Essential amino acid residues and catalytic mechanism of trans-epoxysuccinate hydrolase for production of meso-tartaric acid.

OBJECTIVES: This study aimed to discuss the essential amino acid residues and catalytic mechanism of trans-epoxysuccinate hydrolase from Pseudomonas koreensis for the production of meso-tartaric acid. RESULTS: The optimum conditions of the enzyme were 45 °C and pH 9.0, respectively. It was strongly inhibited by Zn2+, Mn2+ and SDS. Michaelis-Menten enzyme kinetics analysis gave a Km value of 3.50 mM and a kcat of 99.75 s-1, with an exceptional EE value exceeding 99.9%. Multiple sequence alignment and homology modeling revealed that the enzyme belonged to MhpC superfamily and possessed a typical α/ß hydrolase folding structure. Site-directed mutagenesis indicated H34, D104, R105, R108, D128, Y147, H149, W150, Y211, and H272 were important catalytic residues. The 18O-labeling study suggested the enzyme acted via two-step catalytic mechanism. CONCLUSIONS: The structure and catalytic mechanism of trans-epoxysuccinate hydrolase were first reported. Ten residues were critical for its catalysis and a two-step mechanism by an Asp-His-Asp catalytic triad was proposed.

Isolation of Penicillium expansum WH-3 for the production of L(+)-tartaric acid.

The L(+)-form of tartaric acid (L(+)-TA) exists extensively in nature, and is widely used in the food, chemical, textile, building, and pharmaceutical industries (Su et al., 2001). The main method for L(+)-TA production is microbial transformation by cis-epoxysuccinate hydrolase (CESH), which can catalyze the asymmetric hydrolysis of cis-epoxysuccinic acid or its salts to TA or tartrate (Bao et al., 2019). Seventeen species containing CESH have been isolated so far. However, most species for L(+)-TA production have been reported from bacteria (Xuan and Feng, 2019). The only fungus isolated from soil by our lab recently, that could be used as catalyst for the process under acidic condition, is Aspergillus niger WH-2 (Bao et al., 2020). In order to find strains with new characteristics, this study attempted to isolate a new CESH source from fungi and investigate its application value.

Isolation of a novel strain Aspergillus niger WH-2 for production of L(+)-tartaric acid under acidic condition.

OBJECTIVES: To isolate a novel cis-epoxysuccinate hydrolase (CESH)-producing fungus for production of L( +)-tartaric acid, before this, all strains were selected from bacteria. RESULTS: A CESH-producing fungus was first isolated from soil and identified as Aspergillus niger WH-2 based on its morphological properties and ITS sequence. The maximum activity of hyphaball and fermentation supernatants was 1278 ± 64 U/g and 5.6 ± 0.3 U/mL, respectively, in a 5 L fermenter based on the conditions optimized on the flask. Almost 70% of CESH was present in hyphaball, which maintained 40% residual activity at pH 4.0 and showed a good acid stability (pH 3.0-10.0), high conversion rate (> 98%), and enantioselectivity (EE > 99.6%). However, the reported CESHs from bacteria can't be catalyzed under acidic conditions. CONCLUSIONS: The Aspergillus niger WH-2 was the first reported CESH-producing fungus, which could biosynthesize L ( +)-tartaric acid under acidic conditions and provide an alternative catalyst and process.

Cloning and characterization of an oxiranedicarboxylate hydrolase from Labrys sp. WH-1.

OBJECTIVE: This study aimed to clone and characterize the oxiranedicarboxylate hydrolase (ORCH) from Labrys sp. WH-1. METHODS: Purification by column chromatography, characterization of enzymatic properties, gene cloning by protein terminal sequencing and polymerase chain reaction (PCR), and sequence analysis by secondary structure prediction and multiple sequence alignment were performed. RESULTS: The ORCH from Labrys sp. WH-1 was purified 26-fold with a yield of 12.7%. It is a monomer with an isoelectric point (pI) of 8.57 and molecular mass of 30.2 kDa. It was stable up to 55 °C with temperature at which the activity of the enzyme decreased by 50% in 15 min (T5015) of 61 °C and the half-life at 50 °C (t1/2, 50 °C) of 51 min and was also stable from pH 4 to 10, with maximum activity at 55 °C and pH 8.5. It is a metal-independent enzyme and strongly inhibited by Cu2+, Ag+, and anionic surfactants. Its kinetic parameters (Km, kcat, and kcat/Km) were 18.7 mmol/L, 222.3 s-1, and 11.9 mmol/(L·s), respectively. The ORCH gene, which contained an open reading frame (ORF) of 825 bp encoding 274 amino acid residues, was overexpressed in Escherichia coli and the enzyme activity was 33 times higher than that of the wild strain. CONCLUSIONS: The catalytic efficiency and thermal stability of the ORCH from Labrys sp. WH-1 were the best among the reported ORCHs, and it provides an alternative catalyst for preparation of L(+)-2,3-dihydrobutanedioic acid.

Hydrolysis before Stir-Frying Increases the Isothiocyanate Content of Broccoli.

Broccoli is found to be a good source of glucosinolates, which can be hydrolyzed by endogenous myrosinase to obtain chemopreventive isothiocyanates (ITCs); among them, sulforaphane (SF) is the most important agent. Studies have shown that cooking greatly affects the levels of SF and total ITCs in broccoli. However, the stability of these compounds during cooking has been infrequently examined. In this study, we proved that the half-lives of SF and total ITCs during stir-frying were 7.7 and 5.9 min, respectively, while the myrosinase activity decreased by 80% after stir-frying for 3 min; SF and total ITCs were more stable than myrosinase. Thus, the contents of SF and total ITCs decreased during stir-frying largely because myrosinase was destroyed. Subsequently, it was confirmed that compared to direct stir-frying, hydrolysis of glucosinolates in broccoli for 90 min followed by stir-frying increased the SF and total ITC concentration by 2.8 and 2.6 times, respectively. This method provides large quantities of beneficial ITCs even after cooking.

Single point mutations enhance activity of cis-epoxysuccinate hydrolase.

OBJECTIVES: To enhance activity of cis-epoxysuccinate hydrolase from Klebsiella sp. BK-58 for converting cis-epoxysuccinate to tartrate. RESULTS: By semi-saturation mutagenesis, all the mutants of the six important conserved residues almost completely lost activity. Then random mutation by error-prone PCR and high throughput screening were further performed to screen higher activity enzyme. We obtained a positive mutant F10D after screening 6000 mutations. Saturation mutagenesis on residues Phe10 showed that most of mutants exhibited higher activity than the wild-type, and the highest mutant was F10Q with activity of 812 U mg(-1) (k cat /K m , 9.8 ± 0.1 mM(-1) s(-1)), which was 230 % higher than that of wild-type enzyme 355 U mg(-1) (k cat /K m , 5.3 ± 0.1 mM(-1) s(-1)). However, the thermostability of the mutant F10Q slightly decreased. CONCLUSIONS: The catalytic activity of a cis-epoxysuccinate hydrolase was efficient improved by a single mutation F10Q and Phe10 might play an important role in the catalysis.

Isolation of the stable strain Labrys sp. BK-8 for L(+)-tartaric acid production.

A novel cis-epoxysuccinate hydrolase (CESH) producing strain of Labrys sp. BK-8 for production of L(+)-tartaric acid was isolated and identified. After optimization, a maximum activity of 3597 ± 151 U/g was achieved in batch culture in a 10 L fermentor. When Labrys sp. BK-8 was immobilized on κ-carrageenan, the immobilized cells showed a high conversion rate (>99%), enantioselectivity (EE > 99.5%) and storage stability (>90 d). A conversion rate of 97% was still achieved after 10 repeat batches. The CESH was stable over a broad range of temperatures (up to 45°C) and pH values (4.0-10.0). The Labrys sp. BK-8 isolate provides a new alternative with good stability for the industrial biosynthesis of L(+)-tartaric acid.

Cloning, homology modeling, and reaction mechanism analysis of a novel cis-epoxysuccinate hydrolase from Klebsiella sp.

The gene encoding a novel cis-epoxysuccinate hydrolase, which hydrolyzes cis-epoxysuccinate to L (+)-tartaric acid, was cloned from Klebsiella sp. BK-58 and expressed in Escherichia coli. The ORF was 825 bp encoding a mature protein of 274 amino acids with a molecular mass of 30.1 kDa. Multiple sequence alignment showed that the enzyme belonged to the haloacid dehalogenase-like super family. Homology modeling and site-directed mutagenesis were performed to investigate the structural characteristics of the enzyme. Its overall structure consisted of a core domain formed by six-stranded parallel ß-sheets flanked by seven α-helices and a subdomain that had a four helix bundle structure. Residues D48, T52, R85, N165, K195, Y201, A219, H221, and D224 were catalytically important forming the active pocket between the two domains. An (18)O-labeling study suggested that the catalytic reaction of the enzyme proceeded through a two-step mechanism.

Purification and characterization of a novel cis-epoxysuccinate hydrolase from Klebsiella sp. that produces L(+)-tartaric acid.

A strain of Klebsiella sp. BK-58 that produces cis-epoxysuccinate was screened and identified. This novel enzyme hydrolyzes cis-epoxysuccinate to L(+)-tartaric acid and was purified to homogeneity. Its molecular mass was 30.1 kDa determined by MALDI-TOF-MS analysis. It was stable up to 50 °C and from pH 5 to 11 with optima at 50 °C and pH 8.5. The enzyme was metal-independent and was strongly inhibited by 1 mM Cu(2+) and Ag(+), and 1 % (w/v) SDS. The K m , V max and turnover number (k cat ) values for cis-epoxysuccinate were 19.3, 2.24 mM min(-1) and 220 s(-1), respectively.

[Production of L(+)-tartaric acid by immobilized Rhizobium strain BK-20].

The cis-epoxysuccinate hydrolase (CESH) from Rhizobium strain BK-20 is the key enzyme for L(+)-tartaric acid production. To establish a highly efficient and stable production process, we first optimized the enzyme production from Rhizobium strain BK-20, and then developed an immobilized cell-culture process for sustained production of L(+)-tartaric acid. The enzyme activity of free cells reached (3 498.0 +/- 142.6) U/g, and increased by 643% after optimization. The enzyme activity of immobilized cells reached (2 817.2 +/- 226.7) U/g, under the optimal condition with sodium alginate as carrier, cell concentration at 10% (W/V) and gel concentration at 1.5% (W/V). The immobilized cells preserved high enzyme activity and normal structure after 10 repeated batches. The conversion rate of the substrate was more than 98%, indicating its excellent production stability.

Analysis of essential amino acid residues for catalytic activity of cis-epoxysuccinate hydrolase from Bordetella sp. BK-52.

cis-Epoxysuccinate hydrolase (CESH) from Bordetella sp. BK-52, an epoxide hydrolase (EH), catalyzes the stereospecific hydrolysis of cis-epoxysuccinate to D(-)-tartrate. The enzyme, which shows no homology to other reported EHs, belongs to the DUF849 superfamily of prokaryotic proteins, which have unknown function. Metal composition analysis revealed that the CESH is a Zn(2+)-dependent enzyme with an approximately 1:1 molar ratio of zinc to enzyme. The results of an (18)O-labeling study suggest that the enzyme catalyzes epoxide hydrolysis by means of a one-step mechanism. We evaluated the relationship between the structure and function of the enzyme by means of sequence alignment, modeling, substrate binding, and reaction kinetics studies. The CESH has a canonical (ß/α)8 TIM barrel fold, and we used site-directed mutagenesis to identify eight residues (H47, H49, R51, T82, Y138, N140, W164, and D251) as being localized to the active site or highly conserved. On the basis of these results and theoretical considerations, we identified H47 and H49 as zinc-binding ligands, and we propose that a zinc atom and R51, T82, Y138, N140, W164, and D251 are the catalytic residues and participate in substrate binding. In summary, the structure and catalytic mechanism of the CESH from Bordetella sp. BK-52 differ from those of classic EHs, which have an α/ß hydrolase fold, act via a two-step catalytic mechanism, and do not require cofactors, prosthetic groups, or metal ions.

Cloning and characterization of a novel NAD+ -dependent xylitol dehydrogenase from Gluconobacter oxydans CGMCC 1. 637.

OBJECTIVE: To clone the xylitol dehydrogenase gene from Gluconobacter oxydans CGMCC 1.637, to characterize enigmatic properties of xylitol dehydrogenase and to investigate the induction abilities of various carbon sources on the oxidative activity of xylitol dehydrogenase and the effect of various carbon sources on the bioconversion of d-xylulose to xylitol in G. oxydans CGMCC 1.637. METHODS: Touch-down polymerase chain reaction (PCR) was applied to clone the xylitol dehydrogenase gene from chromosomal DNA of G. oxydans CGMCC 1.637. RESULTS: The 798-bp open reading frame of xylitol dehydrogenase encoded a protein of 265 amino acids, with the molecular mass of 27.95 kDa. Sequence analysis of the putative protein revealed it to be a member of short-chain dehydrogenase/reductase family. Xylitol dehydrogenase showed oxidative activity with xylitol and sorbitol and no activity with other polyols, such as d-arabitol. K(m) and V(max) with xylitol was 78.97 mmol/L and 40.17 U/mg, respectively. The highest oxidative activity of xylitol dehydrogenase for xylitol was only 23.27 U/mg under optimum conditions (pH 10.0, 35 degrees C). However, the activity of its reverse reaction, d-xylulose reduction, reached 255.55 U/mg under optimum conditions (pH 6.0, 30 degrees C), 10-times higher than that of xylitol oxidation. Oxidative activity of xylitol dehydrogenase was induced when G. oxydans CGMCC 1.637 was cultivated on d-sorbitol. D-arabitol, which supported a high cell growth, inhibited the oxidative activity of xylitol dehydrogenase and the bioconversion ability of G. oxydans CGMCC 1.637. CONCLUSIONS: The obtained gene from G. oxydans CGMCC 1.637 was a novel gene encoding xylitol dehydrogenase. Oxidative activity of xylitol dehydrogenase in G. oxydans CGMCC 1.637 and the bioconversion ability of G. oxydans CGMCC 1.637 after grown on d-arabitol were inhibited, which provided a valuable clue for further study to increase xylitol yield from d-arabitol.

Site-directed mutagenesis of epoxide hydrolase to probe catalytic amino acid residues and reaction mechanism.

Epoxide hydrolase from Rhodococcus opacus catalyzes the stereospecific hydrolysis of cis-epoxysuccinate to L(+)-tartrate. It shows low but significant similarity to haloacid dehalogenase and haloacetate dehalogenase (16-23% identity). To identify catalytically important residues, we mutated 29 highly conserved charged and polar amino acid residues (except for one alanine). The replacement of D18, D193, R55, K164, H190, T22, Y170, N134 and A188 led to a significant loss in the enzyme activity, indicating their involvement in the catalysis. Single and multiple turnover reaction studies show that the enzyme reaction proceeded through the two-step mechanism involving the formation of a covalent intermediate. We discuss the roles of these residues and propose its possible reaction mechanism.

Molecular cloning and characterization of a cis-epoxysuccinate hydrolase from Bordetella sp. BK-52.

A cis-epoxysuccinate hydrolase (CESH) from Bordetella sp. BK-52 was purified 51.4-fold with a yield of 27.1% using ammonium sulphate precipitation, ionic exchange, hydrophobic interaction, molecular sieve chromatograph and an additional anion exchange chromatography. The CESH was stable in a broad range of temperature (up to 50 degrees C) and pH (4.0-10.0) with optima of 40 degrees C and pH6.5, respectively. It could be partially inhibited by EDTA-Na2, Ag+, SDS and DTT, while slightly enhanced by Ba2+ and Ca2+. The enzyme exhibited high stereospecificity in D(-)-tartaric acid (enantiomeric excess value higher than 99 %) with Km and Vmax value of 18.67 mM and 94.34 micronM/min/mg for disodium cis-epoxysuccinate, respectively. The Bordetella sp. BK-52 CESH gene, which contained 885 nucleotides (open reading frame) encoding 294 amino acids with a molecular mass of about 32 kDa, was successfully overexpressed in Escherichia coli using a T7/lac promoter vector and the enzyme activity increased 42-times compared to original strain. It may be an industrial biocatalyst for the preparation of D(-)-tartaric acid.

Immobilization of Escherichia coli cells with cis-epoxysuccinate hydrolase activity for D(-)-tartaric acid production.

Immobilization of cis-epoxysuccinate hydrolase-containing E. coli for D(-)-tartaric acid production was screened by various methods. The highest recovery of activity was obtained by entrapment in kappa-carrageenan gel. 23.6 g biomass/l and 43.4 g kappa-carrageenan/l were the best immobilization conditions optimized by response surface methodology with 83% yield (114 U/g). Cell autolysis was observed after immobilization. Immobilized cells showed high pH (5-10) stability, thermal (up to 65 degrees C) stability, conversion rate (>99.5%), enantioselectivity (ee > 99.6%), and were less affected by metal ions and surfactants compared with free cells. Conversion rate for immobilized cells preserved 93% after 10 repeated batches (5% for free cells).

Isolation and identification of a novel cis-epoxysuccinate hydrolase-producing Bordetella sp. BK-52 and optimization of enzyme production.

OBJECTIVE: To isolate and identify a novel strain with cis-epoxysuccinate hydrolase (CESH) activity and to optimize its enzyme production. METHODS: The isolated strain was identified by electron microscopy, Biolog gram negative (GN) test, G+C (guanine plus cytosine) content measurement and 16S rDNA sequence. The purified enzymatic biotransformed product was identified by IR, 1H-NMR, 13C-NMR, MS and optical rotation analysis. Then the fermentation conditions for CESH production were optimized. RESULTS: A novel CESH-producing strain was isolated for biotransforming cis-epoxysuccinate to D(-)-tartaric acid. It was assigned to genus Bordetella and named Bordetella sp. BK-52. The optimal conditions were found to be 30 degrees C, pH 7.0, fermentation time 36 h, carbon source of saccharose, inorganic nitrogen source of ammonium sulfate and enzyme inducer of disodium cis-epoxysuccinate. Under these conditions, the maximum CESH activity reached 764 U/g biomass. CONCLUSION: The isolated Bordetella sp. BK-52 provided a new alternative for biosynthesis of D(-)-tartaric acid from cis-epoxysuccinate.

The apoptotic effect of sarsasapogenin from Anemarrhena asphodeloides on HepG2 human hepatoma cells.

Sarsasapogenin, a kind of mainly effective components of Anemarrhena asphodeloides Bunge (Liliaceae) has the effects of being anti-diabetes and improving memory. However, there are few reports focusing on its anti-tumor effects. In this study, the sarsasapogenin was extracted from rhizomes of A. asphodeloides Bunge and applied to inhibit HepG2 human hepatoma cells. MTT assay showed that sarsasapogenin induced a distinct dose- and time-dependent diminution of cell viability with IC(50) of 42.4+/-1.0microg/ml for 48h. Furthermore, sarsasapogenin-induced apoptosis of HepG2 cells was verified by Hoechst 33258 staining, electron microscopy, DNA fragmentation and PI staining. Flow cytometry analysis showed that sarsasapogenin-induced cell apoptosis was through arrest of cell cycle in G(2)/M phase. Hence we proposed that sarsasapogenin could be used as an anti-liver cancer drug for future studies.