Gamma cyclodextrin glycosyltransferase from evansella caseinilytica: production, characterization and product specificity

Abstract

Alkalohalophilic Evansella caseinilytica produced an extracellular cyclodextrin glycosyltransferase (CGTase) with cyclization activity of 43.5 ± 4.4 U/L in M1 medium containing 1% starch and 6% NaCl in nutrient broth at 37 ºC, pH 9.0, after 48 h. This is the first report of CGTase from this bacterium. 0.1% starch was found to induce CGTase, and further optimization using one variable at a time approach followed by statistical optimization led to 5.5-fold enhancement resulting in 240.5 ± 5.46 U/L. Six parameters were identified as positive signals using Plackett–Burman (PB). Of these, yeast extract, MgSO₄ and tryptone were taken further for Response Surface Methodology (RSM) by disposing beef extract and fixing starch and soya peptone. The optimized M4 medium consisted of tryptone (0.1%, w/v), yeast extract (0.25%, w/v), MgSO₄ (8 mM, w/v), potato starch (0.1%, w/v) and soya peptone (0.2%, w/v). CGTase was further purified with 6.44-fold purification and 19.32% yield employing starch affinity. It was found to be monomeric, corresponding to a size of 68 kDa as estimated by SDS-PAGE and was further confirmed to be 65 kDa by size exclusion chromatography. γ-Cyclodextrins were produced as the major product with a conversion of 5% soluble starch into 20.38% γ-cyclodextrins after 24 h reaction, as determined by HPLC. Peptide fingerprint after LC–MS analysis matched with IPT/TIG domain-containing protein within the genome of E. caseinilytica. Further blastp analysis revealed the closest homology with γ-CGTase from an alkalophilic E. clarkii, thereby confirming CGTase from E. caseinilytica as γ-CGTase.

Introduction

The enzyme Cyclodextrin Glycosyltransferase (CGTase; EC 2.4.1.19) belongs to the GH 13_2 subfamily of the glycosyl hydrolase superfamily, along with other α- and maltogenic amylases based on their sequence similarity (http://www.cazy.org) (Leemhuis et al. 2002; Li et al. 2007; Lombard et al. 2014; Lim et al. 2021). All three enzymes belonging to this subfamily catalyze the hydrolysis and transglycosylation reactions, and are characterized by the presence of three conserved domains A, B, and C. Of these, however, CGTases entail two additional domains D and E, and are multifunctional enzymes (Kelly et al. 2009; Lim et al. 2021). The CGTases are considered as transferase enzyme as they mainly catalyze three transglycosylation reactions viz. cyclization, coupling, and disproportionation, along with minor hydrolysis reaction (Van der Veen et al. 2000; Han et al. 2014). Of these, cyclization is specifically reported to be catalyzed by CGTases, wherein the enzyme acts by cleaving the α-1,4-glycosidic bond of starch or similar α-glucans yielding a glycosyl-intermediate. This intermediate is subsequently transferred to the non-reducing end of the substrate via formation of a new intramolecular α-1,4-glycosidic bond, resulting in the formation of cyclodextrins (Van der Veen et al. 2000).

CGTases find commercial applications in the production of a variety of cyclodextrins and in the synthesis of transglycosylated products (Han et al. 2014; Lim et al. 2021). Cyclodextrins (CDs) are cyclic oligosaccharides and can be mainly classified as α-, β-, and γ-CDs, consisting of six, seven and eight glucose units respectively linked via α-1,4-glycosidic bonds (Qi et al. 2007). These CDs have been studied extensively and are uniquely arranged as hollow cylinders, with a hydrophilic exterior and an internal hydrophobic cavity that can accommodate various hydrophobic carrier molecules. This feature has widened their applicability in various industrial sectors like food, pharmaceutical, plastics, cosmetics, agro-chemical, etc. (Li et al. 2007; Wang et al. 2013; Del Valle 2004). The production of CDs using microbial CGTases is a much-explored field, and a variety of microbes including bacteria, archaea, and fungi have been reported to produce CGTase enzyme in the extracellular medium (Abd-Aziz et al. 2007; Leemhuis et al. 2010; Lim et al. 2021). CGTase mediated CD synthesis usually yields a mixture of α-, β-, and γ-CDs, and depending on the ratio of the product formed, the enzyme is further classified into individual subgroups as α-, β- or γ-CGTases (Wu et al. 2012). Most of the well-characterized CGTases are from different strains belonging to the genus Bacillus and are largely β-CGTases, followed by α-CGTases, and a very few γ-CGTases (Abd-Aziz et al. 2007; Lim et al. 2021). All known CGTases synthesize a mixture of cyclodextrins including α-, β- or γ-CDs and large-ring CDs (Larsen et al. 1998; Yang et al. 2001; Cao et al. 2005; Sonnendecker et al. 2019; Wang et al. 2020).

Currently, the demand for γ-CGTases is continuously growing as γ-CDs are more desirable in food and pharmaceutical industries as compared to α- and β-CDs; their higher water solubility and biodegradability, as well as larger hydrophobic cavity allows the formation of inclusion complexes with the larger organic molecules in concentrated form (Li et al. 2007). However, most of the wild-type γ-CGTases and mixed type β/γ CGTases screened till date exhibit low product specificity for γ-CD production (Li et al. 2007) except γ-CGTases from E. clarkii (Takada et al. 2003) and B. thuringiensis GU-2 (Goo et al. 2014), where γ-CD is reported to be the major product formed. This mandates additional time-consuming and expensive steps of separation and purification of highly water soluble γ-CDs from cyclodextrin mixtures, resulting in low yields and higher prices, thus limiting their market value (Szejtli 2004). Therefore, γ-CGTases capable of specifically producing γ-CDs or an improved ratio of γ-CDs are desired to increase their market share.

Hence, the present study was undertaken, wherein an alkalo halophilic bacterial strain Evansella caseinilytica SP^T [(Bacillus caseinilyticus SP^T; Reddy et al. 2015) Gupta et al. 2020] was evaluated for extracellular γ-CGTase production, followed by its ability to synthesize γ-CDs. Peptide fingerprints obtained through LC–MS analysis of the purified protein were used to identify the protein sequence within the proteomic database of E. caseinilytica. Protein BLAST (blastp) was further performed to identify its closest homolog among known CGTases.

Materials and methods

Culture

Evansella caseinilytica SP^T, (Bacillus caseinilyticus; Reddy et al. 2015) Gupta et al. (2020), an alkalohalophilic strain was purchased as lyophilized powder from the Microbial Culture Collection Centre (NCMR, Pune, India). It was revived and cultivated in nutrient broth (pH 9.0) containing 6% NaCl at 37 ºC/200 rpm as per the prescribed protocol (Reddy et al. 2015).

Chemicals and Reagents

Soluble starch was purchased from Sigma-Aldrich (USA). Cyclodextrin standards (α, β, and γ) and culture media components were procured from Himedia (India). Solvents were purchased from Qualigens (USA) and HPLC-grade acetonitrile was procured from Merck (USA). Dyes bromocresol green, xylene cyanol, and phenolphthalein were purchased from SRL. All other chemicals used were of analytical grade.

Standard protocol for the production of extracellular CGTase from E. caseinilytica SP^T

Seed inoculum was cultured in nutrient broth in the presence of 6% NaCl (pH 9.0; adjusted with 1 N NaOH before autoclaving) at 37 ºC/200 rpm for 24 h. Enzyme production was carried out by inoculating 2% seed inoculum into medium containing 1% starch (M1 medium) in a total volume of 50 ml. The culture was incubated at 37 ºC/200 rpm for 48 h following which it was centrifuged at 4350 × g for 30 min. The cell-free supernatant was harvested and further assayed for the γ-CGTase activity.

Qualitative plate assays for CGTases

Chromogenic media used for gel diffusion assays were prepared according to standard methods that have been widely reported in the literature (Hamaker and Tao 1993; Mennoci et al. 2008). M1 medium with 2% agar was supplemented with 0.01% congo red and 0.001% xylene cyanol for γ-CGTase (Hamaker and Tao 1993), and 0.05% phenolphthalein for β-CGTase activity (Mennoci et al. 2008). The final pH was adjusted to pH 9.0 with 1 N NaOH before autoclaving. The media plates were prepared, and wells were bored in the middle of the plates using a cork borer. 100 µl of the production broth was added to the well and 50 mM Tris–Cl pH 9.0 buffer was used as a control. The plates were incubated at 37 ºC for 48 h or till a clear halo was observed.

Quantitative assays for CGTases

Quantitative assays for determining the β- and γ-CGTase activity in the production broth were performed as per the standard protocol described earlier respectively by Higuti et al. (2004) and Takada et al. (2003), with slight modifications. For β-CGTase activity, the reaction was set using 0.5 ml crude enzyme and 0.5 ml 1.5% potato starch prepared in 50 mM Tris–Cl buffer pH 9.0 and incubated at 60 ºC for 20 min. Following incubation, 1 ml 0.02% phenolphthalein dye prepared in 25 mM Na₂CO₃ was added in order to stop the enzymatic reaction. The reaction was further diluted with 8 ml distilled water immediately after adding dye and absorbance was measured at 550 nm.

The reaction for γ-CGTase activity was set similarly as mentioned above for assaying the β-CGTase activity. Herein, the reaction was terminated by adding 100 µl of 5 mM bromocresol green dye prepared in 20% ethanol. The reaction mixture was incubated for 20 min at room temperature (25–30º C) following which, 2 ml of 1 M acetate buffer (pH 4.2) containing 30 mM citric acid was added before measuring absorbance at 630 nm.

Substrate control and enzyme control were set in the same manner. The substrate control served as blank and enzyme control values were subtracted from the test readings. CGTase activity was expressed as µmoles of respective cyclodextrin synthesized per min and it was calculated using the regression equation determined for the respective cyclodextrin standard (y = 0.312x + 0.0123 for γ-CD and y = 0.2236x + 0.0022 for β-CD).

Dextrinizing activity

The dextrinizing activity of γ-CGTase was performed following the iodine binding assay described by Gigras et al. (2002) with some modifications. A total reaction mixture of 1 ml consisted of 0.5 ml appropriately diluted enzyme and 0.5 ml 1% soluble potato starch prepared in 50 mM Tris–Cl pH 9.0 as substrate. The reaction was incubated at 60 ºC for 10 min and was terminated by immediately keeping the tubes on ice after incubation. The absorbance was then measured at 620 nm. Substrate control and enzyme control were also prepared similarly. A 10% decline in OD620 nm relative to the substrate control was considered as 1 dextrinizing unit under standard assay conditions.

Cell growth and protein estimation

Protein concentration was determined using Bradford reagent and BSA as a standard (regression equation y = 0.016x). Optical density (OD) of the cells at 600 nm was used to measure the bacterial growth.

Medium optimization for γ-CGTase production: One variable at a time (OVAT) approach and statistical optimization methods

The medium optimization for γ-CGTase production was done using the standard protocol described earlier. Variations if any are described in the respective experiments.

Effect of different starch concentrations and alternate inducers

In M1 medium, the concentration of starch was varied from 0.0–1.0%, and culture was incubated at 37 ºC/ 200 rpm for 48 h, and the resulting medium was termed as M2. This was followed by assessing the effect of various alternate inducers by replacing starch in the M2 medium with 0.1% of various alternate inducers. The results were compared with the M2 medium as control.

Optimization of physiological parameters

Various physiological parameters (pH, temperature and, salt concentration) were standardized for optimal γ-CGTase production in the M2 medium. The standardized conditions were taken further for statistical optimization of the enzyme production.

Statistical optimization

Screening of signal parameters was done among nine variables using Plackett–Burman (PB) design in M2 medium following standard incubation conditions. The maximum and minimum range of each factor was selected to generate a set of 12 experiments using Design expert^® software 6.0 and their effect on γ-CGTase production was analyzed by Pareto graph based on the p- and E-values. The interaction between three signal factors viz. tryptone, MgSO₄, and yeast extract was further evaluated by Response Surface Methodology (RSM) employing Box-Behnken design using Design expert^® software 6.0. The optimum concentration and interaction of various parameters were studied by ANOVA analysis. The predictions were validated by performing random within and out of design experiments, and the final medium was termed as M4.

Time kinetics for growth and production of enzyme in optimized medium

The bacterial growth and associated enzyme production was studied as a function of time over a period of 6 to 120 h in the M4 medium. 2 ml culture broth sample was withdrawn at regular intervals and further processed for γ-CGTase activity and growth measurement (OD600 nm).

Purification of γ-CGTase using starch affinity method

The γ-CGTase purification was achieved using the standard starch adsorption method (Kitayska et al. 2011) with slight modifications. The enzyme sample (1.5 L) was concentrated by ultrafiltration using a 5 kDa molecular weight cutoff membrane filter and the obtained retentate (220 ml) was allowed to adsorb for 1 h on 10% (w/v) insoluble corn starch mixed with 30% (w/v) ammonium sulphate, under continuous stirring on a magnetic stirrer at 4 ºC. γ-CGTase bound starch was separated by centrifugation at 4,350 × g for 20 min. The pellet was further washed (twice) with 50 mM potassium phosphate buffer (pH 7.0) followed by centrifugation at 4,350 × g for 20 min. Elution was achieved by suspending the pellet in 25 ml of 1 mM β-CD prepared in phosphate buffer and incubating at 37 ºC for 30 min. The elution step was repeated twice to achieve complete desorption. The final sample was additionally centrifuged at 4,350 × g for 20 min and purified γ-CGTase was obtained in the supernatant. It was then passed through a 0.45 µm syringe filter and further concentrated (1 mg/ml) using centrifugal filters with a 10 kDa molecular cut-off value (Pall Corporation, New York, USA).

Molecular weight determination of purified enzyme

The molecular weight of purified γ-CGTase was determined by size exclusion chromatography using Hiload 16/600 Superdex^™ 200 pg column equipped on AKTA Prime plus system (GE Healthcare Biosciences AB, Sweden). The protein sample was loaded at a concentration of 1 mg/ml. The elution volume Ve of the positive fraction was used to determine the molecular weight from a standard curve prepared using the elution volume of standard molecular weight markers (29-443 kDa; Sigma-Aldrich USA) as per the manufacturer’s protocol. The positive fractions were collected, pooled, and concentrated before analyzing γ-CGTase activity, and its purity was examined on a 10% SDS-PAGE (Laemmli 1970) gel. The purified γ-CGTase was further subjected to LC–MS analysis (Central Instrumentation Facility, University of Delhi South Campus, New Delhi) and the obtained mass spectrometric data was evaluated for identifying the peptides within the proteomic database of E. caseinilytica. The identified peptides were subjected to BLAST analysis using the blastp program (http://www.ncbi.nlm.nih.gov/BLAST/) and the complete protein sequence was obtained. The close homologs of the protein thereby identified were obtained similarly, and it was further subjected to conserved domain search tool of NCBI and the CGTase specific domains were marked.

Native PAGE and activity staining

For zymogram analysis, γ-CGTase sample was run on a 10% Native PAGE-gel following which, the gel was equilibrated with the assay buffer (50 mM phosphate buffer, pH 9.0) for 10 min. It was then overlaid onto a 1% starch agar plate containing 0.01% congo red and 0.001% xylene cyanol dyes prepared in 50 mM Tris–Cl pH 9.0. The plate was then incubated at 37 ºC overnight and the zone of starch hydrolysis was visualized.

Enzymatic conversion of starch to cyclodextrin (CD)

The reaction was set by solubilizing 1 g soluble potato starch in 19 ml buffer (50 mM Glycine–NaOH pH 11.0) and adding 1 ml appropriately diluted enzyme corresponding to a final enzyme concentration of 5 U/g starch. The reaction was incubated at 60 ºC for time intervals of 2 h and 24 h. The γ-CD production was then analyzed by TLC (Chung et al. 1998) followed by product quantification using HPLC.

For HPLC analysis, the samples were centrifuged at 7,000 × g for 2 min followed by filtration through a 0.45 μm syringe filter. The 2 h sample was directly injected onto HPLC, whereas the 24 h sample was further diluted 1:10 in the mobile phase solvent, constituted by acetonitrile and water in a ratio of 65:35 (v/v), before loading. Product analysis was done using a Sugar-D column (Cosmosil; 4.6 mm I.D. X 250 mm) employing a Refractive Index Detector (RID). Analytical method for CD detection was used as per the column specifications under an isocratic flow of the mobile phase. The flow rate was maintained at 1 ml/min and the oven temperature was set at 30 ºC. The type of CD synthesized in the enzymatic reaction was identified upon comparison with the commercial standards. A standard curve of different γ-CD concentrations (mM) vs peak area was regressed and was utilized in the quantification of γ-CD synthesis (y = 685.73x + 1727.5).

Effect of pH and temperature on enzyme activity

The enzyme activity as a function of pH and temperature was determined in a pH range of 7.0–12.0 (50 mM phosphate buffer for pH 7.0, 50 mM Tris–Cl buffer for pH 8–10 and 50 mM Glycine–NaOH buffer for pH 11–12), and 30–80 ºC following the standard assay protocol.

The thermal stability of γ-CGTase was determined by incubating the enzyme at varying temperatures for prolonged time intervals of 3 h in sealed tubes to prevent any loss due to evaporation. The samples were withdrawn at regular time intervals and the % residual γ-CGTase activities were determined under standard assay conditions considering the activity obtained at 0 h as 100%. The thermal stability of γ-CGTase in presence of salt was also determined in a similar manner by incubating the enzyme sample in presence of 1 M and 2 M NaCl at 70 ºC. The natural logarithmic values of % residual activity was then plotted vs time to obtain the inactivation curve; the slope of the plot indicated the dissociation constant (Kd) and the t_1/2 value was calculated as t_1/2 = 0.693/Kd.

Effect of salt (NaCl) concentration on activity and stability of γ-CGTase

Effect of NaCl concentration on the γ-CGTase activity was determined in the presence of 0–10% NaCl as per the standard BCG assay. The salt stability of γ-CGTase was additionally assessed by performing steady state kinetics in a Perkin-Elmer Fluorescence Spectrophotometer (Model L55). 20 µg of the purified γ-CGTase (prepared in 50 mM phosphate buffer pH 7) was incubated in the presence of different (0–10%) NaCl concentrations and incubated overnight (18–20 h) at room temperature (25–28 ºC). The enzyme was excited at 280 nm in a 0.2 cm path length cell and the emission spectrum was recorded from 300 to 500 nm at 25 ºC at each salt concentration.

Results and discussion

Confirmation of γ-CGTase activity and Product Identification

The production of γ-CGTase was carried out in M1 medium (nutrient broth containing 6% salt and 1% starch; pH 9.0) at 37 ºC, 200 rpm for 48 h. γ-CGTase activity in the cell-free culture supernatant was initially detected qualitatively by gel diffusion assay. A clear visible zone of hydrolysis was obtained on congo red- xylene cyanol starch- agar plates (Fig. 1A) within 12 h of incubation, while a very faint zone was obtained on phenolphthalein starch agar plates (Fig. 1B), even after prolonged intervals of up to 72 h. Congo red- xylene cyanol specifically identifies γ-CDs (Hamaker and Tao 1993) whereas phenolphthalein is utilized in specifically detecting β-CDs (Mennoci et al. 2008). A distinct hydrolysis zone on congo red- xylene cyanol plates ascertained extracellular production of γ-CGTase by E. caseinilytica which converts starch preferably to γ-cyclodextrin (γ-CD), with slight conversion to β-cyclodextrin (β-CD) as well.

Fig. 1.

Qualitative plate assay for CGTase activity; A congo red- xylene cyanol starch-agar plate for γ-CGTase activity; B phenolphthalein starch-agar plate for β-CGTase activity. Buffer (50 mM Tris–Cl pH 9.0) was considered as control and 100 µl of each sample was loaded in the wells

Further, specific spectrophotometric assays were performed to determine both β and γ-CGTase activity in the cell-free culture supernatant. These assays are based on the formation of inclusion complex of γ and β-CDs with their respective dyes bromocresol green and phenolphthalein (Higuti et al. 2004; Takada et al. 2003). A total of 43.52 ± 4.39 U/L γ-CGTase activity was determined in the cell-free culture supernatant while a very low β-CGTase activity was observed; color development in this reaction was detected only upon overnight incubation of the reaction mixture. The γ-CGTase units were also correlated with dextrinizing units, and 1 cyclizing or γ-CGTase unit was estimated to equate 70 dextrinizing units.

The results indicated that the CGTase present in the extracellular broth of E. caseinilytica is possibly a γ-CGTase, which largely synthesizes γ-CDs using starch. This is the very first report of γ-CGTase from E. caseinilytica, which specifically produces γ-CDs along with negligible amounts of β-CDs. There are very few previous reports of a CGTase exhibiting relatively high product specificity for γ-CDs. Such CGTases include: γ-CGTase from E. clarkii (Takada et al. 2003) which has been reported to produce a mixture of all three CDs constituted majorly by γ-CD (79% of the total CDs); γ-CGTase from Bacillus sp. G-825–6 (Hirano et al. 2006; Sonnendecker al. 2019) reported to produce only β-, and γ-CD with no α-CD formation; and γ-CGTase from B. thuringiensis GU-2 (Goo et al. 2014) which has been reported to produce > 95% γ-CD of the total CD yield. However, owing to its large industrial potential, γ-CGTase production in E. caseinilytica was further optimized employing different strategies described below.

Medium optimization for γ-CGTase production

Production optimization of extracellular γ-CGTase was done by One Variable at a Time (OVAT) approach and statistical optimization including Plackett–Burman (PB) and Response Surface Methodology (RSM).

Optimization of starch concentration and inducer selection

The presence of starch at a concentration of 0.1% in the medium favored highest γ-CGTase production (84.52 ± 2.27 U/L) while it decreased by almost 50% as the starch concentration was increased to 1% (Fig. 2A). Also, the enzyme production was very low (24.08 ± 1.36 U/L) in the absence of starch indicating its role as an inducer, and is in confirmation with various earlier reports available in the literature (Takada et al. 2003; Kitcha et al. 2008). Lower yields of enzyme in the presence of high starch concentrations may perhaps be a result of catabolite repression as suggested earlier by Yap et al. (2010) where 0.1% sago starch favored maximum production while higher starch concentrations were inhibitory for CGTase production by Bacillus lehensis S8. Hence, the concentration of starch was fixed to 0.1% in the production medium, and the resulting medium was termed as the M2 medium.

Fig. 2.

Production profile of γ-CGTase as a function of A starch concentration in M1 medium and B alternate inducers in M2 medium

The effect of various alternate inducers viz. glucose, maltose, type IV dextrin and β-CD on the production of γ-CGTase was studied in comparison to starch in the M2 medium (Pocsi et al. 1998; Gawande et al. 2003; Abd-Aziz et al. 2007; Ibrahim et al. 2013). It was observed that starch supported maximum γ-CGTase production (85.83 ± 2.97 U/L) followed by dextrin (69.36 ± 3.55 U/L), while maltose exhibited no significant effect on γ-CGTase production (Fig. 2B). This is in consistence with earlier reports where starch and dextrin have been reported to induce CGTase production (Freitas et al. 2004; Higuti et al. 2004; Abd-Aziz et al. 2007; Mora et al. 2012). Therefore, based on the highest activity obtained, 0.1% starch was retained as an inducer and M2 medium was taken further.

Physiological parameters for maximum enzyme production

The effect of temperature, pH and NaCl concentration on γ-CGTase production from E. caseinilytica was investigated in the M2 medium within a temperature range of 28–60 ºC, pH range of 8–11, and at NaCl concentrations of 0–10%. It was observed that the enzyme production and growth exhibited similar pattern (Fig. 3). The bacterium could grow well within a temperature range of 28-45 ºC, co-relating with higher enzyme production, while the lowest enzyme titres were obtained at 60 ºC (10.38 ± 2.19 U/L) at which the bacterium was observed to grow as suspended pellets. The highest γ-CGTase production was obtained at 37 ºC and 45 ºC with respective values of 84.52 ± 2.27 U/L and 81.70 ± 2.57 U/L, while a relatively lower production of 64.17 ± 2.61 U/L was observed at 28 ºC (Fig. 3A). The organism could grow upto pH 11.0, with optimal growth at pH 9.0, at which the maximum γ-CGTase production (84.52 ± 2.27 U/L) was also obtained (Fig. 3B). Similar γ-CGTase production was obtained at pH 10.0 and 11.0 with respective values of 53.49 ± 0.91 U/L and 48.86 ± 1.22 U/L. The highest γ-CGTase production was obtained at pH 8.0 with titres of 68.70 ± 1.49 U/L. Further, maximum γ-CGTase production was observed in the medium supplemented with 6% salt (84.52 ± 2.27 U/L) followed by 3% salt (77.67 ± 3.56 U/L) (Fig. 3C), which again corresponded to optimal growth conditions. Overall, the maximal γ-CGTase production was achieved at 37 ºC, pH 9.0 and in presence of 6% NaCl.

Fig. 3.

Effect of various physiological parameters A temperature, B pH and C NaCl concentration on growth and γ-CGTase production in M2 medium

The results are in agreement with previous reports where optimal γ-CGTase production was obtained at alkaline pH and mesophilic temperature. Maximum CGTase production from Bacillus alkalophilic CGII was reported in presence of 1% Na₂CO₃ (~ pH 10.2) at 37 ºC (Frietas et al. 2004). Similarly, CGTase production in Bacillus clausii E16 was reported to be highest at pH 10.1 and 37 ºC (Alves-Prado et al. 2007). Further, 37 ºC and pH 10.15 were reported optimal for the CGTase production from Bacillus sp. MK6 (Ai-Noi et al. 2008) whereas CGTase from Bacillus sp. ND1 was maximally produced at pH 8.9 and 30.5 ºC (Upadhyay et al. 2019). Likewise, maximum CGTase production from Bacillus circulans ATCC 21783 was reported at pH 9.7 and 36 ºC (Pinto et al. 2007). Further, Mennoci et al. (2008) isolated three strains of Bacillus sp. wherein maximum CGTase production was achieved at 40 ºC. Amongst all strains, CGTase from BACRP and BACAR showed maximum specific activity at pH 7 and pH 10 respectively, in presence of 2% salt. Whereas, the CGTase from strain BACNC-1 exhibited highest specific activity at pH 10 in absence of salt. Furthermore, γ-CGTase production from B. thuringiensis GU-2 was also reported to be highest at 37 ºC and pH 8.5 (Goo et al. 2014).

Plackett–Burman (PB) design

The effect of nine variables namely yeast extract, starch, peptone/tryptone, beef extract, triton X-100, MgSO₄, K₂HPO₄, CaCl₂, and soya peptone was investigated on γ-CGTase production from E. caseinilytica (Table 1). Variables were selected on the basis of the composition of M2 medium and other media reported in literature for CGTase production (Mahat et al. 2004; Zain et al. 2007; Rajput et al. 2016). Amongst all, six variables viz. starch, MgSO₄, yeast extract, tryptone, beef extract and soya peptone were observed to be positive signal factors, whereas K₂HPO₄, CaCl₂ and triton X-100 were found to be negative signal factors (Fig. S1), as determined by calculating the E-value of each factor (Table 2). Contrastingly, starch and K₂HPO₄ were the only positive and negative signal factors identified by the p-value. Based on the results, a new M3 medium was designed wherein starch was fixed at 0.1%, and among the two additive nitrogen sources, soya peptone was selected at a fixed value of 0.2%, while beef extract was removed. Moreover, the negative signal factors like triton X-100, K₂HPO₄ and CaCl₂ were omitted, and the final M3 medium contained tryptone (0.3%; w/v), MgSO₄ (5.0 mM), yeast extract (0.5%; w/v), soya peptone (0.2%; w/v), starch (0.1%; w/v) and NaCl (6%; w/v).

Table 1.

Plackett–Burman design showing γ-CGTase activity obtained in various experimental runs

Run	Yeast extract (%, w/v)	Starch (%, w/v)	Tryptone/Peptone* (%, w/v)	Beef extract (%, w/v)	Triton X-100** (%, v/v)	MgSO4 (mM)	K2HPO4 (%, w/v)	CaCl2 (mM)	Soya Peptone (%, w/v)	γ-CGTase (U/L)
1	0.10	0.02	0.5P	0.10	0.05	5.00	0.00	5.00	0.20	44.7 ± 9.3
2	0.50	0.02	0.5 T	0.00	0.00	0.00	0.20	5.00	0.20	27.7 ± 6.4
3	0.50	0.10	0.5 T	0.00	0.05	5.00	0.00	5.00	0.05	133.1 ± 9.6
4	0.10	0.10	0.5 T	0.10	0.00	5.00	0.20	0.00	0.20	137.5 ± 6.4
5	0.10	0.02	0.5 T	0.10	0.05	0.00	0.20	5.00	0.05	13.4 ± 0.8
6	0.50	0.02	0.5 T	0.10	0.00	5.00	0.00	0.00	0.05	54.0 ± 7.7
7	0.10	0.10	0.5P	0.00	0.00	5.00	0.20	5.00	0.05	63.0 ± 4.9
8	0.10	0.10	0.5 T	0.00	0.05	0.00	0.00	0.00	0.20	77.7 ± 4.4
9	0.10	0.02	0.5P	0.00	0.00	0.00	0.00	0.00	0.05	64.1 ± 10.2
10	0.50	0.10	0.5P	0.10	0.00	0.00	0.00	5.00	0.20	106.0 ± 9.3
11	0.50	0.02	0.5P	0.00	0.05	5.00	0.20	0.00	0.20	37.5 ± 3.0
12	0.50	0.10	0.5P	0.10	0.05	0.00	0.20	0.00	0.05	93.0 ± 2.6

γ-CGTase units were then correlated with dextrinizing units (1 cyclizing Unit/ CGTase Unit = 70 Units of dextrinizing activity)

*Denotes α-numeric variable where T stands for tryptone and P stands for peptone

**Triton X-100 was added in the respective medium 24 h post-inoculation

Table 2.

Analysis of variables using Plackett–Burman design

Run	Variable	Range coding level		E-value*	p-value**	Remarks
		Maximum	Minimum
1	Yeast extract (%, w/v)	0.50	0.10	8.49	0.2846	Positive
2	Starch (%, w/v)	0.10	0.02	58.17	0.0023	Positive
3	Tryptone/ Peptone (0.5%, w/v)	Tryptone	Peptone	5.86	0.4136	Positive
4	Beef extract (%, w/v)	0.10	0.00	7.60	0.3290	Positive
5	Triton X-100 (%, v/v)	0.05	0.00	− 8.82	0.1926	Negative
6	Soya peptone (%, w/v)	0.20	0.05	1.74	0.4744	Positive
7	MgSO4 (mM, w/v)	5.00	0.00	14.66	0.3056	Positive
8	K2HPO4 (%, v/v)	0.20	0.00	− 17.91	0.0164	Negative
9	CaCl2 (mM, w/v)	5.00	0.00	− 12.63	0.2971	Negative

E-value* is an average of difference between the sum of all negative and positive responses for each variable p-value** is calculated by unpaired student t-test which denotes the effect of variable on the response and p < 0.05 was considered significant

As shown earlier in the study, starch is required to induce γ-CGTase production and was thus obtained as a positive signal. This is in corroboration with CGTases from several Bacillus sp. where CGTase production has been reported to increase in presence of various types of starch (Zain et al. 2007; Blanco et al. 2012; Rajput et al. 2016).

Among the nitrogen sources, yeast extract and soya peptone were studied as numeric variables, whereas tryptone and peptone were studied as alpha numeric variables. Among all, yeast extract was obtained as the highly positive signal which is in agreement with earlier reports where it has been well recognized as a major nitrogen source for supporting both, cellular growth and CGTase production (Zain et al. 2007; Rajput et al. 2016). Requirement of peptone/ tryptone along with yeast extract has also been reported for optimal CGTase production from Bacillus oshimensis (Kamble and Gupte 2014) and Amphibacillus sp. NPST-10 (Ibrahim et al. 2013). And as examined in the present study, tryptone was found to be favorable for γ-CGTase production among the two (Fig. S1). Further, beef extract and soya peptone were studied at low concentrations as additional nitrogen sources, and both were observed to be the positive signal factors for γ-CGTase production. However, soya peptone was further retained as an additive nitrogen source and beef extract was excluded due to ethical reasons. Therefore, a final combination of three nitrogen sources, yeast extract, tryptone and soya peptone was selected for RSM studies. There are several reports in literature where a combination of nitrogen sources has been reported to support higher CGTase production, due to variations in the overall amino acid composition of different nitrogen sources (Abd-Aziz et al. 2007; Higuti et al. 2004; Rauf et al. 2008).

Several reports have also shown enhanced CGTase production in the presence of divalent cations, especially magnesium ion (Rosso et al. 2002; Rajput et al. 2016). In line with these reports, among the two divalent cations studied, magnesium was found to be a positive signal factor for γ-CGTase production, while calcium posed a negative impact. This is in contrast with earlier report on bacterial CGTase production, wherein calcium supplemented in the production medium was shown to enhance the stability of CGTase (Rajput et al. 2016).

Phosphates acts as buffering agents and as a source of phosphorus, therefore they are added to the media to improve enzyme production (Rajput et al. 2016). But, in the present study, phosphate was found to be a negative signal and was thereby omitted from the media. Further, triton X-100 was also added to the media in order to enhance the extracellular secretion of the enzyme. It was supplemented in the stationary phase (24 h) at which the CGTase production has been reported to be maximum (Zain et al. 2007). However, its addition was insignificant, and it was observed to be a negative signal factor.

Therefore, six factors favoring enzyme production were ranked in the order as starch > MgSO₄ > yeast extract > beef extract > tryptone > soya peptone according to their E-values. Out of these, MgSO₄, yeast extract and tryptone were selected for further optimization (in RSM). While soya peptone and starch were fixed at maximum level, as they were observed to be essential for the γ-CGTase production. Even though starch exhibited a highly positive effect, it was not taken further for optimization in RSM as increasing its concentration resulted in lower γ-CGTase activity during the OVAT study.

Response Surface Methodology (RSM)

The interactions between the signal factors in M3 medium selected from the PB design were evaluated by Response Surface Methodology (RSM). Here, fixed values of 0.1% soluble starch and 0.2% soya peptone was used, whereas yeast extract, MgSO₄ and tryptone concentrations were varied over three levels (Table S1) using Box-Behnken design. A set of 17 experiments with five center points [Tryptone (0.3%; w/v), MgSO₄ (5.0 mM) and yeast extract (0.5%; w/v)] were designed and the results (Table 3) were studied by ANOVA analysis using a reduced quadratic model.

Table 3.

Results of RSM carried out using Box-Behnken design with three independent variables and five center points

Run No.	A/ Tryptone (%, w/v)	B/ MgSO4 (mM)	C/ Yeast Extract (%, w/v)	Predicted CGTase activity (U/L)	Observed CGTase activity (U/L)
1	0	0	0	182.08	170.2 ± 3.1
2	+ 1	− 1	0	169.71	171.9 ± 1.9
3	− 1	− 1	0	156.86	154.1 ± 1.2
4	0	− 1	+ 1	173.57	175.5 ± 2.1
5	0	+ 1	− 1	207.45	207.9 ± 2.3
6	0	− 1	− 1	161.21	166.0 ± 7.8
7	− 1	+ 1	0	221.99	222.8 ± 6.4
8	− 1	0	− 1	207.84	207.7 ± 4.3
9	0	0	0	182.08	192.5 ± 8.4
10	0	0	0	182.08	186.6 ± 8.2
11	− 1	0	+ 1	187.88	189.9 ± 8.6
12	+ 1	+ 1	0	197.07	202.9 ± 5.8
13	0	0	0	182.08	189.2 ± 5.5
14	0	+ 1	+ 1	219.81	218.8 ± 9.0
15	+ 1	0	− 1	169.49	164.4 ± 6.7
16	+ 1	0	+ 1	214.17	211.2 ± 7.3
17	0	0	0	182.08	165.7 ± 5.7

Upon regression analysis of the experimental data, the following quadratic equation was obtained for the γ-CGTase production (Y):

$$Y=182.08-3.02\ast A+23.12\ast B+6.18\ast C+4.33\ast A^2+8.44\ast C^2-9.44\ast A\ast B+16.16\ast A\ast C$$

where A, B and C represent tryptone, MgSO₄ and yeast extract respectively.

The reduced quadratic model indicated that B and AC were significant for the γ-CGTase production. The quadratic term AC of the model was significant for the responses, indicating that the γ-CGTase production was majorly dependent on the interplay of tryptone and yeast extract. The model coefficients determined by multiple linear regression and analysis of variance (ANOVA) are presented in the Table S2.

The F value and R² (efficient of correlation) value of 11.85 and 0.9021 respectively for γ-CGTase production indicated that the model was significant. This ensured satisfactory adjustment of the quadratic model to the experimental data and indicated that approximately 90.21% of variability in the responses (γ-CGTase production) could be explained by the model. An adjusted R² value of 0.8259 was also in agreement with the predicted R² value. In addition, the model had an adequate precision value of 10.761 for γ-CGTase production, suggesting that the model can be used to navigate the design space. The relative effect of tryptone and yeast extract on γ-CGTase production at fixed MgSO₄ concentrations (– 1, 0, + 1), as determined by three-dimensional response surface plots, suggests the need of higher concentration of MgSO₄ at minimum concentrations of tryptone and yeast extract to achieve higher γ-CGTase production (Fig. 4). This indicates that the γ-CGTase production is directly proportional to the concentration of MgSO₄, while lower concentrations of both tryptone and yeast extract favored maximum γ-CGTase production.

Fig. 4.

3D curves for maximum γ-CGTase production showing effect of interaction between tryptone and yeast extract at different MgSO₄ concentrations A 2 mM, B 5 mM and C 8 mM

Various authors have also reported that the CGTase production is maximum at low concentrations of nitrogen sources due to its repression in presence of excessive nitrogen source including CGTases from Bacillus lehensis (Blanco et al. 2012), Bacillus sp. TS1 (Zain et al. 2007), Bacillus sp. SM-02 (Coelho et al. 2016). Further, γ-CGTase production was found to significantly enhance with increasing MgSO₄ concentrations which is in corroboration with a report by Rosso et al. (2002) for CGTase production from Bacillus circulans DF 9R. For determining the model accuracy, the predictions drawn from the model were validated successfully with five random experiments and the experimentally determined production values were in agreement with the values predicted by the model and the data has been presented in Table S3.

Hence, based on the response surface curves and validation experiments, tryptone and yeast extract were fixed at their minimum concentrations, and maximum MgSO₄ concentration was chosen for optimal γ-CGTase production by E. caseinilytica SP^T. This resulted into a new M4 medium constituted by tryptone (0.1%; w/v), yeast extract (0.25%; w/v), MgSO₄ (8 mM; w/v), potato starch (0.1%; w/v) and soya peptone (0.2%; w/v) at pH 9.0 (set with 1 N NaOH). A total γ-CGTase production of 240 ± 5.46 U/L was achieved in the M4 medium resulting into an enhancement of 2.8-fold over the OVAT study, and 5.6-fold over the initial M1 medium (Table 4). The fold enhancement is in agreement with some previous reports on CGTase production wherein nearly fivefold enhancement over the basal medium was observed (Ai-Noi et al. 2008) following medium optimization.

Table 4.

Summary of strategies adopted for maximum γ-CGTase production from E. caseinilytica

Production strategy	Production time (h)	CGTase (U/L)	Protein (mg/mL)	Fold enhancement
Unoptimized medium (M1 medium)	48	43.5 ± 4.4	0.05 ± 0.005	1
One-variable-at-a-time (OVAT)/ M2 medium	48	84.5 ± 2.27	0.07 ± 0.004	1.94
Response surface methodology/ M4 medium	24	240.5 ± 5.46	0.27 ± 0.07	5.53

After medium optimization, the effect of various types of starch from different sources on γ-CGTase production was evaluated, as they have been reported to result in variable CGTase production levels (Rosso et al. 2002; Kulshreshtha et al. 2020). It was observed that E. caseinilytica SP^T was able to significantly produce γ-CGTase using all types of starch (soluble and insoluble), suggesting that any cheaper source of starch could be utilized in the production of γ-CGTase. Maximum enzyme production was however achieved in the presence of potato starch (240.15 ± 0.63 U/L) and was followed by rice starch (226.15 ± 1.72 U/L) (Fig. S2). In similar studies from B. macerans and B. licheniformis, CGTase production was reported to be maximum in the presence of soluble potato starch (Dalmotra et al. 2016; Bonilha et al. 2006).

Time kinetics for growth and production of enzyme in optimized medium (M4)

The time kinetics for production of extracellular γ-CGTase from E. caseinilytica was conducted in the final optimized M4 media (Fig. 5). The production of γ-CGTase instigated in the log phase (6 h), following which, the titers increased vividly at the early stationary phase (14 h). In the late stationary phase, the enzyme titers gradually increased upto 24 h and were thereafter maintained at the same level upto 120 h. This is in contrast to CGTase production profile from Bacillus sp. ND1 where enzyme activity dropped as the bacterial growth reached the stationary phase (Upadhyay et al. 2019). And, the enzyme production profile observed in the present study is similar to previous report of CGTase production from Bacillus sp. MK6 (Ai-Noi et al. 2008). Further, bacterial growth profile followed similar pattern and both, maximum growth and γ-CGTase production, were obtained at 24 h. In a similar report from B. clausii E16, the maximum CGTase production was achieved after 24 h in an optimized medium (Alves-Prado et al. 2007). Therefore, in the subsequent studies, production time for γ-CGTase was reduced to 24 h in the optimized M4 medium, after which the culture supernatant was harvested.

Fig. 5.

Time kinetics of γ-CGTase production alongside growth profile of E. caseinilytica

Purification of γ-CGTase by starch-affinity method

The crude culture supernatant obtained after 24 h of production was initially concentrated by ultrafiltration using a 5 kDa molecular weight cut-off membrane. A specific activity of 5.23 U/mg was obtained in the crude enzyme broth after medium optimization, and was nearly 30 fold higher than the specific activity reported for the similar enzyme from E. clarkii (Takada et al. 2003). Following ultrafiltration, maximum enzyme was obtained in the retentate with 57.32% recovery (Table 5). The retentate was then subjected to one-step purification employing the starch affinity method (Kitayska et al. 2011). The protein was eluted using 1 mM β-CD in 50 mM potassium phosphate buffer (pH 7.0), resulting in a purification fold of 6.75 and 19.32% overall yield (Table 5). In several earlier reports, starch affinity method has been utilized in the purification of CGTases from Bacillus sp. (Takada et al. 2003; Kitayska et al. 2011; Rajput et al. 2016; Sonnendecker et al. 2017).

Table 5.

Purification scheme for extracellular γ-CGTase from E. caseinilytica

Fraction	Total γ-CGTase (U)	Total protein (mg)	Specific activity (U/mg)	Fold purification	% Yield
Culture broth (crude)	348.75	66.65	5.23	1	100
Retentate	199.90	22.20	9.00	1.72	57.32
Starch affinity	67.40	2.00	33.70	6.75	19.32

Complete procedure was repeated thrice (S_d of specific activities are ± 0.03, 0.10 and 0.20 at each respective step) and their mean values are presented. All values are rounded off to significant digits

The homogenous purification of the enzyme was confirmed by SDS-PAGE analysis, where a single band of approximately 68 kDa (Fig. S3) was observed. Further, the purified enzyme was subjected to gel filtration chromatography and it eluted as a single peak at retention volume of 75 ml, corresponding to a molecular weight of approximately 65 kDa (Fig. S4), thus confirming the monomeric nature of γ-CGTase. The microbial CGTases have been reported to be monomeric in nature with their molecular weight ranging from 64 to 80 kDa (Li et al. 2007). Gel filtration was followed by Native PAGE and zymogram analysis of the eluted protein, wherein an active band of approximately 55 kDa corresponding to γ-CGTase was observed (Fig. S5). The difference observed in the molecular weight of the enzyme under non-reducing conditions (native gel) could be due to the shape of the protein which might affect its mobility. This is in agreement with previous reports on bacterial enzymes showing variable mobility and molecular weights on native and SDS-PAGE (L’Hocine et al. 2000; Yang et al. 2011).

γ-CD production using γ-CGTase from E. caseinilytica SP^T

The γ-CD produced using the purified enzyme was initially visualized on thin layer chromatography where spots correlating to γ-CDs were observed (Fig. 6). For further HPLC analysis, the commercially available α, β and γ-CDs were used as standards. Their respective retention peaks were observed at different times of 5.35, 5.94 and 7.08 min on a sugar column (Cosmosil; 4.6 mm I.D. X 250 mm) using RID detector (Fig. 7A, 7B, and 7C). The reaction mixture when analyzed on HPLC showed a product peak at approximately 7 min corresponding to γ-CD (Fig. 7D and 7E). Upon quantification of the product peak, formation of 3.1 mg/ml γ-CD was determined. This corresponded to 6.2% conversion of soluble potato starch to γ-CD in 2 h at pH 11.0 and 60 ºC, in a 20 ml reaction. The enzymatic conversion of starch to γ-CDs further increased when the reaction was prolonged till 24 h, where 203.8 mg γ-CD was obtained and it corresponded to 20.38% starch conversion. The starch conversion obtained in the present study agrees with the earlier reported conversion yield of 20% by Takada et al. (2003). They utilized γ-CGTase from E. clarkii in a reaction containing 1% soluble potato starch (48 h), but the yields achieved in the present study are much higher. The γ-CD yields (10.1 mg/ml) are comparable to earlier report of γ-CD production using CGTase from Bacillus sp. G-825–6 (γ-CGTase 825–6), where around 7 mg/ml of γ-CD production was reported using 78 mU/g soluble starch (Hirano et al. 2006).

Fig. 6.

Product analysis of enzymatic conversion of starch to CD by TLC Synthesis of γ-CD was analyzed on TLC using a mobile phase (Butanol: Ethanol: Water: Ammonia) in the ratio of 5:3:4:2. TLC was developed using 50% (v/v) sulfuric acid in methanol. 5 µl from 5 mg/ml stock of each standard α, β and γ-CD; and 20 µl of each reaction sample viz. enzyme control (EC), substrate control (SC) and test (T) were spotted on TLC plate

Fig. 7.

HPLC chromatograms of commercial A α, B β and C γ-CD, and reaction products after D 2 h and E 24 h of enzymatic conversion, where a peak corresponding to γ-CD was obtained 10 µl of standard and reaction products were loaded on the column

Previous reports in the literature have shown enhancement in the cyclodextrin yields upon optimization of the reaction conditions, or by addition of solvents as complexing agents, and by site-directed mutagenesis. Hirano et al. (2006) optimized the pH and time for attaining maximum CD yields, and obtained around 7 mg/ml γ-CD at pH 10 using 78 mU/g soluble starch. Alves-Prado et al. (2007) screened different starch sources and achieved maximum conversion of around 80% (mixture of α, β and γ-CD) using 1% soluble starch. Further, Wu et al. (2012) optimized various enzymatic conditions and reported maximum conversion of 30% at pH 12, 60 ºC using 5 U/g of soluble starch in a non-solvent process. The conversion was further increased to 57% in a solvent-process containing 2% glycyrrhizic acid. In a similar study, Wang et al. (2013) reported 50.4% CD yield by employing γ-CGTase from E. clarkii in the presence of cyclododecanone and 15% potato starch, following stepwise optimization of various reaction parameters. The CD yields were further enhanced to 72.6% when an enzyme variant Y186W was employed for γ-CD production under optimized conditions in presence of cyclododecanone (Wang et al. 2020). Further, Nakagawa et al. (2006) performed site- directed mutations at the residues Ala223 and Gly255 in the γ-CGTase from E. clarkii. They reported enhancement in the γ-CGTase activity upon substitution of Ala223 with basic amino acids (lysine, arginine and histidine), while replacement of Gly255 with any of these residues resulted in a significant reduction.

The cyclodextrin yields are often limited because of the end product inhibition and coupling activity of enzyme which results in breakdown of CDs into linear oligosaccharides (Biwer et al. 2002; Li et al. 2007; Leemhuis et al. 2010). The lower CD yields obtained in the current study could have resulted due to the coupling activity of γ-CGTase which was observed upon its incubation with 1% γ-CD for 24 h. Here, almost 50% γ-CD hydrolysis was observed, which perhaps is the reason for obtaining lower product yields (Fig. S6). In a similar study, the γ-CGTase from Bacillus sp. G-825–6 was reported to exhibit end product degradation (Hirano et al. 2006). This is in contrast to γ-CGTase from E. clarkii which has been reported to exhibit no such coupling activity, however, the enzyme gets inhibited in presence of γ-CDs which accumulate during the reaction (Takada et al. 2003).

Identification of γ-CGTase protein sequence in proteome of E. caseinilytica

The peptide fragments obtained after LC–MS analysis (Fig. S7) of the purified enzyme were subjected to protein BLAST analysis against the proteome available for E. caseinilytica at NCBI. The peptide sequences showed maximum identity to a protein annotated as “IPT/TIG domain containing protein” (WP_090889670.1) from E. caseinilytica (Table S4).

Further, blastp analysis revealed that the protein sequence shared close homology to cyclodextrin glycosyltransferase protein from E. clarkii with maximum identity of 99.86% in a 100% query coverage. While, other proteins among the obtained results shared 50–70% homology (Table S5). The protein sequence was further subjected to NCBI conserved domain analysis and respective domains were marked (Fig. S8) according to the previous reports from E. clarkii (Takada et al. 2003; Akita et al. 2004). Among five domains (A-E), the domains A, B and C are characteristic to all GH-13 family of enzymes, and additional two domains (D and E) are specific to CGTases (Han et al. 2014; Sonnendecker et al. 2019; Lim et al. 2021).

It is noteworthy that the primary sequence of γ-CGTase from an alkalohalophile E. caseinilytica shared 100% homology to a non-halophilic bacterium E. clarkii. Furthermore, the enzyme production conditions from the present strain were halophilic; the enzyme was produced in presence of 6% NaCl while the γ-CGTase from E. clarkii has been reported to be produced in absence of salt (Takada et al. 2003). The presence of salt has been reported to result in distinct conformational changes in the protein (Wohl et al. 2021). And in accordance with this, salinity of the production medium might have resulted in an altered conformation of the γ-CGTase reported in the current study. Hence to elucidate this, biochemical and biophysical attributes of the present γ-CGTase in terms of pH, temperature and salt stress were studied and compared with that of already reported γ-CGTase from E. clarkii (Takada et al. 2003).

Effect of pH, temperature, and salt on enzyme activity and stability

The γ-CGTase from E. caseinilytica exhibited maximum relative activity at pH 11.0, and less than 40% relative activity was observed below pH 7.0 (Fig. S9A), indicating that the enzyme is optimally active in an alkaline environment. It was found to be stable in a pH range of 6–12 after incubation at room temperature for 1 h (Fig S9B). Further, γ-CGTase exhibited maxima at 60 ºC with more than 50% relative activity within the range of 40–70 ºC (Fig. S9C). No enzyme activity was observed at 30 ºC whereas less than 20% relative activity was observed at 80 ºC. The pH and temperature optima were found to be in agreement with an earlier study of γ-CGTase from E. clarkii, where maximum activity was also reported at pH 10.5–11.0 and 60 ºC (Takada et al. 2003). The temperature optima for majority of the previously reported CGTases from different Bacillus species was found to be within the range of 45–65 ºC (Hirano et al. 2006; Li et al. 2007; Pishtiyski et al. 2008; Elbaz et al. 2015; Rajput et al. 2016). In contrast to this, most of the CGTases reported in literature are optimally active at near neutral pH (Gawande and Patkar 2001; Pishtiyski et al. 2008; Arce-Vázquez et al. 2016; Kitayska et al. 2011; Elbaz et al. 2015; Rajput et al. 2016) and few at moderately alkaline pH, including CGTase from Brevibacterium sp. strain 9605 (Mori et al. 1994) and γ-CGTase from Bacillus sp. strain G-825–6 (Hirano et al. 2006). However, most of the CGTases including γ-CGTase from E. clarkii have been reported to be stable in neutral to alkaline pH range (Takada et al. 2003; Lim et al. 2021).

The present γ-CGTase was highly thermostable as it retained 100% native activity for more than 3 h at 60 ºC (Fig. 8A) and a t_1/2 of nearly 24 min at 70 ºC was determined (Fig. 8B). This contrasts with the similar protein reported from E. clarkii which was found to be thermolabile and it exhibited 50% loss in activity upon incubation at 55 ºC for 15 min (Takada et al. 2003). However, majority of CGTases from different Bacillus species are reported to be stable in the range of 40–70 ºC (Pishtiyski et al. 2008; Elbaz et al. 2015; Rajput et al. 2016). Thermostable γ-CGTases are advantageous as they can withstand the high temperatures of various industrial processes. Such higher temperatures facilitate solubility of the starch substrates by gelatinization, thereby reducing viscosity of the solution and lowering the risk of contamination, resulting in improved yields during the enzymatic conversions (Van der veen et al. 2000; Biwer et al. 2002).

Fig. 8.

Effect of temperature on γ-CGTase activity; A Thermal stability of γ-CGTase based on residual enzyme activity as a function of temperature; 100% activity corresponded to 35.29 ± 0.43 U/mg and B Thermal inactivation curve of γ-CGTase obtained at 70 ^◦C; slope of thermal inactivation curve was taken as the Kd value and t_1/2 was calculated as 0.693/Kd

The γ-CGTase from E. caseinilytica exhibited > 95% relative activity even in the presence of 10% NaCl (Fig. 9A). The steady-state fluorescence studies indicated that the tertiary structure of the protein remained unaltered when subjected to salt stress for prolonged intervals of time (Fig. 9B). This could be attributed to the saline environment wherein protein production was carried out. Further, the present γ-CGTase exhibited a higher t_1/2 of nearly 46.5 min and 239 min at 70 ºC in presence of 1 M and 2 M salt respectively, (Fig. S10) as compared to 24 min determined under the salt-free conditions. The enzymes derived from halophilic organisms are generally known to optimally function and tolerate high salt and high temperatures (Sinha and Khare 2014; Cira-Chávez et al. 2018), and in accordance with these reports, higher thermostability of the present γ-CGTase was observed in presence of salt. Similar effect of increase in thermostability in the presence of salt has not been reported for CGTases including γ-CGTase from E. clarkii. (Takada et al. 2003; Lim et al. 2021).

Fig. 9.

Effect of different salt concentrations (0–10%) on A γ-CGTase activity, relative activity at each concentration was plotted considering activity obtained in absence of salt as 100%; and B intrinsic fluorescence scans of γ-CGTase acquired in presence of various salt concentrations

Conclusion

This is the first report of γ-CGTase from an Indian isolate E. caseinilytica (Reddy et al. 2015) Gupta et al. 2020. Medium optimizations resulted in an overall 5.5-fold increase in the enzyme production corresponding to 240.5 ± 5.46 U/L. The purified enzyme cyclized soluble starch majorly to γ-CDs. Peptide fingerprints of the purified protein shared high homology with the well-established γ-CGTase from E. clarkii. γ-CGTase from E. caseinilytica was highly thermostable and retained its activity even in the presence of 10% salt. The presence of salt masked the effect of high temperature (70 ºC), wherein the t_1/2 increased from 24 to 239 min in presence of 2 M NaCl. Further, tertiary structure analysis showed no alterations even after prolonged salt stress. Thus, the present enzyme is robust and is ideal for the starch industry which generally requires thermostable enzymes due to the solubility and gelatinization of starch at high temperatures.

Abbreviations

%

Percentage

˚C

Degree Celsius

H

Hours

Min

Minutes

U

Units

µl; ml

Microlitres; Millilitres

L

Litres

Rpm

Revolutions per minute

w/v

Weight by volume

v/v

Volume by volume

nm

Nanometer

M

Molar concentration

µm

Micron

kDa

Kilodaltons

mg

Milligrams

NaCl

Sodium Chloride

NaOH

Sodium Hydroxide

Na₂CO₃

Sodium Carbonate

BSA

Bovine Serum Albumin

SDS

Sodium Dodecyl Sulphate

MgSO₄

Magnesium Sulphate

CaCl₂

Calcium Chloride

K₂HPO₄

Dipotassium hydrogen phosphate

PAGE

Polyacrylamide Gel Electrophoresis

RID

Refractive Index Detector

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.