Engineering of a fungal β-galactosidase to remove product inhibition by galactose

β-galactosidase is an enzyme administered as a digestive supplement to treat lactose intolerance, a genetic condition prevalent in most world regions. The gene encoding an acid-stable β-galactosidase potentially suited for use as a digestive supplement was cloned from Aspergillus niger van Tiegh, sequenced and expressed in Pichia pastoris. The purified recombinant protein exhibited kinetic properties similar to those of the native enzyme and thus was also competitively inhibited by its product, galactose, at application-relevant concentrations. In order to alleviate this product inhibition, a model of the enzyme structure was generated based on a Penicillium sp. β-galactosidase crystal structure with bound β-galactose. This led to targeted mutagenesis of an Asp²⁵⁸-Ser-Tyr-Pro-Leu-Gly-Phe amino acid motif in the A. niger van Tiegh enzyme and isolation from the resultant library of a mutant β-galactosidase enzyme with reduced sensitivity to inhibition by galactose (K _i of 6.46 mM galactose, compared with 0.76 mM for the wildtype recombinant enzyme). The mutated enzyme also exhibited an increased K _m (3.76 mM compared to 2.21 mM) and reduced V _max (110.8 μmol min⁻¹ mg⁻¹ compared to 172.6 μmol min⁻¹ mg⁻¹) relative to the wild-type enzyme, however, and its stability under simulated fasting gastric conditions was significantly reduced. The study nevertheless demonstrates the potential to rationally engineer the A. niger van Tiegh enzyme to relieve product inhibition and create mutants with improved, application-relevant kinetic properties for treatment of lactose intolerance.     

d-Amino acid dehydrogenase from Helicobacter pylori NCTC 11637

Helicobacter pylori is a microaerophilic bacterium, associated with gastric inflammation and peptic ulcers. d-Amino acid dehydrogenase is a flavoenzyme that digests free neutral d-amino acids yielding corresponding 2-oxo acids and hydrogen. We sequenced the H. pylori NCTC 11637 d-amino acid dehydrogenase gene, dadA. The primary structure deduced from the gene showed low similarity with other bacterial d-amino acid dehydrogenases. We purified the enzyme to homogeneity from recombinant Escherichia coli cells by cloning dadA. The recombinant protein, DadA, with 44 kDa molecular mass, possessed FAD as cofactor, and showed the highest activity to d-proline. The enzyme mediated electron transport from d-proline to coenzyme Q₁, thus distinguishing it from d-amino acid oxidase. The apparent K _m and V _max values were 40.2 mM and 25.0 μmol min⁻¹ mg⁻¹, respectively, for dehydrogenation of d-proline, and were 8.2 μM and 12.3 μmol min⁻¹ mg⁻¹, respectively, for reduction of Q₁. The respective pH and temperature optima were 8.0 and 37°C. Enzyme activity was inhibited markedly by benzoate, and moderately by SH reagents. DadA showed more similarity with mammalian d-amino acid oxidase than other bacterial d-amino acid dehydrogenases in some enzymatic characteristics. Electron transport from d-proline to a c-type cytochrome was suggested spectrophotometrically.     

NADH oxidation drives respiratory Na+ transport in mitochondria from Yarrowia lipolytica

It is generally assumed that respiratory complexes exclusively use protons to energize the inner mitochondrial membrane. Here we show that oxidation of NADH by submitochondrial particles (SMPs) from the yeast Yarrowia lipolytica is coupled to protonophore-resistant Na⁺ uptake, indicating that a redox-driven, primary Na⁺ pump is operative in the inner mitochondrial membrane. By purification and reconstitution into proteoliposomes, a respiratory NADH dehydrogenase was identified which coupled NADH-dependent reduction of ubiquinone (1.4 μmol min⁻¹ mg⁻¹) to Na⁺ translocation (2.0 μmol min⁻¹ mg⁻¹). NADH-driven Na⁺ transport was sensitive towards rotenone, a specific inhibitor of complex I. We conclude that mitochondria from Y. lipolytica contain a NADH-driven Na⁺ pump and propose that it represents the complex I of the respiratory chain. Our study indicates that energy conversion by mitochondria does not exclusively rely on the proton motive force but may benefit from the electrochemical Na⁺ gradient established by complex I. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning, biochemical characterisation, tissue localisation and possible post-translational regulatory mechanism of the cytosolic phosphoglucose isomerase from developing sunflower seeds

Lipid biosynthesis in developing sunflower (Helianthus annuus L.) seeds requires reducing power. One of the main sources of cellular NADPH is the oxidative pentose phosphate pathway (OPPP), generated from the oxidation of glucose-6-phosphate. This glycolytic intermediate, which can be imported to the plastid and enter in the OPPP, is the substrate and product of cytosolic phosphoglucose isomerase (cPGI, EC 5.3.1.9). In this report, we describe the cloning of a full-length cDNA encoding cPGI from developing sunflower seeds. The sequence was predicted to code for a protein of 566 residues characterised by the presence of two sugar isomerase domains. This cDNA was heterologously expressed in Escherichia coli as a His-tagged protein. The recombinant protein was purified using immobilised metal ion affinity chromatography and biochemically characterised. The enzyme had a specific activity of 1,436 μmol min⁻¹ mg⁻¹ and 1,011 μmol min⁻¹ mg⁻¹ protein when the reaction was initiated with glucose-6-phosphate and fructose-6-phosphate, respectively. Activity was not affected by erythrose-4-phosphate, but was inhibited by 6-P gluconate and glyceraldehyde-3-phosphate. A polyclonal immune serum was raised against the purified enzyme, allowing the study of protein levels during the period of active lipid synthesis in seeds. These results were compared with PGI activity profiles and mRNA expression levels obtained from Q-PCR studies. Our results point to the existence of a possible post-translational regulatory mechanism during seed development. Immunolocalisation of the protein in seed tissues further indicated that cPGI is highly expressed in the procambial ring.     

Biochemical characterization of the carotenoid 1,2-hydratases (CrtC) from <Emphasis Type="Italic">Rubrivivax gelatinosus</Emphasis> and <Emphasis Type="Italic">Thiocapsa roseopersicina</Emphasis>

Two carotenoid 1,2-hydratase (CrtC) genes from the photosynthetic bacteria Rubrivivax gelatinosus and Thiocapsa roseopersicina were cloned and expressed in Escherichia coli in an active form and purified by affinity chromatography. The biochemical properties of the recombinant enzymes and their substrate specificities were studied. The purified CrtCs catalyze cofactor independently the conversion of lycopene to 1-HO- and 1,1′-(HO)₂-lycopene. The optimal pH and temperature for hydratase activity was 8.0 and 30°C, respectively. The apparent K _m and V _max values obtained for the hydration of lycopene were 24 μM and 0.31 nmol h⁻¹ mg⁻¹ for RgCrtC and 9.5 μM and 0.15 nmol h⁻¹ mg⁻¹ for TrCrtC, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed two protein bands of 44 and 38 kDa for TrCrtC, which indicate protein processing. Both hydratases are also able to convert the unnatural substrate geranylgeraniol (C20 substrate), which functionally resembles the natural substrate lycopene.     

High expression of recombinant <Emphasis Type="Italic">Streptomyces</Emphasis> sp. S38 xylanase in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by codon optimization and analysis of its biochemical properties

In recent years, the biotechnological use of xylanases has grown remarkably. To efficiently produce xylanase for food processing and other industry, a codon-optimized recombinant xylanase gene from Streptomyces sp. S38 was synthesized and extracellularly expressed in Pichia pastoris under the control of AOX1 promoter. SDS-PAGE and activity assay demonstrated that the molecular mass of the recombinant xylanase was estimated to be 25 kDa, the optimum pH and optimum temperature were 5.5 and 50°C, respectively. In shake flask culture, the specific activity of the xylanase activity was 5098.28 U/mg. The K _m and V _max values of recombinant xylanase were 11.0 mg/ml and 10000 μmol min⁻¹ mg⁻¹, respectively. In the presence of metal ions such as Ca²⁺, Cu²⁺, Cr³⁺ and K⁺, the activity of the enzyme increased. However, strong inhibition of the enzyme activity was observed in the presence of Hg²⁺. This is the first report on the expression properties of a recombinant xylanase gene from the Streptomyces sp. S38 using Pichia pastoris. The attractive biochemical properties of the recombinant xylanase suggest that it may be a useful candidate for variety of commercial applications.     

Genetic and biochemical characterization of a protease-resistant mesophilic β-mannanase from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. S27

A β-mannanase gene, designated as man5S27, was cloned from Streptomyces sp. S27 using the colony polymerase chain reaction (PCR) method and expressed in Escherichia coli BL21 (DE3). The open reading frame consisted of 1,161 bp and encoded a 386-amino-acid polypeptide (Man5S27) with calculated molecular mass of 37.2 kDa. The encoded protein comprised a putative 38-residue signal peptide, a family 5 glycoside hydrolase domain, and a family 10 carbohydrate-binding module. Purified recombinant Man5S27 had high specific activity of 2,107 U mg⁻¹ and showed optimal activity at pH 7.0 and 65°C. The enzyme remained stable at pH 5.0–9.0 and had good thermostability at 50°C. The K _m values for locust bean gum and konjac flour were 0.16 and 0.41 mg ml⁻¹, with V _max values of 3,739 and 1,653 μmol min⁻¹ mg⁻¹, respectively. Divalent metal ions such as Mn²⁺, Zn²⁺, Ca²⁺, Pb²⁺, and Fe²⁺ enhanced the enzyme activity, but Ag⁺ and Hg²⁺ strongly inhibited the activity. Man5S27 also showed resistance to various neutral proteases (retaining >95% activity after proteolytic treatment for 2 h).     

Characterization of PSI recovery after chilling-induced photoinhibition in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) leaves

By simultaneously analyzing the chlorophyll a fluorescence transient and light absorbance at 820 nm as well as chlorophyll fluorescence quenching, we investigated the effects of different photon flux densities (0, 15, 200 μmol m⁻² s⁻¹) with or without 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on the repair process of cucumber (Cucumis sativus L.) leaves after treatment with low temperature (6°C) combined with moderate photon flux density (200 μmol m⁻² s⁻¹) for 6 h. Both the maximal photochemical efficiency of Photosystem II (PSII) (F _v/F _m) and the content of active P700 (ΔI/I _o) significantly decreased after chilling treatment under 200 μmol m⁻² s⁻¹ light. After the leaves were transferred to 25°C, F _v/F _m recovered quickly under both 200 and 15 μmol m⁻² s⁻¹ light. ΔI/I _o recovered quickly under 15 μmol m⁻² s⁻¹ light, but the recovery rate of ΔI/I _o was slower than that of F _v/F _m. The cyclic electron transport was inhibited by chilling-light treatment obviously. The recovery of ΔI/I _o was severely suppressed by 200 μmol m⁻² s⁻¹ light, whereas a pretreatment with DCMU effectively relieved this suppression. The cyclic electron transport around PSI recovered in a similar way as the active P700 content did, and the recovery of them was both accelerated by pretreatment with DCMU. The results indicate that limiting electron transport from PSII to PSI protected PSI from further photoinhibition, accelerating the recovery of PSI. Under a given photon flux density, faster recovery of PSII compared to PSI was detrimental to the recovery of PSI or even to the whole photosystem.     

A Novel β-Agarase with High pH Stability from Marine Agarivorans sp. LQ48

A novel endo-type β-agarase gene, agaA, was cloned from a newly isolated marine bacterium, Agarivorans sp. LQ48. It encodes a protein of 457 amino acids with a calculated molecular mass of 51.2 kDa. The deduced protein contains a typical N-terminal signal peptide of 25 amino acid residues, followed by a catalytic module, which is homologous to that of glycoside hydrolase family 16. A sequence similar to a carbohydrate-binding module is found in the C-terminal region of the enzyme. The overall amino acid sequence shares a highest identity of 73% with the sequence of beta-agarase AgaB from Pseudoalteromonas sp. strain CY24. The mature agarase was highly expressed extracellularly in Escherichia coli. At pH 7.0 and 40°C, the purified recombinant AgaA had a high specific activity of 349.3 μmol min⁻¹ mg⁻¹, a K _m of 3.9 mg ml⁻¹, and a V _max of 909.1 μmol min⁻¹ mg⁻¹ for agarose. The recombinant enzyme hydrolyzed the β-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main products. Enzyme activity analysis revealed that the optimal temperature and pH of the recombinant AgaA were 40°C and 7.0, respectively. Notably, AgaA still retained more than 95% activity after incubation at pH 3.0–11.0 for 1 h, a characteristic much different from other agarases reported. It is the first agarase identified to have so wide a pH range stability. This favorable property could make AgaA to be attractive to the food, cosmetic, and medical industrial applications.     

Characterization of Xyn10A, a highly active xylanase from the human gut bacterium Bacteroides xylanisolvens XB1A

A xylanase gene xyn10A was isolated from the human gut bacterium Bacteroides xylanisolvens XB1A and the gene product was characterized. Xyn10A is a 40-kDa xylanase composed of a glycoside hydrolase family 10 catalytic domain with a signal peptide. A recombinant His-tagged Xyn10A was produced in Escherichia coli and purified. It was active on oat spelt and birchwood xylans and on wheat arabinoxylans. It cleaved xylotetraose, xylopentaose, and xylohexaose but not xylobiose, clearly indicating that Xyn10A is a xylanase. Surprisingly, it showed a low activity against carboxymethylcellulose but no activity at all against aryl-cellobioside and cellooligosaccharides. The enzyme exhibited K _m and V _max of 1.6 mg ml⁻¹ and 118 μmol min⁻¹ mg⁻¹ on oat spelt xylan, and its optimal temperature and pH for activity were 37°C and pH 6.0, respectively. Its catalytic properties (k _cat/K _m = 3,300 ml mg⁻¹ min⁻¹) suggested that Xyn10A is one of the most active GH10 xylanase described to date. Phylogenetic analyses showed that Xyn10A was closely related to other GH10 xylanases from human Bacteroides. The xyn10A gene was expressed in B. xylanisolvens XB1A cultured with glucose, xylose or xylans, and the protein was associated with the cells. Xyn10A is the first family 10 xylanase characterized from B. xylanisolvens XB1A.     

Molecular cloning and characterization of the novel acidic xylanase XYLD from Bispora sp. MEY-1 that is homologous to family 30 glycosyl hydrolases

We cloned and sequenced a xylanase gene named xylD from the acidophilic fungus Bispora sp. MEY-1 and expressed the gene in Pichia pastoris. The 1,422-bp full-length complementary DNA fragment encoded a 457-amino acid xylanase with a calculated molecular mass of 49.8 kDa. The mature protein of XYLD showed high sequence similarity to both glycosyl hydrolase (GH) families 5 and 30 but was more homologous to members of GH 30 based on phylogenetic analysis. XYLD shared the highest identity (49.9%) with a putative endo-1,6-β-d-glucanase from Talaromyces stipitatus and exhibited 21.1% identity and 34.3% similarity to the well-characterized GH family 5 xylanase from Erwinia chrysanthemi. Purified recombinant XYLD showed maximal activity at pH 3.0 and 60 °C, maintained more than 60% of maximal activity when assayed at pH 1.5–4.0, and had good thermal stability at 60 °C and remained stable at pH 1.0–6.0. The enzyme activity was enhanced in the presence of Ni²⁺ and β-mercaptoethanol and inhibited by some metal irons (Hg²⁺, Cu²⁺, Pb²⁺, Mn²⁺, Li⁺, and Fe³⁺) and sodium dodecyl sulfate. The specific activity of XYLD for beechwood xylan, birchwood xylan, 4-O-methyl-d-glucuronoxylan, and oat spelt xylan was 2,463, 2,144, 2,020, and 1,429 U mg⁻¹, respectively. The apparent K _m and V _max values for beechwood xylan were 5.6 mg ml⁻¹ and 3,622 μmol min⁻¹ mg⁻¹, respectively. The hydrolysis products of different xylans were mainly xylose and xylobiose.     

Desulforubrerythrin from <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>, a novel multidomain protein

A novel multidomain metalloprotein from Campylobacter jejuni was overexpressed in Escherichia coli, purified, and extensively characterized. This protein is isolated as a homotetramer of 24-kDa monomers. According to the amino acid sequence, each monomer was predicted to contain three structural domains: an N-terminal desulforedoxin-like domain, followed by a four-helix bundle domain harboring a non-sulfur μ-oxo diiron center, and a rubredoxin-like domain at the C-terminus. The three predicted iron sites were shown to be present and were studied by a combination of UV–vis, EPR, and resonance Raman spectroscopies, which allowed the determination of the electronic and redox properties of each site. The protein contains two FeCys₄ centers with reduction potentials of +240 mV (desulforedoxin-like center) and +185 mV (rubredoxin-like center). These centers are in the high-spin configuration in the as-isolated ferric form. The protein further accommodates a μ-oxo-bridged diiron site with reduction potentials of +270 and +235 mV for the two sequential redox transitions. The protein is rapidly reoxidized by hydrogen peroxide and has a significant NADH-linked hydrogen peroxide reductase activity of 1.8 μmol H₂O₂ min⁻¹ mg⁻¹. Owing to its building blocks and its homology to the rubrerythrin family, the protein is named desulforubrerythrin. It represents a novel example of the large diversity of the organization of domains exhibited by this enzyme family.     

Purification and characterization of an endoglucanase from <Emphasis Type="Italic">Aspergillus terreus</Emphasis> highly active against barley β-glucan and xyloglucan

An endoglucanase (1, 4-β-d glucan glucanohydrolase, EC 3.2.1.4) which was catalytically more active and exhibited higher affinity towards barley β-glucan, xyloglucan and lichenin as compared to carboxymethylcellulose (CMC) was purified from Aspergillus terreus strain AN₁ following ion-exchange and hydrophobic interaction chromatography and gel filtration. The purified enzyme (40-fold) that apparently lacked a cellulose-binding domain showed a specific activity of 60 μmol mg⁻¹ protein⁻¹ against CMC. The purified enzyme had a molecular weight of 78 and 80 KDa as indicated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and gel filtration, respectively, and a pI of 3.5. The enzyme was optimally active at temperature 60°C and pH 4.0, and was stable over a broad range of pH (3.0–5.0) at 50°C. The endoglucanase activity was positively modulated in the presence of Cu²⁺, Mg²⁺, Ca²⁺, Na⁺, DTT and mercaptoethanol. Endoglucanase exhibited maximal turn over number (K _cat) and catalytic efficiency (K _cat/km) of 19.11 × 10⁵ min⁻¹ and 29.7 × 10⁵ mM⁻¹ min⁻¹ against barley β-glucan as substrate, respectively. Hydrolysis of CMC and barley β-glucan liberated cellobiose, cellotriose, cellotetraose and detectable amount of glucose. The hydrolysis of xyloglucan, however, apparently yielded positional isomers of cellobiose, cellotriose and cellotetraose as well as larger oligosaccharides.     

Molecular characterization of phenylalanine ammonia lyase gene from <Emphasis Type="Italic">Cistanche deserticola</Emphasis>

We cloned the gene, CdPAL1, from Cistanche deserticola callus using RACE PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes from other plant species. The gene shows high homology to other known PAL genes registered in GenBank. The recombinant protein exhibited Michaelis–Menten kinetics with a K _m of 0.1013 mM, V _max of 4.858 μmol min⁻¹, K _cat of 3.36 S⁻¹, and K _cat/K _m is 33,168 M⁻¹ S⁻¹. The enzyme had an optimal pH of 8.5 and an activation energy of 38.92 kJ mol⁻¹ when l-Phenylalanine was used as a substrate; l-tyrosine cannot be used as substrate for this protein. The optimal temperature was 55°C, and the thermal stability results showed that, after a treatment at 70°C for 20 min, the protein retained 87% activity, while a treatment at 75°C for 20 min resulted in a loss of over 85% of the enzyme activity. Treatment with heavy metal ions (Hg²⁺, Pb²⁺, and Zn²⁺) showed remarkable inhibitory effects. Among the intermediates from the lignin (cinnamyl alcohol, cinnamyl aldehyde, coniferyl aldehyde, coniferyl alcohol), phenylpropanoid (cinnamic acid, coumaric acid, caffeic acid, and chlorogenic acid) and phenylethanoid (tyrosol and salidroside) biosynthetic pathways, only cinnamic acid showed strong inhibitory effects against CdPAL1 activity with a K _i of 8 μM. Competitive inhibitor AIP exhibited potent inhibition with K _i = 0.056 μM.     

Stability of lipase immobilized on <Emphasis Type="Italic">O</Emphasis>-pentynyl dextran

O-Pentynyl dextran (PyD), an amphiphilic polysaccharide derivative with a degree of substitution (DS) of 0.43 was compared with ion exchange resins Lewatit VP OC 1600, Amberlite XAD 761 and Duolite A568 for immobilization of Lipase from Rhizopus arrhizus by adsorption method. The immobilized enzymes were employed for esterification of octanoic acid with geraniol in n-hexane as model reaction. PyD showed higher lipase adsorption and with 249 μmol min⁻¹ g⁻¹ significant higher esterification activity than the other supports (67–83 μmol min⁻¹ g⁻¹). Biocatalysts from all types of supports except PyD became completely inactive within 8 weeks storing at −10 °C while lipase immobilized on PyD retained its full esterification activity for at least 14 weeks. In repeated use, yield decreased rapidly after two cycles for all supports except for PyD. For this biopolymeric support, constantly 90% yield was achieved even after eight cycles, when the biocatalyst was washed with n-hexane and water and then freeze-dried. To achieve this yield, prolonged reaction times were required, partly on the account of an increasing delay period, probably to adapt active conformation, until the reaction starts.     

A highly stable Cu/Zn superoxide dismutase from <Emphasis Type="Italic">Withania somnifera</Emphasis> plant: gene cloning,expression and characterization of the recombinant protein

A gene from Withania somnifera (winter cherry), encoding a highly stable chloroplastic Cu/Zn superoxide dismutase (SOD), was cloned and expressed in Escherichia coli. The recombinant enzyme (specific activity of ~4,200 U mg⁻¹) was purified and characterized. It retained ~90 and ~70% residual activities after 1 h at 80 and 95°C, respectively. At 95°C, thermal inactivation rate constant (K _d) of the enzyme was 2.46 × 10⁻³ min⁻¹ and half-life of heat inactivation was 4.68 h. The enzyme was stable against a broad pH range (2.5–11.0). It also showed a high degree of resistance to detergent, ethanol and protease digestion. This recombinant Cu/Zn SOD could therefore have useful applications.     

A unique β-agarase,AgaA, from a marine bacterium, <Emphasis Type="Italic">Vibrio</Emphasis> sp. strain PO-303

The agaA gene encoding β-agarase-a (AgaA) was cloned from the chromosomal DNA of a marine bacterium, Vibrio sp. strain PO-303. The nucleotide sequence of the agaA gene consists of 2,958 bp and encodes a protein of 985 amino acids with a molecular mass of 106,062 Da. The deduced enzyme protein contains a typical N-terminal signal peptide of 29 amino acid residues, followed by a 266 amino acid sequence that is homologous to catalytic module of family 16 glycoside hydrolases, a bacterial immunoglobulin group 2 (Big-2)-like domain of 52 amino acid residues, two carbohydrate-binding modules of family 6 separated from Big-2-like domain by nine times repeated GDDTDP amino acid sequence. AgaA is the first agarase that was identified to possess a Big-2-like domain. The recombinant AgaA (rAgaA) expressed in Escherichia coli exhibited maximal activity around 40°C and pH 7.5, with a specific activity of 16.4 units mg⁻¹, a K _m of 1.10 mg ml⁻¹, and a V _max of 22.5 μmol min⁻¹ mg⁻¹ for agarose. The rAgaA hydrolyzed neoagarohexaose, but did not act on neoagarotetraose and neoagarobiose.     

Interaction Between Nanoparticulate Anatase TiO<Subscript>2</Subscript> and Lactate Dehydrogenase

In order to study the mechanisms underlying the effects of TiO₂ nanoparticles on lactate dehydrogenase (LDH, EC1.1.1.27), Institute of Cancer Research region mice were injected with nanoparticulate anatase TiO₂ (5 nm) of various doses into the abdominal cavity daily for 14 days. We then examined LDH activity in vivo and in vitro and direct evident for interaction between nanoparticulate anatase TiO₂ and LDH using spectral methods. The results showed that nanoparticulate anatase TiO₂ could significantly activate LDH in vivo and in vitro; the kinetics constant (Km) and Vmax were 0.006 μM and 1,149 unit mg⁻¹ protein min⁻¹, respectively, at a low concentration of nanoparticulate anatase TiO₂, and 3.45 and 0.031 μM and 221 unit mg⁻¹ protein min⁻¹, respectively, at a high concentration of nanoparticulate anatase TiO₂. By fluorescence spectral assays, the nanoparticulate anatase TiO₂ was determined to be directly bound to LDH, and the binding constants of the binding site were 1.77 × 10⁸ L mol⁻¹ and 2.15 × 10⁷ L mol⁻¹, respectively, and the binding distance between nanoparticulate anatase TiO₂ and the Trp residue of LDH was 4.18 nm, and nanoparticulate anatase TiO₂ induced the protein unfolding. It was concluded that the binding of nanoparticulate anatase TiO₂ altered LDH structure and function.