Biochemical studies of the multicopper oxidase (small laccase) from Streptomyces coelicolor using bioactive phytochemicals and site‐directed mutagenesis

Multicopper oxidases can act on a broad spectrum of phenolic and non‐phenolic compounds. These enzymes include laccases, which are widely distributed in plants and fungi, and were more recently identified in bacteria. Here, we present the results of biochemical and mutational studies of small laccase (SLAC), a multicopper oxidase from Streptomyces coelicolor (SCO6712). In addition to typical laccase substrates, SLAC was tested using phenolic compounds that exhibit antioxidant activity. SLAC showed oxidase activity against 12 of 23 substrates tested, including caffeic acid, ferulic acid, resveratrol, quercetin, morin, kaempferol and myricetin. The kinetic parameters of SLAC were determined for 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulphonic acid), 2,6‐dimethoxyphenol, quercetin, morin and myricetin, and maximum reaction rates were observed with myricetin, where k_cat and K_m values at 60°C were 8.1 (± 0.8) s^?1 and 0.9 (± 0.3) mM respectively. SLAC had a broad pH optimum for activity (between pH 4 and 8) and temperature optimum at 60–70°C. It demonstrated remarkable thermostability with a half‐life of over 10 h at 80°C and over 7 h at 90°C. Site‐directed mutagenesis revealed 17 amino acid residues important for SLAC activity including the 10 His residues involved in copper coordination. Most notably, the Y229A and Y230A mutant proteins showed over 10‐fold increase in activity compared with the wild‐type SLAC, which was correlated to higher copper incorporation, while kinetic analyses with S929A predicts localization of this residue near the meta‐position of aromatic substrates.     

来源于嗜热氢化杆菌的新型对苯二甲酸双(羟乙)酯水解酶的表达纯化与酶学性质

石化来源的聚对苯二甲酸乙二酯(polyethylene terephthalate,PET)被广泛用于矿泉水瓶、食品包装和纺织品等领域,因其在自然界中不易分解,大量使用后的PET废弃物造成了严重的环境污染与资源浪费。使用生物酶法对PET废弃物进行解聚,并对解聚产物进行升级循环利用是进行塑料污染治理的重要方向之一,其中关键的是PET水解酶的解聚效率。对苯二甲酸双(羟乙基)酯(bis(hydroxyethyl)terephthalate,BHET)是PET生物酶解的中间产物,其累积是限制PET水解酶催化效率的一个重要因素,BHET水解酶和PET水解酶的联用能提升PET的整体水解效率。来源于嗜热氢化杆菌(Hydrogenobacter thermophilus)的双烯内酯酶(HtBHETase)对BHET有显著水解效果,将该酶在大肠杆菌(Escherichia coli)中进行重组表达并纯化后,对其酶学性质进行了研究。结果显示,HtBHETase对短碳链的酯类如对硝基苯酚乙酸酯催化活性较高,HtBHETase以BHET为底物时的最适反应pH值和最适反应温度分别为5.0和55℃;该酶有较好的热稳定性,经80℃的条件处理1 h仍能保持80%以上活性,显示出了良好的热稳定性,HtBHETase有在PET塑料生物解聚中使用的潜力,本研究为推动生物酶法降解PET提供了新的参考。     

Production, properties and application to biocatalysis of a novel extracellular alkaline phenol oxidase from the thermophilic fungus Scytalidium thermophilum

Scytalidium thermophilum produces an extracellular phenol oxidase on glucose-containing medium. Certain phenolic acids, specifically gallic acid and tannic acid, induce the expression of the enzyme. Production at 45°C in batch cultures is growth-associated and is enhanced in the presence of 160 μM CuSO₄.5 H₂O and 3 mM gallic acid. The highest enzyme activity is observed at pH 7.5 and 65°C, on catechol. When incubated for 1 h at pH 7 and pH 8, 95% and 86% of the activity is retained. Thermostability decreases gradually from 40°C to 80°C. Estimated molecular mass is c. 83 kDa, and pI is acidic at c. 5.4. Substrate specificity and inhibition analysis in culture supernatants suggest that the enzyme has unique properties showing activity towards catechol; 3,4-dihydroxy-l-phenylalanine (l-DOPA); 4-amino-N, N-diethylaniline (ADA); p-hydroquinone; gallic acid; tannic acid and caffeic acid, and no activity towards l-tyrosine, guaiacol, 2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulphonic acid) (ABTS) and syringaldazine. Inhibition is observed in the presence of salicyl hydroxamic acid (SHAM) and p-coumaric acid. Enzyme activity is enhanced by cetyltrimethylammonium bromide (CTAB) and polyvinylpyrrolidone (PVP), and the organic solvents dimethyl sulfoxide (DMSO) and ethanol. No inhibition is observed in the presence of carbon monoxide. Benzoin, benzoyl benzoin and hydrobenzoin are converted into benzil, and stereoselective oxidation is observed on hydrobenzoin. The reported enzyme is novel due to its catalytic properties resembling mainly catechol oxidases, but displaying some features of laccases at the same time.     

Molecular cloning and characterization of a new cold-active esterase from a deep-sea metagenomic library

A clone which conferred lipolytic activity at low temperature was identified from a fosmid library constructed from a South China Sea marine sediment sample. The gene responsible, estF, consisted of 1,080 bp that encoded 359 amino acid residues, with a typical N-terminal signal peptide of 28 amino acid residues. A phylogenetic analysis of amino acid sequence with other lipolytic enzymes revealed that EstF and seven closely related putative lipolytic enzymes comprised a unique clade in the phylogenetic tree. Moreover, these hypothetic esterases showed unique conservative sites in the amino acid sequence. The recombinant EstF was overexpressed and purified, and its biochemical properties were partially characterized. The optimal substrate for EstF to hydrolyze among a panel of p-nitrophenyl esters (C2 to C16) was p-nitrophenyl butyrate (C4), with a K _m of 0.46 mM. Activity quickly decreased with substrates containing an acyl chain length longer than 10 carbons. We found that EstF was active in the temperature range of 0–60°C, showed the best activity at 50°C, but was unstable at 60°C. It exhibited a high level of activity in the pH range of 7.0–10.0 showing the highest activity at pH 9.0.     

Purification and Properties of a Dehyrodicaffeic Acid Dilactone-forming Enzyme from a Mushroom,Inonotus sp. K-1410

A dehydrodicaffeic acid dilactone-forming enzyme was purified from the mycelia of a mushroom, Inonotus sp. K-1410 by calcium acetate treatment, ammonium sulfate precipitation and column chromatography on Sephadex G-100, DEAE-Sephadex A-50 and caffeic acid-bound AH-Sepharose 4B. The enzyme was purified about 1200-fold from a crude extract and shown to be almost completely homogeneous by polyacrylamide gel electrophoresis. The molecular weight of this enzyme was estimated by gel filtration on Sephadex G-100 to be approximately 39,000. The optimal pH for the enzymic conversion of caffeic acid to dehydrodicaffeic acid dilactone is around 6.0. The enzyme is stable up to 60°C and preincubation of the enzyme at 40°C for 10 min gives 1.5-fold activation compared with preincubation at 0°C. The optimal temperature for the enzyme reaction is 40°C.     

Alkaline proteases produced by <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> RP1 grown on shrimp wastes: Application in chitin extraction,chicken feather-degradation and as a dehairing agent

The current increase in the amount of shrimp wastes produced by the shrimp industry has led to the need in finding new methods for shrimp wastes disposal. In this study, Bacillus licheniformis RP1 was shown to produce proteases when grown in media containing shrimp wastes powder as a sole carbon and nitrogen source, indicating that this bacteria could obtain its carbon and nitrogen requirements directly from shrimp wastes. The maximum protease production was obtained when the strain was grown in a medium containing (g/L): shrimp wastes powder 30, KCl 1.5, K₂HPO₄ 0.5, and KH₂PO₄ 0.5. Using casein zymography, the crude protease preparation was found to produce at least seven proteases. The proteases of B. licheniformis RP1 were tested for shrimp waste deproteinization in the preparation of chitin. The percent of protein removal after 3 h hydrolysis at 60°C and at an enzyme/substrate (E/S) ratio of 0.5 and 5 (Unit of enzyme/mg of protein) were about 68 and 81%, respectively. Additionally, B. licheniformis RP1 showed important feather degrading activity. Complete solubilisation of whole feathers was observed after 24 h of incubation at 50°C. More interestingly, the RP1 proteolytic preparation demonstrated powerful dehairing capabilities for hair removal from skin. Collagen, which is the major leather-forming protein, was not significantly degraded. Considering its promising properties, B. licheniformis RP1 enzymatic preparation may be considered a potential candidate for future use in several biotechnological processes.     

Purification and characterization of the exopolygalacturonase produced by Aspergillus giganteus in submerged cultures

Polygalacturonases are pectinolytic enzymes that catalyze the hydrolysis of the plant cell-wall pectin backbone. They are widely used in the food industry for juice extraction and clarification. Aspergillus giganteus produces one polygalacturonase (PG) on liquid Vogel medium with citrus pectin as the only carbon source. In specific applications, such as those used in the food and medicine industries, the PG must be free of substances that could affect the characteristics of the product and the process, such as color, flavor, toxicity, and inhibitors. We present here an efficient, simple, and inexpensive method for purifying the A. giganteus PG and describe the characteristics of the purified enzyme. Purified PG was obtained after two simple steps: (1) protein precipitation with 70% ammonium sulfate saturation and (2) anion-exchange chromatography on a DEAE-Sephadex A-50 column. The final enzyme solution retained 86.4% of its initial PG activity. The purified PG had a molecular weight of 69.7 kDa, exhibited maximal activity at pH 6.0 and 55–60°C, and was stable in neutral and alkaline media. It had a half-life of 115, 18, and 6 min at 40, 50 and 55°C, respectively. Purified PG showed its highest hydrolytic activity with low-esterified and nonesterified substrates, releasing monogalacturonic acid from substrate, indicating that it is an exopolygalacturonase. PG activity was enhanced in the presence of β-mercaptoethanol, dithiothreitol, Co²⁺, Mn²⁺, Mg²⁺, NH₄ ⁺, and Na⁺ and was resistant to inhibition by Pb²⁺.     

Characterization of recombinant endo-1,4-β-xylanase of Bacillus halodurans C-125 and rational identification of hot spot amino acid residues responsible for enhancing thermostability by an in-silico approach

Increased demand of enzymes for industrial use has led the scientists towards protein engineering techniques. In different protein engineering strategies, rational approach has emerged as the most efficient method utilizing bioinformatics tools to produce enzymes with desired reaction kinetics; physiochemical (temperature, pH, half life, etc) and biological (selectivity, specificity, etc.) characteristics. Xylanase is one of the widely used enzymes in paper and food industry to degrade xylan component present in plant pulp. In this study endo 1,4-β-xylanase (Xyl-11A) from Bacillus halodurans C-125 was cloned in pET-22b (+) vector and expressed in Escherichia coli BL21 (DE3) expression strain. The enzyme had Michaelis constant K_m of 1.32 mg ml^?1 birchwoodxylan (soluble form) and maximum reaction velocity (V_max) 73.53 mmol min^?1 mg^?1 with an optimum temperature of 75 °C and pH 9.0. The thermostability analysis showed that enzyme retained more than 80% of its residual activity when incubated at 75 °C for 2 h. In addition, to increase Xyl-11A thermostability, an in-silico analysis was performedto identify the hot spot amino acid residues. Consensus-based amino acid substitution was applied to evaluate multiple sequence alignment of homologs and identified 20 amino acids positions by following Jensen-Shnnon Divergence method. 3D models of 20 selected mutants were analyzed for conformational transition in protein structures by using NMSim server. Two selected mutants T6K and I17M of Xyl-11A retained 40, 60% residual activity respectively, at 85 °C for 120 min as compared to wild type enzyme which retained 37% initial activity under same conditions, confirming the enhanced thermostability of mutants. The present study showed a good approach for the identification of promising amino acid residues responsible for enhancing the thermostability of enzymes of industrial importance.

     

Purification, Characterization of GH11 Endo-β-1,4-xylanase from Thermotolerant Streptomyces sp. SWU10 and Overexpression in Pichia pastoris KM71H

We have previously described two forms of an endo-β-1,4-xylanase (XynSW2A and XynSW2B) synthesized by thermotolerant Streptomyces sp. SWU10. Here, we describe another xylanolytic enzyme, designated XynSW1. The enzyme was purified to homogeneity from 2 L of culture filtrate. Its apparent molecular mass was 24 kDa. The optimal pH and temperature were pH 5.0 and 40 °C, respectively. The enzyme was stable in a wide pH ranges (pH 1–11), more than 80 % of initial activity remained at pH 2–11 after 16 h of incubation at 4 °C and stable up to 50 °C for 1 h. Xylobiose and xylotriose were the major xylooligosaccharides released from oat spelt xylan by the action of XynSW1, indicating of endo-type xylanase. The complete xynSW1 gene contains 1,011 bp in length and encode a polypeptide of 336 with 41 amino acids of signal peptide. The amino acid sequence analysis revealed that it belongs to glycoside hydrolase family 11 (GH11). The mature xynSW1 gene without signal peptide sequence was overexpressed in Pichia pastoris KM71H. The recombinant XynSW1 protein showed higher molecular mass due to the differences in glycosylation levels at the six N-glycosylation sites in the amino acid sequence and exhibited better physicochemical properties than those of the native enzyme including higher optimal temperature (60 °C), and specific activity, but lower optimal pH (4.0). Because of their stability in a wide pH ranges, both of native and recombinant enzymes of XynSW1, may have potential application in several industries including food, textile, biofuel, and also waste treatment.     

Thermostable α‐Amylase and Glucoamylase from Thermomyces lanuginosus F1

An α‐amylase and a glucoamylase produced by Thermomyces lanuginosus F₁ were separated by ion‐exchange chromatography on Q‐Sepharose fast flow. The enzymes were further purified to electrophoretic homogeneity by chromatography on Sephadex G‐100 and Phenyl‐Sepharose CL‐4B.The molecular weights and isoelectric points of the enzymes were 55,000 Da and pHi 4.0 for α‐amylase and 70,000 Da and pH_i 4.0 for glucoamylase, respectively. The optimum pH and temperatures for the enzymes were found to be 5.0 and 60 °C for α‐amylase, and 6.0 and 70 °C for glucoamylase,respectively. Both enzymes were maximally stable at pH 4.0 and retained over 80% of their activity between pH 5.0 and 6.0 for 24 h. After incubation at 90 °C (1 h), the α‐amylase and glucoamylase retained only 6% and 16% of their activity, respectively. The enzymes readily hydrolyzed soluble starch, amylose, amylopectin and glycogen but hydrolyzed pullulan very slowly. Glucoamylase and α‐amylase had highest affinity for soluble starch with K_M values of 0.80 mg/ml and 0.67 mg/ml, respectively. The α‐amylase hydrolyzed raw starch granules with a predominant production of glucose and maltose. The activities of α‐amylase and glucoamylase increased in the presence of Mn²⁺, Co²⁺, Ca²⁺, Zn²⁺ and Fe²⁺, but were inhibited by guanidine‐HCl, urea and disodium EDTA. Both enzymes possess pH and thermal stability characteristics that may be of technological significance.     

Purification and characterization of thermostable pectate-lyases from a newly isolated thermophilic bacterium, Thermoanaerobacter italicus sp. nov.

A novel thermophilic spore-forming anaerobic microorganism (strain Ab9) able to grow on citrus pectin and polygalacturonic acid (pectate) was isolated from a thermal spa in Italy. The newly isolated strain grows optimally at 70°C with a growth rate of 0.23 h⁻¹ with pectin and 0.12 h⁻¹ with pectate as substrates. Xylan, starch, and glycogen are also utilized as carbon sources and thermoactive xylanolytic (highest activity at 70°–75°C), amylolytic as well as pullulolytic enzymes (highest activity at 80°–85°C) are formed. Two thermoactive pectate lyases were isolated from the supernatant of a 300-l culture of isolate Ab9 after growth on citrus pectin. The two enzymes (lyases a and b) were purified to homogeneity by ammonium sulfate treatment, anion exchange chromatography, hydrophobic chromatography and finally by preparative gel electrophoresis. After sodium dodecylsulfate (SDS) gel electrophoresis, lyase a appeared as a single polypeptide with a molecular mass of 135 000 Da whereas lyase b consisted of two subunits with molecular masses of 93 000 Da and 158 000 Da. Both enzymes displayed similar catalytic properties with optimal activity at pH 9.0 and 80°C. The enzymes were very stable at 70°C and at 80°C with a half-life of more than 60 min. The maximal activity of the purified lyases was observed with orange pectate (100%) and pectate-sodium salt (90%), whereas pectin was attacked to a much lesser extent (50%). The K _m values of both lyases for pectate and citrus pectin were 0.5 g·l⁻¹ and 5.0 g·l⁻¹, respectively. After incubation with polygalacturonic acid, mono-, di-, and tri-galacturonate were detected as final products. A 2.5-fold increase of activity was obtained when pectate lyases were incubated in the presence of 1 mM Ca²⁺. The addition of 1 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of the enzymes. These heat-stable enzymes represent the first pectate-lyases isolated and characterized from a thermophilic anaerobic bacterium. On the basis of the results of the 16S rRNA sequence comparisons and the observed phenotypic differences, we propose strain Ab9 as a new species of Thermoanaerobacter, namely Thermoanaerobacter italicus sp. nov. Received: May 25, 1997 / Accepted: June 5, 1997     

Purification and Characterization of a Thermostable Alkaline Protease from Alkalophilic Thermoactinomyces sp. H8682

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatograhies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25, 000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0.

Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu²⁺ and Hg²⁺. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37°C for 60 min. The optimum pH was pH 11.5–13.0 at 37°C and the optimum temperature was 70°C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl₂, it showed maximum proteolytic activity at 80°C and stability from pH 4–12.5 at 60°C and below 75°C at pH 11.5. The stabilization by Ca²⁺ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of Microbiol serine protease, although alanine of the NH₂-terminal amino acid was deleted.     

Activities of digestive enzymes of polar and subtropical echinoderms

Activities and characteristics of digestive enzymes of polar and subtropical echinoderms are similar. Specific adaptations to facilitate digestion at low temperature were not observed. Levels of chymotrypsin and β-glucosidase of digestive tissues of the Antarctic asteroids Odontaster validus and Odontaster meridionalis, and the Antarctic echinoid Sterechinus neumayeri, are comparable to levels of the asteroids Henricia downii and Astropecten articulatus from the Gulf of Mexico. α-Glucosidase activities are similar for S. neumayeri and A. articulatus. The pH of maximal activity is 7.5 for disaccharidases and 8.5–9.5 for chymotrypsin for both polar and subtropical species. Affinities for substrates were higher at 25°C than at 0°C for disaccharidases of all species. However, K_m values for chymotrypsin increased from 0 to 25°C. Lack of sufficient adaptation by polar echinoderms to facilitate digestion at low temperature may reduce their capacity to digest food. Received: 24 July 1996 / Accepted: 31 January 1997     

Production of cellulases by Thermomucor indicae-seudaticae: characterization of a thermophilic β-glucosidase

Abstract

The current study evaluated the production and characterization of β-glucosidase by the thermophilic fungus Thermomucor indicae-seudaticae in solid-state fermentation of wheat bran. Isolated fungi have significant amounts of β-glucosidase, an enzyme that may be applied to different industrial processes, such as the production of fuels, food, and other chemical compounds. Maximal enzyme activity occurred in pH 3.5–4.5 and at 70?°C. The enzyme exhibited high thermostability, for 1?h, up to 60?°C, and good tolerance to glucose (10?mM) and ethanol (10%). The optimization of fermentative parameters on the production of β-glucosidase was carried out by evaluating the best supplementary nutrient source, pH of nutrient solution, initial substrate moisture and fermentation temperature. The optimization of the above fermentation parameters increased enzyme activity by 120.0%. The highest enzymatic activity (164.0?U/g) occurred with wheat bran containing 70% initial moisture, supplemented with 1.0% (NH₄)₂SO₄ solution at pH 5.5–6.0 and fungus incubated at 40?°C. A more detailed study of β-glucosidase suggested that Sulfur is an important component of the main amino acid present in this enzyme. The enhancer of the enzyme activity occurred when the fungus was grown on wheat bran supplemented with a sulfur-containing solution. In fact, increasing the concentration of sulfur in the solution increased its activity.     

Structure‐based replacement of methionine residues at the catalytic domains with serine significantly improves the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica

The alkaline amylase requires high resistance towards chemical oxidation for use in the detergent and textile industries. This work aims to improve the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica by site‐directed mutagenesis based on the enzyme structure model. Five mutants were created by individually replacing methionine at positions 145, 214, 229, 247, and 317 in the amino acid sequence of alkaline amylase with oxidative‐resistant serine. The pH stability of the mutant enzymes was almost the same as that of the wild‐type (WT) enzyme (pH 7.0–11.0). The stable temperature range of the mutant enzymes M145S and M247S decreased from <50°C of the WT to <40°C, while the thermal stability of the other three mutant enzymes (M214S, M229S, and M317S) was almost the same as that of the WT enzyme. The catalytic efficiency (k_cat/K_m) of all the mutant enzymes decreased when compared to WT enzyme. The mutant enzymes showed increased activity in the presence of surfactants Tween‐60 and sodium dodecyl sulfate. When incubated with 500 mM H₂O₂ at 35°C for 5 h, the WT enzyme retained only 13.3% of its original activity, while the mutant enzymes M145S, M214S, M229S, M247S, and M317S retained 55.6, 70.2, 54.2, 62.5, and 46.4% of the original activities, respectively. The results indicated that the substitution of methionine residues at the catalytic domains with oxidative‐resistant serine can significantly improve the oxidative stability of alkaline amylase. This work provides an effective strategy to improve the oxidative stability of amylase, and the high oxidation resistance of the mutant enzymes shows their potential applications in the detergent and textile industries. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Isolation and characterization of a cold-active,alkaline, detergent stable α-amylase from a novel bacterium Bacillus subtilis N8

A cold-active alkaline amylase producer Bacillus subtilis N8 was isolated from soil samples. Amylase synthesis optimally occurred at 15°C and pH 10.0 on agar plates containing starch. The molecular weight of the enzyme was found to be 205?kDa by performing SDS-PAGE. While the enzyme exhibited the highest activity at 25°C and pH 8.0, it was highly stable in alkaline media (pH 8.0–12.0) and retained 96% of its original activity at low temperatures (10–40°C) for 24?hr. While the amylase activity increased in the presence of β-mercaptoethanol (103%); Ba²⁺, Ca²⁺, Na⁺, Zn²⁺, Mn²⁺, H₂O₂, and Triton X-100 slightly inhibited the activity. The enzyme showed resistance to some denaturants: such as SDS, EDTA, and urea (52, 65, and 42%, respectively). N8 α-amylase displayed the maximum remaining activity of 56% with 3% NaCl. The major final products of starch were glucose, maltose, and maltose-derived oligosaccharides. This novel cold-active α-amylase has the potential to be used in the industries of detergent and food, bioremediation process and production of prebiotics.     

Investigation of some characteristics of enzymes effecting the process of membrane digestion in the paddlefish <Emphasis Type="Italic">Polyodon spathula</Emphasis>

For the first time, a complex investigation of characters of some enzymes effecting the membrane hydrolysis of food in the paddlefish Polyodon spatula is made. The zone of optimum values of temperature of alkaline phosphatase, maltase, and casein-lytic proteinases of the intestinal mucosa is determined within the range from 50 to 60°C, in α-amylase it is shifted towards lower temperatures—10–30°C. A high thermal stability is noted in all investigated enzymes. The maximum level of activity for α-amylase, alkaline phosphatase, and maltase of the intestinal mucosa in the paddlefish Polyodon spatula is within pH 7–9, 9–10, and 6–8, respectively; for casein-lytic proteinases the optimum is at pH 11. In the paddlefish Polyodon spatula, the interaction of nutrients and enzymes of the intestinal mucosa may cause a significant change in the activity level of the enzymes.     

Characterization of a heat stable anti-listerial bacteriocin produced by vancomycin sensitive Enterococcus faecium isolated from idli batter

Lactic acid bacteria (LAB) are known to produce various types of bacteriocins, ribosomally synthesized polypeptides, which have antibacterial spectrum against many food borne pathogens. Listeria monocytogenes, a pathogenic bacterium, is of particular concern to the food industry because of its ability to grow even at refrigeration temperatures and its tolerance to preservative agents. Some of the bacteriocins of LAB are known to have anti-listerial property. In the present study, the bacteriocin produced by vancomycin sensitive Enterococcus faecium El and J4 isolated from idli batter samples was characterized. The isolates were found to tolerate high temperatures of 60°C for 15 and 30 min and 70°C for 15 min. The bacteriocin was found to be heat stable and had anti-listerial activity. The bacteriocin did not lost anti-listerial activity when treated at 100°C for 30 min or at 121°C for 15 min. The bacteriocin lost its antimicrobial activity after treating with trypsin, protinase-K, protease and peptidase.     

Novel thermoactive glucoamylases from the thermoacidophilic Archaea Thermoplasma acidophilum,Picrophilus torridus and Picrophilus oshimae

The thermoacidophilic Archaea Thermoplasma acidophilum (optimal growth at 60 °C and pH 1–2), Picrophilus torridus and Picrophilus oshimae (optimal growth at 60 °C and pH 0.7) were able to utilize starch as sole carbon source. During growth these microorganisms secreted heat and acid-stable glucoamylases into the culture fluid. Applying SDS gel electrophoresis activity bands were detected with appearent molecular mass (Mw) of 141.0, 95.0 kDa for T. acidophilum, 133.0, 90.0 kDa for P. torridus and 140.0, 85.0 kDa for P. oshimae. The purified enzymes were incubated with various polymeric substrates such as starch, pullulan, panose and isomaltose. The product pattern, analyzed by HPLC, showed that in all cases glucose was formed as the sole product of hydrolysis. The purified glucoamylases were optimally active at pH 2.0 and 90 °C and have an isoelectric points (pI) between 4.5 and 4.8. Enzymatic activity was detected even at pH 1.0 and 100 °C. The glucoamylases were thermostable at elevated temperature with a half-life of 24 h at 90 °C for both P. torridus and T. acidophilum, and 20 h at 90 °C for P. oshimae. The enzyme system of T. acidophilum has a lower K _m value for soluble starch (1.06 mg/ml) than the enzymes from P. oshimae and P. torridus (4.35 mg/ml and 2.5 mg/ml), respectively. Enzyme activity was not affected by Na⁺, Mg⁺⁺, Ca⁺⁺, Ni⁺⁺, Zn⁺⁺, Fe⁺⁺, EDTA and DTT. This revised version was published online in August 2006 with corrections to the Cover Date.     

Purification and biochemical characterization of feruloyl esterases from Aspergillus terreus MTCC 11096

Aspergillus terreus MTCC 11096 isolated from the soils of agricultural fields cultivating sweet sorghum was previously identified to produce feruloyl esterases (FAEs). The enzymes responsible for feruloyl esterase activity were purified to homogeneity and named as AtFAE‐1, AtFAE‐2, and AtFAE‐3. The enzymes were monomeric having molecular masses of 74, 23 and 36 kDa, respectively. Active protein bands were identified by a developed pH‐dependent zymogram on native PAGE. The three enzymes exhibited variation in pH tolerance ranging between pH 5–8 and thermostability of up to 55°C. Inhibition studies revealed that the serine residue was essential for feruloyl esterase activity; moreover aspartyl and glutamyl residues are not totally involved at the active site. Metal ions such as Ca²⁺, K⁺, and Mg²⁺ stabilized the enzyme activity for all three FAEs. Kinetic data indicated that all three enzymes showed catalytic efficiencies (k_cat/K_m) against different synthesized alkyl and aryl esters indicating their broad substrate specificity. The peptide mass fingerprinting by MALDI/TOF‐MS analysis and enzyme affinity toward methoxy and hydroxy substituents on the benzene ring revealed that the AtFAE‐1 belonged to type A while AtFAE‐2 and AtFAE‐3 were type C FAE. The FAEs could release 65 to 90% of ferulic acid from agrowaste substrates in the presence of xylanase. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:924–932, 2013