New Biotechnology Volume 00, Number 00 August 2010
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Research Paper
Transgalactosylation of lactose for synthesis of galacto-oligosaccharides using Kluyveromyces marxianus NCIM 3551 Anita Srivastava, Saroj Mishra and Subhash Chand Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz-Khas, New-Delhi 110016, India
Among a number of yeast strains screened for whole cell transgalactosylating activity, Kluyveromyces marxianus NCIM 3551 was found to be most suitable biocatalyst for production of galactooligosaccharides (GOS). Cell permeabilization lead to an efficient bioconversion by b-galactosidase resulting in synthesis of GOS. A maximum GOS yield of 36% (w/w) of total sugars was achieved and the products consisted of tri- and tetra-galacto-oligosaccharides. A lactose conversion rate of 80% and productivity of 24 g/L/h was obtained under the optimum conditions at lactose concentration of 20% (w/v), temperature 408C, pH 6.5 and enzyme units after 3 h of reaction time. Tetrasaccharides were the main component of the reaction mixture. The products were quantitated by HPLC and structurally characterized by mass spectrometry.
Introduction Prebiotic oligosaccharides and their use in food industry has lead to the development of various biotechnological processes and search for new microorganisms for their synthesis. World wide interest in prebiotics stems from their nutritional and health benefits. Enzymatic routes are preferred over chemical routes for oligosaccharides synthesis due to their high specificity, efficiency, and environmental friendly status [1]. The commonly known prebiotics are lactulose, raffinose, malto-oligosaccharides, inulin, fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS). Among these, galacto-oligosaccharides possess the ability to stimulate the proliferation of Bifidobacteria inside the human intestine, hence these are known as ‘Bifidus growth factors’ [2]. GOS are widely used as food stuff additives and perform different functions inside the human body such as maintenance of balance of intestinal microflora, enhancing the digestibility of milk products by improving the lactose tolerance and reducing the serum cholestrol level [3]. GOS are galactose containing oligosaccharides where 2–5 units of galactose are attached to glucose by glycosidic linkages which are produced by transgalactosylation of lactose using b-galactosidase [4–7]. During the process of transgalactosylation, Corresponding author: Srivastava, A. ([email protected], [email protected]) http://dx.doi.org/10.1016/j.nbt.2015.04.004 1871-6784/ß 2015 Published by Elsevier B.V.
lactose serves as galactosyl acceptor, forming a chain of di-, triand tetra-saccharides or even higher polymers of galacto-oligosaccharides. The transgalactosylation reaction involved in GOS formation is a kinetically controlled reaction and the final yield of oligosaccharides is determined by the competition between hydrolysis and galactosyl an acceptor. The transgalactosylation process depends largely on the source of b-galactosidase, the initial substrate concentration and the thermodynamic activity of water [8,9]. Commercially available sources for b-galactosidase are mostly yeast, but because of the intracellular nature of b-galactosidase, expensive enzyme extraction, downstream processing and low stability, these have not been extensively used. An important alternative strategy for economic production of GOS would be the use of whole cells. The advantage of using whole cells over the purified enzymes is their easy availability, reusability, low cost of production and stability, continuous processing and economic viability. However, the permeability barrier of the cell results in lowered reaction rates as less substrate is available for conversion. To overcome this bottle neck and increase the volumetric activity, permeabilization of cells has been reported [10,11]. The permeabilizing agent reduces the phospholipid content in the membrane of the cell, and allows the transfer of low molecular weight compounds in and out of the www.elsevier.com/locate/nbt
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cells. Although ethanol-permeabilized Kluyveromyces lactis cells have been used for bioprocesses [1,12–14], no report appears on the use of Kluyveromyces marxianus NCIM 3551 for GOS synthesis. The aim of the present work was to evaluate the transgalactosylation efficiency of the cell bound b-galactosidase of K. marxianus for synthesis of GOS using ethanol permeabilized cells. Analysis of the products were carried out to identify the major oligosaccharides synthesized during whole cell transformation.
Research Paper
Materials and methods Growth, b-galactosidase activity and galacto-oligosaccharide synthesis K. marxianus NCIM 3465, K. marxianus NCIM 3551 were obtained from National culture collection, Pune. K. lactis MTCC 4063 was procured from MTCC, IMTECH Chandigarh and K. marxianus 1078 was from Department Culture Collection Unit, IIT Delhi. The growth medium consisted of (g/L): yeast extract 17.0; ammonium sulphate 9.0; potassium dihydrogen phosphate 5.0; magnesium sulphate: 0.4; lactose: 30.0 in 50 mM sodium phosphate buffer pH 6.5. The cultures were incubated in an orbital shaker (200 rpm) at 288C for 24 h. The medium was inoculated with 10% (v/v) inoculum and growth carried out at 288C for 48 h. Samples were removed every 8 h for measurement of cell biomass and bgalactosidase activity. Lactose, used for induction in growth medium, was filter sterilized using 0.22 mm syringe filter and added at the beginning of the experiments. The cultures were analyzed for cell associated b-galactosidase activity by incubating with o-nitrophenyl-b-D galactopyranoside (oNPGal). For measurement of activity in the permeabilized cells, the washed cells were permeabilized by incubating in 50% (v/v) ethanol and sodium phosphate buffer, pH 6.5 for 15 min at 258C. Cells were separated from the solvent by centrifugation at 5000 rpm for 5 min and washed twice with 50 mM sodium phosphate buffer. Finally, these were suspended in the same buffer and stored at 48C until used for further enzyme analysis and GOS synthesis [15]. The potential of the cells for galacto-oligosaccharide synthesis was evaluated by adding the cells to 30% (w/v) lactose at 508C in an orbital shaker for different time intervals. Samples were qualitatively analyzed for production of higher oligosaccharides by thin layer chromatography.
Cell associated hydrolytic activities of the selected strain Cell associated hydrolytic activities of the selected strain were determined using 10 mM aryl-glucosides and -galactosides, and, disaccharides prepared in 50 mM sodium phosphate buffer, pH 6.5. The aryl substrates used were o-nitrophenyl-a-D-galactopyranoside, o-nitrophenyl-b-D-galactopyranoside, o-nitrophenyl-a-Dglucopyranoside, o-nitrophenyl-b-D-glucopyranoside. The disaccharides tested were lactose, sucrose, maltose, cellobiose and gentiobiose. Aryl glycosidase activity was determined by measuring the release of o-nitrophenol at 420 nm whereas the enzyme activity towards disaccharides was determined by measuring glucose in the reaction mixture [16].
New Biotechnology Volume 00, Number 00 August 2010
b-galactosidase enzyme). Reactions were carried out with shaking (150 rpm) in an orbital shaker at 508C for 8 h. Aliquots (100 ml) were collected at a regular interval of 1 h and the reaction was stopped by boiling the samples at 1008C for 5 min. Samples were centrifuged at 5000 rpm and supernatant diluted with 50 mM sodium phosphate buffer. This was qualitatively analyzed by thin layer chromatography and quantitatively by high performance liquid chromatography (HPLC). Effect of lactose concentration (10–50%, w/v), temperature (20–608C), pH (5.5–7.5) and enzyme units (0.4–1.6) was investigated at the optimum time determined from previous study.
Analytical methods b-Galactosidase activity Hydrolytic activity of b-galactosidase was monitored using oNPGal as a substrate. To 480 ml of 22 mM oNPGal in 50 mM sodium phosphate buffer, pH 6.5, 20 ml of permeabilized cells (equivalent to 0.18 mg of dry cell mass) were added followed by incubation at 308C for 10 min. After the incubation, 750 ml of 0.4 M Na2CO3 was added to quench the reaction. The absorbance was then measured at 420 nm. One unit of b-galactosidase activity was defined as the amount of enzyme catalyzing the formation of 1 mmol of o-nitrophenol per min under the assay conditions.
Thin layer and high performance liquid chromatography TLC was carried out using silica gel 60 plates (20 cm 20 cm) using n-butanol:ethyl acetate:water (7:1:2, v/v/v) as a mobile phase. An aliquot (1 ml) of the reaction mixture was spotted and air dried on the TLC plate along with the standard GOS (allolactose, galactosyllactose and galacto-tetraose). After completion of the run, plates were dried and the compounds were visualized by spraying with sulphuric acid:ethanol (35:65, v/v) followed by heating at 958C for 5 min. Quantitative analysis of the synthesized carbohydrates was carried out using a refractive index detector in HPLC (Agilent 1100 series). The column used was Rezex RNM carbohydrate column (300 mm 7.8 mm). HPLC grade water was used as a mobile phase with a flow rate of 0.4 ml/min and injection volume of 20 ml. The compounds in the mixture were identified by comparing the retention time with the standard sugars which were glucose, galactose, lactose, allolactose, galacto-triose and galactotetraose. Quantitative analysis of each peak was performed using calibration curves of standard sugars.
Mass spectrometry The synthesized GOS were characterized by positive-ion mass spectra (MS) using electron spray ionization (ESI) technique. The observed ESI-MS and product ion spectra were obtained using an LXQ ion-trap mass spectrometer. GOS fractions were dissolved in methanol/water (1:1) and injected in mass spectrometer and monitored in positive ion mode. Voltage applied was 5.5 kV and nitrogen was used as the nebulizing and the drying gas. Full scan mass spectra ranging from m/z 100 to 1500 were acquired in the positive mode.
Galacto-oligosaccharide synthesis and analysis Reaction conditions were optimized for maximum GOS synthesis in a 15 ml glass vial containing lactose (2.5 ml, 30%, w/v) and ethanol permeabilized cells (0.5 ml corresponding to 0.8 units of 2
Statistical analysis The data shown in the present work are the mean values of three independent experiments with the standard deviation.
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Several yeast strains were tested for their ability to produce bgalactosidase and galacto-oligosaccharides after permeabilizing them with ethanol (Table 1). All yeast strains showed b-galactosidase activity with good cellular growth. High b-galactosidase activity of 245 U/g dry wt was obtained with K. marxianus 1078 after 40 h followed by K. marxianus NCIM 3465 (150.4 U/g dry wt), K. lactis MTCC 4063 (133.1 U/g dry wt), K. marxianus NCIM 3551 (129 U/g dry wt) as shown in Fig. 1. Screening for transgalactosyla was performed on ethanol treated permeabilized whole cells (b-galactosidase activity of 0.8 units). A maximum GOS yield of 29% (w/w of substrate) was obtained with K. marxianus NCIM 3551 followed by K. marxianus 1078 (14%) and K. marxianus NCIM 3465 (13%). The reaction products were qualitatively analyzed by TLC (supplementary Fig. 1). GOS were identified from their respective Rf values and quantitated by HPLC (supplementary Fig. 2). Mass spectrometry of the reaction products indicated peaks at m/z 365.11, m/z 527.16 and m/z 707.22 which corresponded to presence of galacto di-, tri- and tetra-saccharides (M+, Na+) respectively (Fig. 2a–c). These were assigned to lactose or allolactose (m/z 365.11, Na+ adduct), triose (m/z 527.16, Na+ adduct) and to galacto-tetraose (m/z 707.22) (Fig. 2d). Several other molecular species were also detected at m/z values of 101.00, 203.06, 281.09, 437.19 and 614.14 indicating formation of other by-products. However, these could not be assigned any structures. The ion at m/z 203.05 represented a disaccharide moiety with loss of hexose residue. The percent yield (29%) obtained with K. marxianus 3551 was quite comparable to the GOS yield of 35% (from 500 g/L lactose) using whole cells of Bifidobacterium bifidum NCIMB 41171 [2] and 44% with whole cells of K. lactis using 400 g/L lactose [15]. A GOS yield of 16.5% using permeabilized cells of K. marxianus with 500 g/L lactose conc. at 458C [17] and 32.5% from 400 g/L of lactose solution using Bifidobacterium longum BCRC 15708 have also been reported [7]. All this data indicates the feasibility of using whole cell biotransformations for GOS synthesis.
300 250 200 150 100 50 0
0
8
16
24 32 Time (h)
40
48
56
FIGURE 1
Growth and cell associated b-galactosidase activity of K. marxianus 1078 , K. marxianus 3465 , K. marxianus 3551 , and K. . marxianus 4063
activity of 153 U/g dry cell wt was obtained on lactose (b1!4) and 155 U/dry cell wt on cellobiose (b1!4). Relatively high activities were also detected on sucrose and maltose. This analysis indicated the presence and accessibility of a variety of a- and b-glucosidase activity with whole cells that can be used in biotransformation activities. Such activities have been reported to be used for synthesis of alkyl a-glucosidases [18], alkyl b-glucosides [19] and gluco-oligosaccharides [20] using different bacterial and yeast systems. Since the objective of the present study was to synthesize GOS, lactose was used as a substrate.
Evaluation of conditions that affect GOS yield Time course of GOS formation using whole cells Fig. 4 shows the time course profile of GOS synthesis using 30% (w/ v) lactose. High consumption of lactose was accompanied by a high rate of GOS formation during the first hour. Maximum GOS yield of 29% (w/w) was reached after 3 h of reaction time. The glucose in the reaction medium was found to be higher than galactose indicating transgalactosylation to be important for GOS formation. With longer incubations, no significant increase in GOS concentration was obtained but glucose levels increased and this was attributed to hydrolysis of the synthesized GOS. At the time of maximum GOS concentration (3 h), the reaction mixture contained total GOS (290 g/L) comprising 22% tetraose and 7% triose. While residual lactose was 26%, glucose and galactose were present at 24% and 21% respectively. The data also
Profile of hydrolytic activities of K. marxianus NCIM 3551 The other carbohydrate hydrolyzing activities were also assayed using permeabilized cells. Fig. 3a and b shows good hydrolysis of both the aryl glycosides and di-saccharides. Maximum activity of 245 U/g cell dry wt was obtained with oNPbGal followed by pNPbGal. Five fold less activity was demonstrated on oNPaGal and oNPbGlu indicating preference for sugar as well as the type of glycosidic linkage of sugars. Among the disaccharides, high
TABLE 1
Galactooligosaccharide yield and b-galactosidase activities in different yeast strains Microorganisms
*
*
Transgalactosylating activity
K. marxianus 1078
245 10.15
14 0.57
+
K.marxianus NCIM 3465
150 6.51
13 0.57
+
K. lactis MTCC 4063
133 6.57
K. marxianus NCIM 3551
129 5.97
*
b-Galactosidase activity (U/g dry cell wt)
GOS yield percent (w/w of substrate) SD
– 29 1.0
+
Data are mean values of three independent experimental repeats standard. www.elsevier.com/locate/nbt 3 Please cite this article in press as: Srivastava, A. et al., Transgalactosylation of lactose for synthesis of galacto-oligosaccharides using Kluyveromyces marxianus NCIM 3551, New Biotechnol. (2015), http://dx.doi.org/10.1016/j.nbt.2015.04.004
Research Paper
β -galactosidase activity (U/g dry wt. of cells)
Results and discussion Screening of b-galactosidase producing microorganisms with potential of transforming lactose into galacto-oligosaccharides
NBT-786; No of Pages 7 RESEARCH PAPER
New Biotechnology Volume 00, Number 00 August 2010
Research Paper FIGURE 2
ESI-MS analysis of (a) standard allolactose, m/z 365.11 (sodium adduct); (b) standard galacto-triose, m/z 527.16 (sodium adduct); (c) standard galacto-tetraose, m/z 707.22; (d) reaction products showing lactose or allolactose at m/z 365.11, galacto-triose at m/z 527.16 and galacto-tetraose at m/z 707.22.
indicated high transgalactosylation activity in the beginning of the reaction, resulting in higher GOS concentration, while the bgalactosidase hydrolytic activity increased on longer incubations. While similar results have been obtained in some other studies [7,10,17], our study reports higher productivity of 24.0 g/L/h compared to 0.99 g/L/h for Aspergillus oryzae b-galactosidase [22], 20.0 g/L/h for Bifidobacterium b-galactosidase [23] and 17.5 g/L/h for BgaP412 b-galactosidase [24]. Maximum GOS concentration of 7.6 g/L (16% w/w of total sugar) has been reported with commercial b-galactosidase (Biolactase) from Bacillus circulans after 1.5 h with lactose conversion of 50% (w/v). With bgalactosidase of K. lactis (Lactozyme), 7.0 g/L of GOS was obtained after 4.5 h. Importantly, higher lactose conversion of 95% (w/v) with a yield of 4.5 g/L has been reported with b-galactosidase (Lactase F) from A. oryzae. A maximum amount of GOS (5.5% of total sugars) using B. circulans purified b-galactosidase with 39% lactose conversion has also been reported [26]. The formation of galacto-tetraose as a major product, instead of triose, is advantageous due to its resistance towards hydrolysis by salivary and digestive enzymes. This also facilitates proliferation of health promoting bacteria in the intestine compared to galacto-triose as reported in various studies. 4
Effect of initial lactose concentration on GOS synthesis Initial lactose concentration plays a significant role in transgalactosylation reactions [27–29]. Its effect was studied on GOS yield and the results in Fig. 5a indicate that the yield of GOS increased with an increase in lactose concentration up to 20%, after which it decreased. A maximum GOS yield of 32% (w/w) and lactose conversion of 76% was attained at a lactose concentration of 20% (Fig. 5a). This compared well to the GOS yield of 37% (w/w of substrate) obtained at low substrate concentration (10.8%, w/v) using b-galactosidase from A. oryzae in aqueousorganic co-solvent mixture [22]. A yield of 25.1% (w/w) was attained at 10% lactose concentration using K. marxianus var. lactis OE-20, corresponding to 25.1 g/L of GOS concentration [30]. A 41% (w/w) yield with initial lactose concentration of 20.5% (w/v) was reported with recombinant b-galactosidase from Lactobacillus plantarum [31]. In contrast to this, several authors have reported maximum GOS at high (30–40%) lactose concentration [7,23,24,32]. This was attributed to high availability of both the donor and the acceptor molecules for transgalactosylation, suppression of water activity thereby reducing hydrolysis [24]. In the present study, the b-galactosidase produced by the selected yeast performed well at lower lactose
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concentration producing significant amount of GOS as compared to other studies.
a 275 225
Effect of temperature on GOS synthesis
200
Fig. 5b shows the effect of reaction temperature on GOS synthesis using 20% (w/v) initial lactose concentration. GOS yield and lactose conversion increased with an increase in temperature and maximum GOS yield of 33% (w/w) and lactose conversion of 77% was achieved at 408C. An increase in temperature to 608C resulted in a slight reduction in the GOS which was attributed to inactivation of the cell bound enzyme. Similar results have been reported [7] using crude intracellular b-galactosidase obtained after ultra sonic treatment of B. longum BCRC 15708 cells, demonstrating that an increase in reaction temperature from 25 to 458C also increased the GOS yield from 13 to 32.5% and lactose conversion rate from 24.1% to 59.4%. In another report, maximum GOS yield of about 9% (w/w), corresponding to 4.5 g/L, was reported at 408C using A. oryzae b-galactosidase [25]. The results were in conformity with that of b-galactosidase BgaP412 isolated and screened from a metagenomic library, expressed in Pichia pastoris indicating that an increase in temperature leads to higher solubility of lactose, and a reduction in viscosity, which in turn, increased the rate of reaction. Transgalactosylation was thus favored over hydrolytic activity [24] as also seen with Bifidobacterium infantis RW-8120 [5] for GOS synthesis. Other report revealed the best GOS production at 508C using purified b-galactosidase of Penicillium simplicissimum, whereas, at high temperature of 558C inactivation of the enzyme occurred leading to lowered production of GOS [33].
175 150 125 100 75 50 25 0 oNPβGal
oNPαGal
pNPβGal
oNPαGlu
oNPβGlu
Substrates (10 mM)
b Hydrolytic activity (U/g dry cell wt.)
180 160 140 120 100 80 60 40 20 0 Genobiose
Lactose
sucrose
maltose
cellobiose
Substrates (10 mM)
FIGURE 3
Hydrolytic activity of permeabilized whole cells towards 10 mM (a) oNPaGal, oNPbGal, pNpbGal, oNPaGlu, oNPbGlu and (b) gentiobiose, lactose, sucrose, maltose and cellobiose.
120 tetraose
100
GOS yield (%w/w)
triose lactose
80
glucose galactose
60
40
20
Effect of pH on GOS synthesis Fig. 5c shows the effect of pH on GOS synthesis. Maximum GOS yield of 33% and lactose conversion of 77% was obtained at pH 6.5. Although a noticeable increase in GOS yield was observed from 28% to 32% as pH increased from 5.5 to 6.0, a significant decrease (30% to 17%, w/w) occurred between pH 7.0 and 7.5. These results were supported by the b-galactosidase of B. longum where maximum GOS yield was observed at 6.8 [7]. Similar observations of high GOS yield at pH 7.0 were observed for a b-galactosidase derived from a metagenomic library [24]. In general, b-galactosidase from yeast or bacterial origin have more neutral pH optima between 6.0 and 7.5, while b-galactosidases from filamentous fungus, such as A. oryzae, show maximum GOS formation at low pH [24]. Drastic reduction in GOS yield was reported for A. oryzae when pH was greater than 6.0. At high pH of 8.0, GOS content was negligible [22]. In some cases, pH had no effect on GOS production as in the case of b-galactosidase from Bacillus singularis [34,35]. Thus, pH optimum for transgalactosylation varies depending on the source of b-galactosidase.
0 0
1
2
3
4 5 Time (h)
6
7
8
9
FIGURE 4
Time course of GOS synthesis using permeabilized whole cells of K. marxianus NCIM 3551 at 508C. An initial lactose concentration of 30% was used with cells containing 0.8 units of b-galactosidase. Data presented are mean SD of three independent experiments. GOS yield refers to the quantities of galactotriose and -tetraose as determined by HPLC.
Effect of enzyme units on GOS yield Availability of higher amounts of catalyst is expected to increase the rate of transgalactosylation. For this, enzyme concentration was varied from 0.40 to 1.6 units. Maximum GOS yield of 36% (w/w) and lactose conversion of 80% (w/v) was obtained with 1.2 units of b-galactosidase. There was no significant change in GOS synthesis between 0.40 and 0.80 units. As enzyme concentration increased to 1.6 enzyme units, percent conversion was increased to
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Research Paper
Hydrolytic activity (U/g dry cell wt)
250
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New Biotechnology Volume 00, Number 00 August 2010
Research Paper FIGURE 5
Optimization of reaction conditions for maximum GOS synthesis using permeabilized cells (a) lactose concentration, (b) temperature, (c) pH, (d) enzyme units.
82% with only 30% GOS yield. Maximum GOS concentration of 360 g/L with 1.2 units of enzyme, in the present study, is promising when compared to GOS concentration of 4.5 g/L using 2.0 units of A. oryzae b-galactosidase [25]. A GOS yield of 33% using 0.2 units (0.025 mg) of purified b-galactosidase after 48 h of reaction time has also been reported [22] whereas yield of 49.2% was reported with permeabilized whole cells of Lactococcus lactis which has hyperthermophilic b-galactosidase [36]. A high yield of 64% (w/w) has also been reported using 22.5 g (wet wt) of whole cells of Rhodotorula minuta IFO879 after 55 h of incubation time [21].
41% yield with commercial Lactozym and Maxilact LGX 5000 respectively [15]. A GOS yield of 16%, 15% and 9% (w/w) of total sugars was obtained with 12.3, 29.3 and 2.0 units of commercial b-galactosidase/g of lactose with the B. circulans, K. lactis and A. oryzae respectively [25]. These are significantly lower than the GOS produced in the present work.
GOS yield with permeabilized cells GOS yield with commercial β -galactosidase Residual lactose with permeabilized cells
6
120
90 GOS yield (%w/w)
Fig. 6 shows a comparison of the time profile of GOS synthesis using the permeabilized K. marxianus cells and commercial bgalactosidase from A. oryzae. The maximum GOS yield of 36% (w/w), lactose conversion of 80% was obtained after 3 h of reaction time with K. marxianus 3551 permeabilized cells. As opposed to this, a maximum GOS yield of 24% (w/w) and lactose conversion of 55% was obtained after 10 h with the commercial b-galactosidase. Thus, while a productivity of 24.0 g/L/h was obtained with the permeabilized yeast cells, a productivity of only 4.8 g/L/h was obtained with commercial b-galactosidase. Analysis of the reaction mixture indicated a mixture of galacto-tetraose (27%), galacto-triose (9%), monosaccharides (44%), lactose (20%) with permeabilized cells. Only 12% GOS were obtained with the A. oryzae system. A GOS yield of 44% (w/w) was reported for the K. lactis system, which was quite comparable to 42% and
Residual lactose with commercial β - galactosidase
105
100
75
80
60 60 45 40
30
Residual lactose (%w/v)
Comparison of GOS production using permeabilized cells and commercial b-galactosidase from A. oryzae
20
15 0
0 0
1
2
3
4
5
6
7
8
9
10
Time (h)
FIGURE 6
Comparison of GOS synthesis between permeabilized yeast cells and commercial b-galactosidase.
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Conclusions
Acknowledgments
This work describes the use of permeabilized cells of K. marxianus NCIM 3551 for synthesis of GOS. The novelty of the work lies in the use of permeabilized whole cells using low substrate concentrations and shorter reaction time which in turn increased the productivity of the process. Use of whole cells makes this process economical. Maximum GOS yield of 36% (w/w) was obtained at an initial lactose concentration of 20% (w/v). The reaction mixture was composed of 27% tetrasaccharides and 9% trisaccharides with a productivity of 24.0 g/L/h. This level of productivity was much higher than that attained with other whole cell systems.
The authors are grateful to Department of Science and Technology, New Delhi (under Govt. of India) for financial support for the project number SR/WOS-A/LS-437/2011 under Women Scientist Scheme-A.
Appendix A. Supplementary data
References ¨ ck M, Kulbe KD, Lorenz W, Haltrich D. [1] Splechtna B, Nguyen TH, Steinbo Production of prebiotic galacto-oligosaccharides from lactose using b-galactosidases from Lactobacillus reuteri. J Agric Food Chem 2006;54(14):4999–5006. [2] Tzortzis G, Goulas A, Gibson G. Synthesis of prebiotic galactooligosaccharides using whole cells of a novel strain, Bifidobacterium bifidum NCIMB 41171. Appl Microbiol Biotechnol 2005;68(3):412–6. [3] Gosling A, Stevens GW, Barber AR, Kentish SE, Gras SL. Recent advances refining galactooligosaccharide production from lactose. Food Chem 2010;121(2):307–18. [4] Akiyama K, Takase M, Horikoshi K, Okonogi S. Production of galactooligosaccharides from lactose using a beta-glucosidase from Thermus sp. Z-1. Bio Biotechnol Biochem 2001;65(2):438–41. [5] Roy D, Daoudi L, Azaola A. Optimization of galacto-oligosaccharide production by Bifidobacterium infantis RW-8120 using response surface methodology. J Indus Microbiol Biotechnol 2002;29(5):281–5. [6] Matella NJ, Dolan KD, Lee YS. Comparison of galactooligosaccharide production in free-enzyme ultrafiltration and in immobilized-enzyme systems. J Food Sci 2006;71(7):C363–8. [7] Hsu CA, Lee SL, Chou CC. Enzymatic production of galactooligosaccharides by b-galactosidase from Bifidobacterium longum BCRC 15708. J Agric Food Chem 2007;55(6):2225–30. [8] Chen SX, Wei DZ, Hu ZH. Synthesis of galacto-oligosaccharides in AOT/ isooctane reverse micelles by b-galactosidase. J Mol Catal B: Enzym 2001;16(2):109–14. [9] Chen CW, Ou-Yang CC, Yeh CW. Synthesis of galactooligosaccharides and transgalactosylation modeling in reverse micelles. Enzym and Microb Technol 2003;33(4):497–507. [10] Chockchaisawasdee S, Athanasopoulos VI, Niranjan K, Rastall RA. Synthesis of galacto-oligosaccharide from lactose using b-galactosidase from Kluyveromyces lactis: studies on batch and continuous UF membrane-fitted bioreactors. Biotechnol Bioeng 2005;89(4):434–43. [11] Prenosil JE, Stuker E, Bourne JR. Formation of oligosaccharides during enzymatic lactose hydrolysis and their importance in a whey hydrolysis process: Part II. Exp Biotechnol Bioeng 1987;30(9):1026–31. [12] Fontes EAF, Passos FML, Passos FJV. A mechanistical mathematical model to predict lactose hydrolysis by b-galactosidase in a permeabilized cell mass of Kluyveromyces lactis: validity and sensitivity analysis. Proc Biochem 2001;37(3):267–74. [13] Genari AN, Passos FV, Passos FML. Configuration of a bioreactor for milk lactose hydrolysis. J Dairy Sci 2003;86(9):2783–9. [14] Panesar R, Panesar PS, Singh RS, Kennedy JF, Bera MB. Production of lactosehydrolyzed milk using ethanol permeabilized yeast cells. Food Chem 2007;101(2):786–90. [15] Rodriguez-Colinas B, de Abreu MA, Fernandez-Arrojo L, de Beer R, Poveda A, Jimenez-Barbero J, et al. Production of galacto-oligosaccharides by the b-galactosidase from Kluyveromyces lactis: comparative analysis of permeabilized cells versus soluble enzyme. J Agric Food Chem 2011;59(19):10477–84. [16] Miller GL. Use of nitrosalisylic acid reagent for determination of reducing sugar. Anal Chem 1959;31(3):426–8. [17] Manera AP, Costa FADA, Rodrigues MI, Kalil SJ, Maugeri Filho F. Galactooligosaccharides production using permeabilized cells of Kluyveromyces marxianus. Int J Food Eng 2010;6(6). [18] Ojha S, Mishra S, Kapoor S, Chand S. Synthesis of hexyl a-glucoside and apolyglucosides by a novel Microbacterium isolate. Appl Microbiol Biotechnol 2013;97(12):5293–301.
[19] Rather MY, Mishra S, Chand S. b-Glucosidase catalysed synthesis of octyl-b-Dglucopyranoside using whole cells of Pichia etchellsii in micro aqueous media. J Biotechnol 2010;150:490–6. [20] Ojha S, Mishra S, Chand S. Production of iso-maltooligosaccharides using cell bound a-glucosidase of Microbacterium sp. LWT – Food Sci Technol 2015;60: 486–94. [21] Onishi N. Yokozeki and galacto-oligosaccharide production by Rhodotorula minuta IFO879. J Ferment Bioeng 1996;82(2):124–7. [22] Srisimarat W, Pongsawasdi P. Enhancement of the oligosaccharide synthetic activity of b-galactosidase in organic solvents by cyclodextrin. Enzym Microbiol Technol 2008;43(6):436–41. [23] Rabiu B, Jay A, ibson GR, Rastall RA. Synthesis and fermentation properties of novel galacto-oligosaccharides by beta-galactosidases from Bifidobacterium species. Appl Environ Microbiol 2001;67:2526–30. [24] Wang K, Lu Y, Liang WQ, Wang SD, Jiang Y, Huang R, et al. Enzymatic synthesis of galacto-oligosaccharides in an organic–aqueous biphasic system by a novel bgalactosidase from a metagenomic library. J Agric Food Chem 2012;60(15): 3940–6. [25] Rodriguez B, Fernandez L, Ballesteros AO, Plou FJ. Galactooligosaccharides formation during enzymatic hydrolysis of lactose: towards a prebiotic enriched milk. Food Chem 2014;145:338–94. [26] Mozaffar Z, Nakanishi K, Matsuno R. Formation of oligosaccharides during hydrolysis of lactose in milk using b-galactosidase from Bacillus circulans. J Food Sci 1984;50:1602–6. [27] Albayrak N, Yang ST. Production of galacto-oligosaccharides from lactose by Aspergillus oryzae b-galactosidase immobilized on cotton cloth. Biotechnol Bioeng 2002;77(1):8–19. [28] Boon MA, Janssen AEM, van’t Riet K. Effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides. Enzym Microb Technol 2002;26(2–4):271–81. [29] Jorgensen F, Hansen O, Stougaard P. High efficiency synthesis of oligosaccharides with a truncated b-galactosidase from Bifidobacterium bifidum. Appl Microbiol Biotechnol 2001;57(5–6):647–52. [30] Kim JH, Lee DH, Lee JS. Production of galactooligosacharide by b-galactosidase from Kluyveromyces marxianus lactis OE-20. Biotechnol Bioproc Eng 2001;6:337–40. [31] Iqbal S, Nguyen TH, Maishberger T, Haltrich D. b-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides. Carbohy Res 2010;345:1408–16. [32] Urrutia P, Rodriguez-Colinas B, Fernandez-Arrojo L, Ballesteros AO, Wilson L, Illanes As Plou FJ. Detailed analysis of galactooligosaccharides synthesis with bgalactosidase from Aspergillus oryzae. J Agric Food Chem 2013;61(5):1081–7. [33] Cruz R, Cruz VDA, Belote JG, Khenayfes MDO, Dorta C, Oliveira LHDS, et al. Production of transgalactosylated oligosaccharides (TOS) by galactosyltransfrase activity from Penicillium simplissimum. Bioresour Technol 1999;70:165–71. [34] Shin HJ, Park JM, Yang JW. Continuous production of galacto-oligosaccharides from lactose by Bullera singularis b-galactosidase immobilized in chitosan beads. Proc Biochem 1998;33(8):787–92. [35] Iwasaki KI, Nakajima M, Nakao SI. Galacto-oligosaccharide production from lactose by an enzymic batch reaction using b-galactosidase. Proc Biochem 1996;31(1):69–76. [36] Yu L, Sullivan JO. Production of galactooligosaccharides using a hyperthermophilic b-galactosidase in permeabilized whole cells of Lactococcus lactis. J Dairy Sci 2014;97:694–703.
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.nbt.2015.04. 004.
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Research Paper
Transgalactosylation of lactose for synthesis of galacto-oligosaccharides using Kluyveromyces marxianus NCIM 3551 Anita Srivastava, Saroj Mishra and Subhash Chand Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz-Khas, New-Delhi 110016, India
Among a number of yeast strains screened for whole cell transgalactosylating activity, Kluyveromyces marxianus NCIM 3551 was found to be most suitable biocatalyst for production of galactooligosaccharides (GOS). Cell permeabilization lead to an efficient bioconversion by b-galactosidase resulting in synthesis of GOS. A maximum GOS yield of 36% (w/w) of total sugars was achieved and the products consisted of tri- and tetra-galacto-oligosaccharides. A lactose conversion rate of 80% and productivity of 24 g/L/h was obtained under the optimum conditions at lactose concentration of 20% (w/v), temperature 408C, pH 6.5 and enzyme units after 3 h of reaction time. Tetrasaccharides were the main component of the reaction mixture. The products were quantitated by HPLC and structurally characterized by mass spectrometry.
Introduction Prebiotic oligosaccharides and their use in food industry has lead to the development of various biotechnological processes and search for new microorganisms for their synthesis. World wide interest in prebiotics stems from their nutritional and health benefits. Enzymatic routes are preferred over chemical routes for oligosaccharides synthesis due to their high specificity, efficiency, and environmental friendly status [1]. The commonly known prebiotics are lactulose, raffinose, malto-oligosaccharides, inulin, fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS). Among these, galacto-oligosaccharides possess the ability to stimulate the proliferation of Bifidobacteria inside the human intestine, hence these are known as ‘Bifidus growth factors’ [2]. GOS are widely used as food stuff additives and perform different functions inside the human body such as maintenance of balance of intestinal microflora, enhancing the digestibility of milk products by improving the lactose tolerance and reducing the serum cholestrol level [3]. GOS are galactose containing oligosaccharides where 2–5 units of galactose are attached to glucose by glycosidic linkages which are produced by transgalactosylation of lactose using b-galactosidase [4–7]. During the process of transgalactosylation, Corresponding author: Srivastava, A. ([email protected], [email protected]) http://dx.doi.org/10.1016/j.nbt.2015.04.004 1871-6784/ß 2015 Published by Elsevier B.V.
lactose serves as galactosyl acceptor, forming a chain of di-, triand tetra-saccharides or even higher polymers of galacto-oligosaccharides. The transgalactosylation reaction involved in GOS formation is a kinetically controlled reaction and the final yield of oligosaccharides is determined by the competition between hydrolysis and galactosyl an acceptor. The transgalactosylation process depends largely on the source of b-galactosidase, the initial substrate concentration and the thermodynamic activity of water [8,9]. Commercially available sources for b-galactosidase are mostly yeast, but because of the intracellular nature of b-galactosidase, expensive enzyme extraction, downstream processing and low stability, these have not been extensively used. An important alternative strategy for economic production of GOS would be the use of whole cells. The advantage of using whole cells over the purified enzymes is their easy availability, reusability, low cost of production and stability, continuous processing and economic viability. However, the permeability barrier of the cell results in lowered reaction rates as less substrate is available for conversion. To overcome this bottle neck and increase the volumetric activity, permeabilization of cells has been reported [10,11]. The permeabilizing agent reduces the phospholipid content in the membrane of the cell, and allows the transfer of low molecular weight compounds in and out of the www.elsevier.com/locate/nbt
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cells. Although ethanol-permeabilized Kluyveromyces lactis cells have been used for bioprocesses [1,12–14], no report appears on the use of Kluyveromyces marxianus NCIM 3551 for GOS synthesis. The aim of the present work was to evaluate the transgalactosylation efficiency of the cell bound b-galactosidase of K. marxianus for synthesis of GOS using ethanol permeabilized cells. Analysis of the products were carried out to identify the major oligosaccharides synthesized during whole cell transformation.
Research Paper
Materials and methods Growth, b-galactosidase activity and galacto-oligosaccharide synthesis K. marxianus NCIM 3465, K. marxianus NCIM 3551 were obtained from National culture collection, Pune. K. lactis MTCC 4063 was procured from MTCC, IMTECH Chandigarh and K. marxianus 1078 was from Department Culture Collection Unit, IIT Delhi. The growth medium consisted of (g/L): yeast extract 17.0; ammonium sulphate 9.0; potassium dihydrogen phosphate 5.0; magnesium sulphate: 0.4; lactose: 30.0 in 50 mM sodium phosphate buffer pH 6.5. The cultures were incubated in an orbital shaker (200 rpm) at 288C for 24 h. The medium was inoculated with 10% (v/v) inoculum and growth carried out at 288C for 48 h. Samples were removed every 8 h for measurement of cell biomass and bgalactosidase activity. Lactose, used for induction in growth medium, was filter sterilized using 0.22 mm syringe filter and added at the beginning of the experiments. The cultures were analyzed for cell associated b-galactosidase activity by incubating with o-nitrophenyl-b-D galactopyranoside (oNPGal). For measurement of activity in the permeabilized cells, the washed cells were permeabilized by incubating in 50% (v/v) ethanol and sodium phosphate buffer, pH 6.5 for 15 min at 258C. Cells were separated from the solvent by centrifugation at 5000 rpm for 5 min and washed twice with 50 mM sodium phosphate buffer. Finally, these were suspended in the same buffer and stored at 48C until used for further enzyme analysis and GOS synthesis [15]. The potential of the cells for galacto-oligosaccharide synthesis was evaluated by adding the cells to 30% (w/v) lactose at 508C in an orbital shaker for different time intervals. Samples were qualitatively analyzed for production of higher oligosaccharides by thin layer chromatography.
Cell associated hydrolytic activities of the selected strain Cell associated hydrolytic activities of the selected strain were determined using 10 mM aryl-glucosides and -galactosides, and, disaccharides prepared in 50 mM sodium phosphate buffer, pH 6.5. The aryl substrates used were o-nitrophenyl-a-D-galactopyranoside, o-nitrophenyl-b-D-galactopyranoside, o-nitrophenyl-a-Dglucopyranoside, o-nitrophenyl-b-D-glucopyranoside. The disaccharides tested were lactose, sucrose, maltose, cellobiose and gentiobiose. Aryl glycosidase activity was determined by measuring the release of o-nitrophenol at 420 nm whereas the enzyme activity towards disaccharides was determined by measuring glucose in the reaction mixture [16].
New Biotechnology Volume 00, Number 00 August 2010
b-galactosidase enzyme). Reactions were carried out with shaking (150 rpm) in an orbital shaker at 508C for 8 h. Aliquots (100 ml) were collected at a regular interval of 1 h and the reaction was stopped by boiling the samples at 1008C for 5 min. Samples were centrifuged at 5000 rpm and supernatant diluted with 50 mM sodium phosphate buffer. This was qualitatively analyzed by thin layer chromatography and quantitatively by high performance liquid chromatography (HPLC). Effect of lactose concentration (10–50%, w/v), temperature (20–608C), pH (5.5–7.5) and enzyme units (0.4–1.6) was investigated at the optimum time determined from previous study.
Analytical methods b-Galactosidase activity Hydrolytic activity of b-galactosidase was monitored using oNPGal as a substrate. To 480 ml of 22 mM oNPGal in 50 mM sodium phosphate buffer, pH 6.5, 20 ml of permeabilized cells (equivalent to 0.18 mg of dry cell mass) were added followed by incubation at 308C for 10 min. After the incubation, 750 ml of 0.4 M Na2CO3 was added to quench the reaction. The absorbance was then measured at 420 nm. One unit of b-galactosidase activity was defined as the amount of enzyme catalyzing the formation of 1 mmol of o-nitrophenol per min under the assay conditions.
Thin layer and high performance liquid chromatography TLC was carried out using silica gel 60 plates (20 cm 20 cm) using n-butanol:ethyl acetate:water (7:1:2, v/v/v) as a mobile phase. An aliquot (1 ml) of the reaction mixture was spotted and air dried on the TLC plate along with the standard GOS (allolactose, galactosyllactose and galacto-tetraose). After completion of the run, plates were dried and the compounds were visualized by spraying with sulphuric acid:ethanol (35:65, v/v) followed by heating at 958C for 5 min. Quantitative analysis of the synthesized carbohydrates was carried out using a refractive index detector in HPLC (Agilent 1100 series). The column used was Rezex RNM carbohydrate column (300 mm 7.8 mm). HPLC grade water was used as a mobile phase with a flow rate of 0.4 ml/min and injection volume of 20 ml. The compounds in the mixture were identified by comparing the retention time with the standard sugars which were glucose, galactose, lactose, allolactose, galacto-triose and galactotetraose. Quantitative analysis of each peak was performed using calibration curves of standard sugars.
Mass spectrometry The synthesized GOS were characterized by positive-ion mass spectra (MS) using electron spray ionization (ESI) technique. The observed ESI-MS and product ion spectra were obtained using an LXQ ion-trap mass spectrometer. GOS fractions were dissolved in methanol/water (1:1) and injected in mass spectrometer and monitored in positive ion mode. Voltage applied was 5.5 kV and nitrogen was used as the nebulizing and the drying gas. Full scan mass spectra ranging from m/z 100 to 1500 were acquired in the positive mode.
Galacto-oligosaccharide synthesis and analysis Reaction conditions were optimized for maximum GOS synthesis in a 15 ml glass vial containing lactose (2.5 ml, 30%, w/v) and ethanol permeabilized cells (0.5 ml corresponding to 0.8 units of 2
Statistical analysis The data shown in the present work are the mean values of three independent experiments with the standard deviation.
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Several yeast strains were tested for their ability to produce bgalactosidase and galacto-oligosaccharides after permeabilizing them with ethanol (Table 1). All yeast strains showed b-galactosidase activity with good cellular growth. High b-galactosidase activity of 245 U/g dry wt was obtained with K. marxianus 1078 after 40 h followed by K. marxianus NCIM 3465 (150.4 U/g dry wt), K. lactis MTCC 4063 (133.1 U/g dry wt), K. marxianus NCIM 3551 (129 U/g dry wt) as shown in Fig. 1. Screening for transgalactosyla was performed on ethanol treated permeabilized whole cells (b-galactosidase activity of 0.8 units). A maximum GOS yield of 29% (w/w of substrate) was obtained with K. marxianus NCIM 3551 followed by K. marxianus 1078 (14%) and K. marxianus NCIM 3465 (13%). The reaction products were qualitatively analyzed by TLC (supplementary Fig. 1). GOS were identified from their respective Rf values and quantitated by HPLC (supplementary Fig. 2). Mass spectrometry of the reaction products indicated peaks at m/z 365.11, m/z 527.16 and m/z 707.22 which corresponded to presence of galacto di-, tri- and tetra-saccharides (M+, Na+) respectively (Fig. 2a–c). These were assigned to lactose or allolactose (m/z 365.11, Na+ adduct), triose (m/z 527.16, Na+ adduct) and to galacto-tetraose (m/z 707.22) (Fig. 2d). Several other molecular species were also detected at m/z values of 101.00, 203.06, 281.09, 437.19 and 614.14 indicating formation of other by-products. However, these could not be assigned any structures. The ion at m/z 203.05 represented a disaccharide moiety with loss of hexose residue. The percent yield (29%) obtained with K. marxianus 3551 was quite comparable to the GOS yield of 35% (from 500 g/L lactose) using whole cells of Bifidobacterium bifidum NCIMB 41171 [2] and 44% with whole cells of K. lactis using 400 g/L lactose [15]. A GOS yield of 16.5% using permeabilized cells of K. marxianus with 500 g/L lactose conc. at 458C [17] and 32.5% from 400 g/L of lactose solution using Bifidobacterium longum BCRC 15708 have also been reported [7]. All this data indicates the feasibility of using whole cell biotransformations for GOS synthesis.
300 250 200 150 100 50 0
0
8
16
24 32 Time (h)
40
48
56
FIGURE 1
Growth and cell associated b-galactosidase activity of K. marxianus 1078 , K. marxianus 3465 , K. marxianus 3551 , and K. . marxianus 4063
activity of 153 U/g dry cell wt was obtained on lactose (b1!4) and 155 U/dry cell wt on cellobiose (b1!4). Relatively high activities were also detected on sucrose and maltose. This analysis indicated the presence and accessibility of a variety of a- and b-glucosidase activity with whole cells that can be used in biotransformation activities. Such activities have been reported to be used for synthesis of alkyl a-glucosidases [18], alkyl b-glucosides [19] and gluco-oligosaccharides [20] using different bacterial and yeast systems. Since the objective of the present study was to synthesize GOS, lactose was used as a substrate.
Evaluation of conditions that affect GOS yield Time course of GOS formation using whole cells Fig. 4 shows the time course profile of GOS synthesis using 30% (w/ v) lactose. High consumption of lactose was accompanied by a high rate of GOS formation during the first hour. Maximum GOS yield of 29% (w/w) was reached after 3 h of reaction time. The glucose in the reaction medium was found to be higher than galactose indicating transgalactosylation to be important for GOS formation. With longer incubations, no significant increase in GOS concentration was obtained but glucose levels increased and this was attributed to hydrolysis of the synthesized GOS. At the time of maximum GOS concentration (3 h), the reaction mixture contained total GOS (290 g/L) comprising 22% tetraose and 7% triose. While residual lactose was 26%, glucose and galactose were present at 24% and 21% respectively. The data also
Profile of hydrolytic activities of K. marxianus NCIM 3551 The other carbohydrate hydrolyzing activities were also assayed using permeabilized cells. Fig. 3a and b shows good hydrolysis of both the aryl glycosides and di-saccharides. Maximum activity of 245 U/g cell dry wt was obtained with oNPbGal followed by pNPbGal. Five fold less activity was demonstrated on oNPaGal and oNPbGlu indicating preference for sugar as well as the type of glycosidic linkage of sugars. Among the disaccharides, high
TABLE 1
Galactooligosaccharide yield and b-galactosidase activities in different yeast strains Microorganisms
*
*
Transgalactosylating activity
K. marxianus 1078
245 10.15
14 0.57
+
K.marxianus NCIM 3465
150 6.51
13 0.57
+
K. lactis MTCC 4063
133 6.57
K. marxianus NCIM 3551
129 5.97
*
b-Galactosidase activity (U/g dry cell wt)
GOS yield percent (w/w of substrate) SD
– 29 1.0
+
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β -galactosidase activity (U/g dry wt. of cells)
Results and discussion Screening of b-galactosidase producing microorganisms with potential of transforming lactose into galacto-oligosaccharides
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New Biotechnology Volume 00, Number 00 August 2010
Research Paper FIGURE 2
ESI-MS analysis of (a) standard allolactose, m/z 365.11 (sodium adduct); (b) standard galacto-triose, m/z 527.16 (sodium adduct); (c) standard galacto-tetraose, m/z 707.22; (d) reaction products showing lactose or allolactose at m/z 365.11, galacto-triose at m/z 527.16 and galacto-tetraose at m/z 707.22.
indicated high transgalactosylation activity in the beginning of the reaction, resulting in higher GOS concentration, while the bgalactosidase hydrolytic activity increased on longer incubations. While similar results have been obtained in some other studies [7,10,17], our study reports higher productivity of 24.0 g/L/h compared to 0.99 g/L/h for Aspergillus oryzae b-galactosidase [22], 20.0 g/L/h for Bifidobacterium b-galactosidase [23] and 17.5 g/L/h for BgaP412 b-galactosidase [24]. Maximum GOS concentration of 7.6 g/L (16% w/w of total sugar) has been reported with commercial b-galactosidase (Biolactase) from Bacillus circulans after 1.5 h with lactose conversion of 50% (w/v). With bgalactosidase of K. lactis (Lactozyme), 7.0 g/L of GOS was obtained after 4.5 h. Importantly, higher lactose conversion of 95% (w/v) with a yield of 4.5 g/L has been reported with b-galactosidase (Lactase F) from A. oryzae. A maximum amount of GOS (5.5% of total sugars) using B. circulans purified b-galactosidase with 39% lactose conversion has also been reported [26]. The formation of galacto-tetraose as a major product, instead of triose, is advantageous due to its resistance towards hydrolysis by salivary and digestive enzymes. This also facilitates proliferation of health promoting bacteria in the intestine compared to galacto-triose as reported in various studies. 4
Effect of initial lactose concentration on GOS synthesis Initial lactose concentration plays a significant role in transgalactosylation reactions [27–29]. Its effect was studied on GOS yield and the results in Fig. 5a indicate that the yield of GOS increased with an increase in lactose concentration up to 20%, after which it decreased. A maximum GOS yield of 32% (w/w) and lactose conversion of 76% was attained at a lactose concentration of 20% (Fig. 5a). This compared well to the GOS yield of 37% (w/w of substrate) obtained at low substrate concentration (10.8%, w/v) using b-galactosidase from A. oryzae in aqueousorganic co-solvent mixture [22]. A yield of 25.1% (w/w) was attained at 10% lactose concentration using K. marxianus var. lactis OE-20, corresponding to 25.1 g/L of GOS concentration [30]. A 41% (w/w) yield with initial lactose concentration of 20.5% (w/v) was reported with recombinant b-galactosidase from Lactobacillus plantarum [31]. In contrast to this, several authors have reported maximum GOS at high (30–40%) lactose concentration [7,23,24,32]. This was attributed to high availability of both the donor and the acceptor molecules for transgalactosylation, suppression of water activity thereby reducing hydrolysis [24]. In the present study, the b-galactosidase produced by the selected yeast performed well at lower lactose
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concentration producing significant amount of GOS as compared to other studies.
a 275 225
Effect of temperature on GOS synthesis
200
Fig. 5b shows the effect of reaction temperature on GOS synthesis using 20% (w/v) initial lactose concentration. GOS yield and lactose conversion increased with an increase in temperature and maximum GOS yield of 33% (w/w) and lactose conversion of 77% was achieved at 408C. An increase in temperature to 608C resulted in a slight reduction in the GOS which was attributed to inactivation of the cell bound enzyme. Similar results have been reported [7] using crude intracellular b-galactosidase obtained after ultra sonic treatment of B. longum BCRC 15708 cells, demonstrating that an increase in reaction temperature from 25 to 458C also increased the GOS yield from 13 to 32.5% and lactose conversion rate from 24.1% to 59.4%. In another report, maximum GOS yield of about 9% (w/w), corresponding to 4.5 g/L, was reported at 408C using A. oryzae b-galactosidase [25]. The results were in conformity with that of b-galactosidase BgaP412 isolated and screened from a metagenomic library, expressed in Pichia pastoris indicating that an increase in temperature leads to higher solubility of lactose, and a reduction in viscosity, which in turn, increased the rate of reaction. Transgalactosylation was thus favored over hydrolytic activity [24] as also seen with Bifidobacterium infantis RW-8120 [5] for GOS synthesis. Other report revealed the best GOS production at 508C using purified b-galactosidase of Penicillium simplicissimum, whereas, at high temperature of 558C inactivation of the enzyme occurred leading to lowered production of GOS [33].
175 150 125 100 75 50 25 0 oNPβGal
oNPαGal
pNPβGal
oNPαGlu
oNPβGlu
Substrates (10 mM)
b Hydrolytic activity (U/g dry cell wt.)
180 160 140 120 100 80 60 40 20 0 Genobiose
Lactose
sucrose
maltose
cellobiose
Substrates (10 mM)
FIGURE 3
Hydrolytic activity of permeabilized whole cells towards 10 mM (a) oNPaGal, oNPbGal, pNpbGal, oNPaGlu, oNPbGlu and (b) gentiobiose, lactose, sucrose, maltose and cellobiose.
120 tetraose
100
GOS yield (%w/w)
triose lactose
80
glucose galactose
60
40
20
Effect of pH on GOS synthesis Fig. 5c shows the effect of pH on GOS synthesis. Maximum GOS yield of 33% and lactose conversion of 77% was obtained at pH 6.5. Although a noticeable increase in GOS yield was observed from 28% to 32% as pH increased from 5.5 to 6.0, a significant decrease (30% to 17%, w/w) occurred between pH 7.0 and 7.5. These results were supported by the b-galactosidase of B. longum where maximum GOS yield was observed at 6.8 [7]. Similar observations of high GOS yield at pH 7.0 were observed for a b-galactosidase derived from a metagenomic library [24]. In general, b-galactosidase from yeast or bacterial origin have more neutral pH optima between 6.0 and 7.5, while b-galactosidases from filamentous fungus, such as A. oryzae, show maximum GOS formation at low pH [24]. Drastic reduction in GOS yield was reported for A. oryzae when pH was greater than 6.0. At high pH of 8.0, GOS content was negligible [22]. In some cases, pH had no effect on GOS production as in the case of b-galactosidase from Bacillus singularis [34,35]. Thus, pH optimum for transgalactosylation varies depending on the source of b-galactosidase.
0 0
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FIGURE 4
Time course of GOS synthesis using permeabilized whole cells of K. marxianus NCIM 3551 at 508C. An initial lactose concentration of 30% was used with cells containing 0.8 units of b-galactosidase. Data presented are mean SD of three independent experiments. GOS yield refers to the quantities of galactotriose and -tetraose as determined by HPLC.
Effect of enzyme units on GOS yield Availability of higher amounts of catalyst is expected to increase the rate of transgalactosylation. For this, enzyme concentration was varied from 0.40 to 1.6 units. Maximum GOS yield of 36% (w/w) and lactose conversion of 80% (w/v) was obtained with 1.2 units of b-galactosidase. There was no significant change in GOS synthesis between 0.40 and 0.80 units. As enzyme concentration increased to 1.6 enzyme units, percent conversion was increased to
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Research Paper
Hydrolytic activity (U/g dry cell wt)
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NBT-786; No of Pages 7 RESEARCH PAPER
New Biotechnology Volume 00, Number 00 August 2010
Research Paper FIGURE 5
Optimization of reaction conditions for maximum GOS synthesis using permeabilized cells (a) lactose concentration, (b) temperature, (c) pH, (d) enzyme units.
82% with only 30% GOS yield. Maximum GOS concentration of 360 g/L with 1.2 units of enzyme, in the present study, is promising when compared to GOS concentration of 4.5 g/L using 2.0 units of A. oryzae b-galactosidase [25]. A GOS yield of 33% using 0.2 units (0.025 mg) of purified b-galactosidase after 48 h of reaction time has also been reported [22] whereas yield of 49.2% was reported with permeabilized whole cells of Lactococcus lactis which has hyperthermophilic b-galactosidase [36]. A high yield of 64% (w/w) has also been reported using 22.5 g (wet wt) of whole cells of Rhodotorula minuta IFO879 after 55 h of incubation time [21].
41% yield with commercial Lactozym and Maxilact LGX 5000 respectively [15]. A GOS yield of 16%, 15% and 9% (w/w) of total sugars was obtained with 12.3, 29.3 and 2.0 units of commercial b-galactosidase/g of lactose with the B. circulans, K. lactis and A. oryzae respectively [25]. These are significantly lower than the GOS produced in the present work.
GOS yield with permeabilized cells GOS yield with commercial β -galactosidase Residual lactose with permeabilized cells
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90 GOS yield (%w/w)
Fig. 6 shows a comparison of the time profile of GOS synthesis using the permeabilized K. marxianus cells and commercial bgalactosidase from A. oryzae. The maximum GOS yield of 36% (w/w), lactose conversion of 80% was obtained after 3 h of reaction time with K. marxianus 3551 permeabilized cells. As opposed to this, a maximum GOS yield of 24% (w/w) and lactose conversion of 55% was obtained after 10 h with the commercial b-galactosidase. Thus, while a productivity of 24.0 g/L/h was obtained with the permeabilized yeast cells, a productivity of only 4.8 g/L/h was obtained with commercial b-galactosidase. Analysis of the reaction mixture indicated a mixture of galacto-tetraose (27%), galacto-triose (9%), monosaccharides (44%), lactose (20%) with permeabilized cells. Only 12% GOS were obtained with the A. oryzae system. A GOS yield of 44% (w/w) was reported for the K. lactis system, which was quite comparable to 42% and
Residual lactose with commercial β - galactosidase
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Residual lactose (%w/v)
Comparison of GOS production using permeabilized cells and commercial b-galactosidase from A. oryzae
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FIGURE 6
Comparison of GOS synthesis between permeabilized yeast cells and commercial b-galactosidase.
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Conclusions
Acknowledgments
This work describes the use of permeabilized cells of K. marxianus NCIM 3551 for synthesis of GOS. The novelty of the work lies in the use of permeabilized whole cells using low substrate concentrations and shorter reaction time which in turn increased the productivity of the process. Use of whole cells makes this process economical. Maximum GOS yield of 36% (w/w) was obtained at an initial lactose concentration of 20% (w/v). The reaction mixture was composed of 27% tetrasaccharides and 9% trisaccharides with a productivity of 24.0 g/L/h. This level of productivity was much higher than that attained with other whole cell systems.
The authors are grateful to Department of Science and Technology, New Delhi (under Govt. of India) for financial support for the project number SR/WOS-A/LS-437/2011 under Women Scientist Scheme-A.
Appendix A. Supplementary data
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Research Paper
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.nbt.2015.04. 004.
