A new L-arabinose isomerase with copper ion tolerance is suitable for creating protein–inorganic hybrid nanoflowers with enhanced enzyme activity and stability

Abstract

Protein–inorganic hybrid nanoflowers were prepared using Cu²⁺, PBS buffer, and a copper ion tolerant L-arabinose isomerase that was derived from Paenibacillus polymyxa (PPAI). The expensive rare sugar L-ribulose or D-tagatose was prepared utilizing these nanoflowers with L-arabinose or D-galactose as the substrate, respectively. The PPAI hybrid nanoflowers showed better enzyme stabilities in both thermal and acidic conditions. Meanwhile, long term batch production was successful showing high stability using these hybrid nanoflowers, which achieved a conversion rate of 61.8% from L-arabinose to L-ribulose.

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L-Ribose is an important pharmaceutical intermediate for the production of L-form sugar based antivirus drugs including those targeting human immunodeficiency virus (HIV), hepatitis B/C virus (HBV/HCV), and human cytomegalovirus (HCMV).^1–3 L-Ribose does not exist in nature and it can only be obtained through chemical synthesis. However, its synthetic route is complicated, which composed of 5–6 steps and the final yield is low.^1,2 Recently, biological production of L-ribose was established using a two-step reaction: L-arabinose, as the starting raw material, was firstly converted into its ketose isomer L-ribulose by L-arabinose isomerase (namely AI, EC 5.3.1.4), and then isomerized into L-ribose by mannose-6-phosphate isomerase (namely MPI, EC 5.3.1.8).^4,5 This method achieved a conversion rate of 23.6% from L-arabinose to L-ribose and a volumetric productivity of 39.3 g L⁻¹ h⁻¹.⁵ Considering that liquid L-arabinose was easily extracted from corncob fibres hydrolysates, this method is economical. On the other hand, AI was widely reported to be effective in producing another monosaccharide – D-tagatose, which is rarely existed in nature but showing advantages for human beings such as low calorie, stabilizing blood sugar levels, preventing tooth decay, and promoting growth of intestinal probiotics.^6–8 In 2003, the U. S. Food and Drug Administration (FDA) certified D-tagatose as a generally recognized as safe (GRAS) material and permitted it using in foods, beverages, and dietary supplements.

For industrial biocatalytic processes, immobilization of enzymes is a requisite for their use. Enzyme immobilization permits the reuse ability of the enzymes and simplifies the overall design and performance control of the bioreactors.^9–12 Thus, it is importance to develop novel immobilization techniques using various materials. The current enzyme immobilization techniques rely on the interactions between the proteins and solid surfaces, which include physical adsorption, electrostatic forces, specific recognition, and covalent bonding. Feasibility and price are the basic criterion for the immobilization material selection. Previously reported enzyme immobilization method for AI used alginate, glutaraldehyde, and aminopropyl glass, which are relatively cheap.^13–15 However, these materials have disadvantages such as poor stability at high temperatures and protein leakages after a long-term use. To fulfill the industrial requirements, chasing better catalytic stability is one of the essential targets. Thus, how to improve the stability of AI emerges as a key problem. Recently, the protein–inorganic hybrid nanoflowers were reported to be beneficial in improving enzymatic stabilities.¹⁶ This technique might reduce enzyme dissociation and has cooperative effects between enzyme and the immobilization material. This nanoflower consists of Cu₃(PO₄)₂·3H₂O and enzymes, which are formed after mixture of Cu²⁺ ion, PBS buffer, and the enzyme solution. Besides Cu²⁺, few reports showed that other metal ions are suitable for crystallizing these nanoflowers. Unfortunately, Cu²⁺ was commonly reported to be a strong inactivator for the enzyme activity of AI, such as observed in AIs derived from Shewanella sp., Anoxybacillus flavithermus, Bacillus stearothermophilus US100, Bacillus stearothermophilus IAM11001, Bacillus subtilis, Lactobacillus sakei 23K, and Lactobacillus fermentum CGMCC2921.^17–23 Thus, it is not suitable to prepare nanoflowers using AI as the component enzyme. Recently, the metal ion-independent AI enzyme was discovered such as AIs derived from Bacillus stearothermophilus US100, B. halodurans, and Arthrobacter sp. 22c.^19,24,25 However, these metal ion-independent AIs can also be inactivated by Cu²⁺ at very low concentrations.¹⁹ In this study, we screened and identified a copper ion tolerant AI and successfully prepared protein–inorganic hybrid nanoflowers, which are suitable for the batch production of L-ribulose and D-tagatose.

The open reading frame (ORF) of PPAI consists of 1425 base pair nucleotides corresponding to 474 amino acids with a theoretical molecular weight of 53 125 Da and an isoelectric point of 5.02. The amino acid sequence of PPAI was aligned with previously reported various AIs (Fig. S1†). Results showed that PPAI shares the highest sequence identity (91.1%) to BLAI (AI that derived from Bacillus licheniformis²⁶). The conserved catalytic residues of AI were also found in PPAI including residues E306, E331, H350, and H450. For other AIs such as those derived from Bacillus stearothermophilus US100, Alicyclobacillus acidocaldarius, Thermotoga maritima, Lactobacillus sakei 23K, Bacillus subtilis, and Lactobacillus fermentum CGMCC2921, the sequence identities to PPAI are 53.6%, 53.3%, 47.4%, 60.5%, 50.1%, and 55.1%, respectively.^{19,21–23,27,28} The recombinant PPAI was firstly purified by nickel affinity chromatography. The elusion buffer containing 200 mM imidazole eluted a major band and the protein was collected and loaded onto a Superdex S-200 16/60 size exclusion column. The recombinant protein was purified to homogeneity. SDS-PAGE analysis revealed a single band corresponding to purified recombinant PPAI protein with an estimated molecular weight of approximately 57 kDa (Fig. S2†), which is in accordance with the theoretical molecular weight (56 945 Da, including his-taq and vector part). The purified PPAI enzyme exhibited a specific activity of 57 U mg⁻¹ for L-arabinose and 16 U mg⁻¹ for D-galactose, demonstrating the encoding gene was successfully expressed in E. coli. The molecular weight of the PPAI whole enzyme was determined by Superdex S-200 16/60 column using various protein standards (Bio-Rad). Results showed that PPAI is a tetramer with a whole enzyme molecular weight of 232.4 kDa and 58.1 kDa for each subunit (Fig. S3-A and -B†). Previously reported BLAI was characterized to possess a tetrameric folding while BSAI was a dimer in gel filtration and might be homodimer in solution.^21,26 The optimal temperature and pH of PPAI were determined and results were shown in Fig. S4.† The optimal temperature of PPAI is 50 °C while it retained over 80% original activity at the range of 40–55 °C. Above 55 °C, the enzyme activity decreased rapidly. The optimal pH of PPAI is 7.5 while it showed a very broad optimal pH range that retains 80% activity at the pH profile of 6.5 to 10.0. The k_cat values of PPAI free enzyme over different temperature and pH were also determined and showed in Table S2.† Copper ion was reported to be a strong inhibitor for known AIs.¹⁹ However, PPAI showed a high tolerance towards Cu²⁺. 1 mM Cu²⁺ caused no influence on PPAI enzyme activity while 10 mM Cu²⁺ slightly decreased activity to 93% of its original level. Meanwhile, Mg²⁺ increased activity to 113% of the wild-type, which is the highest among tested ions (Table S1†). Surprisingly, the commonly reported activator such as Mn²⁺ and Co²⁺ showed subtle influences on activity. In addition, different concentrations of metal ion caused subtle difference as compared by results of 1 mM and 10 mM ions. These results demonstrate that PPAI showed no requirement for divalent metal ions for its enzyme activity. The blue precipitate was observed at the bottom of the tube after 3 days after mixing PPAI enzyme, PBS buffer, and CuCl₂ solution. The SEM results of the precipitate showed the flower-like structures. With a decreasing concentration of protein (0.5, 0.1, and 0.02 mg mL⁻¹), the number of nucleation sites decreases, resulting in the increased size of hybrid nanoflowers and changes in morphology varied from small buds to blooming flowers (Fig. 1). To carry out a comparison to PPAI, we used the E. coli AI (ECAI) to prepare hybrid nanoflowers. Similar to PPAI, the ECAI nanoflowers were successfully formed but no activity was detected, which might be due to the strong inhibition of Cu²⁺ on ECAI enzyme activity (data not shown). The optimal temperature and pH of the hybrid nanoflowers were the same of the free enzymes (50 °C and pH 7.5). However, it showed higher activities at various temperatures and pH, as shown in Fig. S5.† After immobilization, the activity of the immobilized enzyme for L-arabinose was 45 U mg⁻¹ and that for D-galactose was 9.7 U mg⁻¹, respectively. Thus, the recovery rate of immobilization for L-arabinose was 79% and that for D-galactose was 61%. As a comparison of the kinetic parameters of free and immobilized enzymes, the k_cat for PPAI free enzyme was 9184 min⁻¹ and K_m was 167 mM. For immobilized enzyme, k_cat was 77 695 min⁻¹ and K_m was 1036 mM. Both of the values were determined under optimal conditions. Finally, we determined the thermal and pH stabilities of the PPAI–inorganic hybrid nanoflowers. As shown in Fig. 2, the half-life of nanoflowers at 50 °C (pH 7.5) is 262 h while the free enzyme is 22.6 h (with the addition of 1 mM Mn²⁺). In addition, the half-life under pH 5.0 was determined to be 92 h while the free enzyme quickly lost activity at this condition (half-life is 1.2 h). These results showed that the creation of protein–hybrid nanoflowers significantly improved enzymatic properties of PPAI in both thermal and acidic environments, which are beneficial for industrial applications of AI. Storage stability results showed that the catalytic activity of hybrid nanoflowers retained its original levels for over 31 days. The nanoflowers were stored at 4 °C for 30 days and the activity was observed to decrease slightly (∼9%). The conversion rate utilizing PPAI–hybrid nanoflower after 48 h reaction achieved 61.8% using L-arabinose as substrate while it is 39.9% using D-galactose as substrate (Fig. 3). HPLC analysis revealed the presence of the L-ribulose peak, as shown in Fig. S6.†

Fig. 1 SEM images of hybrid nanoflowers made from PPAI and Cu₃(PO₄)₂. The nanoflowers were obtained from an aqueous solution of 0.02 (a), 0.1 (b), and 0.5 (c) mg mL⁻¹ PPAI enzyme, 120 mM Cu²⁺, 200 mM PBS at pH 7.4 and incubated at 25 °C for three days. (d) Proposed mechanism of the synthesis of hybrid nanoflowers. Yellow spheres indicate protein molecules.

Fig. 2 Catalytic stability of PPAI–hybrid nanoflowers determined at 50 °C and pH 7.5. The red line consists of (■) represents the residual activity of PPAI–hybrid nanoflowers while black line with (●) represents the residual activity of PPAI free enzyme. Determination of the enzyme activity was carried out by the cysteine–carbazole–sulfuric acid method and HPLC.

Fig. 3 Conversion profile of PPAI–hybrid nanoflowers in 48 h using D-galactose or L-arabinose as substrate. The black line (□) represents the conversion rate for L-arabinose and the red line (●) represents the conversion rate for D-galactose.

The protein–inorganic hybrid nanoflower was firstly created by Ge et al. and several enzymes were used to prepare such nanoflowers, including α-lactalbumin, laccase, carbonic anhydrase, and lipase.¹⁶ However, isomerases have not been used to prepare nanoflowers. AI, one of the endol–keto isomerases, was successfully immobilized as protein–inorganic nanoflowers and showed stable catalytic stability. The successful formation of sugar isomerase hybrid nanoflowers showed stable catalytic activities for L-ribulose and D-tagatose production, which can solve the problem of AI stability. This is the first report of protein–inorganic hybrid nanoflower formation using AI. The hybrid nanoflower overcome these problems and showed feasibility.

Recent publications claimed that Ca²⁺ is also suitable for enzyme–inorganic hybrid nanoflowers preparation.²⁹ However, we failed to create PPAI–Ca₃(PO₄)₂ hybrid nanoflowers by the reported method, as shown in Fig. S7.† This implied that protein hybrid nanoflower preparation methods show requirements for the types of participating proteins. Jebors et al. reported an enzyme immobilization method using chemically synthesized Noria and NoriaPG materials to immobilize AI enzyme.³⁰ These materials are proved very effective in enhancing enzyme stability. However, they are also difficult to synthesize and the making process required organic solvents.

Successful attempts have been made to prepare protein–inorganic hybrid nanoflowers utilizing Paenibacillus polymyxa AI as the macromolecular component. The SEM results showed flower like PPAI–Ca₃(PO₄)₂ hybrid nanoflowers, which exhibited better thermal and pH tolerance than the free enzymes. By using these nanoflowers, L-ribulose and D-tagatose were efficiently produced in 24 h. To date, this study showed the utilization of nano-grade inorganic materials to immobilize sugar isomerase, which maintained activity and enhanced catalytic stability.

Acknowledgements

We gratefully acknowledge the financial support from the National Natural Science Foundation of China (31400053 to Z. Xu and 21376203 to J. Gao), the Natural Science Foundation of Jiangsu Province (BK20140933 to Z. Xu), the Jiangsu Province Science and Technology Support Plan Project (BE2015366 to H. Xu), and the State Key Laboratory of Materials-Oriented Chemical Engineering (KL14-01 to B. Chi and KL12-02 to B. Chen).

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Footnotes

† Electronic supplementary information (ESI) available: Experimental details and additional figures. See DOI: 10.1039/c5ra27035a

‡ These authors contribute equally to this work.

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