Synthesis and Characterization of Microbial Fructosyltransferase (FTase) from endophytic Bacillus stercoris S1 for Fructoligosaccharide production

doi:10.21203/rs.3.rs-5793453/v1

Purpose: Presently, there is a rising awareness of the additional health benefits and market potential for functional foods. Among various functional foods short chain Fructooligosaccharides (FOS) have make its prestige in food industry. Therefore, FOS producing enzymes seems very promising as in the future, FOS markets are expected to thriving worldwide. Keeping in view the potential of microbial Fructosyltransferase in FOS production, an attempt has been made to produce FTase of microbial origin followed by its partial purification and characterization.

Methods: Stevia rebaudiana was exploited to isolate Fructosyltransferase (FTase) enzyme producing endophytic bacteria. Preliminary screening to select FTase producers was done by Triphenyl Tetrazolium Chloride (TTC) plate assay. Fructosyltransferase producer was identified by morphological, biochemical techniques followed by 16S rRNA gene technique. Fructosyltransferase production of B. stercoris S1 was enhanced by optimization of inoculum size, incubation time, temperature, pH of medium, carbon source concentration by following one variable at time method. Partial purification of FTase was achieved by ammonium sulphate precipitation at 30-60%. Partially purified enzyme was characterized for its temperature, pH and shelf stability.

Results: In total 07 bacterial isolates were isolated. The bacterial isolate S1 was selected as it exhibited maximum zone of hydrolysis (22 mm) in TTC plate assay. Quantitative screening was done in terms of transfructosylating activities. Isolate S1 exhibited 50.06 U/ml. Maximum Fructosyltransferase activity 119.55 U/ml was recorded in nutrient broth supplemented with 60 % sucrose at 72 h with an optimized pH of 6.0 at 40 °C. FTase titres after partial purification were 161.25 U/ml with specific activity 497.68 U/mg, purification fold and recovery percent 1.73 and 73.6 % respectively. Partially purified FTase were found active in a temperature range 30⁰C to 80⁰C and in pH range of 5.0 to 9.0. FTase was found stable at -20⁰C for 45 days.

Conclusion: The results obtained showed that the B. stercoris S1 represents a promising source for FTase enzyme that can be efficiently utilized for FOS production.

Fructosyltransferase

Prebiotics

Fructooligosaccharides

Bacillus stercoris

Characterization

Presently, there is a rising awareness of the additional health benefits and market potential for functional foods. Among various functional foods, short chain Fructooligosaccharides (FOS) are in great demand in food industry. These constitute an important class of carbohydrates that have health beneficial effects include activation of the human immune system, resistance to infection, enhanced mineral absorption in the gastrointestinal tract, synthesis of vitamin B-complex, lowering serum cholesterol etc. Furthermore, they beneficially affect the health of host by selectively stimulating the growth and /or activity of Bifidobacterium and Lactobacilli (Yadav et al. 2022).

FOS are substituent for sugar and fat in food products. Low sweetness of FOS is useful in those foods where the use of sucrose is restricted. These can also be used as fat replacers because they enhance the stability of foams, emulsions and mouthfeel by increasing unctuousness in food. As food ingredient, FOS are extensively employed in infant formula to stimulate the infant microbiota. They are also added in dairy products, beverages, bakery products (Nobre et al. 2022). Fructooligosaccharides are defined as a combination of three sugar molecules: 1-kestose (GF2), nystose (GF3) and frutofuranosylnystose (GF4) where the units Fructosyltransferase (F) are combined with sucrose (GF) at the β position (2 →1) (De la Rosa et al. 2022). Fructooligosaccharides represent one of the main oligosaccharide classes with prebiotic characteristics. Prebiotics are defined as a substrate that is selectively utilized by host microorganisms conferring health benefit (Bed Ferrari et al 2022). FOS fulfills the criteria for prebiotic classification as these are resistance to gastric acidity to hydrolysis by mammalian enzymes, and to gastrointestinal absorption, fermented by intestinal microflora; and these selectively stimulate of the growth and activity of those intestinal bacteria that contribute to health and well-being (Chen et al. 2020).

FOSs are naturally found in commonly consumed fruits and vegetables (De La Rosa). However, the production of FOS using Fructosyltransferase (FTase) derived from microorganisms has attracted attention in last decade. Fructosyltransferase (FTases EC 2.4.1.9.) possess transfructosylating activity acting on the β-1-2 link of sucrose and transfer a fructose molecule to an acceptor such as other sucrose molecule leading to generation of FOS and release glucose in the reaction (Muñiz-Márquez et al., 2016). Plant Fructosyltransferases are known to have various region selectivities and each of them could produce oligosaccharides with different structure. The production of plant FTases is limited by seasonal conditions and the production yield of FOS prepared by these enzymes takes a longer production path as it acts as a series of enzymes that work together to synthesize FOS in relatively small amount. Whereas, FTases of microbial origin remain unaffected by seasonal conditions and only single enzyme produces FOS.

Microbial FTase are extracellular as well as intracellular. However, until now a complex information about properties and preparation of Fructosyltransferases is still missing. Though, with the popularity of functional foods the future of FOS producing enzymes seems very promising as in the future, FOS markets are expected to thriving worldwide. Keeping in view the popularity of microbial FTases in FOS production an attempt has been made in the present investigation to isolate and screen potential FTase of microbial origin followed by its partial purification and characterization.

Isolation of Fructosyl Transferase producer

Fructosyltransferase producing strain was isolated from a medicinal plant Stevia rebaudiana (Stevia). Isolation was carried out on Nutrient agar under aerobic conditions by spread plate method. The bacterial colonies obtained on were purified by streaking. The pure cultures were preserved at −20 °C on Nutrient broth containing 30 % glycerol (v/v) in deep freezer.

Preliminary screening of Fructosyltransferase producing microbes

Preliminary screening of isolated bacterial samples were done by Triphenyl Tetrazolium Chloride (TTC) plate assay (Ojwach et al.2020).

Streak plate method

24 h old each bacterial isolate was streaked on Czapek Dox Agar (CDA) plate supplemented with 3% sucrose. These plates were incubated for 48 h at 37ºC. After incubation, these streaked plates were sprayed with TTC dye which produced a red color surrounding the streaks due to reduction of TTC in presence of FTase.

Well Diffusion Method

Growth of Bacterial isolates

Each bacterial isolate was grown in 50 ml of nutrient broth supplemented with 3% sucrose. Isolates were incubated at 37±2°C for 48 h till substantial growth of isolate was observed in the broth. Isolate was centrifuged at 10,000 rpm at 4°C for 20 min. The supernatant of each bacterial isolate was collected in a sterilized test tube and pellet was discarded.

Assay

Wells of 7mm diameter at 5mm depth were cut with the help of borer on Czapek Dox Agar plates supplemented with 3% sucrose. 200 μl of culture supernatant was poured into the well. The plates were then incubated at 37°C for 24 h. After 24 h of incubation TTC dye was sprayed over well. Clear reddish colored zones were observed visually around the well. Fructosyltransferase producing isolates were selected on the basis of the diameter of zone of hydrolysis (Red coloured) surrounding the well.

Quantitative screening of fructosyltransferase

FTase Assay (Ojwach et al. 2020)

Reagents

i) 5 % Sucrose in citrate phosphate buffer (0.1M, pH 6.5)

ii) Dinitro salicylic acid (DNSA) Reagent: NaOH: 1.0 g, Phenol: 0.2 g, Sodium potassium tartrate : 20.0 g, Sodium sulphate : 0.05g, DNSA reagent : 1.0g, Distilled water : 100ml

iii) Standard solution of glucose (1mg/ml)

Procedure

The reaction mixture contains 1 ml culture supernatant and 2 ml of 5% sucrose in citrate phosphate buffer (0.1M, pH 6.5). The reaction mixture was incubated at 50°C for 20 minutes. After incubation 1 ml of reaction mixture was added to 3ml of DNSA reagent and tubes were boiled in water bath for 15 minutes. With the exception of the enzyme, a control experiment was conducted. When the solution was still warm, 1 ml of sodium potassium tartrate (Rochelle Salt) was added and tubes were cool to ambient temperature. The O.D. was measured at 540 nm using a spectrophotometer and compared to the reagent blank, which consisted of 1 ml of distilled water, 3 ml of DNSA reagent and 1 ml of sodium potassium tartrate (Rochelle Salt). In order to create the standard curve, a stock solution of glucose (1 mg/ml) was used. The enzyme activity was expressed in terms of U/ml and specific Activity U/mg.

i) U/ml of enzyme activity represents μ moles of glucose released/min/ml of enzyme.

ii). Specific Activity (SA) represents μ moles of glucose released/min/mg of protein

Determination of Protein concentration

Proteins were measured by using Bovine Serum Albumin as a standard protein (Lowry et al. 1951)

Statistical analysis

The data recorded on microbiological studies were statistically analysed by using MS-Excel and OPSTAT packages. The mean values of data were used for the analysis of variance (ANOVA) by using Complete Randomized Design (Sheoram et al. 1998).To improve reliability and accuracy of results three replications were done with each experiment.

Molecular identification of isolate exhibiting FTase activity:

The selected isolate S1 identified at molecular level using 16S rRNA technique. The identification of isolates was carried out at the sequencing facility of National Centre for Microbial Resource (NCMR), National Centre for Cell Science, Pune. On the basis of 16S r RNA gene technique S1 was identified Bacillus stercoris. The sequences so obtained were submitted in National Centre for Biotechnology Information (NCBI) to get an accession number.

Optimization of process parameters for fructosyltransferase by screened enzyme producing isolates by one variable at a time approach (OVAT).

Different growth parameters viz. inoculum size, incubation time, temperature, pH and sucrose concentration were studied for B. stercorisS1 to monitor their effect on FTase enzyme production.

Effect of Inoculum Size

B. stercoris S1 was grown separately in 50ml of nutrient broth and incubated at 37±2°C for 24 h to achieve substantial growth. 10 ml inoculum of OD 0.5, 1.0, 1.5, 2.0 and 2.5 was added in each 250 ml of Erlenmeyer flask containing 90 ml of nutrient broth supplemented with 10 % sucrose. The flasks were incubated at 37°C, 100 rpm. After 24 h, the culture contents of each flask were centrifuged at 10,000 rpm for 15 min at 4°C. Supernatants were collected and stored at 4°C in refrigerator for further use. The quantitative tests were performed with the supernatant. FTase activity and proteins were measured as given above.

Effect of incubation time

In total 90 ml of nutrient broth was seeded with 10 ml of B. stercoris S1 (1.0 OD). The flasks were incubated at different incubation times ranging from 0 h, 24 h, 48 h, 72 h, 96 h,120 h and 144 h at 100 rpm. CFU and enzyme activity was calculated periodically after every 24 h. For enzyme activity the culture contents of each flask was centrifuged at 10,000 rpm for 15 min at 4°C. Supernatants were collected and stored at 4°C in refrigerator for further use. The quantitative tests were performed with the supernatant as mentioned above.

Effect of temperature

The flasks containing B. stercoris S1(1.O OD, 10 %) were incubated at different temperature ranging from 30°C, 35°C, 40°C, 45°C, 50°C and 55°C at 100 rpm. B. stercoris S1 was incubated for 72 h. The quantitative tests were performed with the supernatant as mentioned above.

Effect of pH

The media was prepared by adding 90 ml nutrient broth each in 6 different flasks with different pH ranging from 3, 4, 5, 6, 7 and 8 respectively. Each flask was inoculated with 10 ml of inoculum (1.0 OD) and incubated at 100 rpm for 72 h. After incubation followed by centrifugation enzyme assay was performed as mentioned above.

Effect of sucrose concentration

The nutrient broth supplemented with different sucrose concentration ranging from 3%, 5%, 10%, 20 %, 30%, 40%, 50%, 60% and 70% were inoculated with 10% inoculum of (1.0 OD) of B. stercoris S1. After incubation and centrifugation supernatant was collected and the quantitative tests were performed as mentioned above. Kinetic parameters such as maximum reaction rate (V_max ) and Michaelis constant (K_m ), and catalytic constant (K cat) were determined by plotting the lineweaver burk plot.

Partial purification of fructosyltransferase (FTase) enzyme

B. stercoris S1 having OD 1.0 was inoculated with 10% concentration in nutrient broth containing 60% sucrose, pH 6.0. Inoculated flasks were kept at 40ºC, 100 rpm for 24 h and 72 h respectively. Different concentrations of ammonium sulfate i.e. 0-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90% were evaluated to attain saturation point for FTase from B. stercoris S1. The preparations were kept at 4°C for overnight and then centrifuged that resulted in separation of pellets and supernatants. FTase was precipitated at 30-60% level of saturation of ammonium sulfate. Precipitates of each fraction so obtained were dissolved in phosphate buffer (0.1 M, pH 7.2) and were refrigerated until further use. Enzyme activity and protein concentration was measured as given above.

Characterization of partially purified FTase of Bacillus stercoris S1

Effect of pH on activity of partially purified FTase of Bacillus stercoris S1

1 ml aliquot of partially purified FTase enzyme was added to 2 ml of freshly prepared appropriate buffers of different pH in the range 4, 5, 6, 7, 8, 9 and 10 viz. citrate phosphate buffer 0.1 M for pH 4.0-6.0, phosphate buffer 0.1M for pH 7.0, Tris HCL 0.1 M for pH 8.0 and Glycine-Sodium Hydroxide buffer 0.1 M for pH 9.0-10.0. These preparations were incubated for 20 minutes at 50°C.Enzyme assay and protein concentration was measured as described above.

Effect of Temperature on activity of partially purified FTase of Bacillus stercoris S1

1 ml of partially purified FTase preparation was added into 2 ml of citrate phosphate buffer pH 6.5 in sterilized test tube separately. Each test tube then treated at different temperature of 30°C, 40°C,50°C …………. 90°C for 20 minutes. These preparations were incubated for 20 minutes at 50°C.Enzyme assay and protein concentration was measured as described above.

Shelf stability

The partially purified FTase was stored at -20°C and stability was determined at intermittent intervals viz. 15, 30, 45 days.

Fructooligosaccharide (FOS) Production:

FOS production was carried out using 60% sucrose w/v. Partially purified FTase 1ml each from B. stercoris S1 were mixed with 9 ml of sucrose and incubated at 55 ºC in an incubator for 24 h.

Analysis of reaction products

Fourier Transform Infra-Red Spectroscopy (FTIR)

End products of sucrose bio trans formation were analyzed using FTIR. Samples were subjected to FTIR analysis (Shimadzu 8400S FTIR Spectrometer, equipped with KBr beam splitter) Spectrophotometer was operated at a special range of 4000-400 cm^-1 with a maximum resolution of 0.85cm^-1

Isolation, Screening and identification of FTase producing bacterial isolate

In total 07 isolates were isolated from Stevia rebaudiana which is also called as sweet leaf. Screening of FTase producing bacterial and fungal isolates have been done by Triphenyl Tetrazolium Chloride (TTC) plate assay. Maximum zone of hydrolysis was recorded for isolate S1 i.e.22 mm, rest 06 isolated bacteria showed zones of hydrolysis with in 5mm to 7mm.Therefore, S1 was selected for further studies. The zones of hydrolysis by an enzyme in the agar plate containing Czapek dox agar with 5% (w/v) sucrose as carbon source pH 7.0 stained with TTC reagent after overnight incubation at 37⁰C (Fig. 1). In the presence of reducing sugars, the TTC is reduced to a red water-insoluble compound, Triphenyl formazan. The appearance of zones of hydrolysis around the well and color intensity indicated positive FTase activity (Ojwach et al 2020). The zone of hydrolysis developed after TTC staining recorded for FTase production have been done and reported by many authors.

Selected isolate was identified on the basis of their phenotypic, biochemical characteristics. (Table 1) is depicting morphological and biochemical characteristics of S1.Further identification was done by using 16S rRNA gene technique. The identification of isolates was carried out at the sequencing facility of National Centre for Microbial Resource (NCMR), National Centre for Cell Science, Pune Maharashtra. Sequences of isolate S1 so obtained were submitted to NCBI database and matched with already existing sequences. Sequence similarity search was done by using Basic Local Alignment Search Tool (BLAST). Isolate S1 showed 99% of homology with nucleotide sequence of Bacillus stercoris 1200 BP with accession number JCM30051.The 16SrRNA sequences of S1 have been registered to Gene bank data base with accession number OR553400. Bacterial 16S rRNA gene sequence has emerged as a preferred genetic technique. A phylogenetic tree of Bacillus stercoris and related species was constructed by Maximum Likelihood Estimation method as displayed in (Fig. 2). This method was chosen to explore different tree topologies and branch lengths to find the one with the highest likelihood. The bootstrap values for critical nodes, including the clade containing Bacillus stercoris S1 and Bacillus stercoris JCM30051, were maximally supported 86–92%. So, we classify S1 as Bacillus stercoris S1. To best of researcher’s knowledge this is the first report of FTase production by isolate B. stercoris S1. Although, In literature other studies pertaining to FTase production by Bacillus sp viz. Bacillus cereus, B. subtilis, B. coagulans, B. amyloliquifaciens etc. have been reported by Babu et al., (2008) and Fan et al., (2021).

Quantitative assay for Fructosyltransferase production

Quantitative assay for FTase production was carried out by FTase enzyme assay (Ojawach et al. 2020). The culture supernatant was taken with 5% sucrose solution as a substrate at pH 6.5 (0.1 M citrate phosphate buffer). The mixture was incubated at 50ºC for 20min. DNSA was added followed by boiling. Rochelle salt was added and O.D was measured at 540 nm. The enzyme activity was expressed in terms of U/ml (µmoles of Glucose released/min/ml of enzyme). Bacillus stercoris S1 showed highest FTase activity i.e. 51.62 U/ml with 78.09 U/mg specific activity. Authors (Belorkar et al. 2015) measured enzyme activity quantitatively FTase activity i.e. 36.88 U/mg for Aspergillus niger, 21.45 U/mg for Aspergillus flavus and 23.78 U/mg for Aspergillus versicolor. EI-Beih (2009) reported (37.04U/ml) extracellular FTFase enzyme by B. cereus in submerged fermentation. Babu et al., (2008) reported 78.92 U mL^-1 FTase production by Bacillus subtilis NCIM 2439.

Optimization of Process Parameters for Fructosyltransferase (FTase) by Screened Hyperenzyme Producing Isolates by One Variable at a Time (OVAT) Approach

Effect of inoculum size

Effect of inoculum size on FTase production from selected bacterial isolate i.e. Bacillus stercoris S1 was studied. The different inoculum size viz., 0.5, 1.0, 1.5, 2.0 and 2.5 OD₆₀₀ nm with 10% concentration were used for assessing enzyme activity. It has been revealed from the experiment that highest FTase production by B. stercoris S1 was with the inoculum size of 1.0 OD at 10% concentration which has been depicted in (Table 2).Maximum enzyme activity i.e. 50.06 ± 0.57 U/ml was observed with 1.0 OD of inoculum size, whereas minimum enzyme activity and reducing sugars were obtained with inoculum size of OD 2.5 i.e. 42.57 ± 1.15 U/ml. Authors (Ganaie et al. 2017) studied the effect of inoculum size on FTase production based on spore count. It was observed that as the spore size was intensified, a marked increase of FTase activity (197.11 ± 2.02 U/gds) was noticed. The treatment of large inoculum size enhances intra specific competition of microbial cells which leads to scarcity of nutrients availability thus lessen the FTase production. Thus, it is important to provide an optimum inoculum level in fermentation process.

Effect of Incubation time

Effect of incubation time on FTase production from selected bacterial isolates i.e. Bacillus stercorisS1 were studied. The different incubation time (Hours) viz., 0, 24, 48, 72, 96, 120 and 144 were used for assessing enzyme activity. CFU/ml was measured after each 24 h of interval along with enzyme activity. Maximum FTase activity for Bacillus stercorisS1 was 68.86 U/ml was observed at 72h with specific activity i.e. 140.53 U/mg whereas minimum FTase enzyme production for B. stercorisS1 was found at 0 h i.e. 6.65 U/ml with specific activity i.e. 13.85 U/mg. (Fig. 3). Incubation time enables the microorganisms to undergo cell division and increase their number which will enhance the amount of enzyme produced (Rolfe et al. 2012) Similar finding has been reported in literature in one of the study culture conditions utilizing the carbon and nitrogen supplies to determine the time course of FTase synthesis by A. carbonariusPC-4. FTase was produced using pineapple crown (1.0% w/v) as the carbon source and soybean protein (0.2% w/v) as the nitrogen source (Do Nascimento et al. 2019).

Effect of Temperature

Effect of temperature on FTase production from selected bacterial isolates i.e. Bacillus stercoris S1 were studied. The different incubation temperature viz., 30°C, 35°C, 40°C,45°C and 55°C were used for assessing enzyme activity. Effect of temperature on Bacillus stercorisS1 for FTase enzyme production has been shown in (Table 3).Maximum FTase activity for Bacillus stercorisS1 was observed at 40°C i.e. 73.27 ± 0.57 U/ml with specific activity i.e. 133.70 U/mg, whereas least FTase enzyme production for Bacillus stercorisS1 was found at 30°C i.e. 42.51 U/ml with specific activity i.e. 75.23 ± 1.15 U/mg. Temperature of the process is a parameter that influences cellular metabolism and the kinetics of enzymes. The reaction rates and collisions, the strength of molecular interactions and other physico-chemical characteristics are affected at varied temperature (Lopez et al. 2023). Effect of temperature on Fructosyltransferase enzyme production from Lactobacillus plantarum was determined by (Naganandhini et al. 2014). The inoculated flasks with L. plantarum were incubated at temperature range from 28–37°C for 48 h and the maximum activity of 160.54 ± 12.6 U/ml was exhibited at temp of 32°C.

Effect of pH

Effect of pH on FTase production from selected bacterial isolates i.e. Bacillus stercoris S1 were studied using batch culture fermentation. The different pH viz., 3, 4, 5, 6, 7, and 8 were used for assessing enzyme activity. Effect of pH on Bacillus stercorisS1 for FTase enzyme production has been shown in (Table 4). Maximum FTase activity for Bacillus stercorisS1 was observed at 6 pH i.e. 77.27 ± 1.15 U/ml with specific activity i.e. 124.02 ± 1.15U/mg whereas least FTase enzyme production for Bacillus stercorisS1 was found at 3 pH i.e. 41.51 ± 0.57 U/ml with specific activity i.e. 76.16 ± 0.57U/mg. Similar reports have been reported by many authors in literature. In one of the investigation an initial pH of 6.0 yielded the highest levansucrase activity (39.5 U/ml) and maximal polysaccharide production (28.2 g/l) (Youssef et al.2021). Deviations above or below this pH value had an adverse effect on enzyme activity. Remarkably, the enzyme activity remained stable within the pH range of 6.0 to 8.0.

Effect of sucrose concentration and determination of kinetic parameters

Effect of sucrose concentration on FTase production from selected bacterial isolates i.e. B. stercoris S1 was studied using batch culture fermentation. The different sucrose concentration (%) viz., 3, 5, 10, 20, 40, 50, 60 and 70% were used for assessing enzyme activity. Effect of sucrose concentration on B. stercoris S1 for FTase enzyme production has been shown in (Table 5). Maximum FTase activity for B. stercoris S1 was observed at 60% sucrose concentration i.e. 115.11 ± 0.57U/ml with specific activity i.e. 178.18 ± 0.57 U/mg, whereas least FTase enzyme production for B. stercoris S1 was found at 3% sucrose concentration i.e. 36.66 ± 0.57 U/ml with specific activity i.e. 68.01 ± 0.57U/mg. It has been cited in literature that high transfructosylating activity level achieved with high sucrose concentration. Researchers have reported FTase production at high sucrose concentration. FTase are able to catalyze transfructosylation reaction synthesizing FOS at higher sucrose concentration. Sucrose is used as a fructosyl donor and acceptor to complete the elongation of glycosidic chain and formation of FOS (Rustiguel et al. 2021). FTases cleaves the β 1–2 linkage of sucrose and transfers the fructosyl group leading to FOS formation.

Kinetic parameters

Kinetic parameters such as maximum reaction rate (V_max ) and Michaelis constant (K_m), and catalytic constant (K cat) were determined by lineweaver burk plot.

1. X – intercept of Lineweaver -Burk plot:

The equation of the Lineweaver- Burk plot is:

$$\:\frac{1}{v}=\frac{{k}_{m}}{{V}_{max}}.\frac{1}{S}+\frac{1}{{V}_{max}}$$

The x- intercept of the plot corresponds to -1/k_m, so:

x- intercept =$\:\frac{-1}{Km}$ = -0.015

K_m= $\:\frac{1}{0.015}=66.6\:\text{m}\text{g}/\text{m}\text{l}$

2. Lineweaver- Burk equation and calculation of V:

The equation can also be expressed as:

$$\:\frac{1}{v}=\frac{{k}_{m}}{{V}_{max}}.\frac{1}{S}+\frac{1}{{V}_{max}}$$

The y- intercept of the plot is $\:\frac{1}{{V}_{max}}$

V_max= 0.0081

Therefore by solving, V_max= $\:\frac{1}{0.0081}=123.45\:\text{U}/\text{m}\text{l}$

Kcat = Vmax/Et = 123.45/161.2 = 0.765 s^− 1

We have obtained values of Km and Vmax equal to 66.6 mg/ml and 123.45 U/ml for sugar as substrate. Kcat was 0.765s^− 1. Xu et al (2015) obtained values of Km and Vmax equal to 56.05 56.05 g L^− 1 and 800.1 U mg^− 1 for sucrose as substrate, respectively, for the purified FTase of Penicillum oxalicum.

The statistical analysis of the study

Analysis was performed using analysis of variance (ANOVA) to evaluate the significance of differences among various inoculum sizes, incubation times, temperatures, pH levels, sucrose concentrations on FTase production by B. stercoris S1. Each experiment was conducted in triplicate (three replications per treatment) to ensure reliability and statistical robustness (Table 6).

Partial Purification of Fructosyltransferase Enzyme from B. stercoris S1

Partial purification of FTase was attained by ammonium sulphate precipitation. Bacillus stercorisS1 was grown under optimized conditions. B. stercoris was centrifuged at 10,000 rpm at 4ºC for 20 min. Cell free supernatants was taken in beakers and then subjected to sequential ammonium sulphate precipitations. Maximum FTase activity was observed with 30–60% concentration of ammonium sulphate. The enzyme activity of partially purified FTase was 161.25 U/ml with specific activity of 497.68 U/mg, Purification fold was 1.73 with recovery 73.86% (Table 6). The results are in close conformity with FTase from A. oryzae S719 where partial purification of enzyme was achieved at 95% of ammonium sulphate precipitation and enzyme activity was 310000 U with specific activity 156, purification fold 2.4 and recovery % was 85% (Han et al. 2020).

Characterization of Partially Purified Fructosyltransferase (FTase) Enzyme

Effect of Temperature

The effect of temperatures regime on the enzyme activity of partially purified FTase has been presented in Fig. 4 ranging from 30°C, 40°C …...90°C for 20 min. It was found that the enzyme exhibited maximum activity at 50°C for B. stercorisS1 i.e. 161.25 U/ml. The specific activity at 50°C for B. stercorisS1 is 497.68 U/mg. Minimum enzyme activity for B. stercorisS1 was 39.71 U/ml. with specific activity 136.46 U/mg at temperature 80ºC. When the temperature increased above 50°C the activity of the enzyme was affected negatively and gradually reduced. As the denaturation of the enzymatic protein occurs at elevated temperatures therefore, after certain level of temperature above 50°C, the enzyme activity decreased rapidly. In nutshell, the enzyme was most efficient at 50°C as compared with other tested temperatures. Purified FTase showed thermal stability over a wide range of temperature 40°C and 60°C (Ojawach et al.2020).

Effect of pH on activity of partially purified FTase from Bacillus stercoris S1

Figure 5 exhibits the impact of varying pH on the partially purified FTase activity with pH values ranging from (4, 5, 7...9). It was observed that pH variations had a significant impact on the enzyme's activity. Within the range of 6.0 to 8.0, the enzyme showed considerable activity. The highest enzyme activity was found at pH 7.0 161.25 U/ml B. stercorisS1 with specific activity 462.03 U/mg. The minimum enzyme activity was obtained at pH 4.0 i.e. 1.06 U/ml for FTase from B. stercorisS1 with specific activity 4.90 U/mg. The enzyme activity declined when the pH of enzyme medium altered on either side of optimum range from acidic to alkaline, a decline in enzyme activity was observed. The majority of enzyme exhibit activity at limited pH range and if this pH is exceeded or lowered the enzyme activity decreases. Similar reports have been reported in literature by many authors. Partially purified FTase from A. niger showed optimum activity at pH 6.0 (Ojawach etal. 2020). The relative activity declined below pH 4.0 and above pH 7.5 (Silva et al. 2021).

Shelf stability partially purified FTase from Bacillus stercoris S1

The partially purified FTase was stored at -20ºC in deep freezer. The enzyme activity was checked at intervals after a time duration of i.e. 15, 30, 45 days. The enzyme was stable upto 45 days. The enzyme activity for B. stercoris S1 on 15, 30 and 45was 161.07, 160.98 and 160.62 U/ml with the specific activity 508.10, 507.82 and 508.29U/mg respectively as shown in Table 7. The shelf stability of enzyme is an important character for enzyme activity. Enzymes have limited stability during long term storage. This is due to the deleterious effects of the environmental moisture and microbial contamination. Also, due to a variety of intramolecular and intermolecular chemical reactions including hydrolysis, aggregation, deamidation, oxidation, β-elimination, and changes in conformation enzymes in aqueous solutions are inherently unstable. This may result in a loss of its biological activity Fernandez (2010).

Analysis of FTase and sucrose reaction products by Fourier Transform Infra-Red Spectroscopy (FTIR)

FTIR spectra was used to identify preliminary structures in the functional groups of oligosaccharides. The functional group of oligosaccharides based on peak vibration values in the region of infra red radiations. The FTIR spectra demonstrated that the graphs of oligosaccharides were consistent with typical carbohydrate vibrational bands. Data pertaining to FTIR analysis has been depicted in Table 9 and Fig. 6. The FTIR spectra of the sample was recorded in the range of 3559 to 689 cm^− 1 and the peaks were compared. Starting from 3300 − 3200 cm^− 1, bands have shown the presence of intermolecular bonded O-H stretching, which indicates the presence of hydroxyl groups, carboxylic acids and water. Sample exhibit visible peaks at 3300 − 3200 cm^− 1, which indicates the presence of hydroxyl O-H stretching. The spectrum between 1600 and 1670cm-1 represents NH2 bending and C = O stretching, which shows the presence of primary, secondary amides and carbohydrates indicated the presence of binding peptides in these oligosaccharides (Yang et al. 2020).The absorption between 1300 and 1600 cm-1 depicts the coupled stretching and bending of carbohydrates. The absorption between 1090–1140 indicates sucrose consumption and glycosidic bond formation due to transfructosylation due to FTase action. This region is called fingerprint region of sugars (Santos et al., 2014). The wavelength at 650–1000 represents = C–H out-of-plane bending and depicts the presence of alkene compound. Similar observations regarding FTIR spectra for FOS generation by FTase have been reported by many researchers in literature. In one of the report (Choukade et al.2019). transformed sucrose to FOS by using FTase from Aspergillus tamari NKRC 1229.Authors characterized FOS using FTIR. They revealed that evolution of peaks at 996, 1025 and 1122 cm^_1 during 1–24 h of FTase action indicated sucrose consumption and glycosidic bond formation due to transfructosylation.

In the present investigation an attempt has been made to isolate most efficient FTase producing potential bacteria from stevia its screening, identification, optimization to enhance the maximum enzyme production, partial purification followed by its characterization. The present study thus evaluates the feasibility of using partially purified extracellular FTase of bacterial origin in the enzymatic synthesis of biofunctional fructooligosaccharides due to its stability, high activity at varied temperature and pH range. The enzyme FTase is partially purified in this study that can be further purified and immobilized for improved FOS generation and can be extensively useful in food industries because of their functional properties. In this study sucrose has been utilized for FTase and FOS production which can be replaced by low cost substrates viz., agriculture/ horticulture waste. By utilizing these waste to produce microbial based FTase enzymes would be a cost- effective, and scalable method for producing health-promoting prebiotic FOS. This strategy supports waste valorization and aligns with circular economy principles

Author contributions

Conceptualization: Dr Neha Gautam ; Methodology : Dr Neha Gautam; Formal analysis and investigation: Puneet Kumar; Dr Neha Gautam, Vikas Kumar and Shruti Gupta ; Writing original draft preparation Dr Neha Gautam and Stuti Sharma;

supervision: Dr Neha Gautam

Funding :

No funding

Data availability

All data generated or analyzed during this study are included in this published article.

Consent for publication

All of the authors consent to the publication of this manuscript in Annals of Microbiology.

Competing interests

The authors declare no competing interests in publishing this manuscript.

Conflict of interest statement

The authors have no competing interests to declare that are relevant to the content of this article.

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Table 1. Morphological and biochemical characteristics of FTase producer

Cell morphology	S1
Color	White
Texture	Rough
Form	Irregular
Elevation	Flat
Margin	Irregular
Gram Staining
Gram +/-	+ve
Shape	Rod
Biochemical Assay
Catalase	+
Fermentation with glucose	+
Casein hydrolysis	+
Citrate utilization	-
Indole	-
MR-VP	+
H₂S production	-
Urease	-
Nitrate reductase	+
Growth conditions	Aerobic

Table 2. Effect of inoculum size on FTase production by B. stercoris S1

Inoculum size OD_(600nm) (10%)	Fructosyltransferase (FTase)		Protein (mg/ml)
Inoculum size OD_(600nm) (10%)	Enzyme activity (U/ml) *	Specific activity (U/mg) **	Protein (mg/ml)
0.5	45.41 ± 1.15	109.95 ± 0.57	0.413 ± 0.00
1.0	50.06 ± 0.57	71.92 ± 0.57	0.696 ± 0.05
1.5	47.63 ± 0.57	73.73 ± 0.57	0.646 ± 0.05
2.0	44.77 ± 0.57	72.09 ± 0.57	0.621 ± 0.05
2.5	42.57 ± 0.57	77.96 ± 0.57	0.546 ± 0.00
CD_0.05	2.30	1.81	0.14
SE (Mean)	1.6	1	0.00

* Enzyme activity (U/ml): mmoles of Glucose released/min/ml of enzyme.

**Specific activity: enzyme activity/mg of protein

Data presented along with mean (±) Standard Error

Table 3. Effect of temperature on FTase production by B. stercoris S1

Temperature Degree Celsius	FructosylTransferase (FTase)		Protein (mg/ml)
Temperature Degree Celsius	Enzyme activity (U/ml) *	Specific activity (U/mg) **	Protein (mg/ml)
30ºC	42.51 ± 0.57	75.23 ± 1.15	0.565 ± 0.00
35ºC	58.15 ± 0.57	81.55 ± 0.57	0.713 ± 0.00
40ºC	73.27 ± 0.57	133.70 ± 1.15	0.548 ± 0.00
45ºC	62.07 ± 0.57	105.20 ± 0.57	0.590 ± 0.00
50ºC	61.49 ± 0.57	99.82 ± 0.57	0.616 ± 0.00
55ºC	46.19 ± 0.57	65.89 ± 1.15	0.701 ± 0.00
CD_0.05	1.77	2.81	0.00
SE (Mean)	1	2.5	0.00

* Enzyme activity (U/ml): mmoles of Glucose released/min/ml of enzyme.

**Specific activity: enzyme activity/mg of protein

Data presented along with mean (±) Standard Error

Table 4. Effect of pH on FTase production by B. stercoris S1

pH	Fructosyltransferase (FTase)		Protein (mg/ml)
pH	Enzyme activity (U/ml) *	Specific activity (U/mg) **	Protein (mg/ml)
3	41.51 ± 0.57	76.16 ± 0.57	0.545 ± 0.00
4	45.41 ± 1.15	109.95 ± 0.57	0.413 ± 0.00
5	51.55 ± 0.57	93.05 ± 1.15	0.554 ± 0.00
6	77.27 ± 1.15	124.02 ± 1.15	0.623 ± 0.00
7	59.03 ± 0.57	106.93 ± 0.57	0.552 ± 0.00
8	53.48 ± 0.57	107.17 ± 0.57	0.499 ± 0.00
CD_0.05	2.51	2.51	0.01
SE (Mean)	2	2	0.00

* Enzyme activity (U/ml): mmoles of Glucose released/min/ml of enzyme.

**Specific activity: enzyme activity/mg of protein

Data presented along with mean (±) Standard Error

Table 5. Effect of sucrose concentration on FTase production by B. stercoris S1

Sucrose Concentration %	Fructosyltransferase (FTase)		Protein (mg/ml)
Sucrose Concentration %	Enzyme activity (U/ml) *	Specific activity (U/mg) **	Protein (mg/ml)
3	36.66 ± 0.57	68.01 ± 0.57	0.539 ± 0.00
5	61.30 ± 577	108.11 ± 0.57	0.567 ± 0.00
10	75.83 ± 0.57	199.95 ± 0.57	0.539 ± 0.00
20	91.66 ± 0.57	239.32 ± 0.57	0.383 ± 0.00
30	95.61 ± 0.57	221.83 ± 0.57	0.431 ± 0.00
40	97.33 ± 0.57	228.47 ± 0.57	0.426 ± 0.00
50	104.89 ± 0.57	232.05 ± 0.57	0.452 ± 0.00
60	115.11 ± 0.57	178.18 ± 0.57	0.646 ± 0.00
70	111.64 ± 0.57	248.64 ± 0.57	0.449 ± 0.00
CD_0.05	1.71	1.71	0.01
SE (Mean)	1	1	0.00

* Enzyme activity (U/ml): mmoles of Glucose released/min/ml of enzyme.

**Specific activity: enzyme activity/mg of protein

Data presented along with mean (±) Standard Error

Table 6. Statistical significance of factors affecting FTase production by B. stercoris S1

Factor	Significant Difference observed (p≤0.05)	Most effective condition	CD_0.05
Inoculum Size	Yes	1.0 OD(50.06 U/ml)	2.30
Incubation Time	Yes	72h (68.86 U/ml)	1.62
Temperature	Yes	40⁰C (73.27 U/ml)	1.77
pH	Yes	6.0 (77.27 U/ml)	2.51
Sucrose Concentration	Yes	60% (115.11 U/ml)	1.71

Table7. Partial Purification of FTase from B. stercoris S1

Steps	Volume (ml)	FTase			Protein (mg/ml) *	Purification fold***	Recovery (%)
Steps	Volume (ml)	Enzyme activity^# (U/ml)	Total activity	Specific activity (U/mg)**	Protein (mg/ml) *	Purification fold***	Recovery (%)
Crude culture supernatant	200	119.11	23822	287.01	0.415	1	100
Ammonium sulphate precipitation (30-60%)	15	161.25	2418	497.68	0.324	1.73	73.86

^#Enzyme activity (U/ml): mmoles of Glucose released/min/ml of enzyme.

* Protein concentration was determined by Lowry’s method

**Specific activity is the activity unit/ protein

***Purification fold is increase in specific activity

****Recovery % is remaining protein concentration as % of the initial protein

Table 8. Shelf stability of partially purified FTase from B. stercoris S1

Days	*Bacillus stercoris* S1
Days	Enzyme activity (U/ml) *	Specific activity (U/mg) **	Protein concentration (mg/ml) ***
15	161.07 ± 0.57	508.10 ± 0.57	0.317 ± 0.00
30	160.98 ± 0.57	507.82 ± 0.59	0.317 ± 0.00
45	160.62 ± 0.57	508.29 ± 0.57	0.316 ± 0.00
CD_0.05	1	1	1
SE (Mean)	1	1	0.00

^*Enzyme activity (U/ml): mmoles of Glucose released/min/ml of enzyme.

**Specific activity is the activity unit/ protein

*** Protein concentration was determined by Lowry’s method

Data presented along with mean (±) Standard Error

Table 9. Analysis of FTase and sucrose reaction products by Fourier Transform Infra-Red Spectroscopy

Wave numbers	S1	Functional group	Compound
829	0.12	=C–H out-of-plane bending	Alkene
900-100	0.14	=C–H out-of-plane bending	Alkene
1049–1043	0.25	C-O bending	Hydroxyl
1140-1090	0.13	C-O stretching	Phenols
1600-1300	0.15	Coupled stretching and bending	Carbohydrates
1670-1600	-	NH2 bending and C=O stretching	Primary amide and Secondary amide
3300-3200	0.17	O-H Stretching	Carboxylic acids, Water

Synthesis and Characterization of Microbial Fructosyltransferase (FTase) from endophytic Bacillus stercoris S1 for Fructoligosaccharide production

Status:

Journal Publication

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Abstract

Figures

Background

Materials and Methods

Results and Discussion

Isolation, Screening and identification of FTase producing bacterial isolate

Quantitative assay for Fructosyltransferase production

Effect of inoculum size

Effect of Incubation time

Effect of Temperature

Effect of pH

Effect of sucrose concentration and determination of kinetic parameters

Kinetic parameters

1. X – intercept of Lineweaver -Burk plot:

2. Lineweaver- Burk equation and calculation of V:

The statistical analysis of the study

Characterization of Partially Purified Fructosyltransferase (FTase) Enzyme

Effect of Temperature

Analysis of FTase and sucrose reaction products by Fourier Transform Infra-Red Spectroscopy (FTIR)

Conclusion

Declarations

References

Tables

Status:

Journal Publication

Version 1