Purification and characterization of a glucoamylase from Rhizopus oryzae

Glucoamylase (EC 3.2.1.3) of Rhizopus oryzae NRRL 395 was purified approximately sevenfold by sequential ammonium sulfate fractionation, Biogel P-100 gel filtration, Q-Sepharose anion exchange and S-Sepharose cation exchange. The pH and temperature optima were 4·8 and 60°C, respectively. Enzyme was stable at temperatures up to 40°C and pH values between 3 and 8. The molecular weight was 67 000 daltons as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the pI was 8·7 as determined by chromatofocusing. The K_m for amylopectin and soluble starch were 0·98 and 1·34 mg/ml, respectively. The V_max for amylopectin and soluble starch were 782 and 136 μmoles of glucose produced per mg of protein per min, respectively. The enzyme activity was inhibited by Hg²⁺, Pb²⁺ and Cd²⁺, but not by EDTA.     

Purification and characterization of a second aminopeptidase (pepC-like) from Lactobacillus delbrueckii subsp. bulgaricus B14

A second aminopeptidase was purified from cell-free extracts of Lactobacillus delbrueckii subsp. bulgaricus B14 by ammonium sulphate precipitation and two steps of anion-exchange chromatography. After SDS polyacrylamide-gel electrophoresis in the presence of β-mercaptoethanol, one protein band was detected at 54 kDa. The same molecular mass was estimated by gel filtration. SDS polyacrylamide-gel electrophoresis in the absence of β-mercaptoethanol resulted in a single band at 220 kDa, indicating that the enzyme forms complexes of four molecules under non-reducing conditions. Activity was markedly increased by reducing and metal-chelating agents. Thiol-group inhibitors, such as iodoacetic acid, inhibited the enzyme strongly. In contrast to Mg²⁺ and Ca²⁺, which had slightly activating effects, other divalent cations reduced enzyme activity at a concentration of 1 mM. The aminopeptidase showed highest activity at 50°C and pH 6·5–7 and hydrolyzed a wide range of di- and tripeptides. The most suitable substrates were Leu-Gly, Leu-Gly-Gly, Ala-Ala-Ala, and Met-Gly-Gly. For Leu-Gly and Leu-Gly-Gly, K_m-values of 1·81 mM and 2·17 mM and turnover numbers of 870 s⁻¹ were calculated, with a maximal rate of hydrolysis (V_max) of 4600 and 2780 μmol/min per mg of protein, respectively. The aminopeptidase did not cleave Lys-pNA, a substrate hydrolyzed by all type-‘N’ aminopeptidases from lactic acid bacteria with high velocities. It compared well, however, with pepC found in Lactococcus.     

Purification and properties of an acid phosphatase from Lactobacillus curvatus DPC2024

An acid phosphatase was purified to homogeneity from a cell-free extract of Lactobacillus curvatus DPC2024 by chromatography on diethylaminoethyl-Sephacel, Phenyl Sepharose, chelating Sepharose Fast Flow and MonoQ. The purified enzyme was a tetramer with a subunit molecular mass of 26 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel filtration chromatography. Its optimum activity was at pH 4.5 and 70°C, with more than 65% of its activity retained after pre-heating for 30 min at 70°C. The enzyme was strongly inhibited by NaF (0.1 m ) and ZnCl₂ (1.0 m ); slightly inhibited by hexametaphosphate, tripolyphosphate or pyrophosphate at 1.0 m concentrations; but unaffected by 10 m ascorbic acid. The acid phosphatase hydrolysed a number of phosphate esters but not bis(p-nitrophenyl)phosphate nor uridine-5′-monophosphate. The N-terminal amino acid sequence of the first 20 residues of this enzyme showed 65% homology with an acid phosphatase from Lactobacillus plantarum DPC2739 and some homology with other phosphatases from mammals, yeasts and Escherichia coli.     

Trypsin-like enzyme from intestine and pyloric caeca of spotted goatfish (Pseudupeneus maculatus)

Trypsin-like enzyme was partially purified from the intestine and pyloric caeca of spotted goatfish (Pseudupeneus maculatus) by a simple three steps procedure: heat treatment, ammonium sulphate precipitation and Sephadex G-75 filtration. The enzymes from the intestine and pyloric caeca were 96- and 57.7-fold purified with yield values of 68.1% and 26.1%, respectively. The pyloric caeca enzyme collected from the Sephadex G-75 filtration showed a single band in SDS–PAGE (24.5 kDa). Both enzymes presented identical optima pH (9.0) and temperature (55 °C). After incubation at 45 °C for 30 min, enzymes obtained from intestine remained fully activity while a loss of activity (10%) of enzyme extracted from pyloric caeca was registered. Michaelis constant was not significantly different for trypsin-like enzyme from pyloric caeca (1.82 ± 0.19 mM) and that from the intestine (1.94 ± 0.45 mM) acting on benzoyl-dl-arginine-p-nitroanilide (BAPNA). Finally, their activities were inhibited by the following ions in decreasing order: Al³⁺ > Zn²⁺ > Hg²⁺ = Cu²⁺ > Cd²⁺. The effects of Ca²⁺, Mg²⁺, Mn²⁺, Ba²⁺, K¹⁺, Li¹⁺ and Co²⁺ showed to be less intensive. The similarities between them provide basis for the proposition of obtaining an attractive protease preparation from the tons of intestine and pyloric caeca, that are usually discarded, from this fish which is an important species exported by North-eastern Brazilian fishery industry.     

Thermal inactivation of lectins (PHA) isolated from Phaseolus vulgaris

The influence of the thermal process on the loss of ability to bind a carbohydrate target was studied on lectins (PHA) purified from Phaseolus vulgaris seeds. Thermal inactivation of aqueous solutions of pure PHA occurred according to a biphasic first-order mechanism, the thermodynamic parameters, at pH 7·3, being as follows: ΔH*₁ = ΔH*₂ = 86·2 kcal mole⁻¹, ΔS*₁ = − 54·04 cal deg⁻¹ and ΔS*₂ = − 56·71 cal deg⁻¹. The first-order rate constants appeared to be dependent on pH (minimal around 7) and divalent cations. All different subunits constituting the whole PHA were inactivated at the same rate. The biphasic nature of this process is independent of the presence of 10 m Ca⁺⁺ or Mg⁺⁺ and appeared to indicate a discrete aggregation of PHA molecules.     

酪蛋白铁螯合肽的分离纯化及结构解析

为探讨酪蛋白铁螯合肽螯合机制,对比固定金属亲和层析和阴离子交换层析初分再结合Sephacryl S-100 HR凝胶过滤层析分离对酪蛋白铁螯合肽进行分离纯化效果,并通过紫外光谱、傅里叶变换红外光谱和液相色谱-质谱联用方法对纯化后多肽结构进行解析。结果表明,亲和层析与凝胶过滤层析法结合分离得到的酪蛋白多肽组分的铁螯合活性高于阴离子交换层析组分,其铁螯合活性可达39.56?μg/mg。紫外光谱和红外光谱检测证明酪蛋白肽和铁离子形成螯合物,羧基位点螯合前后发生变化;质谱鉴定出3?条肽段,氨基酸序列为HIQKEDVPSER、ITVDDKHYQK和TRLHPVQER,肽段中Glu、Asp、Gln出现频率高,侧链均含有C＝O键,推测多肽的羰基位是铁离子的主要螯合位点。     

Purification and characterization of an endopeptidase from Lactobacillus delbrueckii subsp. bulgaricus B14

An intracellular endopeptidase was purified from cell-free extracts of Lactobacillus delbrueckii subsp. bulgaricus B14 by anion exchange chromatography on DEAE-Sepharose, hydroxyapatite chromatography, second anion exchange chromatography on Mono-Q, and metal-chelating affinity chromatography. The endopeptidase was a monomer with a molecular mass of approximately 70 kDa determined by SDS-PAGE and gel filtration. Various oligopeptides (e.g. Met-enkephalin, bradykinin) were hydrolysed by the endopeptidase. Exopeptidase activity and cleavage of dipeptides or tripeptides was not observed. The K_M value for the cleavage of Metenkephalin was 1.2 mM. Temperature and pH optima were 47 °C and pH 7.7, respectively. The endopeptidase was inhibited by the classical agents for metal-dependent (EDTA) and serine (DFP) enzymes. Activity was increased by Co²⁺ and Mg²⁺, no effect was observed with Ca²⁺. After inhibition with EDTA, enzyme activity could be restored fully by Co²⁺. Activity was inhibited by Zn²⁺, Mn²⁺, Fe²⁺, Cu²⁺, Cd²⁺ and Hg²⁺. The N-terminal sequence of the endopeptidase was determined as: H₂N-Val-Arg-Gly-Gly-Ser-Gly-Asp-Thr-Thr-Val-0H.     

Purification, partial characterisation and mode of action of enterococcin EFS2, an antilisterial bacteriocin produced by a strain of Enterococcus faecalis isolated from a cheese

Enterococcus faecalis strain EFS2, isolated from the surface of a traditional cheese, produced a bacteriocin active against Gram-positive bacteria including Listeria spp. and some Staphylococcus aureus strains. The bacteriocin, named enterococcin EFS2, has been purified to homogeneity by ammonium sulphate precipitation and reversed-phase high performance liquid chromatography (RP-HPLC). The molecular weight was determined by mass spectrometry to be 7149.6. The amino acid composition of enterococcin EFS2 revealed that it contained 67 amino acid residues and had a blocked amino-terminal end. Enterococcin EFS2 induced viability loss, efflux of K⁺ ions and ATP, and cell lysis. Kinetic study of bactericidal activity of enterococcin EFS2 on Listeria innocua strain LIN 11 indicated slower cell destruction than by nisin. At pH 7.0, the activity of enterococcin EFS2 was the highest at 35 °C and was lost at 15 °C. The bacteriocin was more active against L. innocua strain LIN11 in broth adjusted to pH 6.0, 7.0 and 8.0 than to pH 4.5 at 30 °C.     

Purification and characterization of a dipeptidase from Lactobacillus curvatus DPC2024

A dipeptidase was purified to homogeneity from a cell-free extract of Lactobacillus curvatus DPC2024 by chromatography on diethylaminoethyl-sephacel, phenyl sepharose, chelating sepharose fast flow and MonoQ. The purified dipeptidase was a monomer of ∼52 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel filtration chromatography. The enzyme was optimally active at pH 8 and 50°C and retained ∼10% of its maximum activity after pre-heating for 10 min at 70°C. The enzyme was a metallopeptidase, strongly inhibited by 0.1 mM ethylenediaminetetraacetic acid and o-phenanthroline and reactivated by a number of divalent metal ions. The enzyme was also inhibited by p-chloromercuribenzoate and β-mercaptoethanol. The enzyme was a strict dipeptidase, capable of hydrolysing a range of dipeptides but not tri-, tetra- or pentapeptides, p-nitroanilide derivatives of amino acids nor N- and C-terminal-blocked dipeptides. The N-terminal amino acid sequence of the first 20 residues showed significant homology with dipeptidases from Lactobacillus delbrueckii subsp. lactis DSM 7290 and Lactococcus lactis subsp. cremoris MG1363.     

The peptide hydrolase system of Lactobacillus reuteri.

Peptide hydrolase system of Lactobacillus reuteri CRL 1098, a lactic acid bacteria of sourdough origin, was investigated. This microorganism has a broad range of peptidases consisting of an active aminopeptidase, X-Prolyl-dipeptidylaminopeptidase, dipeptidase and tripeptidase. Aminopeptidase, iminopeptidase and endopeptidase are most likely located in the cytoplasmic fraction showing no detectable association with the cell membrane, while dipeptidase and tripeptidase are mainly associated with the latter fraction. The peptidases are metalloenzymes activated by Co²⁺ and inhibited by Cu²⁺, Hg²⁺, Cd²⁺ and by metal-complexing reagents. The aminopeptidase activity inhibited by EDTA can be restored by Mn²⁺ while that of di- and tripeptidase treated with 1,10-phenantroline can be restored by Zn²⁺ and Co²⁺, respectively.     

Isolation of angiotensin I converting enzyme (ACE) inhibitor from fermented oyster sauce, Crassostrea gigas

Angiotensin I converting enzyme (ACE) inhibitor was isolated from fermented oyster sauce (FOS) and purified. Oyster was fermented with 25% NaCl (w/w) at 20 °C for 6 months. FOS was passed through a 40-mesh sieve, desalted using an electrodialyzer and then lyophilized. ACE inhibitory activity of FOS was investigated, and the IC₅₀ value was determined to be 2.45 mg/ml. ACE inhibitor from FOS was purified using SP-Sephadex C-25 ion exchange chromatography, Sephadex G-50 gel chromatography, high-performance liquid chromatography (HPLC) on a gel permeation chromatography (GPC) column and reversed-phase HPLC on a C₁₈ column. The purified inhibitor had an IC₅₀ value of 0.0874 mg/ml, and it exhibited competitive inhibition against ACE. The purified peptide was evaluated for its antihypertensive effect in spontaneously hypertensive rats (SHRs) following oral administration. Rat blood pressure significantly decreased after inhibitor injection.     

Identification of halothane genotypes by calcium accumulation and their meat quality using live pigs

The three halothane genotypes (NN, Nn, and nm) were identified by measuring the capacity for Ca²⁺ accumulation by sarcoplasmic reticulum in whole muscle homogenate preparations of M. longissimus dorsi with a Ca²⁺ specific electrode at 35°C. Significant differences (P < 0·001) in deterioration (%) of Ca²⁺ accumulation, 12% for NN, 35% for Nn, and 81% for nn pigs, were observed after ageing the whole muscle homogenate preparations for 24 h in ice.

Predictions of meat quality in live pigs (n = 34) based on the values for water-holding capacity, assessed as fluid (g/0·5 g wet wt LD), and pH (fluid) by using small biopsy LD samples (Cheah et al. 1993) were performed on all the halothane genotypes. The halothane genotype NN (n = 11) showed a fluid value of 0·37 ± 0·01 and a pH (fluid) value of 6·62 ± 0·03 as compared with 0·61 ± 0·02 and 5·84 ± 0·04, respectively, for the halothane genotype nn (n = 13). The Nn pigs (n = 10) showed fluid (0·49 ± 0·03) and pH (fluid) (6·19 ± 0·11) values between those values observed for the two homozygotes (NN and nn). Predictions of meat quality in live pigs from biopsy LD muscles were confirmed from assessments on post-mortem LD muscles based on pH₁ and fibre optic probe (FOP) measurements.

The extent of deterioration (%) in Ca²⁺ accumulation showed high correlations with fluid (r = −0·861) and pH (fluid) (r = −0·831) in the biopsy LD samples, and with pH₁ (r = 0·663), FOP (r = −0·812), and drip (%) loss (r = −0·777) in the post-mortem LD samples.     

Effect of pretreatment and temperature on air-drying of Dioscorea alata and Dioscorea rotundata slices

Effects of pretreatment and drying conditions on yam varieties, namely Dioscorea alata and Dioscorea rotundata, in a fabricated laboratory scale hot air drier at temperature range of 50–80 °C and constant air velocity of 1.5 m²/s were investigated. Mass transfer during air-drying of yam slices was described using Fick’s diffusion model. Drying took place entirely in the falling rate period. Temperature dependency of moisture on diffusivity was illustrated by the Arrhenius relationship. Over the range of temperature, moisture diffusivities varied from 9.92 × 10⁻⁸ to 1.02 × 10⁻⁷ and 0.829 × 10⁻⁶ to 1.298 × 10⁻⁵ m²/s for D. alata and D. rotundata, respectively. Activation energy for drying of D. alata and D. rotundata varied from 25.25 to 46.46 and 41.75 to 72.47 kJ/mol, respectively.     

A practical trial of pyrethrins-in-oil surface sprays for the protection of bagged grain against infestation by Cadra cautella (Wlk.) (Lepidoptera, Phycitidae) in Kenya

Two formulations of synergized pyrethrins in technical white oil were tested as monthly protective sprays on stacks of fumigated bagged wheat, primarily against Cadra cautella (Wlk.) but also against Sitophilus oryzae (L) and Tribolium castaneum (Hbst.), under warm-temperate storage conditions in up-country Kenya. The formulations were: 0·4% pyrethrins with 2·0% piperonyl butoxide, applied at 50 ml/m², and 0·4% pyrethrins with 0·4% piperonyl butoxide at 20 ml/m².

Results were assessed by recording infestation in samples taken from each stack after 18 weeks storage and five spray applications.

Both treatments gave reasonably good protection against C. cautella but were not satisfactory against S. oryzae or T. castaneum. There was no evidence of any taint in bread made from the treated grain, but the higher application rate caused excessive staining of the bags.

It is concluded that satisfactory control of reinfestation by C. cautella can be expected in practice using 0·4% pyrethrins in oil with only a minimal quantity of added piperonyl butoxide, and that 20 ml/m² is a suitable rate for application to bagged produce.     

Some properties of the polygalacturonase from four Zimbabwean wild fruits (Uapaca kirkiana, Zizphus mauritiana, Tamarindus indica and Berchemia discolor fruits)

Polygalacturonase was extracted from ripe Uapaca kirkiana, Zizphus mauritiana, Tamarindus indica and Berchemia discolor fruits of Zimbabwe. Protein concentrations and activities of the enzymes in the extracts were determined in the four fruit extracts. The protein concentrations in the enzyme extracts ranged from 0.82 ± 0.17 to 1.97 ± 0.13 mg/ml and enzyme activities from 1.99 ± 0.13 to 6.64 ± 0.38 mmol min⁻¹ mg⁻¹ in the four fruit extracts. Optimum pH of the enzyme ranged from 4.5 to 5 and optimum temperature from 25 to 37 °C. The enzyme extracts reduced the viscosity of 1% pectin solutions in an experiment which was done together with assay for reducing sugars to prove the activities of the enzyme extracts in the four wild fruits. The K_m and V_max ranged from 0.115 to 0.252 mg/ml and 0.0057 to 0.0119 mmol reducing groups/min/mg protein, respectively, in the four polygalacturonase extracts. Calcium chloride and sodium chloride activated the PG from all sources to a greater extend than magnesium chloride and barium chloride. PG from the other three fruits had very little effect on the polysaccharide from U. kirkiana.     

The effect of carbon dioxide gas alone or in combinations on the mortality of Tribolium castaneum (Herbst) and T. confusum du Val (Coleoptera, Tenebrionidae)

Adults of Tribolium castaneum (Herbst) and T. confusum du Val exposed to various mixtures of N₂ or He and O₂ were killed when the O₂ concentration reached to 1·7% or below, whereas the adults exposed to CO₂ : O₂ mixtures were killed mostly due to the deleterious effects of CO₂ itself. At 26·7°C and 38 ± 6%r.h., 95 per cent mortality of T. confusum adults was obtained by an exposure of 271 hr to 45% CO₂ : 55% air mixture; 58 hr to 62% CO₂ : 38% air mixture; and 47·5 hr to 80% CO₂ : 20% air mixture, while for T. castaneum adults 95 per cent mortality required 192, 60, and 44 hr respectively. Data obtained by exposing mature and immature stages of both species to 100% CO₂ suggest that adults were the most susceptible, followed by larvae, eggs and pupae. Generally speaking, inactive stages (egg and pupal) were more tolerant to CO₂ than active stages (larval and adult). Increasing the temperature from 15·6° to 26·7°C resulted in increased insect mortalities; the degree of response varying in different stages. Increasing r.h. decreased the susceptibility of adult insects to 100% CO₂.

Under airtight conditions 200 adult T. castaneum with 8 g of food medium sealed in 1·2 1. glass flasks depleted the O₂ supply from 20·9% to a critical 1·7% level in 7 days, and adults of T. confusum depleted to the 1·6% level in 5 days of airtightness, and both species produced about 20% of carbon dioxide gas.     

Mitogenicity of isolectins isolated from Phaseolus vulgaris L. var. athropurpurea in rat spleen lymphocytes

The lymphoblastic transformation capacity of rat spleen lymphocytes has been evaluated by stimulating the cells with isolectins isolated from raw seeds of Phaseolus vulgaris L. var. athropurpurea (PHVa). PHVa albumin and globulin fractions (AF and GF) and the isolectins E₄+E₃L, E₂L₂, EL₃ and L₄+albumins (where E and L indicate erythroagglutinating and leucoagglutinating activities respectively) were isolated, purified and identified by gel chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).It has been found, first, that the four isolectins were exclusively contained in PHVa AF; second, that the highest mitogenic activity displayed by these glycoproteins was obtained after three days of incubation; and, finally, that all the isolectins showed a similar mitogenic activity, the classical gaussian curves for isolectin dilutions versus mitogenic activity being similar for all the isolectins tested, regardless of the predominance of either the E or L subunits.     

Purification and properties of an arginyl aminopeptidase from Debaryomyces hansenii

A metallo arginyl aminopeptidase (EC 3.4.11.6) activated by Co(2+) was isolated from Debaryomyces hansenii CECT 12487. The enzyme was purified after precipitation with protamine sulphate, followed by a weak anion exchange chromatography, gel filtration chromatography and a strong anion exchange chromatography. The arginyl aminopeptidase (AAP) was purified 337 folds, with a 18% recovery. The AAP appeared to be a dimer with a molecular mass of 101 kDa. The enzyme was active in the pH range from 6 to 9. The optimal activity was detected at pH 7.0 and at 37 degrees C. AAP activity was inhibited by typical aminopeptidase inhibitors (puromycin and bestatin), reducing agents (DTT), chelating agents (EDTA, EGTA and phenantroline) and sulphydryl groups reagents (iodoacetate). Ca(2+), Mn(2+) and Co(2+) activated the enzyme, while Cu(2+), Cd(2+), Hg(2+) and Mg(2+) inhibited it. The K(m) values calculated for Arg-AMC (7-amido-4-methylcoumarin) and Leu-AMC were 0.071 and 0.094 mM, respectively. The enzyme showed maximum specificity for basic amino acids (Arg and Lys), but was also able to hydrolyze non-charged amino acids (Leu, Met and Ala) and, at a minor rate, aromatic amino acids (Phe and Tyr). AAP showed higher activity when an acid residue was located at the C-terminal position of dipeptides.The described purification of an arginyl aminopeptidase from the yeast D. hansenii can contribute to the lack of knowledge about the exopeptidase activity in one of the yeasts more frequently isolated in sausage and to understand its role during the ripening of a fermented sausage.     

一株嗜热菌木糖异构酶的纯化与性质研究

研究了1株嗜热菌(Anoxybacillus flavithermus)所产木糖异构酶的分离纯化以及酶学性质。结果表明,经硫酸铵沉淀、Sephadex G-75凝胶过滤、纤维素DE-52弱阴离子交换柱和Q Sepharose Fast Flow强阴离子交换层析得到的木糖异构酶,分子量约为181 ku,由4个相同分子量的亚基组成。酶反应的最适温度为80℃,最适为pH为7.0且最适pH范围宽泛,pH6.0~11.0酶反应活性能保持80%左右。该酶热稳定性及耐碱性能良好,70℃保温1 h后酶活仍能保持近80%左右;pH5.0~8.0保温1 h后酶活仍能保持近80%以上,甚至pH12.0保温1 h后酶活性仍能保持40%左右。Mn2+和Co2+对酶活性有明显促进作用,Zn2+、Cu2+以及Al3+对酶活性有一定程度的抑制。     

红佳酿酵母β-葡萄糖苷酶的分离纯化及酶学性质

以红佳酿酿酒酵母为出发菌株,通过离子交换法分离纯化β-葡萄糖苷酶,测定酶活力和蛋白质含量,并研究温度、pH值稳定性和金属离子、葡萄糖、酒精度对酶活力的影响。结果表明:粗酶液经过离子交换层析后,纯化倍数为19.41,得率为38.67%;经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gelelectrophoresis,SDS-PAGE)电泳检测为1条谱带,达到电泳纯,分子质量约为45 kD;β-葡萄糖苷酶热稳定性较差,在20~40℃较稳定,在pH 5.0~10.0较稳定;Al3+、Cu2+对酶活力有抑制作用,K+、Mg2+、Ca2+、Zn2+、Na+对酶活力的影响不明显;葡萄糖和酒精对酶活力无明显抑制作用。