Highly Efficient Production of Transfructosylating Enzymes Using Low-Cost Sugarcane Molasses by A. Pullulans FRR 5284

doi:10.21203/rs.3.rs-375469/v1

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Highly Efficient Production of Transfructosylating Enzymes Using Low-Cost Sugarcane Molasses by A. Pullulans FRR 5284

https://doi.org/10.21203/rs.3.rs-375469/v1

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In this study, sugarcane molasses was used to produce transfructosylating enzymes by A. pullulans FRR 5284. It was found that NaNO₃ was a better nitrogen source than yeast extract while exogeneous phosphorous was not needed. Adding only 4.4 g/L NaNO₃ into the molasses medium containing 100 g/L sugars led to the highest total transfructosylating activity of 123.8 U/mL. Scale-up of the enzyme production process from shake flasks to 1 L reactor improved the enzyme activity and productivity to 171.7 U/mL and 3.58 U/mL/h, 39% and 108% higher than the corresponding activity and productivity from shake flasks, respectively. FOS production from 500 g/L sucrose led to the highest yields of ~ 61% using intracellular, extracellular, and total enzymes from shake flasks and the reactor. Enzymes from different sources led to very different FOS profiles, indicating that FOS profiles can be controlled by adjusting intracellular and extracellular enzyme ratios to adjust prebiotic activity.

Biotechnology and Bioengineering

Molasses

Aureobasidium

Transfructosylation

Fructooligosaccharides

Nitrogen

Scale-up

Aureobasidium is an attractive genus of microorganisms for biosynthesis of transfructosylating enzymes, namely β-D-fructofuranosidase (FFase, EC 3.2.1.26) and fructosyltransferase (FTase, EC 2.4.1.9). These enzymes convert sucrose and inulin to fructooligosaccharides (FOS), which are health-beneficiary oligosaccharides and are used as food and feed prebiotics (Bali et al. 2015; Flores-Maltos et al. 2016). Transfructosylating enzymes are often produced using sucrose as a carbon source (Bali et al. 2015; Flores-Maltos et al. 2016). High purity sucrose is commercially produced from sugarcane and sugar beet juice after removal of reducing sugars such as glucose and fructose and other impurities such as protein, minerals, and colorants. Along with sucrose production, molasses, a sucrose-rich by-product is also generated after sucrose crystallization and separation. Although high purity sucrose is a preferred carbon source for producing high purity FOS for human consumption, biosynthesis of transfructosylating enzymes does not always need high purity sucrose as a carbon source.

Nitrogen is an important nutrient affecting microbial growth and product synthesis. The effect of nitrogen source and concentration on transfructosylating enzyme production was previously studied with microorganisms such as Aspergillus strains (Ashok Kumar et al. 2001; Dorta et al. 2006). In addition, optimal nitrogen concentration is also affected by other nutrients such as phosphorus and magnesium (Ashok Kumar et al. 2001; Vandáková et al. 2004). However, such studies have been very limited with Aureobasidium strains. To the authors’ knowledge, the effect of nitrogen sources such as yeast extract and NaNO₃ at different levels and combination ratios on FFase production by A. pullulans CCY 27-1-94 was only reported in one study, in which combination of 10 g/L yeast extract and 10 g/L NaNO₃ led to the maximum FFase activity production using synthetic sucrose media (Vandáková et al. 2004). Moreover, high concentrations of yeast extract and NaNO₃, e.g., 10–20 g/L (each) at various ratios were used for transfructosylating enzyme production from pure sucrose media by Aureobasidium strains (Aung et al. 2019; Jung et al. 2011; Salinas and Perotti 2009; Zhang et al. 2019).

Molasses contains 30% − 60% sucrose, around 6% − 12% glucose, and 6% − 12% fructose (Bortolussi and O’Neill 2006; Dorta et al. 2006; Zhang et al. 2019). Molasses also contains proteins, minerals such as calcium, potassium, magnesium and phosphorus, and vitamins, which are either derived from sugarcane or additives used in the cane juice clarification process which may include the addition of lime and phosphoric acid (Doherty 2011; Thai et al. 2012). Molasses is often directly used as a feed supplement or fermented to bioproducts such as ethanol and microbial oils (Bento et al. 2020; Mordenti et al. 2021). Molasses was also studied for producing transfructosylating enzymes, but the studies were only limited to Aspergillus strains (Dorta et al. 2006; Xie et al. 2017). In one study, it was reported that highest transfructosylating activity by Aspergillus was achieved with addition of up to 15 g/L yeast powder into molasses medium (Dorta et al. 2006). In the studies with Aureobasidium strains, molasses was only used for producing FOS by transfructosylating enzymes derived from Aureobasidium strains (Khatun et al. 2020; Shin et al. 2004; Zhang et al. 2019). To the authors’ knowledge, the use of molasses to produce FOS enzymes by Aureobasidium strains has not been studied. The authors expected that with the use of molasses as a sucrose source, the levels of exogeneous nutrients could be reduced significantly compared to the use of synthetic sucrose media, thus significantly reducing the enzyme production cost.

Transfructosylating enzymes consists of several individual enzymes and these enzymes likely have different transfructosylating and hydrolytic activities (Yoshikawa et al. 2006). These enzymes are generally produced both intracellularly and extracellularly and the compositions of intracellular and extracellular transfructosylating enzymes are likely different because enzymes may have different capabilities to migrate from cells to fermentation broth through cell membranes. Therefore, it is likely that FOS produced from intracellular and extracellular transfructosylating enzymes have different ratios of individual FOS compounds, namely 1-kestose (GF₂), nystose (GF₃), and 1, 1, 1-kestopentaose (GF₄). As a result, a combination of the use of intracellular and extracellular transfructosylating enzymes likely leads to the production of FOS with profiles having optimal prebiotic activities for food and feed applications. In this study, molasses was firstly studied as a low-cost carbon source for producing transfructosylating enzymes by a recently selected novel A. pullulans strain. The effect of exogenous nitrogen type and concentrations of exogeneous nitrogen and phosphorus on transfructosylating activity was investigated in shake flasks. Moreover, under selected conditions, enzyme production was scaled up to a 1 L stirred tank bioreactor and compared with shake flasks. Lastly, FOS production using intracellular, extracellular, and mixed enzymes from both shake flasks and the bioreactors was performed and compared. This study provided important process optimization and scale information on production of transfructosylating enzymes using sugarcane molasses by A. pullulans and indicated a simple strategy for FOS production from sucrose by transfructosylating enzymes with controllable FOS profiles.

Materials

Sugarcane molasses was supplied by the Racecourse Sugar Mill in Mackay, Australia. The molasses contained 41.7 wt% sucrose, 7.4 wt% glucose and 5.9 wt% fructose based on high performance chromatography (HPLC) analysis, which was described in sugar analysis section. The molasses also contained 4.3 g/kg total nitrogen, 1.8 g/kg phosphorus, 2.8 g/kg magnesium, 9.5 g/kg potassium, 2.3 g/kg calcium and others, which were determined using inductively coupled plasma optical emission spectrometry (supplementary material). Sugars, such as sucrose, glucose, fructose, GF₂, and GF₃ were purchased from Sigma-Aldrich (US) while GF₄ was obtained from Megazyme (Ireland). All other chemicals were purchased from Sigma-Aldrich (US), except for yeast extract (Merck, Germany). All the chemicals used in this study were analytical grade or above.

A. pullulans FRR 5284 was purchased from the Australian CSIRO Fungal Culture Collection. The strain was identified as a highly effective FOS enzyme producer in the authors’ previous work (Khatun et al. 2020). The strain was stored in 30% glycerol stock solution at -80 ℃.

Preparation of seed culture

The inoculum medium was prepared based on the authors’ previous study (Khatun et al. 2020). Stock solution of the strain was firstly streaked onto YPD agar plates two days before liquid cultivations. The spores of A. pullulans FRR 5284 were generated after cultivation at 28 ℃ for 2 days and the agar plate was stored at 4 ºC for short-term use within 30 days.

Preculture medium contained 20 g/L glucose, 20 g/L peptone and 10 g/L yeast extract. The medium pH was adjusted to 5.5 by 2 M HCl or 2 M NaOH before sterilization at 121 ℃ for 15 min. A loop of a single colony of the strain was picked and transferred to 10 mL YPD solution in a 50 mL tube. Cultivation was conducted at 28 ºC and 180 rpm for 2 days. After 2 days cultivation, the preculture was collected and centrifuged at 4000 rpm for 10 min. The supernatant was discarded, and the cells were resuspended in (2 × 10 mL) water. The cell solution was re-centrifuged at 4000 rpm for 10 min. The washed cells were resuspended in water and the concentration was controlled based on 2–3 g/L. The cell solution was used as seed culture for next stage shake flask cultivation.

Seed culture for bioreactor inoculation was prepared in a two-step cultivation. The first step was similar to the shake flask inoculation. After the end of the first-step cultivation, 10 mL of inoculum taken from the first-step culture was inoculated into 250 mL shake flasks containing 90 mL diluted molasses (total sugar 20 g/L of sucrose equivalent) with 20 g/L peptone and 10 g/L yeast extract, followed by cultivation at 28 ℃ and 180 rpm for 48 h. After 48 h of cultivation, the cultivation mixture was centrifuged, and cells were collected. The cells were resuspended in water and the final inoculum size was adjusted to 2–3 g/L based on optical density of the yeast solution.

Production of transfructosylating enzymes by A. pullulans FRR 5284 in shake flasks

NaNO₃ and yeast extract combination

Sugarcane molasses was used as a low-cost sucrose source for producing FOS enzymes at a total sugar concentration of 100 g/L (sucrose equivalent) by A. pullulans FRR 5284. Two exogenous nitrogen sources, namely NaNO₃ and yeast extract were added to the molasses medium at NaNO₃ nitrogen (N1)/yeast extract nitrogen (N₂) ratios of 0:0 (control, no exogeneous nitrogen), 1:0, 1:1, and 3:1 at a final nitrogen concentration of 2.48 g/L (equivalent to 15.0 g/L NaNO₃), including 0.92 g/L molasses nitrogen (equivalent to 5.6 g/L NaNO₃). Exogeneous phosphorous source, namely Na₂HPO₄ was supplemented to a final phosphorus concentration of 0.87 g/L (equivalent to 4.0 g/L Na₂HPO₄) including 0.40 g/L molasses phosphorous (equivalent to 1.8 g/L Na₂HPO₄). Other exogeneous nutrients such as magnesium salt and potassium salt were not used as these nutrients were already at relatively high levels (e.g., 3.0 g/L MgSO₄ equivalent, 4.1 g/L KCl equivalent, etc.) in molasses media at 100 g/L sugars.

Cultivations were performed in 250 mL shake flasks containing 50 mL medium. 5 mL seed preculture prepared in 2.2 were inoculated into the shake flasks to start the cultivation. Cultivations was performed at 28 ºC and 180 rpm for 120 h. During cultivation, homogeneous samples of 5 mL were withdrawn at 24 h, 48 h, 72 h, 96 h and 120 h. The shake flask trial was conducted in triplicate. The samples were centrifuged at 4000 g for 15 min. Small portions of the supernatants were collected for determination of residual sugars and FOS by HPLC. The supernatants were stored at -20 ℃ for further determination of activities of extracellular FOS enzymes. The cell biomass was washed with deionized water twice (10 mL water × 2) and freeze-dried for 48 h. The freeze-dried cell biomass was stored at 4 ºC for further determination of activities of intracellular enzymes.

Exogeneous NaNO₃ and Na₂HPO₄

Following the previous section, only NaNO₃ was selected as the sole nitrogen source for further study (as addition of yeast extract did not improve transfructosylating enzyme production) with the use of Na₂HPO₄ as the exogeneous phosphorus source. Three nitrogen levels were selected, namely 0.92 g/L (no exogeneous nitrogen), 1.65 g/L and 2.48 g/L in combination of three phosphorus levels, namely 0.40 g/L, 0.65 g/L and 0.87 g/L, which corresponded to NaNO₃ equivalent levels of 5.6 g/L, 10.0 g/L and 15.0 g/L and Na₂HPO₄ levels of 1.8 g/L, 3.0 g/L and 4.0 g/L, respectively. Other conditions and the experimental procedure were same as those in the previous section.

Scale-up of fructosyltransferase production to 1 L bioreactor

Following determination of exogeneous nutrients required for transfructosylating enzyme production at 100 g/L molasses sugars (sucrose equivalent) by A. pullulans FRR 5284 in shake flasks, the enzyme production was scaled up to a 1 L bioreactor (Sartorius Biostat Q-Plus). The reactor contained 500 mL molasses medium. The prepared seed culture was inoculated to the bioreactor with an initial cell concentration of 2–3 g/L. The pH was controlled at 5.5 by addition of 2 M NaOH or 2 M HCl and the dissolve oxygen level was maintained at above 20% by controlling the agitation speed at 350–500 rpm and the air flow rate of 0.5–0.9 vvm. The cultivation temperature was controlled at 28 ℃. The cultivation was undertaken for 72 h when almost all carbon sources were consumed by A. pullulans FRR 5284 based on a pre-trial. During the cultivation, 15 mL samples were collected at 12 h, 24 h, 36 h, 48 h, and 72 h, respectively. The samples were centrifuged at 4000 rpm for 15 min. Supernatants and cells were processed using similar approach to that for shake flask samples. The supernatant and cell samples were stored for later analysis. All cultivations in the bioreactor were conducted in duplicate.

Enzyme activity assay

Transfructosylating and hydrolyzing activities of intracellular enzymes of cells and extracellular enzymes of supernatants were determined, respectively. For intracellular enzyme activity assay, the reactions were carried out in 5 mL 50% (w/v) sucrose (in 50 mM sodium acetate buffer, pH 5.5) solutions containing 0.5 g/L freeze-dried cells. The extracellular enzyme activity assay was conducted under the same conditions except for replacement of 0.5 g/L freeze-dried cells with 0.1 mL supernatants. The reactions were conducted at 55 ℃ and a magnetic stirring speed of 100 rpm for 1 h, followed by boiling the reaction mixture for 15 minutes to stop the reactions. Residual sucrose, glucose, fructose, and FOS were determined by HPLC. The results were used to calculate the activities of intracellular and extracellular enzymes (Khatun et al. 2020; Zhang et al. 2016).

FOS production from sucrose

FOS production using whole cells, supernatant and the whole fermentation broth containing the cells and supernatant from shake flask culture (under selected conditions) and from bioreactor was conducted. The enzyme-containing cell and supernatant samples were from 72 h shake flaks cultivation and 48 h bioreactor cultivation, respectively, at which the total transfructosylating activities based on the cultivation volume unit (U/mL) were the highest. The total transfructosylating activities of cells, supernatant and the whole fermentation broth used for FOS production were kept at the same level, namely 20 U/mL reaction solution, which were equivalent to approximately 4.34 mg cells/mL reaction solutions, 0.32 mL supernatant/mL reaction solution and 0.16 mL fermentation broth/mL reaction solution, respectively, based on the specific transfructosylating activities of cells and supernatant.

FOS production was performed at 55 ℃ and pH 5.5 with a magnetic stirring speed of 100 rpm for 12 h with pure sucrose solutions containing 500 g/L sucrose. Samples of 0.5 mL were withdrawn at 1 h, 3 h, 6 h, and 12 h for kinetic study of FOS production. The FOS production trial was conducted in triplicate.

Determination of sugars and FOS

Residual sugar concentrations in molasses, and the sugar concentrations in cultivation media were analyzed by HPLC using the methods published in a previous study (Khatun et al. 2020). For FOS production, residual sugar concentrations in FOS media were also referred to the previously reported methods (Khatun et al. 2020).

Calculations

The specific intracellular and extracellular transfructosylating activities were calculated based on per mg cells and per mL supernatant, respectively, using the method published elsewhere (Khatun et al. 2020; Zhang et al. 2016). Intracellular and extracellular transfructosylating activities were also converted to activities based on per unit of cultivation volume (U/mL). The yields of individual FOS and total FOS were calculated by comparison of initial sucrose concentration.

Statistical analysis

All the experiments in this study were undertaken in triplicate unless stated differently. The mean values were reported with standard deviations. Statistical analysis was performed using Student’s t-test with considering a p < 0.05 level as significance.

Effect of exogeneous nitrogen and phosphorous on transfructosylating enzyme production in shake flasks

Effect of NaNO₃-N/yeast extract-N ratio

NaNO₃ and yeast extract are two commonly used nitrogen sources for transfructosylating enzyme production by Aureobasidium strains. In many studies with synthetic sucrose media, these two nitrogen sources were used together at high concentrations of 10–20 g/L with the addition of other nutrients such as phosphorous, magnesium and potassium (Aung et al. 2019; Jung et al. 2011; Salinas and Perotti 2009; Yoshikawa et al. 2006; Zhang et al. 2019). Since molasses is rich in many nutrients, only exogeneous nitrogen and phosphorus were used in the present study. The authors firstly investigated the effect of the nitrogen molar ratios of the two nitrogen sources on transfructosylating enzyme production in the presence of a total of 0.87 g/L phosphorous (4.0 g/L Na₂HPO₄ equivalent).

Figure 1 shows effect of addition of exogeneous nitrogen and the nitrogen molar ratios of the two nitrogen sources on cell biomass production and sugar consumption. Exogeneous nitrogen addition accelerated cell biomass production and sugar consumption and reducing NaNO₃-nitrogen/yeast extract-nitrogen (N1/N2) ratio while maintaining the total nitrogen level led to rapid cell biomass production and sugar consumption especially in the first 96 h. This is understandable as organic nitrogen such as yeast extract can be consumed more readily than inorganic nitrogen source such as NaNO₃. In addition, yeast extract also contains other nutrients such as vitamins, which facilitate the growth of microorganisms. Therefore, in many previous studies, yeast extract is often combined with NaNO₃ for transfructosylating enzyme production by Aureobasidium strains in synthetic sucrose media.

Figure 2 shows the effect of N1/N2 on the transfructosylating activities of intracellular, extracellular, and total enzymes. Specific intracellular activities (U/mg cells) and extracellular activities increased first, peaked at 72 h, and decreased after 72 h. The intracellular activities based on per unit of cultivation volume also increased in the early stage of the cultivation and peaked at different times as cells continued to grow in the 120 h cultivation period (Fig. 1). Overall, the total activities including both intracellular and extracellular activities peaked at 72 h. Table 1 summarizes the experiment conditions and 72 h results for better comparison. Addition of exogeneous nitrogen source increased enzyme activity. However, increasing the portion of yeast extract in the combined exogeneous nitrogen sources did not increase transfructosylating activity. Instead, increasing yeast extract proportion reduced enzyme production despite the increased cell concentration and sugar consumption.

Table 1

Transfructosylating activities of intracellular, extracellular and total enzymes after 72 h of shake flask cultivation with two exogeneous nitrogen sources at different ratios
N1/N2¹	Total nutrient (g/L)		Salt equivalent (g/L)		Cells (g/L)	Transfructosylating activity
N1/N2¹	N	P	NaNO₃	Na₂HPO4	Cells (g/L)	Intracellular (U/mg)	Intracellular (U/mL)	Extracellular (U/mL)	Total (U/mL)
0:0²	0.92	0.87	5.6	4.0	8.7 ± 0.2	2.4 ± 0.0	20.9 ± 0.3	37.8 ± 1.5	58.7 ± 1.8
1:0	2.48	0.87	15.0	4.0	16.2 ± 1.2	3.3 ± 0.0	53.9 ± 1.1	55.9 ± 3.3	109.8 ± 4.4
3:1	2.48	0.87	15.0	4.0	19.8 ± 1.2	1.9 ± 0.0	37.1 ± 2.3	53.8 ± 5.1	90.9 ± 7.4
1:1	2.48	0.87	15.0	4.0	21.5 ± 0.9	1.7 ± 0.0	35.9 ± 0.9	46.6 ± 0.9	82.5 ± 4.7
Note: 1. N1 - NaNO₃ nitrogen, N2 - yeast extract nitrogen, 2. Exogeneous nitrogen sources were not used

Effect of exogenous NaNO₃ and Na₂HPO₄

The effect of exogeneous nutrients on transfructosylating enzyme production using molasses by A. pullulans FRR 5284 was further studied at three nitrogen levels and three phosphorous levels with the addition of different levels of NaNO₃ and Na₂HPO₄. As shown in Fig. S1, in general high levels of nitrogen and phosphorous led to more rapid cell biomass production and sugar consumption which was a similar observation to that by previous investigation (Hayashi et al. 1992; Vandáková et al. 2004). At the highest levels of nitrogen and phosphorous, residual sugars were less than 5.0 g/L compared to approximately 47 g/L sugars left in the molasses medium without addition of exogeneous nutrients.

Figure 2 shows the transfructosylating activities of intracellular, extracellular, and total enzymes with detailed experiment condition shown in Table 2. Specific intracellular transfructosylating activities peaked at 72 h while intracellular and extracellular activities based on per unit of cultivation volume peaked either at 72 h or 96 h because of increasing cell biomass concentrations. Overall, the total transfructosylating activities peaked at 72 h − 96 h. For specific intracellular activity (U/mg), it appeared that high phosphorous level had a negative effect and the highest intracellular activity at the same nitrogen level was achieved without addition of exogeneous phosphorous. For example, the highest intracellular activity of 4.6 U/mg was achieved at 72 h with a total nitrogen concentration of 1.65 g/L (10.0 g/L NaNO₃ equivalent) with addition of only 4.4 g/L NaNO₃ (Mid N, low P). The second highest (4.1 U/mg) was achieved with a total nitrogen of 2.48 g/L (15.0 g/L NaNO₃ equivalent) with addition of 9.4 g/L NaNO₃ (High N, low P). Table 2 summarizes 72 h activity results. The highest intracellular transfructosylating activity of 67 U/mL based on per unit of cultivation volume was achieved with the addition of only 4.4 g/L NaNO₃. The extracellular activity at this condition was at the similar level to other results with medium and high levels of nitrogen. The highest total activity of 123.8 U/mL was achieved with the addition of only 4.4 g/L (mid N, low P), significantly higher than others (p < 0.5).

Table 2

Transfructosylating activities of intracellular, extracellular and total enzymes after 72 h of shake flask cultivation with different levels of nitrogen and phosphorous
Total nutrient (g/L)		Salt equivalent (g/L)		Cells (g/L)	Transfructosylating activity
N	P	NaNO₃	Na₂HPO₄	Cells (g/L)	Intracellular (U/mg)	Intracellular (U/mL)	Extracellular (U/mL)	Total (U/mL)
0.92¹	0.40²	5.6	1.8	6.7 ± 0.6	3.2 ± 0.0	21.4 ± 2.3	25.6 ± 1.7	47.1 ± 4.0
0.92¹	0.65	5.6	3.0	8.2 ± 1.2	2.9 ± 0.0	23.6 ± 1.9	32.5 ± 1.4	56.1 ± 3.3
0.92¹	0.87	5.6	4.0	8.7 ± 0.2	2.4 ± 0.0	20.9 ± 0.3	37.8 ± 1.5	58.7 ± 1.8
1.65	0.40	10.0	1.8	14.7 ± 0.9	4.6 ± 0.2	67.0 ± 0.7	56.8 ± 1.9	123.8 ± 2.6
1.65	0.65	10.0	3.0	15.3 ± 0.6	3.6 ± 0.0	55.8 ± 0.8	57.8 ± 1.8	113.6 ± 2.6
1.65	0.87	10.0	4.0	16.5 ± 0.2	3.4 ± 0.1	55.7 ± 2.5	59.5 ± 1.4	115.2 ± 3.9
2.48	0.40	15.0	1.8	13.7 ± 0.1	4.1 ± 0.1	56.2 ± 0.4	54.3 ± 5.7	110.6 ± 6.1
2.48	0.65	15.0	3.0	15.4 ± 0.5	3.6 ± 0.1	54.8 ± 1.1	55.4 ± 1.1	110.3 ± 2.2
2.48	0.87	15.0	4.0	16.2 ± 1.2	3.3 ± 0.0	53.9 ± 1.1	55.9 ± 3.3	109.8 ± 4.4
Note: 1. Exogeneous nitrogen was not added, 2. Exogeneous phosphorous was not added.

A few studies investigated the effect of nitrogen source on transfructosylating enzyme production using synthetic sucrose with various microorganisms including Aspergillus and Aureobasidium (Hayashi et al. 1992; Nascimento et al. 2019; Vandáková et al. 2004). These studies often led to different conclusions on the best nitrogen source, possibly depending on the strains and other cultivation conditions. Moreover, relatively high concentrations of nitrogen sources (10–20 g/L) and phosphorous sources (~ 5 g/L) together with other nutrients such as magnesium are often used in synthetic sucrose media for producing transfructosylating enzymes (Aung et al. 2019; Dominguez et al. 2012; Jung et al. 2011; Salinas and Perotti 2009; Sangeetha et al. 2004a; Sangeetha et al. 2004b; Shin et al. 2004; Vandáková et al. 2004; Zhang et al. 2019). In the studies of transfructosylating enzyme production by Aureobasidium strains, combination of yeast extract with NaNO₃ is widely used in synthetic sucrose medium (Aung et al. 2019; Jung et al. 2011; Salinas and Perotti 2009; Vandáková et al. 2004; Yoshikawa et al. 2006; Zhang et al. 2019). Some studies reported the production of transfructosylating enzymes using molasses by Aspergillus strains as media but high-cost organic nitrogen sources such as yeast extract and peptone were still used at relatively high concentrations (e.g., 15–30 g/L) with others (Dorta et al. 2006; Xie et al. 2017). The present study, however, showed that addition of yeast extract and phosphorous, and over-dosage of NaNO₃ only increased cell biomass production but did not improve enzyme production. Since molasses already contained nitrogen (a significant portion is derived from sugarcane proteins), phosphorous and other nutrients, only low-cost inorganic nitrogen was required at a relatively low concentration. Over-dosage of nitrogen and phosphorous may lead to the rapid production of cell growth and suppresses the expression of transfructosylating enzymes.

Transfructosylating enzyme production in 1 L bioreactor

Transfructosylating enzyme production was scaled to 1 L bioreactor using molasses medium with addition of only 4.4 g/L exogeneous NaNO₃. Figure 3 shows and compares cell biomass and sugar consumption at shake flaks and the reactor scale. Cultivation in 1 L bioreactor significantly improved cell growth rate and sugar consumption. After 72 h cultivation, only 7.2 g/L residual sugars were left, and cell biomass concentration reached 27.9 g/L compared to residual sugars of 12.6 g/L and cells of 23.8 g/L after 120 h cultivation in shake flask.

Figure 5 compared enzyme production in shake flask and bioreactor. For intracellular enzymes, the maximum specific activity of 4.4 U/mg in the reactor was achieved at only 48 h cultivation compared to 4.6 U/mg after 72 h cultivation in shake flask. The intracellular activity based on per unit of volume reached a platform after 48 h as cell concentration increase offset the specific activity drop. The intracellular, extracellular, and total activities at 48 h reached 96.4 U/mL, 75.3 U/mL, and 171.7 U/mL, 44%, 33% and 39% higher than those corresponding activities achieved at 72 h in shake flasks, respectively. The productivity of total activity was 3.58 U/mL/h, 108% higher than that achieved in shake flasks.

Although the enzyme activity achieved in the present study in the 1 L reactor is still lower than those (550–2100 U/mL) achieved with some engineered strains of Aureobasidium (Zhang et al. 2019), it is much higher than many other studies by wild strains using either sucrose or molasses medium (Shin et al. 2004; Vandáková et al. 2004; Xie et al. 2017). The results from the present study also indicated that production of transfructosylating enzymes from sugarcane molasses by A. pullulans FRR 5284 can be further improved through process optimisation and strain engineering.

Comparison of FOS production using intracellular, extracellular, and total enzymes from shake flasks and reactor.

FOS production was compared using intracellular, extracellular, and total enzymes from both shake flasks (72 h) and the reactor (48 h) was compared at the activity loading of 20 U/mL reaction solution. Table 3 shows that total FOS could be produced rapidly using either the individual or mixed enzymes from shake flasks and the reactor. The total FOS yields with most enzyme samples reached 60% or more after only 3 h reaction. The total FOS yields reduced, which was attributed to the further hydrolysis of FOS to individual reducing sugars.

Table 3

FOS yields using intracellular, extracellular and mixed enzymes from shake flasks and 1 L reactor
Batch	Enzyme type	FOS yield (Xie et al.)
Batch	Enzyme type	1h	3 h	6 h	12 h
Shake flask	Intracellular - cells	58.0 ± 2.0	61.2 ± 0.6	62.0 ± 0.1	54.0 ± 0.5
	Extracellular - broth	55.2 ± 1.0	58.4 ± 0.2	60.2 ± 1.2	56.0 ± 0.9
	Mixed	56.3 ± 1.1	60.1 ± 0.5	61.5 ± 1.0	53.7 ± 0.9
1 L bioreactor	Intracellular - cells	56.3 ± 1.1	60.1 ± 0.5	61.5 ± 1.0	56.5 ± 0.8
	Extracellular - broth	59.2 ± 1.0	61.7 ± 0.5	62.7 ± 0.2	58.2 ± 0.5
	Mixed	57.7 ± 0.6	61.3 ± 0.7	61.4 ± 0.2	57.4 ± 0.6

The prebiotic activity of FOS is also affected by the FOS composition. Understanding the change of GF₂/GF₃/GF₄ ratio will help to determine the reaction time to achieve the preferred ratio of GF₂/GF₃/GF₄. In order to track the ratio changes with different enzymes, relative ratio changes of GF₃ and GF₄ compared to GF₂ (as 1.0) were monitored and the results were shown in Fig. 6. Interestingly, although the total FOS yields were in the similar levels (Table 3), the ratios of GF₃ and GF₄ compared to GF₂ varied using different samples. The use of intracellular enzymes from shake flasks led to the highest GF₃/GF₂ ratios after 3 h reaction while the use of intracellular enzymes resulted in the lowest GF₃/GF₂ ratio. In contrast, the GF₃/GF₂ ratio difference between using intracellular and extracellular enzymes from the reactor was in a much smaller range. Regarding the GF₄/GF₂ ratio, the use of extracellular enzymes from the reactor led to the highest GF₄/GF₂ ratios through the reaction while the use of reactor intracellular enzymes had the least GF₄/GF₂ ratios. In contrast to GF₃/GF₂ ratio, the difference between the GF₄/GF₂ ratios using intracellular and extracellular enzymes from shake flasks was in a much small range. Figure 6 also shows that the use of the mixed enzymes containing both intracellular and extracellular enzymes led to the GF₃/GF₂ and GF₄/GF₂ ratios in between those using the individual intracellular and extracellular enzymes. To the authors’ knowledge, comparison of FOS profiles using intracellular and extracellular transfructosylating enzymes have not been previously reported.

Previously, it was found that FOS from Aspergillus ibericus with GF₂/GF₃/GF₄ ratio of 1:1.28:0.10 had higher activities in promoting the growth of a number of probiotics in an in vitro digestion study compared to FOS from A. pullulans CCY 27-1-94 with GF₂/GF₃/GF₄ ratio of 1:1.61:0.21 (Nobre et al. 2018). Moreover, there are a few commercial FOS products with different FOS composition. For example, Profeed (Tereos, France) and Nutraflora P-95 (Ingredion, Canada) have similar GF₃/GF₂ ratios of 1.43:1 but different GF₄/GF₂ ratios (0.27:1 for Profeed vs 0.36:1 for Nutraflora) according to their product specifications. In contrast, Actilight (Tereos, France) and one FOS product from GCT Nutrition (Nobre et al.) had a similar high level of GF₃/GF₂ ratio of ~ 1.7:1 but different GF₄/GF₂ ratios (0.20:1 for Actilight and 0.30:1 for GCT FOS) (Kaplan and Hutkins 2000; Nobre et al. 2019). Studies also showed probiotics such as Lactobacillus and Bifidobacterium strains had different responses towards FOS products such as NutraFlora P-95, Raftilose P95 and Inulin-S from different companies and/or sources (Huebner et al. 2007). The results from the present study indicated that FOS profiles could be controlled through combination of the activity ratio of intracellular and extracellular enzymes and control of reaction time for various applications. This will be very useful for the production of FOS products requiring different GF₂/GF₃/GF₄ ratios for applications.

In this study, sugarcane molasses was used as a low-cost carbon source for the production of transfructosylating enzymes by a newly identified A. pullulans strain. High transfructosylating activity was achieved with only addition of 4.4 g/L NaNO₃ into molasses. Moreover, the enzyme production using molasses medium supplemented with NaNO₃ was successful scaled-up to a 1 L bioreactor with significant improvement in activity concentration and productivity. This study also demonstrated that FOS composition could be controlled through combination of the activity ratio of intracellular and extracellular enzymes and control of reaction time, which is useful for the production of FOS different compositions for food and feed applications.

Aureobasidium pullulans: A. pullulans; FOS: fructooligosaccharides; GF₂: kestose; GF₃: nystose; GF₄: 1,1,1-kestopentaose; U_t: transfructosylating activity; U_h: hydrolysis activity

Acknowledgements

The work was undertaken as part of the Biorefineries for Profit project which was funded by Sugar Research Australia and the Australian Government Department of Agriculture, Water and the Environment through the Rural R&D for Profit Program and Queensland Government Department of Agriculture and Fisheries, Cotton Research and Development Corporation, Forest & Wood Products Australia, Australian Pork Ltd, Southern Oil Refining, Queensland University of Technology and NSW Department of Primary Industries.

Authors’ contributions

MSK designed and conducted most of experiments, collected data and prepared the manuscript. MH assisted the cultivation in 1 L bioreactor. MDH, RES and IMO reviewed and provided comments on the manuscript. ZZ interpreted the data and finalised the manuscript. All authors read and approved the final manuscript.

Funding

The Biorefineries for Profit project (project no. 2015/902) was funded through the Rural Research and Development for Profit Program.

Availability of data and materials

The data and the materials are all available in this article and the supporting information.

Declarations

Ethics’ approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing financial interests.

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You are reading this latest preprint version

Highly Efficient Production of Transfructosylating Enzymes Using Low-Cost Sugarcane Molasses by A. Pullulans FRR 5284

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Materials

Preparation of seed culture

NaNO₃ and yeast extract combination

Exogeneous NaNO₃ and Na₂HPO₄

Scale-up of fructosyltransferase production to 1 L bioreactor

Enzyme activity assay

FOS production from sucrose

Determination of sugars and FOS

Calculations

Statistical analysis

Results And Discussion

Effect of exogeneous nitrogen and phosphorous on transfructosylating enzyme production in shake flasks

Effect of NaNO₃-N/yeast extract-N ratio

Effect of exogenous NaNO₃ and Na₂HPO₄

Transfructosylating enzyme production in 1 L bioreactor

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1

Highly Efficient Production of Transfructosylating Enzymes Using Low-Cost Sugarcane Molasses by A. Pullulans FRR 5284

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Materials

Preparation of seed culture

NaNO3 and yeast extract combination

Exogeneous NaNO3 and Na2HPO4

Scale-up of fructosyltransferase production to 1 L bioreactor

Enzyme activity assay

FOS production from sucrose

Determination of sugars and FOS

Calculations

Statistical analysis

Results And Discussion

Effect of exogeneous nitrogen and phosphorous on transfructosylating enzyme production in shake flasks

Effect of NaNO3-N/yeast extract-N ratio

Effect of exogenous NaNO3 and Na2HPO4

Transfructosylating enzyme production in 1 L bioreactor

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1

NaNO₃ and yeast extract combination

Exogeneous NaNO₃ and Na₂HPO₄

Effect of NaNO₃-N/yeast extract-N ratio

Effect of exogenous NaNO₃ and Na₂HPO₄