Expression, Characterization, and Immobilization of a Novel D-Lactate Dehydrogenase from Salinispirillum sp. LH 10-3-1

Abstract

Salinispirillum sp. LH 10-3-1 was newly isolated from the alkali lake water samples collected in Inner Mongolia. In this study, a gene coding for D-lactate dehydrogenase from the strain LH 10-3-1 (SaLDH) was cloned and characterized. The recombinant enzyme was a tetramer with a native molecular mass of 146.2 kDa. The optimal conditions for SaLDH to reduce pyruvate and oxidize D-lactic acid were pH 8.0 and pH 5.0, at 25 °C. Cu²⁺ and Ca²⁺ slightly promoted the oxidation and reduction activities of SaLDH, respectively. To improve the stability of SaLDH, the enzyme was immobilized on Cu₃(PO₄)₂-based inorganic hybrid nanoflowers. The results showed that the reduction activity of the hybrid nanoflowers disappeared, and the optimum temperature, specific activity, thermostability, and storage stability of the immobilized SaLDH were significantly improved. In addition, the biotransformation of D-lactic acid to pyruvate catalyzed by SaLDH and the hybrid nanoflowers was investigated. The maximum conversion of D-lactic acid catalyzed by the immobilized SaLDH was 25.7% higher than by free enzymes, and the immobilized SaLDH could maintain 84% of its initial activity after six cycles.

1. Introduction

D-lactate dehydrogenase (LDH, EC 1.1.1.28) is a key enzyme in the fermentative metabolism of lactic acid bacteria (LAB), which catalyzes the reversible conversion of pyruvate to D-lactic acid with concomitant oxidation of NADH [1]. Nowadays, LDH has become the main enzyme for the production of D-phenyllactic acid (PLA) and optically pure D-lactic acid (LC) [2]. The former (PLA) is a novel and ideal antimicrobial agent in the food industry with a broad spectrum against both bacteria and fungi [3]. The latter (LC) is an important industrial material and has extensive applications in the fields of food, medicine, cosmetics, chemical industry, and environmental protection [4]. In addition, LDH, as an enzyme used for diagnostic biosensors, is also of great significance in identifying the diseases associated with elevated D-lactic acid concentration in urine or serum [5]. So far, LDH has only been found in invertebrates, lower fungi, and prokaryotes, and the catalytic properties of the enzyme for the reduction of pyruvate have been investigated in most reported studies. However, the enzyme characterization for the oxidation of D-lactic acid has been neglected [6]. Furthermore, the low stability of LDH has greatly hampered its industrial application [7].

In order to improve the stability of the enzymes, various immobilization methods including adsorption, encapsulation, cross-linking, and covalent binding have been developed [8,9]. Generally, immobilized enzymes exhibit higher stability than free enzymes [10]. However, in most cases, the enzymatic activities decline after immobilization due to the changes in enzyme conformation and mass transfer resistance between the substrates and enzymes [11]. In 2012, Ge et al. reported a new method for preparing immobilized enzymes, which not only improved the stability of immobilized enzymes but also enhanced their activity [12]. The immobilized enzymes were called “enzymes inorganic hybrid nanoflowers”. Until now, about ten enzymes have been immobilized by this method, and several dual-enzyme and triple-enzyme hybrid nanoflowers have also been reported [13]. However, there has been no report on the immobilization of LDH using the hybrid nanoflowers method, nor has there been any study on the effect of this immobilization method on the performance of the enzyme catalyzing a reversible reaction.

In this study, a novel LDH from the haloalkaliphilic bacterium Salinispirillum sp. LH10-3-1 (termed as SaLDH) was cloned and characterized. The recombinant protein was then used as organic component to prepare SaLDH/Cu₃(PO₄)₂ hybrid nanoflowers. Further, the reduction and oxidation activities of the immobilized SaLDH on pyruvate and lactate were examined and compared with those of free enzyme. The results show that the immobilized SaLDH exhibited catalytic activity only for the one-way reaction of D-lactic acid to pyruvate, while the free enzyme could catalyze the two-way reaction of this reversible conversion. As far as we know, this phenomenon of enzyme-based hybrid nanoflowers has never been reported. In addition, the optimal temperature, specific activity, thermostability, and storage stability of the immobilized SaLDH have been greatly improved. To the best of our knowledge, this is the first report on the characterization of an LDH from Salinispirillum sp.

2. Materials and Methods

2.1. Chemicals and Strains

Sodium pyruvate, NADH, NAD⁺, 2-ketobutyric acid, phenylpyruvic acid, benzoic acid, D-lactic acid, L-lactic acid, D-(+)-malic acid, and D-(+)-3-phenyllactic acid were obtained from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Copper phosphate, sodium chloride, potassium chloride, sodium dihydrogen phosphate, zinc chloride, disodium hydrogen phosphate, methanol, ethanol, acetone, and benzene were purchased from Sinopharm Chemical Reagent Corporation (Shanghai, China). All reagents are commercial analytical grade.

Salinispirillum sp. LH10-3-1 (China General Microbiological Culture Collection Center, CGMCC NO: 1.16635 ^T) was isolated from alkali lake water samples collected in Inner Mongolia. Escherichia coli BL21 (DE3) (Qingke Biology Co., Ltd., Qingdao, China) were used as the host for protein expression.

2.2. Primary and Secondary Structure Analysis

The LDH amino acid sequences sharing high similarities with SaLDH were retrieved from the NCBI database using the BLASTP online service. These sequences were aligned with several functional and verified LDH sequences by the ClustalX2 program. Phylogenetic analysis was performed by the neighbor-joining method using MEGA V11, with one thousand bootstrap replicates. ESPript 3.0 was used to reveal the secondary structure of SaLDH based on the aligned sequences containing LDH from Leuconostoc mesenteroides ATCC 8293 (LEUM_1756), with high pyruvate reductase activity, and that of Pediococcus claussenii (MH920335), with higher PPA reductase activity [14].

2.3. Gene Cloning

According to the nucleotide sequence encoding SaLDH (NCBI accession number OQ845910), a pair of primers (LDH_F: 5′-GGAATTCATGAAAATCGCCGTCT-3′ and LDH_R: 5′-CCCAAGCTTTTATATCTTAACGACGTGA-3′) were designed to amplify SaLDH gene from the genomic DNA of Salinispirillum sp. LH10-3-1 by PCR. The amplified DNA fragment was purified and digested with EcoRI and HindIII endonucleases (Takara, Dalian, China). Then, the double-digested DNA fragment was ligated into the pET28a(+) plasmid between the EcoRI and HindIII sites, and the recombinant plasmid was transformed into E. coli BL (DE3) cells for expression.

2.4. Expression and Purification of SaLDH

The recombinant E. coli BL21 (DE3) was cultivated in LB medium with kanamycin (50 mg/L) at 37 °C until the absorbance at 600 nm reached 0.8. Then, IPTG was added to 0.5 mM of the culture, and protein expression was induced at 25 °C for 10 h. The cells harvested by centrifugation (8500× g, 10 min) were washed and resuspended with Tris-HCl buffer (50 mM, pH 8.0). After cell disruption and centrifugation, the recombinant SaLDH with 6×His-tag in the supernatant was purified by affinity chromatography using a Ni Sephrose 6 Fast Flow column (5 mL, GE Healthcare, Uppsala, Sweden). The detailed process of cell disruption and protein purification was described in our previous works [15]. The purity of the recombinant SaLDH was checked by SDS-PAGE under denaturing conditions.

2.5. Molecular Mass Determination

The molecular mass of the native form of SaLDH was determined by gel filtration as previously described [16]. Briefly, four standard proteins (carbonic anhydrase, glucose oxidase, bovine serum albumin and apoferritin) and SaLDH were applied to a Superdex 200 10/300 GL column (GE Healthcare, Sweden). The molecular mass of native SaLDH was estimated based on the deduced molecular mass curve, which was constructed from the logarithms of the molecular masses of standard proteins versus their elution volumes.

2.6. SaLDH Activity Assay and Protein Quantification

For the reduction activity of SaLDH on pyruvate, the assay mixture (total volume, 1 mL) contained 0.2 mM NADH, 0.6 mM sodium pyruvate, and the relevant amounts of enzyme in Tris-HCl buffer (50 mM, pH 8.0). To measure the oxidation activity of SaLDH on lactate, the reaction mixture (total volume, 1 mL) consisted of 1 mM NAD⁺, 50 mM D-lactic acid, and the appropriate enzyme in a citric acid/sodium citrate buffer (50 mM, pH 5.0). Two assays were carried out at 25 °C. The change in absorbance at 340 nm was monitored with a UV–Vis spectrophotometer (UV-2450, SHIMADZU, Kyoto, Japan). One unit of activity was defined as the amount of enzyme that was catalyzing the degradation or formation of 1 μmol NADH per minute under the corresponding reaction conditions. The protein concentration was determined using Bradford’s method with bovine serum albumin as the standard [17].

2.7. Characterization of SaLDH

The optimum pH and temperature for the reduction and oxidation activities of SaLDH were assessed at pH 3.0–12.0 and 15–60 °C, respectively. To generate the pH gradient, sodium citrate (50 mM, pH 3.0–6.0), Tris-HCl (50 mM, pH 6.0–9.0) and glycine-NaOH (50 mM, pH 9.0–12.0) buffer solutions were used. The thermal stability of SaLDH was also tested by incubation of the enzyme at different temperatures for 12 h, and the residual activity was measured under the standard conditions.

The effects of the metal ions (Zn²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Co²⁺, Ca²⁺, Mg²⁺, and Ba²⁺, final concentration 5 mM), chelating agent EDTA (final concentration 5 mM), organic solvents (DMSO (dimethyl sulfoxide), DMF (N,N-dimethyl formamide), methanol, ethanol, acetone, and benzene, final concentration 5% (v/v)), and NaCl concentration (0–4 M) on SaLDH were determined under the standard activity assay conditions.

To study the substrate specificity of the reduction activity of SaLDH, sodium pyruvate, 2-ketobutyric acid, oxaloacetic acid, sodium phenylpyruvate, α-ketoglutaric acid, benzoic acid, and 3-methyl-2-oxobutyric acid were used as substrates with NADH as a coenzyme. Substrates for the oxidation activity of SaLDH included D-lactic acid, D-(+)-malic acid, and D-(+)-3-phenyllactic acid with NAD⁺ as a coenzyme. The kinetic parameters (K_m and V_max) for sodium pyruvate, NADH, D-lactic acid, and NAD+ were determined by incubating the enzyme with substrates of various concentrations (0–50 mM) under optimal conditions.

2.8. Synthesis and Characterization of SaLDH/Cu₃(PO₄)₂ Hybrid Nanoflowers

To synthesize the SaLDH/Cu₃(PO₄)₂ hybrid nanoflowers, 1 mL of copper phosphate aqueous solution (0.1 M) was added into 3 mL of PBS solution (0.2 M, pH 7.4) containing 12 mg SaLDH. After incubation at 4 °C for 36 h, the blue precipitate was collected through centrifugation at 9000× g for 10 min. The obtained precipitate was washed thrice with pure water and dried by vacuum freezing.

The morphology, crystal phase, and chemical components of SaLDH/Cu₃(PO₄)₂ hybrid nanoflowers were analyzed by Scanning Electron Microscope (SEM, S-4800, HITACHI, Tokyo, Japan), X-ray diffraction (XRD, PANalytical Empyrean, Alemlo, Netherlands), and Fourier Transform Infrared Spectroscopy (FT-IR, Nicolet 6700, Madison, WI, USA), respectively.

2.9. Catalytic Properties of SaLDH/Cu₃(PO₄)₂ Hybrid Nanoflowers

The reduction and oxidation activities of the SaLDH/Cu₃(PO₄)₂ hybrid nanoflowers were determined using the standard enzyme assay protocol, but the free enzymes (SaLDH) was replaced by the nanoflowers containing the same amount of enzyme.

The optimal catalytic pH and temperature for SaLDH/Cu₃(PO₄)₂ hybrid nanoflowers were evaluated at pH 3.0–12.0 and 20–70 °C, respectively. To investigate thermostability, the immobilized enzyme was incubated at 20–80 °C for 12 h, and the residual activities were tested. The storage stability of free, the immobilized SaLDH was assessed at 4 °C for 35 days, and the residual activities were measured weekly.

2.10. Biotransformation of D-Lactic Acid by Free and Immobilized SaLDH

The biotransformation of D-lactic acid (50 mM) and NAD⁺ (100 mM) catalyzed by the free SaLDH (0.1 mg/mL) and SaLDH/Cu₃(PO₄)₂ hybrid nanoflowers (enzyme content 0.1 mg/mL) was performed under the optimal conditions of a catalyst. D-lactic acid was quantified by UV absorbance as previously described [18].

In addition, the reusability of the SaLDH/Cu₃(PO₄)₂ hybrid nanoflowers was also investigated. After the reaction was completed, the immobilized enzyme was recovered by centrifugation at 10,000× g for 5 min, and washed with glycine-NaOH (50 mM, pH 8.0) buffer. Then, the washed sample was reused for the next catalytic cycle, and the activity of SaLDH in the first cycle was set as 100%.

3. Results

3.1. Sequence Analysis of SaLDH

The SaLDH gene from Salinispirillum sp. LH10-3-1 consists of 993 base pairs and encodes 330 amino acids (NCBI accession No. OQ845910). Alignment analysis of SaLDH with reported LDHs (Figure 1) revealed the substrate binding sites, His-203, Arg-233, Glu-262, His-294, Asp-257 [7], and the conserved GXGXXG (X represents any of the 20 amino acids) motif to bind dinucleotide at positions 151–157 [14].

To reveal the evolutionary status of SaLDH, a phylogenetic tree based on the LDHs of Salinispirillum sp. LH10-3-1 and other microorganisms was constructed based on the amino sequences using the neighbor-joining method (Figure 2). The LDH sequences with the highest homology were included in the phylogenetic tree analysis along, with several functional and verified LDH enzymes. The results show that SaLDH falls into a group comprising LDHs which have originated from a marine environment, such as a mangrove forest (Hahella sp. CCB-MM4 [19], Mangrovitalea sediminis [20]), sea urchin (Colwellia echini) [21], and intertidal sediment (Marinobacter antarcticus) [22]. SaLDH were classified into a small clade with the LDHs of Natronospirillum operosum sp., which shares the highest identity of 75.45%.

3.2. Enzyme Purification and Molecular Mass Determination

The total reduction activity and oxidation activity from the initial crude extract were 4603.7 U and 3161.4 U, respectively. After the affinity chromatography step, the specific reduction activity and oxidation activity of the purified SaLDH were 107.5 U/mg and 40.9 U/mg, respectively, with activity yields of 36.1% and 20.0% (

Table 1. Comparison of SaLDH specific enzyme activity and yield.

Sample	Specific Activity (U/mg)		Yield (%)
	Reduction Activity	Oxidation Activity	Reduction Activity	Oxidation Activity
Initial crude extract	8.0	5.5	100.0	100.0
Purified SaLDH	107.5	40.9	36.1 a	20.0
Immobilized SaLDH	0	66.3	0	162.1 b

^a The yield = (total reduction activity in initial crude extract/ total reduction activity of purified SaLDH) × 100%. ^b The yield = (specific activity of immobilized SaLDH/specific activity of purified SaLDH) × 100%.

).

SDS-PAGE analysis of the purified SaLDH (Figure 3a) showed a single major band near 37 kDa, which was close to the theoretical value of the enzyme (36.3 kDa). The native molecular mass of SaLDH was determined to be approximately 146.2 kDa on the Superdex 200 10/300 GL column (Figure 3b). Thus, the recombinant SaLDH should be a tetramer.

3.3. Effects of pH and Temperature on SaLDH Activity

The optimal pH values for the reduction and oxidation activity of SaLDH were pH 8.0 and pH 5.0 (Figure 4a), respectively, and the optimal temperature was 25 °C (Figure 4b). In addition, except for the optimal temperature, the relative reduction activity of SaLDH was always higher than its relative oxidation activity, especially in the temperature range of 35 °C to 60 °C. Figure 4c illustrated the thermostability of SaLDH. It can be seen that the thermostability of the enzyme was not ideal, and it would quickly deactivate when the temperature exceeded 35 °C.

3.4. Effects of Metal Ions, Organic Solvents, and NaCl Concentration on SaLDH Activity

The effects of metal ions and organic solvents on the activity of SaLDH were also investigated. As shown in

Table 2. Effect of metal ions, EDTA, and organic solvents on SaLDH activity.

Additive	Relative Activity (%)
	Activity with Pyruvate	Activity with Lactate
Control	100.0 ± 0.0	100.0 ± 0.0
Zn2+	86.3 ± 1.6	78.9 ± 1.5
Cu2+	24.2 ± 0.4	107.9 ± 0.6
Mn2+	52.5 ± 0.3	64.4 ± 0.9
Co2+	102.3 ± 1.2	101.2 ± 0.3
Ca2+	105.8 ± 0.5	31.7 ± 0.4
Mg2+	63.9 ± 1.1	65.3 ± 1.8
Ba2+	61.6 ± 0.5	71.9 ± 0.6
Fe3+	73.0 ± 0.9	73.6 ± 1.3
EDTA	96.5 ± 1.2	95.3 ± 1.4
DMSO	99.9 ± 2.1	98.9 ± 1.9
DMF	99.9 ± 1.8	99.0 ± 2.1
Methanol	99.7 ± 2.5	98.6 ± 1.8
Ethanol	99.3 ± 1.4	97.3 ± 1.7
Acetone	98.8 ± 1.7	97.1 ± 2.1
Benzene	90.8 ± 2.2	91.2 ± 1.9

, Cu²⁺ slightly stimulated the oxidation activity of SaLDH, but strongly inhibited the reduction activity of the enzyme. On the contrary, Ca²⁺ had a promoting effect on the reduction activity of SaLDH but led to a loss of 68.3% in the enzyme’s oxidation activity. In addition, Co²⁺ had little effect on both the reduction and oxidation activities of SaLDH, while the other metal ions had mild or moderate inhibitory effects on enzyme activity. The presence of EDTA did not significantly affect the activity of SaLDH, indicating that the enzyme was not metal-dependent. In the organic solvents studied (Table 2), DMSO, DMF, methanol, ethanol, and acetone had no effect on the activity of SaLDH, while the inhibitory effect of benzene on SaLDH activity was about 10%.

Figure 4d shows the influence of NaCl concentration on SaLDH activity. It can be seen that the enzyme had excellent salt resistance and could still maintain nearly 90% enzyme activity at a NaCl concentration of 4.0 M. Another result obtained from Figure 4d is that the relative reduction activity of SaLDH was higher than its relative oxidation activity within the NaCl concentration range of 0.5–4.0 M.

3.5. Substrate Specificity and Kinetic Parameters of SaLDH

The substrate specificity of SaLDH was examined using various potential substrates. As shown in

Table 3. Substrate specificity of SaLDH.

Substrates	Relative Activity (%)	Specific Activity (U/mg)
Sodium pyruvate	100.0	107.5
2-Ketobutyric acid	97.9	105.2
Oxaloacetic acid	74.2	79.8
Phenylpyruvic acid	30.3	32.6
α-Ketoglutaric acid	79.8	85.8
Benzoic acid	69.2	74.4
D-lactic acid	100.0	40.9
L-lactic acid	0.0	0.0
D-(+)-Malic acid	88.4	36.2
D-(+)-3-Phenyllactic acid	84.6	34.6

, SaLDH had reduction activity toward sodium pyruvate (relative activity, 100%), 2-ketobutyric acid (97.9%), α-ketoglutaric acid (79.8%), benzoic acid (69.2%), oxaloacetic acid (74.2%), and phenylpyruvic acid (30.3%). As for the oxidation activity, D-lactic acid (100%), D-(+)-malic acid (88.4%), and D-(+)-3-phenyllactic acid (84.6%) were substrates of SaLDH, but L-lactic acid was inert.

The kinetic parameters of SaLDH were determined using sodium pyruvate, NADH, D-lactic acid, and NAD+ as substrates (

Table 4. The apparent Km values of LDHs from different microorganisms to pyruvate, NADH, D-lactic acid and NAD⁺.

Strains	Km (mM)				References
	Pyruvate	NADH	D-Lactic Acid	NAD+
Salinispirillum sp. LH 10-3-1	0.34	0.52	1.71	0.46	This work
Bacillus coagulans	5.9	n.d.	5.94	n.d.	[1]
Leuconostoc mesenteroides	0.09	0.05	n.d.	n.d.	[23]
Lacobacillus bulgaricus	1.6	n.d.	133	n.d.	[24]
L. plantarum	1.2	n.d.	7.0	n.d.	[25]
L. helveticus CNRZ 32	0.64	n.d.	68	n.d.	[26]
Staphylococcus sp. LDH-1	0.67	n.d.	8.5	n.d.	[27]

Note: n.d., not determined.

). It can be found that the K_m values of the above four substrates were estimated to be 0.34, 0.52, 1.71, and 0.46 mM, respectively. Therefore, SaLDH had the highest affinity for sodium pyruvate. In addition, the V_max of the four substrates were determined to be 561.7, 240.5, 197.2, and 383.6 U/mg, respectively.

3.6. Synthesis and Characterization of SaLDH/Cu₃(PO₄)₂ Hybrid Nanoflowers

The SaLDH/Cu₃(PO₄)₂ hybrid nanoflowers were synthesized from 3.0 mg/mL SaLDH and 25 mM copper phosphate. The immobilization yields of this immobilization method based on enzyme weight and specific enzyme activity were 73.9% and 162.1%, respectively. After vacuum freeze-drying, the resulting SaLDH/Cu₃(PO₄)₂ hybrid nanoflowers appeared as a blue powder. The morphology of the nanoflowers was determined by SEM. In the low-resolution SEM image shown in Figure 5a, the nanoflowers were spherical, with uniform structure and good monodispersity. However, the high-resolution SEM image (Figure 5b) exhibited a layered flower pattern. In addition, it can also be seen from Figure 4a that the diameter of most nanoflowers was about 5 μm.

To investigate the composition of SaLDH/Cu₃(PO₄)₂ hybrid nanoflowers, FT-IR analysis was performed. The FT-IR spectra of SaLDH, Cu₃(PO₄)₂ particles, and SaLDH/Cu₃(PO₄)₂ hybrid nanoflowers were shown in Figure 5c. As can be seen, the nanoflowers contained the main characteristic absorption peaks of SaLDH and Cu₃(PO₄)₂ particles, and no new absorption peak and obvious peak shift were found. Therefore, SaLDH was immobilized on the nanoflowers by self-assembly instead of covalent bonds [12,28]. Additionally, the absorption peaks of the main functional groups in the FT-IR spectrum are listed in Supplementary Table S1.

Figure 5d illustrates the XRD patterns of the Cu₃(PO₄)₂·3H₂O and SaLDH/Cu₃(PO₄)₂ hybrid nanoflowers. It can be found that all diffraction peaks of the nanoflowers were same as those of Cu₃(PO₄)₂·3H₂O, and the peaks presented in both curves can be indexed to the JCPDS card no. 22-0548. These results indicate that the SaLDH/Cu₃(PO₄)₂ hybrid nanoflowers were well crystallized and the inorganic composition of the nanoflowers was copper phosphate trihydrate [29].

3.7. Catalytic Properties of SaLDH/Cu₃(PO₄)₂ Hybrid Nanoflowers

Before characterizing the properties of the SaLDH/Cu₃(PO₄)₂ hybrid nanoflowers, their catalytic activities for the reversible reaction between pyruvate and D-lactic acid were first studied using a standard enzyme assay. The results showed that the immobilized enzyme exhibited catalytic activity only for the one-way reaction of D-lactic acid to pyruvate. Therefore, the oxidation activity of the nanoflowers was subsequently examined.

Figure 6a,b showed the effects of pH and temperature on the oxidation activity of SaLDH/Cu₃(PO₄)₂ hybrid nanoflowers. It can be seen that the optimum reaction conditions of the immobilized enzyme were pH 8.0 and 50 °C. Under these optimal conditions, the specific activity of the nanoflowers reached 66.3 U/mg (Table 1), which was 1.62 times that of SaLDH (40.9 U/mg). In addition, the nanoflowers maintained over 80% of their maximum oxidation activity between pH 6.0 and 9.0 and from 40 to 55 °C, respectively. Furthermore, the immobilized enzyme exhibited much higher thermostability than free SaLDH (Figure 6c), especially in the temperature range of 40–65 °C. For example, after incubation at 60 °C for 12 h, the nanoflowers retained 72.3% of their initial activity, while the residual activity of free SaLDH was only 9.8%.

The storage stability of the free and immobilized SaLDH was determined at 4 °C for 35 days and the results are shown in Figure 6d. As can be seen, the activity profile of the free SaLDH decreased much faster than that of immobilized enzyme. After 35 days of storage at 4 °C, the free SaLDH lost nearly 90% of its initial activity, while the nanoflowers only lost 47% of their initial activity.

3.8. Biotransformation of D-Lactic Acid by Free and Immobilized SaLDH

The biotransformation of D-lactic acid to pyruvate catalyzed by the free SaLDH (pH 5.0, 25 °C, enzyme content: 0.1 mg/mL) and SaLDH/Cu₃(PO₄)₂ hybrid nanoflowers (pH 8.0, 50 °C, enzyme content: 0.1 mg/mL) was performed. As shown in Figure 7a, the maximum conversion of D-lactic acid catalyzed by the free and immobilized SaLDH was 59.6% and 85.3%, respectively. As for the reusability of the nanoflowers (Figure 7b), it was found that the immobilized enzyme could maintain 93%, 86%, and 78% of its initial activity after 4, 6, and 8 cycles, respectively.

4. Discussion

LDH is not only a critical enzyme in the fermentation metabolism of LAB [1,30], but also an important industrial enzyme that can be used for medical diagnosis [5], and the production of PLA and optically pure LC [3,31]. Presently, most of the reported LDHs are found in LAB strains [32], but due to their low stability, it is necessary to search for new sources [7,33]. In this work, a novel LDH from a haloalkaliphilic bacterium Salinispirillum sp. LH10-3-1 (SaLDH) was cloned, characterized, and immobilized. To our knowledge, this is the first report on the characterization of an LDH from Salinispirillum sp.

Alignment analysis of the SaLDH with reported LDHs indicated that His-203, Arg-233, Glu-262, His-294, and Asp-257 are essential residues for substrate binding and catalytic activity of LDH [20,34]. Their location in the active site of the enzyme has been confirmed by the crystal structure of the LDH from L. helveticus [35]. The pairwise distance analysis indicated that the similarity between SaLDH and the other LDHs ranged from 29% to 75%, and the highest identity LDH with SaLDH came from N. operosum, which was a haloalkaliphilic bacterium isolated from cyanobacterium biomass. The topological structure of the phylogenetic tree indicated that SaLDH was clustered with LDHs that originated from marine habitats. This marine LDH group is located quite distinctly from the LDHs described in the previous publications, including those from lactic acid bacteria, which implies that it could possess unique property traits. At this point, no investigation had been performed regarding the LDH proteins of this marine cluster, therefore research on the enzymatic characteristics of SaLDH might shed some light on the enzymatic property preferences for LDH proteins of this marine cluster.

After molecular mass determination, SaLDH was found to be a tetramer with a molecular mass of 146.2 kDa (gel filtration chromatography) or 37 kDa (SDS-PAGE). The tetrameric structure of LDH is rarely reported, because LDHs mostly exist in the form of homodimers with subunit molecular mass of 32–40 kDa [7,32]. In addition, a dimeric LDH with subunit molecular mass of 48 kDa (beyond the above range) was also found in Sulfolobus tokodaii strain 7 [36].

The optimal pH value for the reduction activity of LDH varies greatly in the literature, ranging from 4.0 to 8.6 [26]. In this work, SaLDH exhibited the highest reduction activity at pH 8.0, within the aforementioned pH range. However, the optimum pH for the oxidation activity of SaLDH (pH 5.0) is the lowest among the known LDHs (optimal pH: 7.8–9.6) [37]. In addition, the optimal temperature of SaLDH (25 °C) was the same as that of LDH from L. murinum [27], and this value is the smallest among the reported LDHs [37]. Regarding the thermostability of LDH, it is difficult to compare the literature data due to different testing methods. SaLDH was only stable below 35 °C, but its thermostability could be improved through gene mutation [38] or immobilization [23].

As for the effect of metal ions on the reduction activity of LDH, it was reported that Mg²⁺ and Mn²⁺ were activators while Ca²⁺ was an inhibitor for the enzyme of Lactobacillus sp. ZX1 [28]. On the contrary, Ca²⁺ slightly stimulated SaLDH activity, while Mg²⁺ and Mn²⁺ moderately inhibited enzyme activity. A similar phenomenon was also observed in the oxidative activity of LDH. For example, Cu²⁺ was found to be a strong inhibitor for the enzyme of S. tokodaii strain 7 [36], while Cu²⁺ had a slight promoting effect on the oxidation activity of SaLDH. These results indicated that the structures of LDHs from different sources might be different under the stimulation of the same metal ions [39].

The influences of organic solvents and NaCl concentration on LDH activity have rarely been reported in the literature. In this study, it was found that DMSO, DMF, methanol, ethanol, and acetone did not affect both reduction and oxidation activities of SaLDH. In addition, even at a NaCl concentration of 4.0 M, the enzyme could maintain nearly 90% of its original activity. The salt tolerance of SaLDH may be related to the fact that Salinispirillum sp. LH10-3-1 is a haloalkaliphilic bacterium [16]. The excellent organic solvent and salt resistance of SaLDH will be beneficial for its application in partially hydrophobic and high salt environments.

In the previous literature, LDH has been reported to have broad substrate specificity [24,32]. This property is shared by SaLDH of Salinispirillum sp. LH10-3-1 (See Table 3). In addition, the apparent K_m value of SaLDH for pyruvate (or D-lactic acid, Table 4) was lower than those from B. coagulans [1], L. bulgaricus [24], L. plantarum [40], L. helveticus CNRZ 32 [26], and Staphylococcus sp. LDH-1 [27]. Therefore, SaLDH possesses better substrate affinity than the aforementioned LDHs.

Considering that the thermostability of SaLDH is not ideal (only stable below 35 °C), the enzyme was immobilized using the hybrid nanoflowers method [28]. The analysis results of SEM, FT-IR, and XRD (Figure 5) confirmed the successful synthesis of SaLDH/Cu₃(PO₄)₂ hybrid nanoflowers [41]. The reason for choosing copper phosphate is that Cu²⁺ not only promotes the oxidation activity of SaLDH, but also strongly inhibits its reduction activity. We expected that through the immobilization process, SaLDH would become the catalyst for an irreversible reaction thus improving the conversion rate of the substrate. The results showed that the reduction activity of immobilized SaLDH completely disappeared as expected. This phenomenon of enzyme-based hybrid nanoflowers has never been reported before. In order to make sure the immobilized SaLDH only have reduction activity, the SaLDH/Ca₃(PO₄)₂ hybrid nanoflowers are currently being synthesized based on the effect of Ca²⁺ on enzyme activity.

In addition, the comparison of catalytic properties between the free SaLDH and hybrid nanoflowers showed that the optimum temperature and specific activity of immobilized SaLDH increased by 25 °C (Figure 6b) and 62%, respectively. Furthermore, the thermostability (Figure 6c) of hybrid nanoflowers was also significantly improved. Similar improvements in enzymatic properties were observed in the hybrid nanoflowers composed of other enzymes such as laccase [29] and urease [42], which may be due to the enhanced stiffness and conformational flexibility of enzyme-based hybrid nanoflowers to prevent conformational changes at high temperatures [11]. Furthermore, the enhanced activity of SaLDH in nanoflowers may be attributed to the high surface area of the nanoflower, high optimal temperature, and the interactions between SaLDH and the nanoscale microenvironment containing Cu²⁺ ions [29].

Since the immobilized SaLDH only had oxidation activity, this study investigated the biotransformation of D-lactic acid to pyruvate catalyzed by the free SaLDH and SaLDH/Cu₃(PO₄)₂ hybrid nanoflowers. The results showed that the maximum conversion of D-lactic acid catalyzed by the immobilized SaLDH was 25.7% higher than by the free enzyme (Figure 7a). In addition, the immobilized SaLDH could maintain 84% of its initial activity after six cycles (Figure 7b), indicating that the hybrid nanoflowers had good operational stability.

In summary, this work provides a novel D-lactate dehydrogenase from Salinispirillum sp. LH10-3-1. After systematic characterization and comparison of the reducing and oxidizing activities of SaLDH, the enzyme was immobilized on the Cu₃(PO₄)₂-based inorganic hybrid nanoflowers. With the disappearance of reducing activity, the specific activity, optimal temperature, thermostability, storage stability and operational stability of the oxidation activity of the immobilized SaLDH was significantly improved. These results will expand people’s understanding of the enzyme and provide new ideas for the selection of metal ions in the synthesis of organic–inorganic nanoflowers. In addition, these outstanding characteristics make immobilized SaLDH a promising biocatalyst for industrial application.