Effects of pork sausage on intestinal microecology and metabolism in mice.

BACKGROUND: Processed meat, as an important part of the human diet, has been recognized as a carcinogen by the International Agency for Research on Cancer (IARC). Although numerous epidemiological reports supported the IARC's view, the relevant evidence of a direct association between processed meat and carcinogenicity has been insufficient and the mechanism has been unclear. This study aims to investigate the effects of pork sausage (as a representative example of processed meat) intake on gut microbial communities and metabolites of mice. Microbial communities and metabolites from all groups were analyzed using 16S rRNA gene sequencing and Ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometer (UPLC-Q-TOF/MS), respectively. RESULTS: The levels of Bacteroidetes, Bacteroides, Alloprevotella, Lactobacillus, Prevotella_9, Lachnospiraceae_NK4A136_group, Alistipes, Blautia, Proteobacteria, Firmicutes, Allobaculum, Helicobacter, Desulfovibrio, Clostridium_sensu_stricto_1, Ruminococcaceae_UCG-014, Lachnospiraceae_UCG-006 and Streptococcus (P < 0.05) were obviously altered in the mice fed a pork sausage diet. Twenty-seven metabolites from intestinal content samples and fourteen matabolites from whole blood samples were identified as potential biomarkers from multivariate analysis, including Phosphatidic acid (PA), Sphingomyelin (SM), Lysophosphatidylcholine (LysoPC), Diglyceride (DG), D-maltose, N-acylamides and so forth. The significant changes in these biomarkers demonstrate metabonomic variations in pork sausage treated rats, especially carbohydrate metabolism, lipid metabolism, and amino acid metabolism. CONCLUSION: The present study provided evidence that a processed meat diet can increase the risk of colorectal cancer and other diseases significantly by altering the microbial community structure and disrupting the body's metabolic pathways. © 2023 Society of Chemical Industry.

Rapid detection of micronutrient components in infant formula milk powder using near-infrared spectroscopy.

In order to achieve rapid detection of galactooligosaccharides (GOS), fructooligosaccharides (FOS), calcium (Ca), and vitamin C (Vc), four micronutrient components in infant formula milk powder, this study employed four methods, namely Standard Normal Variate (SNV), Multiplicative Scatter Correction (MSC), Normalization (Nor), and Savitzky-Golay Smoothing (SG), to preprocess the acquired original spectra of the milk powder. Then, the Competitive Adaptive Reweighted Sampling (CARS) algorithm and Random Frog (RF) algorithm were used to extract representative characteristic wavelengths. Furthermore, Partial Least Squares Regression (PLSR) and Support Vector Regression (SVR) models were established to predict the contents of GOS, FOS, Ca, and Vc in infant formula milk powder. The results indicated that after SNV preprocessing, the original spectra of GOS and FOS could effectively extract feature wavelengths using the CARS algorithm, leading to favorable predictive results through the CARS-SVR model. Similarly, after MSC preprocessing, the original spectra of Ca and Vc could efficiently extract feature wavelengths using the CARS algorithm, resulting in optimal predictive outcomes via the CARS-SVR model. This study provides insights for the realization of online nutritional component detection and optimization control in the production process of infant formula.

Assessing the effect of powder characteristics of infant milk on the compressibility of milk powder compression molding.

Infant formula is an important food for those infants who are deprived of breast milk. However, infant formula powder is prone to fly apart, moisture absorption, sticky spoon, and inaccurate measurement. Block infant formula can solve these problems well. In this study, the characteristics (including particle structure morphology, moisture content, particle size, etc.) of infant formula powder were investigated on the compressive strength and solubility of block infant formula after compression molding with infant formula powder as the object. The results showed that the compressive strength and solubility of the block infant formula made from milk powder with a moisture content of 4.75%, particle size larger than 80 mesh, and morphology of compact grape structure appendages were the best. Therefore, milk powder with this characteristic is the most suitable for the preparation of block infant formula. This study provides referenceable experimental data and theoretical basis for the preparation and application of block infant formula.

Physicochemical, microbiological, and sensory properties of low-lactose yogurt using Streptococcus thermophilus with high ß-galactosidase activity.

BACKGROUND: Lactose maldigestion or intolerance affects a large number of individuals worldwide. If lactose is hydrolyzed by the ß-galactosidase enzyme during the fermentation process, lactose-intolerant individuals can consume milk products without experiencing diarrhea, flatulence, or other symptoms. RESULTS AND CONCLUSION: We isolated and characterized Streptococcus thermophilus, which exhibits high ß-galactosidase activity. This was then used as a starter culture with Lactobacillus delbrueckii subsp. bulgaricus in yogurt to determine the effects of different starter ratios and fermentation temperatures on its organoleptic and physical properties. The ß-galactosidase activity of the isolated strain was 2.60 units mg-1 . The optimal temperature was 42 °C for St. thermophilus to acidify yogurt faster than at other temperatures and it was effective in hydrolyzing the lactose in the media and yogurt. The lactic acid bacteria (LAB) population in 37 °C fermented yogurt was higher than in the other samples, but the starters St. thermophilus and Lb. bulgaricus with a ratio of 2:1 used lactose more effectively than other sample ratios. The lactose content decreased significantly at 37 °C, where it was ~50% hydrolyzed. The acceptability of the sensory properties of yogurt was unaffected by relative lower fermentation temperatures (30 and 37 °C), despite using different ratios of St. thermophilus and Lb. bulgaricus as starter cultures. © 2023 Society of Chemical Industry.

Effect of Apple Polyphenols on the Antioxidant Activity and Structure of Three-Dimensional Printed Processed Cheese.

Additives can influence the processability and quality of three-dimensional (3D)-printed foods. Herein, the effects of apple polyphenols on the antioxidant activity and structure of 3D-printed processed cheese were investigated. The antioxidant activities of processed cheese samples with different contents of apple polyphenols (0%, 0.4%, 0.8%, 1.2%, or 1.6%) were evaluated using 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH) assays. In addition, the rheological properties and structural characteristics of the processed cheeses were investigated using rheometry, Fourier transform infrared spectroscopy, and fluorescence spectroscopy. Then, the final printed products were analyzed for comparative molding effects and dimensional characteristics. it was found that apple polyphenols can significantly improve the antioxidant activity of processed cheese. When the amount of apple polyphenols added was 0.8%, the 3D shaping effect was optimal with a porosity rate of 4.1%. Apple polyphenols can be used as a good antioxidant additive, and the moderate addition of apple polyphenols can effectively improve the antioxidant and structural stability of 3D-printed processed cheese.

High efficiency biosynthesis of gardenia blue and red pigment by lactic acid bacteria: A great potential for natural color pigments.

Current production methods of the food colorants, gardenia blue (GB) and red (GR) pigments have low efficiency. One potential approach involves using lactic acid bacteria (LAB), which produce a high level of ß-glucosidase, produce the GB and GR using non-toxic and harmless process. The isolated strain Lactobacillus plantarum S3 and the reference strain Lb. plantarum KCTC3104 showed high ß-glucosidase activity levels of 1.01 and 1.44 unit/mL, respectively. The 12-h bioconversion yield of geniposide to genipin using two strains were 93.4% and 100%, respectively, which are high conversion percentage. For GB, the maximal production yield obtained using Lb. plantarum S3 and Lb. plantarum KCTC3104 under optimal conditions were 2.17 and 2.18 mg/mL, respectively. For GR, glutamic acid (Glu) with Lb. plantarum S3 is the best combination. To the best of our knowledge, this is the first report of an effective alternative method for the production of natural food colorants using LAB.

Production and evaluation of enzyme-modified cheese adding protease or lipase to improve quality properties.

Enzyme-modified cheese (EMC) produced by enzyme hydrolysis is a natural, cost-effective, and flexible alternative to using natural cheese in industrial applications. The modification of cheese by enzymes can increase their benefits for consumer acceptance and health, and intensify the specific cheese flavor. We evaluated the properties of cheese with added protease (Ep) or lipase (El), including texture, sensory, organic acids, volatile compounds, and free amino acids. As results, the hardness and gumminess of the cheese reached their maximum values when the concentration of protease and lipase was 0.1% and 0.6%, respectively. Interestingly, the bitterness and astringency of the cheese was reduced. The highest scores for odor, taste, and overall acceptability were observed on 0.08% protease in Ep and 0.8% lipase in El. Compared with the anchor cheese, eight new compounds were produced after the addition of protease and nine new compounds were produced after the addition of lipase. Irrespective of the type of enzyme, the content of free amino acids decreased slightly with the increase in enzyme content. From the point of view of adding enzyme species, the free amino acids content of Ep was generally higher than that of El, and glutamic acid and proline contents were high. Acetic acid concentrations (aroma-active compounds) of enzyme-modified cheese using protease and lipase were 482-931 mg/100 g and 30-36 mg/100 g, respectively, which were significantly increased. According to the results obtained in this study, a cheese with higher sensorial and textural acceptability was obtained by adding the appropriate protease or lipase.

Sonochemical effects on fabrication, characterization and antioxidant activities of ß-lactoglobulin-chlorogenic acid conjugates.

The ß-lactoglobulin-chlorogenic acid (LG-CA) conjugate was explored to be formed through ultrasonication, redox-pair method and their combination, the ultrasonication used a probe ultrasonic machine with a 6 mm probe at 270 W, and the frequency was 20-25 kHz. The formation of the conjugate was confirmed by SDS-PAGE with a larger molecular weight. Besides, Fourier infrared spectroscopy (FTIR) and Circular dichroism (CD) indicated changes in the secondary structure of the LG-CA conjugate. The α-helix and ß-sheet contents of LG decreased and the unordered content increased significantly after the formation of covalent complexes. In addition, both the ultrasonic treatment and its combination with redox-pair method could significantly improve the antioxidant properties of LG. The former increased to 23.16 µmol Trolox/g sample, the latter 82-106 µmol Trolox/g sample. Therefore, ultrasonication could be used both individually and in combination with the redox-pair method to produce LG-CA conjugates with stronger antioxidant activities.

Characterization and shelf stability of sweetened condensed milk formulated with different sucrose substitutes during storage.

Sweetened condensed milk (SCM) is a value-added milk product with extended shelf life and high levels of nutrition. The high level of sucrose may lead to health problems. Many studies have focused on the reduction of sucrose but seldomly on different combination of sugar substitutes. This study aims to find an ideal sucrose substitution through physiochemical, microbiological and sensory properties of SCM under different storage times. The results demonstrated that substitution with 20% trehalose, 5% lactulose and 15% erythritol resulted in similar sensory and color as control group. The volatile flavor analysis showed that substitution with 30% trehalose, 5% lactulose and 5% polyols was the most similar and hexanoic acid was the symbolistic flavor. Sucrose replacement increased the antibacterial effect and Staphylococcus, Penicillium, Apiotrichum and Candida were widely present. Substitution with 30% trehalose, 5% lactulose and 5% polyols resulted in the most similar water activity, texture, aroma and microbial diversity.

Extraction, Isolation and Identification of Low Molecular Weight Peptides in Human Milk.

Human milk contains numerous free low molecular weight peptides (LMWPs), which may play an important role in infant health and growth. The bioactivities of LMWPs are determined by their structures, especially the amino acid sequences. In the present study, 81 human milk samples were collected and purified by cation-exchange solid-phase extraction (SPE). Ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used for the separation and detection of free LMWPs in human milk. A total of 56 LMWPs were identified and quantified. These LMWPs were mainly derived from 3 regions of ß-casein, which were the amino acid fragments of 16-40, 85-110, and 205-226. The predominant LMWPs were RETIESLSSSEESITEYK, RETIESLSSSEESITEYKQKVEKVK, ETIESLSSSEESITEYK, TQPLAPVHNPIS, and QPLAPVHNPISV with molecular weights of 2247.9573, 2860.2437, 2091.8591, 1372.7666, and 1271.7212, respectively. The results indicated that the technique based on SPE and UPLC-QTOF-MS might greatly facilitate the analysis of LMWPs in human milk.

Effect of steam explosion pretreatment on the composition and bioactive characteristic of phenolic compounds in Chrysanthemum morifolium Ramat cv. Hangbaiju powder with various sieve fractions.

Steam explosion (SE) pretreatment is an efficient technique to promote the fiber degradation and disrupt materials' cell wall. In this study, the effect of SE pretreatment on the changes in phenolic profile, and the in vitro digestion property of a Chinese indigenous herb "Hangbaiju" (HBJ) powder with various sieve fractions (150-, 180-, 250-, 425-, and 850-µm sieves) were studied. After SE pretreatment, the morphological structure, color attributes, and composition of phenolic compounds were altered significantly (p < .05). The composition and content of phenolic compounds were strongly correlated with particle sizes. The higher extraction yield of phenolic compounds was reached in the intermediate sieve fraction (ca. 250-µm sieves). During in vitro digestion, the changes in phenolic compounds were significant due to the transition from an acidic to the alkaline environment (p < .05). Based on the multivariate statistical analysis, apigenin-7-O-glucoside, luteolin-7-O-glucoside, and linarin, were viewed as the characteristic compounds among various samples. The results highlighted that the phytochemical properties mainly including the composition of phenolic compounds, and in vitro digestion properties of HBJ powder with intermediate sieve fraction could be improved after SE pretreatment.

Ultrasound-assisted assembly of ß-lactoglobulin and chlorogenic acid for non covalent nanocomplex: fabrication, characterization and potential biological function.

It is essential to understand the ultrasound-induced changes in assembly of proteins and polyphenols into non covalent nanocomplex. ß-Lactoglobulin (LG) and chlorogenic acid (CA) with various biological activities can be combined to form food-grade nanocomplexes. This study systematically explored the role of high-intensity ultrasound pretreatment on the binding mechanisms of LG and CA, and the potential biological function for embedding curcumin (Cur). The scanning electron microscopy (SEM) revealed that ultrasound treatment could destroy the structure of LG, and the particle size of the protein was reduced to<50 nm. The change in secondary structure of the protein by ultrasound treatment could be revealed by the fourier transform infrared (FTIR) and fluorescence spectra. Besides, it was found that LG and CA were combined to form a complex under the hydrophobic interaction, and CA was bound in the internal cavity of LG with a relatively extended conformation. The result demonstrated that the ratio of Cur embedded in the ultrasonic sample could be effectively increased by 7% - 10%, the particle size in the emulsion was smaller, and the dispersion was more stable. This work contributes to the development of protein-polyphenol functional emulsion systems with the ability to deliver Cur.

Macronutrient content and fatty acid composition and their positional distribution in human breast milk from Zhejiang Province, China in different lactation periods.

Lactational changes in macronutrient content, lipid profile, fatty acid composition, and positional distribution of human breast milk were investigated in this study. A total of 378 milk samples of six different lactation periods, including 0‒5, 6‒14, 15‒30, 31‒90, 91‒180, and 181‒360 days, were collected cross-sectionally from healthy lactating women in Zhejiang, China. As lactation progressed from 0‒5 to 15‒30 days, the lipid content and the percentages of C10:0, C12:0, C14:0, C18:2n-6, and C18:3n-3 increased significantly, while the protein concentration and the proportions of phospholipids, cholesterols, C16:0, C18:1n-9, C24:1n-9, C20:4n-6, C22:4n-6, C22:5n-3, and C22:6n-3 decreased notably. When lactation was further extended to 181‒360 days, the protein content continued to decrease, and the percentages of C12:0 and C14:0 continued to increase, whereas the levels of other tested nutrients remained stable. Although the triacylglycerol positional distributions of some fatty acids underwent significant lactational variations, C14:0, C16:0, C24:1n-9, C22:4n-6, C22:5n-3, and C22:6n-3 were located mainly at the sn-2 position, while C18:1n-9, C18:2n-6, and C18:3n-3 were primarily distributed at the sn-1,3 positions. Compared with human breast milk reported in Western countries, samples in our study demonstrated higher percentages of C18:2n-6, C18:3n-3, C20:4n-6, and C22:6n-3, but lower proportions of C12:0, C14:0, and C18:1n-9. The results from this study indicated a nutritional composition different from that of the Western countries and may provide useful data for the development of infant formulas closer to Chinese breast milk in terms of the fatty acid composition and its specified positional distribution on triglyceride structure.

Ultrasonic degradation kinetics and isomerization of 3- and 4-O-caffeoylquinic acid at various pH: The protective effects of ascorbic acid and epigallocatechin gallate on their stability.

Caffeoylquinic acids are existed in many plant species with various biological and pharmacological activities. 3-O-caffeoylquinic acid and 4-O-caffeoylquinic acid are two isomers of caffeoylquinic acids, which may be degraded and transformed to their isomers in processing. The present paper found that the stability of 3- and 4-O-caffeoylquinic acid had decreased with the increasing solution alkalinity. 3-O-caffeoylquinic acid was more stable than 4-O-caffeoylquinic acid at the same condition. During degradation, 3- and 4-O-caffeoylquinic acid were partially converted to their isomers. Additionally, ultrasonic effects on the degradation and isomerization of 3- and 4-O-caffeoylquinic acid at different pH were studied. Ultrasound facilitated the degradation and isomerization of these compounds. The degradation kinetics were described by the Weibull equation. The protective effect of ascorbic acid and epigallocatechin gallate were also explored. Ascorbic acid and epigallocatechin gallate could alleviate the degradation of 3- and 4-O-caffeoylquinic acid under certain conditions.

The probiotic Companilactobacillus crustorum MN047 alleviates colitis-associated tumorigenesis via modulating the intestinal microenvironment.

Supplementation of probiotics is a promising method to alleviate colorectal cancer (CRC) via modulating the intestinal microenvironment. This study aimed to assess the potential anti-CRC effect of Companilactobacillus crustorum MN047 on an azoxymethane and dextran sulfate sodium-induced colitis-associated (CA)-CRC mouse model. Mice were gavaged with C. crustorum MN047 once daily (∼1 × 109 CFU per mouse). The CA-CRC ameliorating effect of this strain was investigated based on the gut microbiota, inflammation and intestinal barrier integrity. Results showed that C. crustorum MN047 could significantly attenuate tumorigenesis and inflammation via suppressing the TLR4/NF-κB pathway. Moreover, this probiotic could improve the intestinal barrier integrity by increasing the mRNA level of some tight junction-related proteins and reducing goblet cell loss. In addition, C. crustorum MN047 administration led to an increase in beneficial bacteria and a decrease in harmful bacteria, thereby increasing SCFAs and reducing LPS levels. These results suggested that C. crustorum MN047 could partially ameliorate the formation of CA-CRC by modulating the gut microbiota, attenuating inflammation and enhancing the intestinal barrier integrity. Therefore, C. crustorum MN047 was a promising probiotic supplement for attenuating CA-CRC.

Modeling the effect of vibration on the quality of stirred yogurt during transportation.

When transporting yogurt, vibrations and sharp movements can damage its quality. This study developed a model to connect the changes in yogurt quality with the transportation distance as simulated by the total number of vibrations. Linear regression analysis showed that there was a significant negative correlation between the water holding capacity and hardness of the yogurt over the same transport distance (p < 0.05). The yogurt vibration model was established by combining principal component analysis with a Back-Propagation Artificial Neural Network model. The number of training iterations was 2669, with a correlation coefficient of 0.96611, indicating that the model was reliable. The optimal transportation distance was determined to be within the range from 20 rpm for 8 h to 100 rpm for 4 h.

Determination of four nitrofuran metabolites in gelatin Chinese medicine using dispersive solid phase extraction and pass-through solid phase extraction coupled to ultra high performance liquid chromatography-tandem mass spectrometry.

This study established a validated analytical method for the first time on the determination of nitrofuran metabolites, including semicarbazide (SEM), 1-aminohydantoin (AHD), 3-amino-2-oxazolidinone (AOZ) and 3-amino-5-morpholinomethyl-2-oxazolinone (AMOZ) in gelatin Chinese medicine. A C18 column with the mobile phase consisting of acetonitrile and 5 mmol/L ammonium acetate in water was used to separate these nitrofuran metabolites. The limit of detection of SEM, AHD, AOZ and AMOZ were found to be 0.2 µg/kg, 0.3 µg/kg, 0.2 µg/kg and 0.2 µg/kg, whereas their limit of quantification were 0.6 µg/kg, 0.8 µg/kg, 0.6 µg/kg and 0.5 µg/kg. These nitrofuran metabolites exhibited a good linear standard curve (regression coefficients above 0.99) with a concentration range of 2 µg/L to 100 µg/L. Regarding extraction procedure, gelatin Chinese medicine was pre-treated with pepsin and then extracted using 5% formic acid (v/v) in acetonitrile. The resultant extract was purified through dispersive solid phase extraction using 1000 mg anhydrous sodium sulfate, 300 mg octadecyl carbon silica gel sorbent absorbent and 500 mg ethylenediamine-N-propyl carbon silica gel absorbent, and then further purified on Oasis PRiME HLB cartridges. The matrix effect was effectively eliminated after the clean-up procedure as confirmed by comparing the ratio of standard curves prepared by standards dissolved in both matrix solvent and 5 mmol/L ammonium acetate in water: acetonitrile (95:5, v/v). The recoveries of these nitrofuran metabolites under the 1 µg/kg, 2 µg/kg and 10 µg/kg spiking levels were between 77.4% and 95.6%. These metabolites after the extraction were stable at 4 °C for 24 h. The validated method was used to analyze the residue level of these nitrofuran metabolites in 25 gelatin Chinese medicines. Results showed that only one Colla Corii Asini sample contained SEM (2.52 µg/kg) and AOZ (6.27 µg/kg), whereas one Testudinis Carapacis et Plastri sample had SEM (1.27 µg/kg) and AMOZ (9.53 µg/kg).

Determination of Chloramphenicol, Thiamphenicol and Florfenicol in Chinese Gelatin Medicines using Dispersive Solid-Phase Extraction Coupled with Ultra High-Performance Liquid Chromatography-Mass Spectrometry.

This study established a rapid and reliable method to determine chloramphenicol (CAP), thiamphenicol (TAP) and florfenicol (FF) residues in Chinese gelatin medicines. CAP, TAP and FF were extracted from medicine samples using 2% (v/v) ammonium hydroxide in acetonitrile. Trypsin was used to eliminate the matrix effect caused by protein components in gelatin medicines, whereas anhydrous sodium sulfate, C18-N and NH2-PSA adsorbents were applied to reduce matrix effect induced by other components. The analytical method of these drugs was optimized on ultra high-performance liquid chromatography-mass spectrometer (UHPLC-MS/MS) through the analysis of their standard linearity and regression. The optimized extraction and analytical method were validated in one Chinese gelatin medicine sample (Colla corii asini, E Jiao) with three fortification levels (2, 5 and 10 µg/kg), and the recoveries of these drug residues ranged of 87.6-102.7%. The limit of detection and quantification of CAP, TAP and FF in the sample were 0.2 and 0.5 µg/kg, 0.4 and 1.5 µg/kg, and 0.5 and 1.5 µg/kg, respectively. A total of 30 Chinese gelatin medicine samples were analyzed using the established method. No drug residues were found in these samples except for one Testudinis Carapacis et Plastri (1.67 µg/kg FF) and one turtle shell glue (2.55 µg/kg FF).

Efficient production of gamma-aminobutyric acid by engineered Saccharomyces cerevisiae with glutamate decarboxylases from Streptomyces.

Gamma-aminobutyric acid (GABA) is an industrially valuable natural product. This study was aimed to establish an efficient food-grade production process of GABA by engineering Saccharomyces cerevisiae that is generally recognized as safe (GRAS). GABA can be produced by catalytic decarboxylation of l-glutamate (l-Glu) by glutamate decarboxylase (GAD, EC4.1.1.15). Two GADs, SsGAD from Streptomyces sp. MJ654-NF4 and ScGAD from Streptomyces chromofuscus ATCC 49982, were heterologously expressed in S. cerevisiae BJ5464. The engineered yeast strains were used as whole-cell biocatalysts for GABA production. S. cerevisiae BJ5464/SsGAD exhibited significantly higher efficient catalytic activity than that of S. cerevisiae BJ5464/ScGAD. The optimal bioconversion system consisted of a cell density of OD600 30, 0.1 M l-Glu, and 0.28 mM pyridoxal phosphate in 0.2 M Na2 HPO4 -citric acid buffer with pH 5.4, and the reactions were performed at 50 °C for 12 H. S. cerevisiae BJ5464/SsGAD cells can be reused, and the accumulated GABA titer reached 62.6 g/L after 10 batches with an overall molar conversion rate of 60.8 mol%. This work thus provides an effective production process of GABA using engineered yeast for food and pharmaceutical applications.

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.