Intracellular esterase from Lactobacillus casei LILA: nucleotide sequencing,purification, and characterization

An esterase gene (estC) was isolated from a genomic library of Lactobacillus casei LILA. The estC gene consisted of a 777 bp open reading frame encoding a putative peptide of 28.9 kDa. A recombinant EstC fusion protein containing a C-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of EstC revealed that it was a serine-dependent dimeric enzyme. Optimum temperature, NaCl concentration, and pH for EstC were determined to be 30 degrees C, 0% NaCl, and pH 5.5, respectively. EstC had significant activity under conditions simulating those of ripening cheese (10 degrees C, 4% NaCl, and pH 5.1). Kinetic constants (KM and Vmax) were determined for EstC action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, the previously studied EstA from Lactococcus lactis MG1363 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstC and EstA.     

Accumulation of short n-chain ethyl esters by esterases of lactic acid bacteria under conditions simulating ripening Parmesan cheese

EstA from Lactobacillus helveticus CNRZ32 (Lbh-EstA), EstB, and EstC from Lactobacillus casei LILA, and EstA from Lactococcus lactis MG1363 (Lcl-EstA) were evaluated for their ability to accumulate esters in a model system simulating Parmesan cheese ripening conditions (10 degrees C, 2 to 3% NaCl, pH 5.4 to 5.5, aw = 0.850 to 0.925) using Capalase K from kid goat as a positive control. All of the LAB esterases and Capalase K mediated the accumulation of esters in the model system in an enzyme specific manner, which was influenced by a, and selectivity for fatty acid chain-length. In general, enzyme mediated accumulation of ethyl esters was higher at aw values of 0.850 and 0.900 than at aw of 0.925, demonstrating that aw is a critical parameter influencing ester accumulation. The substrate selectivity of esterases, aw, and enzyme type may be important factors in the development of fruity flavors, as evidenced by results in this model system simulating Parmesan cheese ripening conditions.     

A NaCl-stable serine proteinase from Virgibacillus sp. SK33 isolated from Thai fish sauce

An extracellular proteinase from Virgibacillus sp. SK33, isolated from 1 month-old fish sauce, was purified to electrophoretic homogeneity, using hydrophobic interaction chromatography and hydroxyapatite with purification fold of 2.5 and 7% yield. The anomalous molecular weight (MW) of 19 kDa was obtained from SDS–PAGE, whereas a MW of 33.7 kDa was determined by MALDI-TOF. Optimum conditions for catalytic activity were 55 °C and pH 7.5. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferentially hydrolysed Suc-Ala-Ala-Pro-Phe-AMC, indicating a serine proteinase with subtilisin-like characteristics. K_m and k_cat of the purified proteinase were 27 μM and 12 s⁻¹, respectively. Proteinase activity, toward both synthetic and anchovy substrates, increased with NaCl up to 25%. The proteinase exhibited high stability in both the absence and presence of NaCl up to 25%. Approximately 2.5-fold increase in activity was observed in the presence of divalent cations, including Ca²⁺, Mg²⁺ and Sr²⁺ at 100 mM. MALDI-TOF MS and LC–ESI-MS/MS analyses, as well as N-terminal sequences, revealed that the purified enzyme did not match microbial proteinases in the database, indicating it to be a novel proteinase.     

The processing of high-molecular-weight xylanase (XynE, 110 kDa) from Aeromonas caviae ME-1 to 60-kDa xylanase (XynE60) in Escherichia coli and purification and characterization of XynE60

A xylanase gene (xynE) encoding XynE (110 kDa) was cloned from a lambda phage genomic library of Aeromonas caviae ME-1 which is a multiple-xylanase-producing bacterium. Upon nucleotide sequence analysis, we found that xynE comprises 2823 by and encodes a protein of 941 amino acid residues (104,153 Da), which was similar to endo-beta-1,4-xylanases which are categorized to glycosyl hydrolase family 10. An Escherichia coli transformant that harbored pXED30 carrying xynE produced 110-, 84-, 72-, and 66-kDa xylanases in the cell-free extract, and 72- and 66-kDa xylanases in the culture supernatant. We purified the 66-kDa xylanase to electrophoretic homogeneity from a culture supernatant by a series of column chromatographies. The calculated molecular mass of the purified xylanase determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was 60,154.50 Da, and the xylanase was designated XynE60. Analysis of the N-terminal 10 amino acid residues and the determined molecular mass of XynE60 revealed that XynE60 is a product processed at the Gly26-Gly27, and Thr565-Ala566 sites of XynE by proteolytic cleavage. XynE60 showed optimal activity at 55 degrees C and pH 8.0, and was stable below 45 degrees C and at pH 7.0-8.5. The K(m) and V(max) of XynE60 were calculated to be 8.1 mg/ml and 6897 nkat/mg, respectively.     

Characterization of a thermostable family 10 endo-xylanase (XynB) from Thermotoga maritima that cleaves p-nitrophenyl-beta-D-xyloside

Thermotoga maritima MSB8 possesses two xylanase genes, xynA and xynB. The xynB gene was isolated from the genomic DNA of T. maritima, cloned, and expressed in Escherichia coli. XynB was purified to homogeneity by heat treatment, affinity chromatography and ion-exchange column chromatography. The purified enzyme produced a single band upon SDS-PAGE corresponding to a molecular mass of 42 kDa. At 70 degrees C, the enzyme was stable between pH 5.0 and pH 11.4, and it was stable at temperatures of up to 100 degrees C from pH 7.0 to pH 8.5. At 50 degrees C, XynB displayed an optimum pH of 6.14 and at this pH the temperature for optimal enzyme activity was 90 degrees C. XynB exhibited broad substrate specificity and was highly active towards p-nitrophenyl-beta-D-xylobioside with K(m) and k(cat) values of 0.0077 mM and 5.5 s(-1), respectively, at 30 degrees C. It was also active towards p-nitrophenyl-beta-D-xyloside. The initial product of the cleavage of p-nitrophenyl-beta-D-xyloside was xylobiose, indicating that the major reaction in the cleavage was transglycosylation, not hydrolysis.     

Purification and properties of a low-molecular-weight phytase from Cladosporium sp. FP-1

A fungus producing high levels of phytase was isolated from air and identified as Cladosporium sp. The phytase production was stimulated by phytate in the medium used. The maximum production of phytase (108 U/ml) occurred in a medium containing 1.0 g of phytate per 100 ml. The phytase was purified to electrophoretic homogeneity by ion-exchange chromatography and gel filtration. Based on SDS-PAGE analysis, the molecular weight of the purified phytase was calculated to be approximately 32.6 kDa, and the narrow protein band indicated that this phytase is not glycosylated. The phytase has an optimum pH of 3.5, and an optimum temperature of 40 degrees C. The phytase activity was stimulated by 2-mercaptoethanol and dithiothreitol, and inhibited by Ba2+, Pb2+, iodoacetate, p-chloromercuribenzoate and phenylmethylsulfonyl fluoride. The phytase displayed high affinity for phytate and the Km was 15.2+/-3.1 microM. NMR analyses (1D and 2D) indicated that the end hydrolysis product of phytate was myo-inositol 1,2,5-triphosphate.     

Acidothermus cellulolyticus 11B葡萄糖异构酶基因的克隆、表达及酶活性研究

葡萄糖异构酶是生物法转化葡萄糖为果糖制备果葡糖浆的关键酶。本文克隆到一种葡萄糖异构酶基因xyl,来源于嗜酸耐热型微生物Acidothermus cellulolyticus 11B(ATCC43068)。以pET-22b(+)为载体质粒,E.coliBL21(DE3)为宿主细胞,构建了基因重组菌,IPTG可诱导目的重组葡萄糖异构酶过量表达;经亲和层析纯化的重组蛋白样品进行SDS-PAGE分析,在约43kDa处出现显著的特征蛋白条带;活性检测结果表明该重组葡萄糖异构酶具有较高的果糖转化活性。     

Purification, characterization, and steady-state kinetics of a meta-cleavage compound hydrolase from Pseudomonas fluorescens IPO1

2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl-HODA) hydrolase (CumD), an enzyme of the cumene biodegradation pathway encoded by the cumD gene of Pseudomonas fluorescens IP01, was purified to homogeneity from an overexpressing Escherichia coli strain. SDS-polyacrylamide gel electrophoresis and gel filtration demonstrated that it is a dimeric enzyme with a subunit molecular mass of 32 kDa. The pH optima for activity and stability were 8.0 and 7.0-9.0, respectively. The enzyme exhibited a biphasic Arrhenius plot of catalysis with two characteristic energies of activation with a break point at 20 degrees C. The enzyme has a K(m) of 7.3 microM and a k(cat) of 21 s(-1) for 6-isopropyl-HODA (150 mM phosphate, pH 7.5, 25 degrees C), and its substrate specificity covers larger C6 substituents compared with another monoalkylbenzene hydrolase, TodR Unlike TodF, CumD could slightly hydrolyze 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA). A mutant enzyme as to a putative active site residue, S103A, had 10(5)-fold lower activity than that of the wild-type enzyme.     

Purification and characterization of a novel cyclohexylamine oxidase from the cyclohexylamine-degrading Brevibacterium oxydans IH-35A

Cyclohexylamine oxidase (CHAO) from a cell extract of Brevibacterium grown on cyclohexylamine was purified 50.2-fold, to electrophoretic homogeneity, by serial chromatographies. The molecular mass of the native enzyme was estimated to be approximately 50 kDa by gel filtration and SDS-PAGE. The optimum pH was 7.4 and the stable pH range was 6.0 to 7.0. The enzyme was thermostable up to 30 degrees C. The enzyme was found to be highly specific for the deamination of alicyclic monoamines such as cyclopentylamine, cycloheptylamine, and N-methylcyclohexylamine and aliphatic monoamines, such as sec-butylamine. The apparent K(m) value for cyclohexylamine was 1.23 mM. The enzyme was inhibited by flavin enzyme inhibitors such as quinine and quinacrine. The N-terminal 27 amino acid residues were determined as Gly-Ser-Val-Thr-Pro-Asp-Pro-Asp-Val-Asp-Val-Ile-Ile-His-Gly-Ala-Gly-Ile-Ser-Gly-Ser-Ala-Ala-Ala-Lys-Ala-Leu-, revealing homology to conventional flavin-containing amine oxidases (EC 1.4.3.4).     

Ester synthesis and hydrolysis in an aqueous environment,and strain specific changes during malolactic fermentation in wine with Oenococcus oeni

Previous work has shown that Oenococcus oeni produces esterases that are capable of hydrolysing artificial substrates. Using SPME–GCMS, this study provides evidence that purified O. oeni esterases have the ability to both synthesise and hydrolyse esters. Two purified esterases (EstA2 and EstB28) synthesised ethyl butanoate and ethyl hexanoate to varying degrees. Both purified esterases hydrolysed ethyl butanoate, ethyl hexanoate and ethyl octanoate. Once this dual activity was confirmed, malolactic fermentation (MLF) trials were conducted in wine with O. oeni strains that had been previously observed to have either high or low esterase activity against artificial substrates. Strain specific differences were observed and strains with low esterase hydrolysis activity against artificial substrates had a higher level of total esters measured after MLF. The results demonstrate the impact that O. oeni has on wine aroma and relates this to the ester hydrolysis and synthesis abilities of O. oeni strains.     

Purification and properties of a phytase from Candida krusei WZ-001

A phytase from Candida krusei WZ-001 isolated from soil was purified to electrophoretic homogeneity by ion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The phytase is composed of two different subunits with molecular masses of 116 kDa and 31 kDa on SDS-PAGE (or 120 kDa and 30 kDa on gel chromatography), with the larger subunit having a glycosylation rate of around 35%. The phytase has an optimum pH of 4.6, an optimum temperature of 40 degrees C and a pI value of 5.5. The phytase activity was stimulated by 2-mercapto-ethanol and dithiothreitol (DTT), and inhibited by Zn2+, Mg2+, iodoacetate, pI value of 5.5. The phytase activity was stimulated by 2-mercapto-ethanol ethanol and dithiothreitol (DTT), and inhibited by Zn2+, Mg2+, iodoacetate, p-chroloromercuribenzoate (pCMB) and phenylmethylsulfonyl fluoride (PMSF). The phytase displayed a broad substrate specificity and the K(m) for phytate was 0.03 mM. Phytate was sequentially hydrolyzed by the phytase. Furthermore, 1D and 2D NMR analyses and bioassay of myoinositol indicated that the end hydrolysis product of phytate was myoinositol 2-monophosphate.     

Purification and characterization of the extracellular proteinase of Pseudomonas fluorescens NCDO 2085

Pseudomonas fluorescens NCDO 2085 produced a single heat-stable extracellular proteinase in Na caseinate medium at 20 degrees C and pH 7.0. The proteinase was purified to electrophoretic homogeneity using chromatofocusing, gel filtration and ion-exchange chromatography. The purification procedure resulted in a 158-fold increase in the specific activity and a yield of 3.5% of the original activity. The enzyme is a metalloproteinase containing Zn and Ca, with an isoelectric point at 5.40 +/- 0.05 and a mol. wt of 40 200 +/- 2100. It is heat-stable having D-values at 74 and 140 degrees C of 1.6 and 1.0 min respectively; 40 and 70% of the original activity remained after HTST (74 degrees C/17 s) and ultra high temperature (140 degrees C/4 s) treatments respectively. The amino acid composition of the proteinase was determined and compared with those from other Pseudomonas spp.     

Purification and partial characterization of psychrotrophic Serratia marcescens lipase

Serratia marcescens isolated from raw milk was found to produce extracellular lipase. The growth of this organism could contribute to flavor defects in milk and dairy products. Serratia marcescens was streaked onto spirit blue agar medium, and lipolytic activity was detected after 6 h at 30 degrees C and after 12 h at 6 degrees C. The extracellular crude lipase was collected after inoculation of the organism into nutrient broth and then into skim milk. The crude lipase was purified to homogeneity by ion-exchange chromatography and gel filtration. The purified lipase had a final recovered activity of 45.42%. Its molecular mass was estimated by SDS-PAGE assay to be 52 kDa. The purified lipase was characterized; the optimum pH was likely between 8 and 9 and showed about 70% of its activity at pH 6.6. The enzyme was very stable at pH 8 and lost about 30% of its activity after holding for 24 h at 4 degrees C in buffer of pH 6.6. The optimum temperature was observed at 37 degrees C and exhibited high activity at 5 degrees C. The thermal inactivation of S. marcescens lipase was more obvious at 80 degrees C; it retained about 15% of its original activity at 80 degrees C and was completely inactivated after heating at 90 degrees C for 5 min. Under optimum conditions, activity of the enzyme was maximum after 6 min. The Michaelis-Menten constant was 1.35 mM on tributyrin. The enzyme was inhibited by a concentration more than 6.25mM. Purified lipase was not as heat-stable as other lipases from psychrotrophs, but it retained high activity at 5 degrees C. At pH 6.6, the pH of milk, purified lipase showed some activity and stability. Also, the organism demonstrated lipolytic activity at 6 degrees C after 12 h. Therefore, S. marcescens and its lipase were considered to cause flavor impairment during cold storage of milk and dairy products.     

Transglutaminase from Streptoverticillium ladakanum and application to minced fish product

The transglutaminase (TGase) from Streptoverticillium ladakanum was purified to electrophoretic homogeneity after ammonium sulfate fractionation and Blue Sepharose Fast Flow chromatography. The molecular weight of the purified TGase was 30.5 kDa estimated by Superdex 75HR gel filtration, and 37.5 kDa by SDS-PAGE. This enzyme, with optima at pH at 6.0 and 50°C was very stable at pH 5.0–7.0. It was strongly inhibited by PCMB, PMSF, Pb²⁺, Zn²⁺ and Cu²⁺, but not affected by EDTA and Ca²⁺. This suggested that the purified TGase was calcium-independent and its active center contained cysteine. It catalyzed the crosslinking of fish myosin heavy chain and substantially increased the gel strength of mackerel surimi.     

Purification and some characteristics of a monomeric alanine racemase from an extreme thermophile, Thermus thermophilus

We purified to homogeneity an alanine racemase (EC 5.1.1.1) from Thermus thermophilus HB8, an extreme thermophile. Interestingly, the enzyme possessed a monomeric structure with a molecular weight of about 38,000. The enzyme was most active at pH 8 and 75 degrees C, and remained active after incubation at 80 degrees C for 30 min.     

Purification and characterization of rice alpha-glucosidase, a key enzyme for alcohol fermentation of rice polish

Alpha-glucosidase, a key enzyme for nuka-sake brewing, was purified from Oryza sativa cv. Yamadanishiki, which is widely used for sake brewing. The molecular weight of the purified enzyme was 95 kDa. The optimum pH and temperature were 4.5 and 55 degrees C, respectively. The substrate specificity differed from that of Oryza sativa cv. Shinsetsu, which is a variety of rice consumed as a cereal. The extraction of alpha-glucosidase from the rice was stimulated by lactic acid, which suggests that lactic acid plays an important role not only in preventing bacterial contamination, but also in stimulating the parallel fermentation that occurs in nuka-sake brewing.     

Characterisation of partially purified milk-clotting enzyme from Solanum dubium Fresen seeds

The seeds of Solanum dubium were blended and extracted using different types of buffers. The most reliable, quick, and efficient buffer was found to be 5% NaCl in acetate buffer (pH 5.0) which was used throughout the study. The extract was filtered and fractionated twice with ammonium sulphate. The partially purified enzyme was characterised by SDS–PAGE which showed a band of molecular weight of 66 kDa with the presence of other bands of low density. When compared with other plant enzymes, S. dubium enzyme was found to have higher clotting and proteolytic activities. The activity of the enzyme was steadily increased with enzyme and substrate concentration. The enzyme was found to be very stable against a wide range of pH values as well as a wide range of temperature (20–90 °C). The results of substrate specificity of the enzyme showed that the partially purified enzyme preferred both hydrophilic and hydrophobic amino acid residues at P1 position. The catalytic efficiency of the purified enzyme was enhanced by an aliphatic amino acids (Leu) compared to aromatic residue (Phe) at P1 position at the same site.     

Purification and characterization of intracellular proteinase from Lactobacillus casei ssp. casei LLG

The intracellular proteinase of Lactobacillus casei ssp. casei LLG was isolated in the cytoplasmic fraction with 0.05 M Tris-HCl buffer (pH 7.5). The enzyme was purified by the fast protein liquid chromatography system equipped with ion-exchange and gel filtration chromatographies. This proteinase comprised a single monomeric form and had a molecular weight of about 55 kDa and an isoelectric point near pH 4.9. The optimum pH and temperature for the enzyme activity were determined to be pH 6.5 and 37 degrees C, respectively. The enzyme was inactivated by metal-chelating compounds (EDTA, 1,10-phenanthroline) and less affected by serine proteinase inhibitors (diisopropylfluorophosphate, phenylmethylsulfonyl fluoride). Proteinase activity was increased by Ca++, Mn++, and Co++, and inhibited by Cu++, Mg++, and Zn++. The activity of this enzyme to hydrolyze casein appeared to be more active on beta-casein than alphas1-casein and kappa-casein as monitored by polyacrylamide gel electrophoresis.     

Cloning, over-expression and characterization of an alkali-tolerant endo-β-1,4-mannanase from Penicillium freii F63

A glycosyl hydrolase family 5 endo-β-mannanase gene (man5F63) was cloned from Penicillium freii F63 and overexpressed in Pichia pastoris. man5F63 contained an open reading frame of 1260 bp that encoded a polypeptide of 419 amino acids including a putative 18-residue signal peptide. The recombinant enzyme (rMan5F63) was secreted into the culture supernatant to near electrophoretic homogeneity with a high yield (1.1 gl(-1) in flask). Its apparent molecular weight was approximately 72.0 kDa, 29.0 kDa higher than the theoretical molecular mass. rMan5F63 was optimal at pH 4.5 and 60 °C and exhibited good stability over a broad pH range from acidic to alkaline (>85.0% activity at pH 4.0-9.0, >70.0% activity at pH 10.0 and 43.7% even at pH 12.0). The activity of rMan5F63 was significantly enhanced in the presence of Co(2+), Cu(2+), Mn(2+) and β-mercaptoethanol and was strongly inhibited by Hg(2+) and SDS. The specific activity, K(m) and V(max) values were 47.5 U mg(-1), 7.8 mg ml(-1) and 70.4 μmol min(-1)mg(-1), respectively, for locust bean gum, and 40.3 U mg(-1), 2.3 mg ml(-1) and 61.7 μmol min(-1)mg(-1), respectively, for konjac flour. All these favorable enzymatic properties make it cost-effective to commercialization and valuable in various industries.     

Purification, characterization and functional cloning of inositol oxygenase from Cryptococcus

The enzyme inositol oxygenase (myo-inositol : oxygen oxidoreductase; E.C. 1.13.99.1) is a monooxygenase that converts inositol into glucuronic acid in the presence of molecular oxygen. This enzyme is integrated into a pathway leading to either degradation and energy production or the biosynthesis of precursors for polysaccharides. The enzyme was purified from the yeast Cryptococcus lactativorus by a five-step chromatography procedure. The purified enzyme shows a molecular mass of 37 kDa on SDS-PAGE, similar to the estimation of the size of the native enzyme determined by size exclusion chromatography. Peptides of the inositol oxygenase protein derived from a tryptic digest were sequenced de novo by nanoelectrospray tandem mass spectrometry. Using degenerate oligonucleotides, the corresponding gene was cloned from first strand cDNA. The open reading frame encodes a 315 amino acid polypeptide with a predicted molecular mass of 36.9 kDa. Inositol oxygenase is a single copy gene in C. lactativorus. It has close homologues in other fungi such as Cryptococcus neoformans and Neurospora crassa. Biochemical characterization of the enzyme showed a pH optimum of 6-6.5 and a temperature optimum of 30 degrees C. Myo-inositol is the only accepted substrate with a Km of ca. 5 mM. The enzyme contains a Fe-centre but the enzyme activity is resistant to KCN.