Huaiyuan Zhang

Production, Purification, and Characterization of a Potential Thermostable Galactosidase for Milk Lactose Hydrolysis from Bacillus stearothermophilus

Beta-galactosidase, commonly named lactase, is one of the most important enzymes used in dairy processing; it catalyzes the hydrolysis of lactose to its constituent monosaccharides glucose and galactose. Here, a thermostable beta-galactosidase gene bgaB from Bacillus stearothermophilus was cloned and expressed in B. subtilis WB600. The recombinant enzyme was purified by a combination of heat treatment, ammonium sulfate fractionation, ion exchange, and gel filtration chromatography techniques. The purified beta-galactosidase appeared as a single protein band in sodium dodecyl sulfate-PAGE gel with a molecular mass of approximately 70 kDa. Its isoelectric point, determined by polyacryl-amide gel isoelectric focusing, was close to 5.1. The optimum temperature and pH for this beta-galactosidase activity were 70 degrees C and pH 7.0, respectively. Kinetics of thermal inactivation and half-life times for this thermostable enzyme at 65 and 70 degrees C were 50 and 9 h, respectively, and the K(m) and V(max) values were 2.96 mM and 6.62 micromol/min per mg. Metal cations and EDTA could not activate this thermostable enzyme, and some divalent metal ions, namely, Fe(2+), Zn(2+), Cu(2+), Pb(2+), and Sn(2+), inhibited its activity. Thiol reagents had no effect on the enzyme activity, and sulfhydryl group blocking reagents inactivated the enzyme. This enzyme possessed a high level of transgalactosylation activity in hydrolysis of lactose in milk. The results suggest that this recombinant thermostable enzyme may be suitable for both the hydrolysis of lactose and the production of galactooligosaccharides in milk processing.

Immobilization of recombinant thermostable β-galactosidase from Bacillus stearothermophilus for lactose hydrolysis in milk

A recombinant thermostable beta-galactosidase from Bacillus stearothermophilus was immobilized onto chitosan using Tris(hydroxymethyl)phosphine (THP) and glutaraldehyde, and a packed bed reactor was utilized to hydrolyze lactose in milk. The thermostability and enzyme activity of THP-immobilized beta-galactosidase during storage was superior to that of free and glutaraldehyde-immobilized enzymes. The THP-immobilized beta-galactosidase showed greater relative activity in the presence of Ca(2+) than the free enzyme and was stable during the storage at 4 degrees C for 6 wk, whereas the free enzyme lost 31% of the initial activity under the same storage conditions. More than 80% of lactose hydrolysis in milk was achieved after 2 h of operation in the reactor. Therefore, THP-immobilized recombinant thermostable beta-galactosidase from Bacillus stearothermophilus has the potential for application in the production of lactose-hydrolyzed milk.

Extracellular secretion in Bacillus subtilis of a cytoplasmic thermostable β-galactosidase from Geobacillus stearothermophilus

beta-Galactosidase catalyzes the hydrolysis of beta-galactosides into monosaccharides and is widely used in dairy processing. This study reports the extracellular secretion of a cytoplasmic thermostable beta-galactosidase from Geobacillus stearothermophilus IAM11001 in Bacillus subtilis. This enzyme has potential applications in the dairy industry. It was not secreted in B. subtilis by mediation of 3 general secretory signal peptides, but was secreted extracellularly when it was fused to a twin-arginine signal peptide of B. subtilis phosphodiesterase. Defined and rich culture media were used for recombinant enzyme production, and the extracellular target enzymatic activity reached about 44% of the total enzymatic activity synthesized at 18 h of cultivation in Luria-Bertani medium. As a control of secretion, when the signal peptide coding sequence was absent from the N terminus of the target gene bgaB, the extracellular target enzymatic activity obtained under the same condition of cultivation accounted for less than 7% of the total enzymatic activity synthesized. Results also showed that coexpression of the B. subtilis proteins TatAd and TatCd was indispensable for the secretion of the target enzyme.

(13)C-metabolic flux analysis of lipid accumulation in the oleaginous fungus Mucor circinelloides.

The oleaginous fungus Mucor circinelloides is of industrial interest because it can produce high levels of polyunsaturated fatty acid γ-linolenic acid. M. circinelloides CBS 277.49 is able to accumulate less than 15% of cell dry weight as lipids, while M. circinelloides WJ11 can accumulate lipid up to 36%. In order to better understand the mechanisms behind the differential lipid accumulation in these two strains, tracer experiments with (13)C-glucose were performed with the growth of M. circinelloides and subsequent gas chromatography-mass spectrometric detection of (13)C-patterns in proteinogenic amino acids was carried out to identify the metabolic network topology and estimate intracellular fluxes. Our results showed that the high oleaginous strain WJ11 had higher flux of pentose phosphate pathway and malic enzyme, lower flux in tricarboxylic acid cycle, higher flux in glyoxylate cycle and ATP: citrate lyase, together, it might provide more NADPH and substrate acetyl-CoA for fatty acid synthesis.

In vitro screening of lactobacilli with antagonistic activity against Helicobacter pylori from traditionally fermented foods

Helicobacter pylori may cause stomach diseases such as chronic gastritis, peptic ulcer, and gastric cancer, and several studies reported that lactobacilli have inhibitory effects on H. pylori. In this study, 38 Lactobacillus strains were screened for anti-H. pylori activity using in vitro methods, including survivability under the simulated gastric conditions, agar plate diffusion, urease activity, coaggregation, autoaggregation, and hydrocarbon analysis. The results indicate that 2 Lactobacillus strains showed potential anti-H. pylori activity in vitro. Lactobacillus plantarum 18 had the largest zone of inhibition and markedly reduced the urease activity of H. pylori. Lactobacillus gasseri Chen had higher coaggregation rate (58.15%) and hydrophobicity (59.27%) compared with the other strains. Further research is needed to verify the activities of these strains against H. pylori.

Antibacterial and antifungal activities of various solvent extracts from the leaves and stem bark of Anisophyllea laurina R. Br ex Sabine used as traditional medicine in Guinea

ETHNOPHARMACOLOGICAL RELEVANCE Anisophyllea laurina R. Br ex Sabine is a plant that has been used in the folk medicine to treat malaria, dysentery, diabetes and toothache against bacterial infection. Through this study, most likely we are reporting for the first time, its effectiveness as an antibacterial and antifungal agent. AIM OF THE STUDY To evaluate antibacterial and antifungal activities of various solvent extracts from the leaves and stem bark of A. laurina R. Br ex Sabine. MATERIALS AND METHODS The various solvent extracts of leaves and stem bark were tested for antibacterial and antifungal activities against eight bacteria strains and three fungal strains using the well diffusion and micro-dilution methods. RESULTS Antibacterial and antifungal activities of methanol and ethanol extracts were higher than the ethyl acetate and water extract. Leaves extracts had the highest potential activity against bacteria than stem bark extracts, but showed low antifungal activity compared to stem bark extracts. They also exhibited potent growth inhibitory activity against Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli ATCC 8739 and Bacillus subtilis with MIC value of 125µg/mL. CONCLUSIONS The current study confirmed that the leaves and stem bark extracts of A. laurina R. Br ex Sabine proved to be most effective as antibacterial and antifungal activities.

Identification and Characterization of Diacylglycerol Acyltransferase from Oleaginous Fungus Mucor circinelloides

Acyl-CoA:diacylglycerol acyltransferase (DGAT) is a pivotal regulator of triacylglycerol (TAG) synthesis. The oleaginous fungus Mucor circinelloides has four putative DGATs: McDGAT1A, McDGAT1B, McDGAT2A, and McDGAT2B, classified into the DGAT1 and DGAT2 subfamilies, respectively. To identify and characterize DGATs in M. circinelloides, these four genes were expressed in Saccharomyces cerevisiae H1246 (TAG-deficient quadruple mutant), individually. TAG biosynthesis was restored only by the expression of McDGAT2B, and TAG content was significantly higher in the mutants with McDGAT2B expression than in a S. cerevisiae mutant with endogenous DGA1 expression. McDGAT2B prefers saturated fatty acids to monounsaturated fatty acids and has an obvious preference for C18:3 (ω-6) according to the results of substrate preference experiments. Furthermore, only the mRNA expression pattern of McDGAT2B correlated with TAG biosynthesis during a fermentation process. Our experiments strongly indicate that McDGAT2B is crucial for TAG accumulation, suggesting that it may be an essential target for metabolic engineering aimed at increasing lipid content of M. circinelloides.

Characterization of the triple-component linoleic acid isomerase in Lactobacillus plantarum ZS2058 by genetic manipulation

To assess the mechanism for conjugated linoleic acid (CLA) production in Lactobacillus plantarum ZS2058.

Extract of Syzygium aromaticum suppress eEF1A protein expression and fungal growth

Clove extract has therapeutic potential as an antifungal drug, yet the mechanism of action remains ambiguous. Current study aimed to address the molecular process of the antifungal activity exerted by clove extract.