemented with 1% pectin only or 1% pectin with 2% galacturonic acid and incubated at 28 °C for 72 h. CFCS was collected as described above and assayed for pectinase activity, both in the presence and in the absence of 1% galacturonic acid. The ODs of control tubes were also taken into consideration by adding galacturonic acid in the reaction mixture.

2.4 Effect of the presence of galacturonic acid in production medium on pectinase production

The effect of the presence of simple nutrient, galacturonic acid, was studied by challenging the immobilized and free cells of AA15 with galacturonic acid at different time intervals. Briefly, immobilized and free cells were inoculated in 5 different sets of flasks (4 test sets containing MSM with 1% pectin and 1 control set containing MSM with 1% galacturonic acid) and incubated at 28 °C for 72 h. In 1 test set of flasks, 1% galacturonic acid was added at the start of cultivation (at 0 min). In another set, 1% galacturonic acid was added after 1 h of inoculation, while in the third test set 1% galacturonic acid was transferred after 30 h of inoculation. The samples were withdrawn from all the flasks intermittently and pectinase activity was determined.

A control of CB without yeast cells was also incubated for each set of flasks.

All the experiments were conducted in triplicate and mean values have been presented. Standard deviation was calculated using Microcal Origin 6.0.

3.1 Production of pectinase on different carbon sources

The impact of additional carbon sources such as glucose, galactose, xylose and pectin on pectinase production by G. candidum AA15 was studied. It was observed that the strain produced pectinase in presence of all the tested carbon sources (pectin, xylose, glucose and galactose) suggesting the constitutive expression of the enzyme (Oskay and Yalçin, 2015). Pectinase production by immobilized yeast strain was generally much higher than in the free cells (Table 1). The production was not observed in the control flask (matrix without immobilized cells). Among all the added carbon sources, xylose proved to be best for optimal production of pectinase indicating the role of this sugar as inducer. Moreover, the effect of presence of xylose was not so pronounced in case of free cells compared to immobilized cells.

Whereas, negative effect of glucose on pectinase production by immobilized cells of G. candidum AA15 was evident. It is possible that the presence of glucose caused a repressive effect on pectinase production. In addition, the absence of glucose may have limited the growth and growth-dependant pectinase production was suppressed. In contrast, Poletto et al. (2017) studied the pectinase production by Aspergillus niger LB-02-SF and have suggested that, under a growth limiting condition, pectinase production is increased. It was previously reported that if the monosaccharides (such as glucose) are used over a certain concentration, they have a repression effect on the production of pectinolytic enzymes (Akimitsu et al., 2004). Teixeira et al. (2000) reported that pectin esterase activity from Aspergillus japonicus 586 was susceptible to catabolic repression with high glucose concentrations. Indeed the composition of the pectinase production medium influences the pectinolytic activity.

3.2 Pectinase activity in the presence of pectin and pectin with galacturonic acid

Immobilized cells of G. candidumAA15 produced a titer of 0.308 IU mL⁻¹ in presence of pectin and galacturonic acid compared to the 0.218 IU mL⁻¹ when galacturonic acid was not present in the production medium. However, the activity of pectinase was enhanced to 0.4 IU mL⁻¹ when the enzyme assay was performed in presence of galacturonic acid. It indicated the absence of stringent catabolite repression mechanism and end product tolerance of the enzyme in the given strain that was also found active in some other yeast strains (Moyo et al., 2003). The enzymes with tolerance to their end-products do not lose their activity when the reaction is prolonged and end-product is accumulated, hence, can be exploited for large-scale applications. While the free cells produced 0.137 and 0.241 IU mL⁻¹ of pectinase in absence and in presence of galacturonic acid in MSM, respectively, however, the activity remained unaffected by the presence of galacturonic acid in assay mixture (data not shown).

3.3 Regulation of pectinase production

We studied the effect of the addition of galacturonic acid on the production of the pectinolytic activities by G. candidum AA15. The data revealed that the incorporation of galacturonic acid in production medium at different time interval enhanced the pectinase production by immobilized G. candidum AA15. The production of the pectinolytic activity as measured by reducing groups was not negatively affected when galacturonic acid was added to growing culture of G. candidum AA15 regardless of the time of addition. It indicated that galacturonic acid acts as an inducer for pectinase. Generally, pectinase production by AA15 initiated after 2 h of cultivation of immobilized cells and increase until 5 h, whereupon the pattern varied with the change in presence of carbon source in the production medium (Table 2). When the immobilized cells were challenged with the galacturonic acid after 30 h of cultivation a transient increase in the titers of pectinase was noted. Moreover, the pectinase titers remained higher until 28 h when galacturonic acid was present as sole source of carbon endorsing about the positive effect of galacturonic acid on the ability of immobilized cells to produce pectinase.

Interestingly, pectinase from free AA15 appeared distinct than from immobilized cells as the incorporation of galacturonic acid in pectin containing medium at any stage of fermentation resulted in complete inhibition of the pectinase production by the free cells. The gene expression in immobilized yeast is indeed vary greatly than in free cells as reported earlier for encapsulated cells of Saccharomyces cerevisiae BY4722 (Nagarajan et al., 2014). The authors observed interruption in cell division in immobilized cells, however, extracellular amount of glucose and ethanol varied continuously hinting about metabolic status of the cells. Moreover, productivity of ethanol by immobilized cells was five folds higher than by free cells. In this work, galacturonic acid acted as an inducer for immobilized cells but causes self-catabolite repression in free cells when the concentration is increased beyond the utilization capability of the organism. An optimized production medium is indeed required for the strain; any variation from the optimal concentration of carbon source caused decrease in the productivity of the enzyme (Abdullah et al., 2018).