Self-Assembled Hybrid Nanoflowers of ... - ACS Publications

Research Article Cite This: ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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Self-Assembled Hybrid Nanoflowers of Manganese Phosphate and L‑Arabinose Isomerase: A Stable and Recyclable Nanobiocatalyst for Equilibrium Level Conversion of D‑Galactose to D‑Tagatose Shushil Kumar Rai, Lokesh Kumar Narnoliya, Rajender S. Sangwan, and Sudesh Kumar Yadav* Biotechnology and Synthetic Biology, Center of Innovative and Applied Bioprocessing (CIAB), Sector-81 (Knowledge City), Mohali 140306, India S Supporting Information *

ABSTRACT: We report on the synthesis and characterization of a novel hybrid nanoflower of manganese and L-arabinose isomerase and its application in synthesis of D-tagatose, a rare sugar of high commercial value. An open reading frame of 1425 base pairs from Lactobacillus sakai was used to synthesis recombinant L-arabinose isomerase of 474 amino acids in E. coli. A hierarchical flower-like spherical structure with several nanopetals was self-assembled by using purified recombinant L-arabinose isomerase as the organic component and manganese phosphate as the inorganic component. The hybrid nanoflower was characterized by scanning electron microscopy, high-resolution transmission electron microscopy, confocal laser scanning microscopy, Fouriertransform infrared spectroscopy, X-ray diffraction, and energy-dispersive X-ray spectroscopy. Circular dichroism documented no change in structural properties of Larabinose isomerase assembled in the hybrid nanoflower. Kinetic parameters of Larabinose isomerase were improved in the hybrid nanoflower. L-Arabinose isomerase in the manganese hybrid nanoflower was found to convert D-galactose to D-tagatose with a conversion rate of ∼50%, without an addition of manganese in the reaction mixture. The hybrid nanoflowers exhibited excellent reusability and reproducibility during reaction cycle analysis. Hence, the developed manganese hybrid nanoflowers of L-arabinose isomerase shows promise for commercial production of the rare sugar D-tagatose. KEYWORDS: Nanobiocatalyst, Self-Assembly, Hybrid nanoflowers, L-Arabinose isomerase, D-Galactose, D-Tagatose

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INTRODUCTION D-Tagatose is a rare reducing hexoketose sugar with 92% sweetness and only 30% of the energy content to that of sucrose. D-Tagatose is also known as mal-absorbing rare sugar because of its considerably reduced absorption in the small intestine. However, the unabsorbed fraction of D-tagatose is completely fermented by intestinal microflora in the large intestine.1 D-Tagatose possesses numerous health benefits, including antiobesity properties; prebiotic nature; promotion of weight loss; no glycemic effect; reduction in diabetes-related hyperglycemia; noncarcinogenic, antihalitosis, and antibiofilm properties; and improvement of pregnancy and fetal development.1−3 D-Tagatose is used as a part of nonchronic drugs, mouth wash, tooth paste and an array of foods varieties, beverages, health foods, and dietary supplements. It is used in low-carbohydrate diets, chocolate, candy, chewing gum, yogurt, soft drink, bakery items, milk-based drinks, and confectionery.2−7 D-Tagatose has received Generally Recognized as Safe (GRAS) certification from the U.S. Food and Drug Administration (FDA) and allowed its uses in the food and beverage industry.2 D-Tagatose can be produced from D-galactose by chemical methods using a calcium catalyst. However, such chemical process has disadvantages like complex purification steps and © XXXX American Chemical Society

formation of chemical waste and byproducts. Therefore, a biological process involving enzymes is preferred for producing rare sugars at commercial scale.3−6 The biological production of rare sugar needs various techniques of fermentation technology, molecular biology, and enzyme technology.2 Biological production of rare sugars depends on various enzymes including aldo-keto isomerases, carbohydrate epimerases, and oxidoreductases.3,4,7 L-Arabinose isomerase, an enzyme of aldoketo isomerase group catalyzes the isomerization reaction between D-galactose and D-tagatose. The same enzyme, Larabinose isomerase, also catalyzes the conversion of Larabinose into L-ribulose because of the structural similarity of the substrates.8 A number of L-arabinose isomerase enzymes have been reported from microbes with significant catalytic conversion efficiency for D-galactose to D-tagatose, such as Bacillus stearothermophilus,8 Alicyclobacillus hesperidum,9 Arthrobacter chlorophenolicus,10 Bifidobacterium longum,11 and Lactobacillus sakei 23K.12 Though enzymes are a versatile and sustainable catalyst, immobilization may significantly improves their operational Received: January 9, 2018 Revised: March 6, 2018

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DOI: 10.1021/acssuschemeng.8b00091 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering stability and cost effectiveness.13 Nanomaterials offer enhanced mass transfer reaction and provide greater surface area for high enzyme loading, thus improve the biocatalytic performance of the enzymes over conventional immobilization.14 The potential biocatalysts can be immobilized on different nanomaterials such as nanoparticles, nanofibers, nanoporous media, and carbonbased nanomaterials via different strategies such as adsorption, covalent binding, cross-linking, and through ligand binding.14−16 Recent advancement in nanobiocatalysis led the synthesis of self-assembled organic−inorganic hybrid nanoflowers.17 Synthesis of hybrid nanoflowers of enzymes in controlled shape and size18 has been reported with different metals like copper,19 calcium,20 zinc,21 manganese,22 nickel oxide,23 and zinc oxide.24 Different types of metal ions have been reported for different morphology of lipase-metal phosphate nanoflowers.25 A metalloenzyme, such as laccase, displayed a synergistic effect when incorporated in copper phosphate nanoflowers.17 Importantly, metal−organic frameworks and inorganic crystal nanoflowers promise a solution to chemical modifications on immobilization and offer advantages of enhancing enzyme activity, stability, and selectivity.26 Multienzyme incorporated hybrid nanoflowers have also been developed for efficient biosensing of glucose.27 Here, we report on the use of recombinant L-arabinose isomerase from Lactobacillus sakei produced in E. coli in its native as well as self-assembled form for the conversion of Dgalactose into D-tagatose. For the first time, a manganese-based hybrid nanoflower of L-arabinose isomerase has been prepared and characterized. Though both native and nanoflower forms of biocatalysts were observed to be highly efficient for equilibrium conversion of D-galactose to D-tagatose, still a manganese-based nanoflower form of L-arabinose isomerase has the advantage of significant stability and reusability without requiring any metal ion for multiple cycles.

inorganic component was manganese phosphate, were synthesized by the self-assembly method.17 Developed nanoflowers of L-AI (MnNF@L-AI) showed a protein loading of 65 ± 5%. The possibility of any chemical modification during nanoflower formation was avoided, and the stability of the enzyme was maintained as described earlier.28,29 The synthesis of hybrid nanoflowers of copper phosphate as inorganic and various commercial proteins bovine serum albumin, laccase, and lipase as organic components have been reported at ambient temperature with a three-day incubation period.17 The synthesis mechanism of hybrid nanoflowers comprised a threestep process; (a) nucleation and formation of primary crystals, (b) growth of crystals, and (c) formation of nanoflowers through the coordination reaction of metal salts and proteins.17 Generally, native or recombinant proteins lose their activity and stability very rapidly at ambient temperature. Therefore, organic−inorganic hybrid nanoflowers of manganese incorporated L-AI (MnNF@L-AI) were synthesized and characterized. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize surface morphology of hybrid nanoflowers MnNF@L-AI (Figure 2).

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RESULTS AND DISCUSSION Synthesis and Characterization of Hybrid Nanoflowers of L -Arabinose Isomerase. Recombinant Larabinose isomerase (L-AI) was purified through Ni-NTA column chromatography and its analysis on SDS-PAGE suggested apparent molecular weight ∼54 kDa (Figure 1). The L-AI protein isolated from Lactobacillus sakai 23K has been reported for similar molecular weight.12 Obtained purified recombinant L-AI was used in the synthesis of the hybrid nanoflower. The organic−inorganic hybrid nanoflowers, where the organic component was L-AI enzyme protein and the Figure 2. Morphological characterization of hybrid nanoflower MnNF@L-AI. (A) SEM image of hybrid nanoflowers MnNF@L-AI. (B) Magnified SEM image indicating many petals in the flower structure. (C) TEM image of hybrid nanoflower MnNF@L-AI, singleflower structure focus, and inset depicts crystal structure of nanoflower. (D) Two flowers showing the petals in whirls. (E) Closer look of single flower showing petals. (F) Closer look of flower petals.

SEM image of MnNF@L-AI clearly showed a flowerlike structure (Figure 2A), and the magnified SEM image indicated many petals in the flower structure (Figure 2B). Hybrid nanoflowers were found to be of average size 6 ± 2 μm with a uniform distribution of flowerlike structure. The morphology resembles well with earlier reported hybrid nanoflowers.30 TEM characterization showed aggregation of nanosized petals

Figure 1. SDS-PAGE of Ni-NTA purified recombinant L-arabinose isomerase protein. B


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ACS Sustainable Chemistry & Engineering to form a bloom of flowers (Figure 2C−F). Further, the localization of enzymatic protein L-AI within the nanoflower was monitored through confocal laser scanning microscopy. The fluorescein isothiocyanate (FITC) labeled L-AI was used to synthesize nanoflowers by following the same procedure as described for development of hybrid nanoflowers. Confocal laser scanning microscopy clearly documented the localization of FITC labeled L-AI within the nanoflowers (Figure 3). Similar flowerlike nanostructures of various proteins have been reported earlier with different inorganic counterparts.17,31

hybrid nanoflowers and therefore maintained activity similar or better than that of free L-AI. Energy-dispersive X-ray diffraction spectroscopy (EDS) used for the investigation of the elemental composition of the developed hybrid nanoflowers revealed the presence of elements such as carbon (C), oxygen (O), nitrogen (N), manganese (Mn), phosphorus (P), and copper (Cu) in hybrid nanoflowers MnNF@L-AI (Figure 4C). Data suggested the existence of L-AI enzyme by N element and crystals of manganese phosphate by the presence of Mn and P elements, and the Cu peak was due to a copper grid.34,36 X-ray diffraction analysis suggested the crystalline nature of the developed MnNF@L-AI nanoflowers (Figure 4D; Table S1). The nanoflower structure has exhibited a prismatic crystalline system of P21/a space group.37 The relative peak positions of MnNF@L-AI nanoflowers were found to be in better agreement with previously reported XRD patterns for Mn3(PO4)2·3H2O,38 with an additional peak at 31.5 degrees that might be due to protein of the nanoflowers. To get a clear picture of developed nanoflower MnNF@L-AI, thermal gravimetric analysis (TGA) was conducted. On incubation between 25 and 60 °C, the weight loss of nanoflower MnNF@L-AI was only 6.3%. With a further increase in temperature from 60 to 200 °C, the weight loss in the nanoflower was 8.52% (Figure S1). The initial weight loss between room temperature and 200 °C could be due to loss of crystallization water of Mn3(PO4)2·3H2O in nanoflower MnNF@L-AI. However, with a further increase in temperature from 200 to 325 °C, the weight loss was 8.68% and from 325 to 400 °C, the weight loss was 2.95% (Figure S1). This latter weight loss in nanoflower MnNF@L-AI could be a result of thermal pyrolytic decomposition of L-AI, a protein component of nanoflower MnNF@L-AI. The similar pattern of weight loss has been observed previously for the calcium phosphate nanoflower with α-amylase.36 Optimum Catalytic Activity and Effect of Metal Ions on L-AI Activity. To establish an enzymatic assay for recombinant L-AI enzyme in its purified or nanoflower form, various parameters were optimized by varying one parameter at a time. L-AI enzyme activity was analyzed at different concentrations of enzyme from 5 μg to 2 mg and found the highest activity at 0.5 mg mL−1 concentration for pure L-AI as well as for hybrid nanoflower MnNF@ L-AI. Reaction incubation time was analyzed from 5 min to 120 h, and significant linearity was observed until 5 h only. After 5 h there was an enhancement in activity until equilibration level conversion (∼50%), but the rate of catalysis reaction was slow. The effect of various metal ions was analyzed on catalytic activity of only recombinant L-AI enzyme. Manganese was not required in the reaction conducted with hybrid nanoflowers MnNF@L-AI because it already contained manganese. Enzyme activity without adding any metal ion was used as 100%. Out of 13 metal ions, Mn2+ and Co2+ were found to be significantly increasing L-AI catalytic activity. Mn2+ displayed maximum enhancement of ∼260% and Co2+ increased ∼185% catalytic activity of L-AI enzyme. While, catalytic activity of L-AI was inhibited by various metal ions and remained only 15−25% by Cu2+, Zn2+, Fe3+, and Cr3+. Other metal ions such as Mg2+, Ca2+, K+, Na+, Ni2+, Li+, and NH4+ either had no effect or had slightly inhibitory effect on the catalytic activity (Figure 5A). Various previous reports on the influence of metal ions on activity of L-AI have also documented the enhancing effect of Mn2+ and Co2+ and inhibitory effect of Cu2+.11,12,39−42

Figure 3. Confocal laser scanning microscope image of hybrid nanoflowers MnNF@L-AI showing the localization of FITC labeled LAI within the nanoflower.

Fourier transform infrared spectroscopy (FTIR) was used to investigate functional groups of chemical compounds MnNF@ L-AI or Mn3(PO4)2@L-AI, Mn3(PO4)2, and L-AI protein (Figure 4A). The asymmetric stretching vibration (PO−H) appeared at 1065, 1006, and 952 cm−1 for Mn3(PO4)2 and 1059, 1003, 950 cm−1 for MnNF@L-AI were similar to that reported earlier for BSA and gold nanoparticles incorporated with manganese phosphate nanoflowers.22 The symmetric (P− O) stretching appeared at 673 cm−1 for Mn3(PO4)2 and 669 cm−1 for MnNF@L-AI, and the asymmetric vibration (OP−O) observed at 573 cm−1 for Mn3(PO4)2 and 570 cm−1 for MnNF@L-AI were attributed to the bridging phosphorus, indicating the presence of phosphate groups.22,32 The protein secondary structure spectrum band of amide I (1700−1600 cm−1) and amide II (1600−1500 cm−1) for −CONH group appeared at 1643 and 1539 cm−1 for free L-AI and 1646 and 1539 cm−1 for MnNF@L-AI. The insignificant peak shift for amide I and amide II in MnNF@L-AI in comparison to free LAI indicated the enzyme immobilization through self-assembly in hybrid nanoflowers, instead of covalent conjugation.33,34 Circular dichroism (CD) spectrum was used to visualize changes in secondary structure of L-AI protein in hybrid nanoflower (Figure 4B). Use of CD for such analysis has been shown earlier in lipase conjugated carbon nanotubes.35 CD spectrum of L-AI and MnNF@L-AI was found to be very similar. The α-helix content of hybrid nanoflowers MnNF@LAI was 29.2% compared to that of 28.4% of free enzyme L-AI. Results suggested that L-AI retained its secondary structure in C


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Figure 4. Structural characterization of hybrid nanoflower MnNF@L-AI. (A) FTIR spectra of MnNF (a); hybrid nanoflower MnNF@L-AI (b) and LAI (c). (B) CD spectra of MnNF (a); hybrid nanoflower MnNF@L-AI (b) and L-AI (c). (C) EDS pattern of hybrid nanoflower MnNF@L-AI. (D) XRD pattern of hybrid nanoflower MnNF@L-AI.

reaction was carried out for 5 h. However, previously reported 2+ L-arabinose isomerase has been documented for Mn requirements in the range of 0.8−5 mM.12,40 Importantly, L-AI enzyme was able to exhibit more than 85% catalytic efficiency in the presence of as low as 0.02 mM Mn2+. Generally, a higher concentration of Mn2+ is required to achieve maximum enzymatic activity, whereas L-AI achieved the maximum increase in activity by 260% with the use of 0.2 mM Mn2+. Hence, the requirement for low Mn2+ concentration by purified 2+ L-AI and no additional requirement of Mn by the hybrid nanoflowers MnNF@L-AI could be the key feature of this enzyme for its potential use in further downstream processes. Optimum Temperature and pH for L-AI and Hybrid Nanoflower MnNF@L-AI. The optimum temperature for purified recombinant L-AI and hybrid nanoflower MnNF@L-AI was analyzed to catalyze the isomerization reaction from 20 to 80 °C and found maximal activity at 50 °C (Figure 5C). Hybrid nanoflower MnNF@L-AI exhibited better temperature range than pure L-AI. Both recombinant L-AI and MnNF@L-AI retained its ∼70% catalytic activity at 60 °C, whereas at 70 °C only ∼5% activity was found with L-AI and ∼25% with hybrid nanoflower MnNF@L-AI. Enzyme was almost inactive at 80 °C or higher temperature in case of both L-AI and MnNF@L-AI. Although L-AI enzyme was able to catalyze reactions at low temperature, its catalytic efficiency was significantly low. At 20 °C, L-AI had ∼33% and MnNF@L-AI had 58% catalytic activity. At 40 °C, purified recombinant L-AI was found to retain ∼56% activity and MnNF@L-AI retain ∼63% of activity to that of their maximal relative activity at 50 °C temperature. Results indicated that MnNF@L-AI can efficiently work on lower

Figure 5. Optimization of enzymatic assay for L-AI and hybrid nanoflower MnNF@L-AI. (A) Effect of various metal ions on L-AI activity. (B) Effect of various manganese ion concentration on L-AI activity. (C) Effect of temperature on L-AI and hybrid nanoflower MnNF@L-AI activity. (D) Effect of pH on L-AI and hybrid nanoflower MnNF@L-AI activity.

The optimum concentration of Mn2+ for maximum activity of L-AI was found to be 0.2 mM (Figure 5B). Thus, the optimum concentration of various constituents for the maximum conversion rate was standardized as 0.2 mM MnCl2 and 0.25 mg L-AI enzyme in 0.5 mL of total reaction volume at 50 °C in 50 mM HEPES buffer (pH 6.0), and the D


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(Kcat/Km) were significantly higher to that of reported earlier.12 In contrast, L-AI from Alicyclobacillus hesperidum,9 Pediococcus pentosaceus,44 Bacillus thermoglucosidasius,45 Alicyclobacillus acidocaldarius,46 Geobacillus thermodenitrificans47 have been reported for higher Km from 50 to 647 mM. Whereas close-to Km values of 35 and 25.2 mM for D-galactose have been reported for L-AI from Enterococcus faecium43 and Anoxybacillus flavithermus,48 respectively. Hence, the data of this study suggested highest affinity of hybrid nanoflower MnNF@L-AI for D-galactose with the optimized conditions in comparison to previously reported L-AI enzymes. L-AI vis-à-vis Hybrid Nanoflower MnNF@L-AI Catalyzes an Equilibrium Level Isomeric Conversion of DGalactose to D-Tagatose. The production of D-tagatose from D-galactose was analyzed by TLC (Figure 6A) and HPLC (Figure S3), indicated no additional byproduct formation

temperature as well as higher temperature with notable catalytic conversion efficiency. Temperature optima for both purified recombinant L-AI and hybrid nanoflower MnNF@L-AI was found to be 50 °C. While the optimum temperature of Larabinose isomerase enzyme isolated from Pseudoalteromonas haloplanktis has been reported 40 °C and work efficiently at lower temperature 20−30 °C.12,40 The optimum pH for isomerization reaction of recombinant L-AI enzyme was determined to be pH 6.0 (Figure 5D). Although maximum activity for both L-AI and hybrid nanoflower MnNF@L-AI was obtained at pH 6.0, both forms of the enzyme were observed to retain more than 90% of their catalytic activity between pH 5.0 to 7.0. At pH 4.0, purified recombinant L-AI possessed higher activity compared to hybrid nanoflower MnNF@L-AI. However, at pH 9.0, hybrid nanoflower MnNF@L-AI possessed significantly higher activity than L-AI. Most of the L-arabinose isomerase enzymes from different sources had maximum activity in the pH range of 6 to 10. Broadly, L-arabinose isomerase enzymes from microorganisms have been reported for their efficient activity in the slightly basic environment (pH 7−10) and at 50−70 °C.40 Several Larabinose isomerase enzymes have been isolated from microorganisms such as Alicyclobacillus hesperidum, Bacillus coagulans NL01, Lactobacillus reuteri, Enterococcus faecium, Pediococcus pentosaceus with optimum temperatures of 70, 70, 65, 45−50, and 50 °C, and optimum pH values of 7.0, 7.5, 6, 7.0−7.5, and 6.0, respectively.9,39,42−44 Importantly, the purified recombinant L-AI as well as hybrid nanoflower MnNF@L-AI was found to efficiently work between pH 5.0 to pH 7.0. Results suggested the possible use of the present study enzyme in hybrid nanoflowers for possible conversion of D-galactose in acidic medium into D-tagatose. Kinetic Parameters of L-AI and Hybrid Nanoflower MnNF@L-AI. The kinetic parameters of L-AI and hybrid nanoflower MnNF@L-AI, toward D-galactose were determined according to Michaelis−Menten (Figure S2A) and Lineweaver−Burk plot (Figure S2B) under the optimum reaction conditions of pH 6.0 and temperature 50 °C with different concentrations of D-galactose ranging from 1 to 100 mM. The observed Km of L-AI and hybrid nanoflower MnNF@L-AI for Dgalactose were 22.9 mM and 20 mM and Vmax was 125 IU mg−1 and 142.9 IU mg−1, respectively (Table 1). The turnover Table 1. Kinetic Parameters of Recombinant L-AI and Hybrid Nanoflower MnNF@L-AI kinetic parameter

L-AI

MnNF@L-AI

Vmax Km Kcat Kcat/ Km

125 IU mg−1 22.9 mM 46.7 s−1 2.10 mM−1 s−1

142.9 IU mg−1 20 mM 53.5 s−1 2.7 mM−1 s−1

number (Kcat) for L-AI and hybrid nanoflower MnNF@L-AI were observed to be 46.7 s−1 and 53.5 s−1, respectively. The catalytic efficiency (Kcat/Km) was 2.10 mM−1 s−1 for L-AI and 2.7 mM−1 s−1 for hybrid nanoflower MnNF@L-AI. Kinetic analysis of L-AI enzyme from L. sakei using D-galactose substrate has also been reported previously. However, critical optimization of reaction conditions in this study was favoring the better potential of the L-AI enzyme and hybrid nanoflower MnNF@LAI was found to further improve its catalytic activity. The observed Km of L-AI as well as hybrid nanoflower MnNF@L-AI was significantly lower, while Vmax, Kcat and catalytic efficiency

Figure 6. Analysis of D-galactose to D-tagatose conversion efficiency by pure L-AI and hybrid nanoflower MnNF@L-AI. (A) Thin-layer chromatogram of standards, control and enzymatic assay reaction with D-galactose as substrate [G, D-galactose; T, D-tagatose; C1, control (denatured protein); C2, control (without protein); C3, control (without galactose) and E1−E5, enzyme assay]. (B) Catalytic conversion of D-galactose into D-tagatose using pure L-AI and hybrid nanoflower MnNF@L-AI at different time period. (C) Reusability of hybrid nanoflower MnNF@L-AI for five cycles to enhance product yield. E



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during the reaction catalyzed by L-AI or hybrid nanoflower. The production of D-tagatose from D-galactose by recombinant L-AI and hybrid nanoflower MnNF@L-AI at 50 °C was increased continuously until 24 h and reached its maximum or equilibrium level conversion (50%). Both L-AI and hybrid nanoflower MnNF@L-AI displayed almost similar conversion rate at different time period. The conversion ratio of substrate D-galactose into product D-tagatose was measured to be ∼30%, ∼40%, ∼45%, and ∼50% at incubation time of 6, 12, 18, and 24 h, respectively (Figure 6B). Results suggested that reaction equilibrium for isomeric conversion of D-galactose to D-tagatose was achieved in 24 h. The efficient conversion of D-galactose into D-tagatose by hybrid nanoflower MnNF@L-AI could be ascribed by the allosteric activation due to the close proximity of enzyme active site and manganese phosphate. Similar mechanism for higher activity has been reported for the αamylase encapsulated into calcium phosphate hybrid nanoflowers.36 Several L-AI enzymes have been reported with significant catalytic conversion efficiency of D-galactose to Dtagatose such as Bacillus stearothermophilus (36%),8 Alicyclobacillus hesperidum URH17−3−68 (43%),9 Arthrobacter chlorophenolicus (30%),10 Bifidobacterium longum (36%),11 Lactobacillus sakei 23K (36%),12 and Bacillus thermoglucosidasius (45.6%).45 Under the present standardized conditions, L-AI as well as the newly developed manganese-based hybrid nanoflower of L-AI (MnNF@L-AI) were able to catalyze the ∼50% or equilibrium level isomeric conversion of D-galactose to Dtagatose. Reusability and Stability of Hybrid Nanoflowers. The reusability is a key factor for industrial application of enzymes. The developed hybrid nanoflower MnNF@L-AI was reused consecutively for five cycles (Figure 6C). The most significant aspect of this study was activator regeneration where additional Mn2+ was not required even in the first cycle for catalyzing Dgalactose isomerization into D-tagatose. Hence, the developed hybrid nanoflower MnNF@L-AI shows promise for the industrial applications where reusability and downstream processing is of great significance. The decrease in conversion efficiency of hybrid nanoflower MnNF@L-AI after each cycle might be due to leaching effect of the enzyme from the hybrid nanoflowers and thermal deactivation as the reaction was performed at 50 °C for 24 h in each cycle. The developed hybrid nanoflower was stable for more than one month on storage at 4 °C.

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METHODS

Chemicals, Reagents, and Strains. The strains Escherichia coli Top10, E. coli BL21, and pET-28a expression vector were obtained from Novagen (Darmstadt, Germany). Cloning vector pJET1.2, Pfu DNA polymerase, T4 DNA ligase, restriction endonucleases were purchased from NEB (New England Biolabs Inc.) and Fermentas (ThermoFisher Scientific). All other chemicals were of the highest reagent grade (Sigma, CDH). Ni-NTA resin was obtained from Qiagen (Hilden, Germany). Construct Preparation for L-Arabinose Isomerase. L-Arabinose isomerase (L-AI) gene containing 1425 nucleotides from Lactobacillus sakei was selected from NCBI (accession numberWP_011375537). Selected gene sequence was chemically synthesized in a standard cloning vector pUC57 with Nhe1 and Xho1 restriction site. Vector containing L-AI gene was transformed into E. coli Top10 cells for propagation. Plasmid was isolated, restriction digested with Nhe1 and Xho1 restriction enzyme along with pET28a expression vector, and thereafter, both digested products were ligated by using T4 DNA ligase. Finally, construct pET28a-L-AI was transformed in E. coli BL21 cells and confirmed by colony PCR, restriction digestion, and sequencing. Expression and Purification of Recombinant L-Arabinose Isomerase. The E. coli BL21 cells harboring pET28a-L-AI construct was grown in LB medium with 200 rpm shaking at 37 °C until OD at 600 reached to 0.4−0.6. After that, IPTG was added into the medium with a final concentration of 0.5 mM for the expression of recombinant protein, and the culture was incubated at 16 °C with 150 rpm for 16 h. Thereafter, cells were harvested by centrifugation at 5000g for 5 min and resuspended in 50 mM HEPES buffer pH 6.0. Cell disruption was carried out by sonication at 30% amplitude with 5 min pulse cycle of 5 s on and 10 s off through sonicator (Qsonica Inc.). Sonicated lysate was centrifuged at 10 000g for 25 min at 4 °C to remove insoluble cell debris, and the supernatant was used as crude cell extract. The crude extract was filtered through a 0.22 μm filter and loaded on Ni-NTA His tag column to purify recombinant protein according to manufacturer’s instruction. HEPES buffer (50 mM) was used for protein purification, and lastly, recombinant protein was eluted by 400 mM imidazole. Purified protein was dialyzed to remove salts and imidazole. Salt free pure L-AI protein was visualized by Coomassie Blue R250 staining on 12% SDS-PAGE. Protein concentration was determined through Bradford method using bovine serum albumin as a standard. Purified L-AI recombinant protein was stored at 4 °C for further biochemical assay. Synthesis of Hybrid Nanoflowers. The purified L-arabinose isomerase (0.1 mg mL−1) was dissolved in 10 mM phosphate buffer saline (pH 7.4) and uniformly dispersed with 1.2 mM manganese salt solution and then incubated undisturbed at 4 °C for 72 h. Organic− inorganic hybrid nanoflowers were obtained as a white precipitate at the bottom of the reaction vessel. Synthesized nanoflowers were collected after centrifugation at 10 000g and washed successively 2−3 times with 50 mM HEPES buffer (pH 6) to remove unbound protein and stored at 4 °C for further use. L-AI protein loading in the hybrid nanoflowers was determined using equation: Protein loading (%) = (ILp − Ubp)/ILp × 100; where ILp is the initial loaded protein before immobilization and Ubp is unbound protein after immobilization. Characterization of Hybrid Nanoflowers. Synthesized hybrid nanoflowers of L-AI (MnNF@L-AI) were physicochemically characterized using various modern analytical techniques. For morphological characterization, prepared hybrid nanoflowers were air-dried for overnight at room temperature and then fixed on to the magnetic tape, sputter-coated with gold, and analyzed by SEM (JCM-6000, JEOL) at 10 kV. However, the synthesized hybrid nanoflowers were spread out on the copper grid and dried before imaging in HR-TEM (FEI-Technai 20) operated at 200 kV. The elemental analysis of hybrid nanoflowers was carried out by EDS, an attachment with TEM. To monitor the effect of hybrid nanoflowers formation on the functional groups of L-AI protein, dried nanoflower preparation was analyzed by FTIR (Agilent technologies, Cary 600 series) in attenuated total reflection (ATR) mode at wavenumber range from

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CONCLUSIONS The availability of rare sugar monosaccharides is very limited in nature, and their importance makes them highly valuable. DTagatose is one of them and can be produced through isomerization reaction from D-galactose using L-arabinose isomerase enzyme. We have standardized the optimum parameters for equilibrium level isomeric conversion of Dgalactose into D-tagatose with purified recombinant L-arabinose isomerase vis-à-vis with the newly developed manganese-based hybrid nanoflower of L-AI (MnNF@L-AI). Hybrid nanoflower MnNF@L-AI has been found to be able to produce D-tagatose from D-galactose without use of any manganese in the reaction mixture, which is otherwise required by free L-AI. Also, hybrid nanoflower MnNF@L-AI has been found stable for more than one month and useful in multicycles. Enhanced kinetic characteristics, stability, and recyclability of hybrid nanoflower (MnNF@L-AI) over recombinant L-AI holds promise for its industrial applications. F


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ACS Sustainable Chemistry & Engineering 4000 to 400 cm−1. Influence of hybrid nanoflower formation on the secondary structure of L-AI protein was monitored using CD spectroscopy (Jasco, J-815). The samples (0.1 mg mL−1) were prepared in 10 mM HEPES buffer (pH 6), and spectra were recorded in the range of 240−190 nm with scanning speed 50 nm/min, bandwidth of 2 nm, and three times accumulation at 25 °C using quartz cuvette of 1 cm path length. Secondary structure analysis was performed in spectra manager software (Jasco-815). The crystalline structure of MnNF@L-AI was analyzed via powder X-ray diffraction (Rigaku Ultima IV) with Cu Kα radiation (λ = 1.54056 Å). The 20 mg of lyophilized powder of MnNF@L-AI was loaded into the sample holder, and the diffractometer was operated at 40 kV. XRD patterns were recorded in the range of 5−50 θ with a step width of 0.02° and scan speed of 2°/min. The obtained XRD patterns were compared with previously reported and indexed XRD pattern of Mn3(PO4)2· 3H2O in the Inorganic Crystal Structure Database (ICSD), as JCPDS no. 00-003-0426.38 TGA analysis of 5.2 mg of dried powder of MnNF@L-AI was performed with a TGA instrument (Mettler Toledo, TGA/SDTA 851e), and weight loss of MnNF@L-AI was measured at a range of 25−500 °C with a constant heating rate of 10 °C min−1. FITC Labeling of L-AI and Confocal Microscopy. Fluorescein isothiocynate (FITC; Sigma-Aldrich) was used for labeling the L-AI. For labeling, 2 mg of L-AI in 1 mL of 0.1 M sodium bicarbonate buffer, pH 9 was mixed with 50 μL of FITC (0.1% FITC in DMSO) and incubated for 8 h in dark at 4 °C. Excess unbound FITC was removed by overnight dialysis at 4 °C in 10 mM PBS. FITC-labeled L-AI was used to synthesize nanoflowers following the same procedure as described earlier. Nanoflowers of FITC-labeled L-AI were visualized by confocal laser scanning microscopy (Microscope FV 1000 SPD from Olympus). Activity Assay of Recombinant L-Arabinose Isomerase. The enzyme activity of recombinant L-AI in its purified and hybrid nanoflower form was determined by estimating formation of Dtagatose from D-galactose. Unless otherwise stated, the enzymatic reaction was carried out with 20 mM D-galactose, 0.2 mM MnCl2 (only in case of pure L-AI), and 0.25 mg pure L-AI or 0.50 mg hybrid nanoflower (MnNF@L-AI) in 0.5 mL total reaction volume at 50 °C in 50 mM HEPES buffer (pH 6.0). The reaction was stopped after 5 h by boiling reaction mixture at 100 °C for 5 min. Thereafter, the reaction mixture was centrifuged and filtered out through 0.2 μm filter, and used for product analysis. The qualitative and quantitative analysis of D-galactose and D-tagatose in the reaction samples was performed by using HPLC (Agilent Model-1260) equipped with Hi-plex Ca column and refractive index detector (RID). The sample injection volume was 20 μL. The column was eluted with deionized water at 85 °C and 0.6 mL min−1 flow rate. One unit of L-AI activity is defined as the amount of enzyme producing 1 μmol of D-tagatose per min at 50 °C and pH 6.0. Effect of Temperature, pH, and Metal Ions on L-Arabinose Isomerase Activity. Optimum temperature of L-AI as well as MnNF@L-AI was determined by assaying the enzyme samples over the range of 20−80 °C following the procedures described above in the enzyme assay. In order to study the effect of pH on the enzyme activity, 50 mM acetate buffer (pH 4.0 and 5.0), 50 mM HEPES buffer (pH 6.0 to 8.0), and 50 mM Tris-HCl buffer (pH 9.0) were used. To examine the influence of various metal ions on enzymatic activity, the dialyzed L-AI enzyme was assayed in the presence of various chloride salt of mono, di, and trivalent metal ions at 1 mM concentration, namely, MnCl2, CoCl2, MgCl2, CaCl2, KCl, CuCl2, NaCl, NiCl2, LiCl, NH4Cl, ZnCl2, FeCl3, CrCl3 at 50 °C for 5 h. The L-AI enzyme activity without adding any metal ion was taken as 100%, and all experiments were performed in triplicate. Determination of Kinetic Parameters of L-Arabinose Isomerase. Kinetic parameters of the recombinant L-AI in its purified and hybrid nanoflower form (MnNF@L-AI) were determined at the optimum pH 6.0 (50 mM HEPES buffer) in the presence of 0.2 mM of Mn2+ (only in case of pure L-AI) at 50 °C for 5 h using 1 to 100 mM of D-galactose as the substrate. Kinetic parameters, including the Michaelis−Menten constant (Km), the turnover number (Kcat), and

catalytic efficiency (Kcat/Km), were calculated using the Lineweaver− Burk plot. Determination of Enzymatic Conversion of D-Galactose to DTagatose. The conversion of D-galactose to D-tagatose was conducted at 50 °C with purified recombinant L-AI enzyme or its hybrid nanoflower MnNF@L-AI (1 mg mL−1 protein) in 50 mM HEPES buffer (pH 6.0) using 20 mM D-galactose as substrate. The reaction assay of purified recombinant L-AI enzyme included 0.2 mM Mn2+, while reaction assay of hybrid nanoflower MnNF@L-AI was performed without Mn2+ ion. Time depended enzymatic conversion was calculated using four different time intervals as 6, 12, 18, and 24 h. The concentration of generated D-tagatose in the reaction was determined by HPLC as described above.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.8b00091. XRD analysis of hybrid nanoflower MnNF@L-AI; TGA spectrum of the hybrid nanoflowers MnNF@L-AI; Kinetic parameters of purified recombinant L-AI and hybrid nanoflower MnNF@L-AI enzyme using Dgalactose as substrate; and HPLC chromatogram of enzymatic assay catalyzed by L-AI or MnNF@L-AI (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mails for S.K.Y.: [email protected]; skyt@rediffmail.com. ORCID

Sudesh Kumar Yadav: 0000-0002-3215-0308 Author Contributions

All authors contributed equally to this work Notes

This article does not contain any studies with human participants or animals performed by any of the authors. The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Authors are thankful to the CEO, CIAB, Mohali for his constant support and encouragement. We are grateful to Department of Biotechnology (DBT), GOI and LSRB, DRDO, GOI for financial support to conduct this research. S.K.R. is thankful to DBT, GOI for the award of junior research fellowship and Department of Microbial Biotechnology, Panjab University for Ph.D. registration. We are also thankful to the NIPER, Mohali for the use of their CD, TEM CLSM and EDS facilities and IISER, Mohali for the use of XRD facility. This work has been applied for patent under application number 201711024828.

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REFERENCES

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