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Production, purification and physicochemical characterization of D-xylose/glucose isomerase from Escherichia coli strain BL21.

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  • 1. 1Institute of Industrial Biotechnology (IIB), GC University Kachery Road, Lahore, 54000 Punjab Pakistan.
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    • Fatima B 1
    • Javed MM 1
    (2 authors)

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3 Biotech, 09 Jan 2020, 10(2):39
https://doi.org/10.1007/s13205-019-2036-6 PMID: 31988833 PMCID: PMC6949339

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Abstract 

Cell lysate of Escherichia coli strain BL21 showed significant D-glucose isomerase activity. The rate of glucose conversion was increased up to 40% when cells were induced with 1% D-xylose. E. coli BL21 xylose isomerase (ECXI-BL21) was purified to homogeneity, up to 1.9-fold with overall 10.88% enzyme yield by heat shock, salting out and electro-elution. The molecular mass of ECXI-BL21 was estimated as 43.9 kDa on SDS-PAGE. pHopt. and Topt. of the enzyme were calculated as 7.0 and 50 °C, respectively. Activation energy (E a) of ECXI-BL21 was 45 kJ/mol. Enzyme was stable from 30 to 55 °C and at pH range 6.0-8.0. ECXI-BL21(holo) was activated by 10 mM magnesium (35%), 0.5 mM cobalt (20%) and manganese (25%), and 0.5/10 mM Mn2+/Mg2+ (50%) and Co2+/Mg2+ (30%) as compared to ECXI-BL21(apo). Catalytic affinity (K m) of ECXI-BL21 for D-glucose was calculated as 0.82 mM, while maximum velocity (V max) of the reaction D-glucose(aldo) ⇌ D-fructose(keto) was 108 μmol/mg/min. D-fructose formed was identified on silica gel plate. This thermophilic enzyme, T m = 75 °C, has great potential for high fructose syrup production used in food and soft drink industries.

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3 Biotech. 2020 Feb; 10(2): 39.
Published online 2020 Jan 9. https://doi.org/10.1007/s13205-019-2036-6
PMCID: PMC6949339
PMID: 31988833

Production, purification and physicochemical characterization of D-xylose/glucose isomerase from Escherichia coli strain BL21

Bilqees Fatimacorresponding author1 and Muhammad Mohsin Javed1,2
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Abstract

Cell lysate of Escherichia coli strain BL21 showed significant D-glucose isomerase activity. The rate of glucose conversion was increased up to 40% when cells were induced with 1% D-xylose. E. coli BL21 xylose isomerase (ECXI-BL21) was purified to homogeneity, up to 1.9-fold with overall 10.88% enzyme yield by heat shock, salting out and electro-elution. The molecular mass of ECXI-BL21 was estimated as 43.9 kDa on SDS-PAGE. pHopt. and Topt. of the enzyme were calculated as 7.0 and 50 °C, respectively. Activation energy (Ea) of ECXI-BL21 was 45 kJ/mol. Enzyme was stable from 30 to 55 °C and at pH range 6.0–8.0. ECXI-BL21(holo) was activated by 10 mM magnesium (35%), 0.5 mM cobalt (20%) and manganese (25%), and 0.5/10 mM Mn2+/Mg2+ (50%) and Co2+/Mg2+ (30%) as compared to ECXI-BL21(apo). Catalytic affinity (Km) of ECXI-BL21 for D-glucose was calculated as 0.82 mM, while maximum velocity (Vmax) of the reaction D-glucose(aldo) [right harpoon over left harpoon] D-fructose(keto) was 108 μmol/mg/min. D-fructose formed was identified on silica gel plate. This thermophilic enzyme, Tm = 75 °C, has great potential for high fructose syrup production used in food and soft drink industries.

Keywords: Escherichia coli, Electro-elution, Kinetics, Thin-layer chromatography, D-glucose isomerase, Inducers

Introduction

Rapid depletion of fossil fuels diverted the attention of biotechnologist towards the development of efficient technology for bioconversion of renewable biomass into bio-fuel. Cellulose can be completely hydrolyzed to glucose which subsequently fermented to fuel ethanol. However, the utilization of all the components of hemicelluloses is a big challenge, as D-xylose which is one of the major sugars derived from biomass after glucose and comprised upon 40% of hemicelluloses, remained un-fermented, because the fermenting organisms, i.e., Saccharomyces cerevisiae can ferment hexoses very efficiently but not pentoses (Wang et al. 1980a). However, saprophytic bacteria flourished on rotting wood and decaying plant material used D-xylose(aldo) [right harpoon over left harpoon] D-xylulose(keto) activity as energy source (Bhosale et al. 1996). Hence, this activity catalyzed by D-xylose isomerase (XI) (D-xylose ketol isomerase; EC 5.3.1.5), can be utilized for the production of bio-ethanol from ligno-cellulosic biomass. D-xylulose, the isomerized product of D-xylose, has been fermented by conventional yeasts strains, i.e., S. cerevisiae, Schizosacchromyces pombe and Candida tropicalis, very efficiently (Wang et al. 1980a, b; Chiang et al. 1981a, b; Schneider et al. 1981; Gong et al. 1981; Chan et al. 1989). XI also showed aldo [right harpoon over left harpoon] keto conversion of D-glucose as well, hence also known as glucose isomerase (GI, EC 5.3.1.5), although the catalytic affinity of GI for hexoses is much lower than pentoses. D-glucose(aldo) [right harpoon over left harpoon] D-fructose(keto) activity is extensively utilized in industry for high-fructose corn syrup (HFCS) manufacturing from corn starch. This process is comprised upon three successive enzymatic steps: liquefaction by α-amylase, saccharification by amyloglucosidase and isomerization of D-glucose to D-fructose by GI. HFCS has many advantages over sucrose in food industry, because it does not caused crystallization problem, has 10–20% low price (10–20%) due to 1.3 times higher sweetness power. Therefore, annual world-wide production of HFCS has been exceeded to 13 million tons (Rozanov et al. 2009; Staudigl et al. 2014).

Xylose isomerases (XIs) are intracellular, bacterial enzymes, classified as class I and II, based upon number of amino acids, they possessed in their protein sequences (Vangrysperre et al. 1990). Class I enzymes are shorter (comprised upon ~ 390 amino acids) than class II (comprised upon ~ 440 amino acids) enzymes. Class II enzymes have additional 40–50 amino acidsʹ insert at their N-terminus and are more varied in their source organisms from mesophiles (− 5–20 °C), thermophiles (15–45 °C) to hyperthermophiles (≥ 80 °C) as compared to class I which are more commonly mesophilic enzymes (Sriprapundh et al. 2000, 2003; Vieille and Zeikus 2001; Epting et al. 2005). XIs are tetrameric metallo-proteins, the catalytic site in each monomer is comprised upon one active and two metal-binding sites designated as M1 and M2 (Fig. 1). XIs required divalent metal ions chosen from Mn2+, Co2+ and Mg2+ for their activity and stability (Collyer et al. 1990; Van Bastelaere et al. 1992; Bhosale et al. 1996) Xylose metabolism has been described from a wide array of microorganisms, most of which produced XIs only in the presence of D-xylose. Escherichia coli is a well-known Gram-negative bacterium, established in each laboratory. Hence, extensively used for homologous and heterologous protein production. E. coli is a fast-growing organism that utilized inexpensive growth medium and can metabolize both hexose and pentose sugars as energy source. Hence, this system has been established on large scale and used for pharmaceutical and industrial proteins´ production. A disadvantage of E. coli system is, accumulation of endo-toxins or lipopolysaccharide (LPS) as by product, which render unfit the protein produced for human consumption, because LPS are pyrogenic for human and other mammals. Therefore, the protein produced by this system must be purified subsequently for human use, to become endo-toxin free (Terpe 2006). This is the first report of production, purification and physicochemical characterization of xylose/glucose isomerase from E. coli BL21 (ECXI-BL21).

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Monomer of ECXI, showing relative active site and either of the metal-binding sites residues in activated enzyme–substrate complex. ~ 50 extra amino-acid residues (blue) at N-terminus (not commonly present in class I enzymes) indicating that ECXI is class II enzyme (predicted by PatchDock v.β.1.3 using sequence of ECXI retrieved from NCBI (GenBank accession number E24377A) created by UCSF Chimera v.1.10.2 molecular graphic program)

Materials and methods

Strain and chemicals

The cells of Escherichia coli BL21 (F –ompThsdSB (rB–mB–) gal dcm(DE3)) CodonPlus® ( Cat. #69387-3) Novagen® were stored in 20% glycerol at ultra-low temperature (− 80 °C). All chemicals used of analytical grade and purchased from BDH (UK), E-Merck (Germany), Acros (Belgium), Fluka (Switzerland) and Sigma (USA).

Production of ECXI-BL21

Seed cultures were prepared in 100 ml flasks containing 10 ml of low-salt Luria Bertani (LB) broth (Tryptone 10 g, yeast extract 5 g, NaCl 5 g per liter). The flasks were incubated at 37 ºC, pH 7.0, 150 rpm, overnight, after adding a single colony of E. coli cells which was previously grown on LB agar plates (1.5% bacteriological agar). Fifty milliliters of LB broth (pH 7.0) were sterilized at 121 °C, 15 lbs/in2 pressures for 15 min in 250 ml flasks and cooled to room temperature. Seed cultures were diluted to 2% in to each flask (250 ml) individually and agitated (200 rpm) at 30–45 ºC, pH 4–10 for 12–72 h. Meanwhile, when turbidity (O.D at 600 nm) was reached to 0.5–0.6, cells were induced with D-xylose, D-glucose, D-fructose, glycerol, maltose and lactose (filter (0.22 µm) sterilized separately) by adding 1% of each carbon source in each flask separately. A control (without inducer) was also run parallel.

Purification of ECXI-BL21

Cells were harvested by centrifugation at 6000 × g, 4 °C for 10 min. Pellets were re-suspended in Tris–HCl buffer (100 mM, pH 7.5) and cell lysates were prepared by passing 30 short burst of sound waves at 4 °C, having an amplitude of 75% for 20 s followed by intervals of 40 s, using Hielsher Ultrasound Technology. Cell debris was removed by centrifugation for 15 min, at 4 °C (13,000 rpm). Supernatant was lyophilized to about 50% reduction of the original volume. Then, it was heated for 15 min at 50 °C, 100 rpm and cooled on ice. The precipitated proteins were removed at 4 °C, by centrifugation for 15 min (12,000 × g) and the supernatant (5 µl) was resolved on SDS–PAGE (12% acrylamide, w/v) along with pre-stained protein marker (Laemmli 1970). In supernatant, ammonium sulphate crystals were dissolved at 0 °C with constant stirring and precipitated fractions were collected (13,000 rpm, 4 °C) at 30–90% salt saturation individually. Fraction samples were re-suspended in Tris–HCl buffer (100 mM, pH 7.5) separately, sealed in cellulosic dialysis membrane, having a molecular weight cut-off 12,000–14,000. The membrane was previously treated with 1% acetic acid and basic EDTA solution containing 10−3 M EDTA and 1% Na2CO3 (Boyer 1993). Salt was removed by three replacements of same buffer then dialyzed sample (5 µl) was resolved (12% SDS-PAGE). This gel was used as reference for cutting the band from non-denatured gel. Native PAGE was prepared without wells (Fig. 2, step 1) in accordance to (Driska 1998; Branco et al. 2008; Li et al. 2012) and rest of the whole dialyzed sample was resolved on 10% acrylamide (w/v). The gel was stained with coomassie brilliant blue R-250 (0.25%) and de-stained with methanol (30%) and glacial acetic acid (10%). Bands were cut out with sharp scalpels and gel fragment containing band of interest was bounded in dialysis tube (Fig. 2, step 2 and 3). The tube was assembled in horizontal electrophoretic apparatus filled with electrode-running buffer (pH 8.8) containing 250 mM glycine and 25 mM Tris–HCl. Electro-elution was performed at 100 V constant voltages until all the coomassie brilliant blue had been removed from the gel fragment (Fig. 2, step 4). The clear gel slice was removed from dialysis tube (Fig. 2, step 5) and the eluted protein was dialyzed at 4 °C against the three changes of Tris–HCl buffer (100 mM, pH 7.5) (Fig. 2, step 6). The purity of eluted protein was analyzed by SDS-PAGE (12% acrylamide, w/v). Protein contents of the samples at each step of purification were quantified against BSA (Bradford 1976).

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Purification process of ECXI-BL21. (1) 10% Native PAGE of ECXI-BL21 fraction obtained at 70% ammonium sulphate saturation, (2) Gel fragment containing ECXI-BL21, (3) ECXI slice sealed in dialysis membrane, (4) Electro-elution, (5) Gel slice without ECXI-BL21, (6) Dialysis, (7) SDS-PAGE

Enzyme assays

A reaction mixture of 30 µl enzyme and 20 µl test solution containing 100 mM D-glucose, 0.0003 M MnCl2 in Tris–HCl buffer (100 mM, pH 7.5) was incubated at 40 °C, for 10 min, using 96-well microplate. The plate was incubated on ice for 10 min, to stop the reaction. The amount of fructose produced was estimated in accordance to Schenk and Bisswanger (1998). One hundred and fifty micro-liters of 1:1 mixture (v/v, mixed freshly) of 0.05% resorcinol solution (in absolute ethanol) and 0.216 g/L ferric ammonium sulphate dodecahydrate solution in concentrated hydrochloric acid, were added. Plate was incubated at 80 °C for 45 min. The absorbance of red color developed was read by ELISA reader at 490 nm. One unit (U) of isomerase activity was “the amount of ECXI that produced 1 µmol fructose per ml, per min under specified conditions”. For kinetics constants (Km and Vmax), the concentration of D-glucose was varied (1–10 mM) in the reaction mixture and ECXI-BL21 activities were measured as mentioned above. Optimum pH was measured using different buffers (0.1 M), i.e., glycine–HCl (pH 2.0–3.0), sodium acetate (pH 4.0–5.0), sodium phosphate (pH 6.0–7.0), Tris–HCl (pH 8.0), glycine–NaOH (pH 9.0–10). The ΔpKa/ΔT was considered while calculating the results (Dawson et al. 1969). pH stability was estimated by incubating ECXI-BL21 at 50 °C, for 60 min under various pH (2–10) conditions individually then residual ECXI-BL21 activities were calculated at pH 7.0. Thermophilicity was calculated by performing the isomerase reactions at various temperatures (30–90 °C) separately. lnVmax at each temperature treatment was plotted as Arrhenius plot (linear between 30–50 °C) and activation energy (Ea) was calculated by applying the equation Ea = − slope × R where, R = gas constant (8.314 JK−1 mol−1). Thermostability studies were performed by incubating ECXI-BL21 in the absence of substrate at each temperature 30–90 °C individually for 60 min. Residual ECXI-BL21 activities were calculated at 50 °C. ECXI-BL21 was incubated overnight, in Tris–HCl buffer (100 mM, pH 7.5) containing 10 mM EDTA at 4 °C. It was then dialyzed twice against the same buffer containing 2 mM EDTA (at 4 °C) and finally dialyzed twice without EDTA (at 4 °C). The apo-ECXI-BL21 (metal free) thus obtained was equilibrated individually with 0.3, 0.5, 5, and 10 mM chloride salts of Mn2+, Co2+ and Mg2+ and in combination (0.5/10 mM) Mn2+/Mg2+ and Co2+/ Mg2+ at 30 °C, for 30 min. The percentage increase or decrease in activities of holo-ECXI-BL21 (metal equilibrated enzyme) was calculated at 50 °C, relative to apo-ECXI-BL21. The final product of glucose conversion achieved at optimized reaction conditions was identified by thin-layer chromatography (TLC). All assays were performed in parallel triplicates.

TLC analysis

Silica gel (Kiesel gel 60 F254; Merck) plate was developed by double-development technique using ascending method as described previously (Fatima et al. 2016). D-glucose and D-fructose each of 10 mg·ml−1 were spotted as standards. Air-dried developed, plate was sprayed with 5% conc. H2SO4 (v/v, in methanol) and incubated at 150 °C, for 10 min, in hot air oven. Resultant spots were identified by retention factor (Rf) and compared with standards (Walker et al. 1965).

Statistical analysis

Graph Pad Prism 6 software was used to calculate means and standard errors among the treatments (n = 3).

Results and discussion

Production of ECXI-BL21

The maximum D-glucose isomerase activity (146.67 ± 1.2 U/ml/min) was achieved at 37 °C, pH 7.0 after 48 h (Fig. 3a–c) which was independent of any carbon source induction. However, the enzyme activity was increased when E. coli BL21 cells were induced with different carbon sources (Fig. 3d). As compared to control (146.67 ± 1.3 U/ml/min), a maximum 41.09% increase in enzyme activity (206.67 ± 0.5 U/ml/min) was observed when fermentation medium was supplemented with 1% D-xylose. Similarly, Bacillus megaterium cells showed maximum D-glucose isomerase activity at 37 °C, pH 7.0 when induced with 1% D-xylose (Nguyen and Tran 2018). The first report on xylose transport in E. coli X289 revealed that xylose permease system is specifically induced by D-xylose (David and Weismeyer 1970). D-xylose transport system in E. coli is linked with a protein, which only take up D-xylose as the pH changed to alkaline conditions (Lam et al. 1980). However, E. coli BL21 cells produced highest enzyme titer at pH 7.0. Later on, a periplasmic xylose-binding protein was purified from E. coli K-12 (Ahlem et al. 1982). This protein is released only by osmotic shock and is an integral part of high-affinity xylose transport system reported for closely related enteric bacterium Salmonella typhimurium LT2. However, it is very interesting that this binding protein was produced when S. typhimurium LT2 was grown on medium containing xylose as well as glycerol (Shamannat and Sanderson 1979). Previously, among all the carbon sources used, maximum D-glucose isomerase activity (D-glucose(aldo) [right harpoon over left harpoon] D-fructose(keto)) was achieved when E. intermedia cells were induced by 2% D-glucose. However, cells of E. intermedia not showed any D-xylose isomerase activity (D-xylose(aldo) [right harpoon over left harpoon] D-xylulose(keto)) activity when induced with carbon sources other than D-xylose (Natake and Yoshimura 1964). It might due to repressive effects of other sugars by which they were not transported into E. intermedia cells (Ammar et al. 2018).

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Production of ECXI-BL21. Effect of time of incubation (a), effect of temperature (b), effect of pH (c), effect of inducers (d). Error bars show the standard error among parallel replicates (n = 3)

Purification of ECXI-BL21

ECXI-BL21 purified to homogeneity, up to 1.9-fold by heat treatment, salting out and electro-elution. Overall, 10.88% yield of enzyme was achieved (Table ​(Table1).1). Heat treatment of ECXI-BL21 was very efficient, as more than 50% contaminants (other proteins) were removed at this step (Fig. 4a). Previously, ECXI-K12 was purified to homogeneity with an overall yield of 18% (Schellenberg et al. 1984). The purified ECXI-BL21 was resolved on 12% SDS-PAGE. The molecular mass (Mr) of ECXI-BL21 monomer was calculated as 43.9 kDa using calibration curve (Fig. 4b). Similarly, a thermophilic class I enzyme from Thermobifida fusca MBL 10,003 showed Mr = 43.9 kDa (Kasumi et al. 2012). Among class I, mesophilic enzyme from Streptomyces sp.CH7 has Mr = 43.6 kDa (Chanitnun and Pinphanichakarn 2012), while hyperthermophilic, class II enzyme from Thermotoga maritima MSB8 has Mr = 45 kDa (Brown et al. 1993). It was previously reported that a ~ 1.3 kb (1320 bp) gene (xyl-A) fragment of E. coli K12 encoding for XI of 440 amino acid residues long has a Mr = 44 kDa (Schellenberg et al. 1984). Hence, ECXIs are class II enzymes.

Table 1

Purification of E. coli BL21 xylose isomerase

Purification stepsTotal protein (mg)Total activity (U/mg)Specific activity (U/mg)Overall yield (%)Purification fold
Crude extract52510,333.519.681001
Heat treatment327.768097.624.778.361.25
70% (NH4)2SO435980289.481.42
Electro-elution30112537.510.881.9
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SDS-PAGE of ECXI-BL21. a Lane 1: after heat treatment, Lane 2 and 3: before heat treatment, Lane 4: ACT Gene™ (P/N: ACT-IDWW24) pre-stained protein marker (11–245 kDa). b Lane 1: ACT Gene™ (P/N: ACT-IDWW24) pre-stained protein marker, Lane 2: after electro-elution

Physicochemical characterization of ECXI-BL21

The optimum pH of ECXI-BL21 was calculated as 7.0 and by further increase in pH to 8.0, it showed 15% less activity than optimal (Fig. 5a). Similarly, the maximum catalytic activity of ECXI-K12 was calculated as 6.8 (Rozanov et al. 2009). This value is very close to our finding. The neutral pH optima of ECXIs is a good sign as industries utilizing this enzyme demanded slightly acidic to neutral pH optima of the enzyme for HFCS production (Bhosale et al. 1996). Class II, hyperthermophilic enzyme from T. naphthophila (Fatima et al. 2016), thermophilic enzymes from Caldanaerobacter subterraneus sub sp. yonseiensis (Kim et al. 2010) and B. thuringiensis (El-shora et al. 2016) and among class I, enzymes from Streptomyces sp. CH7 (Chanitnun and Pinphanichakarn 2012) and S. albaduncus (Yassien et al. 2013) also showed optimum activity at pH 7.0. ECXI-BL21 was completely unfolded under highly acidic conditions as the enzyme did not show isomerase activity at pH 2.0 and 3.0. Catalytic activity of ECXI-BL21 was reduced to 30 and 40% at pH 4.0 and 5.0, respectively. ECXI-BL21 was stable at pH range 6.0–8.0 when incubated at 50 °C, in the absence of substrate (Fig. 5a). ECXI-BL21 activity was reduced to 14–15% when it was incubated at pH 9, for 60 min. The hyperthermophilic, class II enzymes from T. neapolitana DSM 5068 and T. naphthophila RKU-10 retained 100 and more than 80% of their catalytic activities at pH range 6.8–7.3 and 6.0–8.0, when incubated for 30 min at 90 and 80 °C, respectively (Vieille et al. 1995; Fatima et al. 2016).

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Effect of reaction conditions on ECXI-BL21 activity and stability. Effect of pH and pH stability (a), effect of temperature and thermostability (b), Arrhenius plot (R2 = 0.941) (c). Error bars show the standard error among parallel replicates (n = 3)

Optimal temperature of ECXI-BL21 was determined as 50 °C (Fig. 5b). Similarly, enzyme from B. thuringiensis has optimum temperature 50 °C which is also a thermophilic, class II enzyme (El-shora et al. 2016). ECXI-BL21 was stable from 30 to 55 °C. By further increase in temperature, enzyme undergoes fast denaturation (Tm = 75 °C), as about 12% reduction in its catalytic activity was noted at 60 °C (only by 5 °C increase in temperature) and it was 80% reduced at 90 °C (Fig. 6b). Similarly, ECXI-K12 commenced for denaturation at 45 °C and its catalytic activity was 100% lost at 65 °C, after 60 min of incubation. However, it was optimally active at 45 °C (Rozanov et al. 2009). Hence, ECXI-BL21 is more active at higher temperature than ECXI-K12. The activation energy of ECXI-BL21 for D-glucose(aldo) [right harpoon over left harpoon] D-fructose(keto) conversion was calculated as 45 kJ/mol (Fig. 5c).

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Effect of divalent metal ions on ECXI-BL21 activity. Effect of Mg2+ (a), effect of Co2+ (b), effect of Mn2+ (c), effect of Mg2+ with Co2+ and Mn2+ (d). Error bars show the standard error among parallel replicates (n = 3)

The catalytic activity of ECXI-BL21(holo) was increased to 35% as compared to ECXI-BL21(apo), when it was equilibrated with 10 mM Mg2+ (Fig. 6a) and it was reduced by 10 and 15% when Mg2+ was replaced with 0.5 mM Co2+ (Fig. 6b) and Mn2+ (Fig. 6c), respectively. However, the catalytic activity of ECXI-BL21(apo)was increased maximum to 15% when it was equilibrated with 0.5 mM Mn2+ and 10 mM Mg2+ simultaneously (Fig. 6d) as compared to Mg2+ alone. Maximum 50% increase in enzyme activity of ECXI-BL21(holo) was achieved by Mn2+/Mg2+ combination as compared to ECXI-BL21(apo). Similarly, 30 mM Mg2+ enhanced the catalytic activity of ECXI-K12 and it was further maximized up to 15–20% when 20 mM Mg2+ along with 10 mM Co2+ was simultaneously present in the reaction mixture (Rozanov et al. 2009). Class II, thermophilic XIs from C. subterraneus sub sp. yonseiensis (CSXI) (Kim et al. 2010), Fervidobacterium gondwanense (FGXI) (Kluskens et al. 2010), Anoxybacillus gonensis G2T (AGXI) (Yanmis et al. 2014) and hyperthermophilic XI from T. maritime MSB8 (TMXI) (Brown et al. 1993) required Co2+/Mg2+ at 1/10 mM concentration for their optimal performance. It is reported that the addition of Co2+ should be avoided due to their health and environmental hazards, because of human consumption of HFCS and industrial effluent, respectively (Bhosale et al. 1996). ECXI-BL21 is superior to ECXI-K12, CSXI, FGXI, AGXI and TMXI as it does not required Co2+ for its optimal performance. On silica gel plate, ECXI-BL21 reaction product, produced under above-mentioned optimized conditions appeared as black spots (Fig. 7). The retention factor of D-glucose (Lane 1) and D-fructose (Lane 4) was calculated as 0.62 and 0.65, respectively. A considerable amount of D-glucose was isomerized to D-fructose as depicted by Fig. 7 (Lane 2). There is scarcity of the literature for the comparison of ECXI-BL21 with same enzyme from other strains of E. coli.

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Separation of reducing sugars on kiesel gel plate. a D-glucose, b D-glucose after isomerization catalyzed by ECXI-BL21, c D-glucose + ECXI-BL21 before isomerization, d D-fructose

Activity data of ECXI-BL21, at different substrate concentrations, were plotted as non-linear fit of Michaelis–Menten equation (Fig. 8a) and then transformed as double reciprocal plot (Fig. 8b) (Lineweaver and Burk 1934), using Graph Pad Prism 6. The Km and Vmax values of ECXI-BL21(holo) at 50 °C, pH 7.0 for glucose(aldo) to fructose(keto) conversion were calculated as 0.82 mM and 108 µmol/mg/min, respectively, when it was equilibrated with 0.5 mM Mn2+ and 10 mM Mg2+. However, Rozanov et al. (2009) reported Vmax = 0.06 µmol.mg−1.min−1 and Km = 0.22 1/mol at 30 °C; Vmax = 0.8 µmol.mg−1.min−1 and Km = 0.5 1/mol at 45 °C of ECXI-K12 for D-glucose(aldo) [right harpoon over left harpoon] D-fructose(keto) conversion at 30 mM Mg2+, pH 6.8. Again, ECXI-BL21 is superior to ECXI-K12 as it showed greater catalytic affinity and maximum velocity at higher temperature than K12enzyme, because high temperature shifts equilibrium of the reaction in forward direction (towards fructose). Currently, immobilized XIs used in industries, under trade name Spezyme IGI, Optisweet 22, Gensweet SGI, AGIS-600, Sweetase, Sweetzyme T and Maxazyme from S. rubiginosus, S. rubiginosus, S, murinus, S. griseofuseus, S. phaeochromogenes, S. murinus and Actinoplanes missouriensis manufactured by Finnsugar, Finland; Miles Kali-Chemie, Germany; Genencor, USA; Godo-Shusei, Japan; Nagase, Japan; Novo- Nordisk, Denmark and Gist-Bocades, Netherlands, respectively (Jensen and Rugh 1987), are all mesophilic, class I enzymes. For D-glucose(aldo) [right harpoon over left harpoon] D-fructose(keto) conversion the Km and Vmax of XIs from S. griseofuseus, S. olivochromogenes, Streptomyces sp CH7 and A. missouriensis were reported as 220, 250, 258 and 230, 290 mM and 17.6, 5.33, 32.42 and 33.9 U/mg, respectively (Suekane et al. 1978; Kasumi et al. 1982; Smith et al. 1991; Jenkins et al. 1992; Van Bastelaere et al. 1995; Chanitnun and Pinphanichakarn 2012). These results showed that ECXI-BL21 showed greater catalytic affinity for D-glucose than all commercially available XIs currently used in industries.

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Kinetics of ECXI-BL21. a Michaelis–Menten plot, b Lineweaver–Burk plot (R2 = 0.996)

Conclusion

In conclusion, ECXI-BL21 can be produced from well-known already-established system of E. coli BL21(DE3) Codon Plus. ECXI-BL21 was purified very efficiently by electro-elution as compared to column chromatography which is expensive and time-consuming technique. ECXI-BL21 showed highest catalytic affinity and maximum velocity, required lowest concentration of Mn2+ and not required Co2+ at all, as compared to other XIs reported to date. The enzyme is thermophilic with neutral pH optima as demanded by industries for HFCS production. Also, it has broad range pH and temperature stability. Hence, ECXI-BL21 could be a best candidate for fructose syrup production.

Acknowledgements

Provision of laboratory facilities by University of Veterinary and Animal Sciences (UVAS) Lahore and Institute of Public Health (IPH) Lahore to undertake this research is gratefully acknowledged.

Author contribution

MMJ conceived and supervised the study. BF designed and performed the experiments, analysed the data and wrote the manuscript. Both authors contributed to the discussion and revision of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest related to this article.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

References

  • Ahlem C, Huisman W, Neslund G, Dahms AS. Purification and properties of a periplasmic D-xylose-binding protein from Escherichia coli K-12. J Biol Chem. 1982;257:2926–2931. [Abstract] [Google Scholar]
  • Ammar EM, Wang X, Rao CV. Regulation of metabolism in Escherichia coli during growth on mixtures of the non-glucose sugars: arabinose, lactose, and xylose. Sci Rep. 2018;8:1–11. 10.1038/s41598-017-18704-0. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
  • Bhosale SH, Rao MB, Deshpande VV. Molecular and industrial aspects of glucose isomerase. Microbiol Rev. 1996;60:280–300. [Europe PMC free article] [Abstract] [Google Scholar]
  • Boyer RF (1993) Modern experimental biochemistry, second. The Benjamin/Cummings Publishing Company, Inc.
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. 10.1016/0003-2697(76)90527-3. [Abstract] [CrossRef] [Google Scholar]
  • Branco AT, dos Ferreira BS, de Souza Filho GA. A paper-based electroelution system for protein recovery from stained sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem. 2008;381:267–269. 10.1016/j.ab.2008.03.025. [Abstract] [CrossRef] [Google Scholar]
  • Brown SH, Sjoholm C, Kelly RM. Purification and characterization of a highly thermostable glucose isomerase produced by the extremely thermophilic eubacterium, Thermotoga maritima. Biotechnol Bioeng. 1993;41:878–886. 10.1002/bit.260410907. [Abstract] [CrossRef] [Google Scholar]
  • Chan E-C, Ueng PP, Chen LF. Metabolism of D-xylose in Schizosaccharomyces pombe cloned with a xylose isomerase gene. Appl Microbiol Biotechnol. 1989;31–31:524–528. 10.1007/BF00270788. [CrossRef] [Google Scholar]
  • Chanitnun K, Pinphanichakarn P. Glucose (xylose) isomerase production by Streptomyces sp. CH7 grown on agricultural residues. Brazil J Microbiol. 2012;43:1084–1093. 10.1590/S1517-83822012000300035. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
  • Chiang LC, Gong CS, Chen LF, Tsao GT. D-xylulose fermentation to ethanol by Saccharomyces cerevisiae. Appl Environ Microbiol. 1981;42:284–289. 10.1128/AEM.42.2.284-289.1981. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
  • Chiang LC, Hsiao HY, P.P., et al (1981b) Ethanol production from xylose by enzymic isomerization and yeast fermentation. Biotechnol Bioeng Symp 11:Web
  • Collyer CA, Henrick K, Blow DM. Mechanism for aldose-ketose interconversion by D-xylose isomerase involving ring opening followed by a 1,2-hydride shift. J Mol Biol. 1990;212:211–235. 10.1016/0022-2836(90)90316-E. [Abstract] [CrossRef] [Google Scholar]
  • David JD, Weismeyer H. Control of xylose metabolism in Escherichia coli X289. Biochem Biophys Acta. 1970;201:497–499. 10.1016/0304-4165(70)90171-6. [Abstract] [CrossRef] [Google Scholar]
  • Dawson RM, Elliott DC, Elliott WH, Jones KM. Data for biochemical research. London: Oxford University Press; 1969. [Google Scholar]
  • Driska SP. Using light scattering to locate less than a microgram of protein per band in polyacrylamide tube gels after isoelectric focusing. Anal Biochem. 1998;260:44–49. 10.1006/abio.1998.2696. [Abstract] [CrossRef] [Google Scholar]
  • El-Shora Hamed M, El-Shobaky Ahmed M, Ghoneim Jehan E. Activity of Glucose Isomerase from Bacillus thuringiensis under Different Treatments. International Journal of Current Microbiology and Applied Sciences. 2016;5(2):579–589. 10.20546/ijcmas.2016.502.065. [CrossRef] [Google Scholar]
  • Epting KL, Vieille C, Zeikus JG, Kelly RM. Influence of divalent cations on the structural thermostability and thermal inactivation kinetics of class II xylose isomerases. FEBS J. 2005;272:1454–1464. 10.1111/j.1742-4658.2005.04577.x. [Abstract] [CrossRef] [Google Scholar]
  • Fatima B, Aftab MN, Haq IU. Cloning, purification, and characterization of xylose isomerase from Thermotoga naphthophila RKU-10. J Basic Microbiol. 2016;56:949–962. 10.1002/jobm.201500589. [Abstract] [CrossRef] [Google Scholar]
  • Gong CS, Chen LF, Flickinger MC, et al. Production of ethanol from D-xylose by using D-xylose isomerase and yeasts. Appl Environ Microbiol. 1981;41:430–436. 10.1128/AEM.41.2.430-436.1981. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
  • Jenkins J, Janin J, Rey F, et al. Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 1. Crystallography and site-directed mutagenesis of metal binding sites. Biochemistry. 1992;31:5449–5458. 10.1021/bi00139a005. [Abstract] [CrossRef] [Google Scholar]
  • Jensen VJ, Rugh S. Industrial-scale production and application of immobilized glucose isomerase. Methods Enzymol. 1987;136:356–370. 10.1016/S0076-6879(87)36035-5. [CrossRef] [Google Scholar]
  • Kasumi T, Hayashi K, Tsumura N. Roles of magnesium and cobalt in the reaction of glucose isomerase from Streptomyces griseofuscus S-41. Agric Biol Chem. 1982;46:31–39. 10.1271/bbb1961.46.21. [CrossRef] [Google Scholar]
  • Kasumi T, Mori S, Kaneko S, Koyama Y. Molecular cloning and characterization of D-xylose isomerase from a novel actinobacteria, Thermobifida fusca MBL10003. J Appl Glycosci. 2012;59:43–46. 10.5458/jag.jag.JAG-2011. [CrossRef] [Google Scholar]
  • Kim BC, Yu SN, Kim KY, et al. Cloning, expression and characterization of xylose isomerase, xyl-A, from Caldanaerobacter subterraneus subsp. yonseiensis. Biotechnol Lett. 2010;32:929–933. 10.1007/s10529-010-0255-3. [Abstract] [CrossRef] [Google Scholar]
  • Kluskens LD, Zeilstra J, Geerling ACM, et al. Molecular characterization of the glucose isomerase from the thermophilic bacterium Fervidobacterium gondwanense. Environ Technol. 2010;31:1083–1090. 10.1080/09593330903486673. [Abstract] [CrossRef] [Google Scholar]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. 10.1038/227680a0. [Abstract] [CrossRef] [Google Scholar]
  • Lam VM, Daruwalla KR, Henderson PJ, Jones-Mortimer MC. Proton-linked D-xylose transport in Escherichia coli. J Bacteriol. 1980;143:396–402. 10.1128/JB.143.1.396-402.1980. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
  • Li GQ, Shao J, Guo CG, et al. A simple monolithic column electroelution for protein recovery from gel electrophoresis. Anal Biochem. 2012;430:24–31. 10.1016/j.ab.2012.07.008. [Abstract] [CrossRef] [Google Scholar]
  • Lineweaver H, Burk D. The determination of enzyme dissociation constants. J Am Chem Soc. 1934;56:658–666. 10.1021/ja01318a036. [CrossRef] [Google Scholar]
  • Natake M, Yoshimura S. Studies of glucose isomerase of bacteria: Part II, The glucose isomerizing activity of Escherichia intermedia strain HN-500. Agri Biol Chem. 1964;28:505–509. [Google Scholar]
  • Thi Nguyen Hoang-Yen, Tran Gia-Buu. Optimization of Fermentation Conditions and Media for Production of Glucose Isomerase from Bacillus megaterium Using Response Surface Methodology. Scientifica. 2018;2018:1–11. 10.1155/2018/6842843. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
  • Rozanov AS, Zagrebelny SN, Beklemishev AB. Cloning of Escherichia coli K12 xylose isomerase (glucose isomerase) gene and studying the enzymatic properties of its expression product. Appl Biochem Microbiol. 2009;45:38–44. 10.1134/s0003683809010062. [Abstract] [CrossRef] [Google Scholar]
  • Schellenberg GD, Sarthy A, Larson AE, et al. Xylose isomerase from Escherichia coli. Characterization of the protein and the structural gene. J Biol Chem. 1984;259:6826–6832. [Abstract] [Google Scholar]
  • Schenk M, Bisswanger H. A microplate assay for D-xylose/D-glucose isomerase. Enzyme Microb Technol. 1998;22:721–723. 10.1016/S0141-0229(98)00003-9. [CrossRef] [Google Scholar]
  • Schneider H, Wang PY, Johnson BF (1981) Current developments in yeast research. Pergamon Press, pp 81–85
  • Shamannat DK, Sanderson KE. Uptake and catabolism of D-xylose in Salmonella typhimurium LT2. J Bacteriol. 1979;139:64–70. 10.1128/JB.139.1.64-70.1979. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
  • Smith CA, Rangarajan M, Hartley BS. D-Xylose (D-glucose) isomerase from Arthrobacter strain N.R.R.L. B3728: Purification and properties. Biochem J. 1991;277:255–261. 10.1042/bj2770255. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
  • Sriprapundh D, Vieille C, Zeikus JG. Molecular determinants of xylose isomerase thermal stability and activity: analysis of thermozymes by site-directed mutagenesis. Protein Eng. 2000;13:259–265. 10.1093/protein/13.4.259. [Abstract] [CrossRef] [Google Scholar]
  • Sriprapundh D, Vieille C, Zeikus JG. Directed evolution of Thermotoga neapolitana xylose isomerase: high activity on glucose at low temperature and low pH. Protein Eng Des Sel. 2003;16:683–690. 10.1093/protein/gzg082. [Abstract] [CrossRef] [Google Scholar]
  • Staudigl P, Haltrich D, Peterbauer CK. L-Arabinose isomerase and D-xylose isomerase from Lactobacillus reuteri: Characterization, coexpression in the food grade host Lactobacillus plantarum, and application in the conversion of D-galactose and D-glucose. J Agric Food Chem. 2014;62:1617–1624. 10.1021/jf404785m. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
  • Suekane M, Tamura M, Tomimura C. Physico-chemical and enzymatic properties of purified glucose isomerases from Streptomyces olivochromogenes and Bacillus stearothermophilus. Agric Biol Chem. 1978;42:909–917. 10.1271/bbb1961.42.909. [CrossRef] [Google Scholar]
  • Terpe K. Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol. 2006;72:211–222. 10.1007/s00253-006-0465-8. [Abstract] [CrossRef] [Google Scholar]
  • Van Bastelaere PB, Callens M, Vangrysperre WA, Kersters-Hilderson HL. Binding characteristics of Mn2+, Co2+ and Mg2+ ions with several D-xylose isomerases. Biochem J. 1992;286:729–735. 10.1042/bj2860729. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
  • Van Bastelaere PBM, Kersters-Hilderson HLM, Lambeir AM. Wild-type and mutant D-xylose isomerase from Actinoplanes missouriensis: Metal-ion dissociation constants, kinetic parameters of deuterated and non-deuterated substrates and solvent-isotope effects. Biochem J. 1995;307:135–142. 10.1042/bj3070135. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
  • Vangrysperre W, Van Damme J, Vandekerckhove J, et al. Localization of the essential histidine and carboxylate group in D-xylose isomerases. Biochem J. 1990;265:699–705. 10.1042/bj2650699. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
  • Vieille C, Zeikus GJ. Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability. Microbiol Mol Biol Rev. 2001;65:1–43. 10.1128/MMBR.65.1.1-43.2001. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
  • Vieille C, Hess JM, Kelly RM, Zeikus JG. xyl-A cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana. Appl Environ Microbiol. 1995;61:1867–1875. 10.1128/AEM.61.5.1867-1875.1995. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
  • Walker G, Ricci BA, Goodwin JC. Thin layer chromatography of sugar beet carbohydrates. J A S S B T. 1965;13:503–508. [Google Scholar]
  • Wang PY, Johnson BF, Schneider H. Fermentation of D-xylose by yeasts using glucose isomerase in the medium to convert D-xylose to D-xylulose. Biotechnol Lett. 1980;2:273–278. 10.1007/BF00239856. [CrossRef] [Google Scholar]
  • Wang PY, Shopsis C, Schneider H. Fermentation of a pentose by yeasts. Biochem Biophys Res Commun. 1980;94:248–254. 10.1016/S0006-291X(80)80213-0. [Abstract] [CrossRef] [Google Scholar]
  • Yanmis D, Karaoglu H, Colak DN, et al. Characterization of a novel xylose isomerase from Anoxybacillus gonensis G2T. Turkish J Biol. 2014;38:586–592. 10.3906/biy-1403-76. [CrossRef] [Google Scholar]
  • Yassien MAM, Jiman-fatani AAM, Hani AZ. Purification, characterization and immobilization of glucose isomerase from Streptomyces albaduncus. Afr J Microbiol Res. 2013;7:2682–2688. 10.5897/AJMR2012.2482. [CrossRef] [Google Scholar]
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