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 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 OF A PROTEASE FROM AN ALKALOPHlLIC BACILLUS SUBTILIS CHZ1 ISOLATED FROM A ZIMBABWEAN HOT SPRING

A proteolytic enzyme produced by Bacillus subtilis CHZ1 was purified using ammonium sulfate precipitation, gel filtration and cationic exchange on S‐Sepharose fast flow column chromatography. Production of the protease was higher when the Bacillus strain was cultured in a synthetic medium, M162, supplemented with 0.3% (w/v) organic compared to inorganic nitrogen sources. Enzyme production was growth dependent and production was highest when tryptone was used as the nitrogen source. When run on SDS‐PAGE gel, the purified enzyme gave a 35 kDa band, suggesting that it consisted of one polypeptide chain. High enzyme activity was observed in the pH range of 6–10 with a maximum value at pH 8.0 when 0.5% (w/v) azocasein was used as the substrate. Optimum temperature for protease activity was found to be 60–80C, and the enzyme had considerable thermal stability for 5.5 h retaining about 90% activity after 5.5 h. At 2.5 mM concentration, PMSF, Ag⁺ and Hg⁺ inhibited activity of the protease. Metal cofactors like Mn²⁺, Mg²⁺ and Fe²⁺ increased the enzyme activity by about 20%. Zn²⁺, Cu²⁺ and Ca²⁺ did not affect the enzyme's activity. The pH and thermal stability as well as high specific activity of this enzyme can be exploited for industrial applications.     

Isolation,purification and properties of the peroxidase from the hull of Glycine max var HH2

Soybean hull peroxidase (EC 1.11.1.7), an acidic peroxidase isolated from soybean (Glycine max var HH2) hulls was purified to electrophoretic homogeneity by a combination of ammonium sulphate fractionation, DEAE‐Sephadex A‐50 chromatography, concanavalin A‐Sepharose 4B affinity chromatography and Bio‐Gel P‐60 gel filtration. The specific activity of purified peroxidase was about 57‐fold higher than that of crude extract. The yield was about 16.4%. The molecular weight of the enzyme was estimated to be 38 000 by SDS‐polyacrylamide gel electrophoresis. The peroxidase was a glycoprotein containing about 18.7% carbohydrate, approximately one‐quarter of which was shown to be glucosamine residues. It was found to have an isoelectric point of 3.9. The enzyme was most active at pH 4.6 and 45°C, and was stable in the pH range 2.5–11.5. The enzyme could tolerate heating for 10 min at 75°C without being inactivated, and at 85°C, it took 40 min to inactivate the enzyme 50%, confirming that the peroxidase was a novel thermostable enzyme. Fe ²⁺, Fe³⁺, Sn²⁺, CN ⁻ and N₃⁻ inhibited enzyme activity, while Hg²⁺, Ag⁺, Pb ²⁺, Cr³⁺, EDTA and SDS were not significantly inhibitory. © 1999 Society of Chemical Industry     

Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8–11

A protease from sorghum malt variety KSV8–11 was purified by a combination of dialysis against 4 M sucrose, ion‐exchange chromatography on Q‐Sepharose (Fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 5‐fold to give a 14.1% yield relative to the total activity in the crude extract and a final specific activity of 1348.9 U mg^?1 protein. SDS‐PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 16 KDa. Using casein as substrate, the purified protease had optimal activity at 50°C and maximal temperature stability between 30°C and 40°C but retained over 64% of its original activity after incubation at 60°C for 30 min. The pH optimum was 5.0 with maximum stability at pH 6.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. The protease was inhibited by Ag⁺, Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺, iodoacetic acid (IAA) and p‐chloromercuribenzoate (p‐CMB), stimulated by Cu²⁺, Sr²⁺, phenylmethylsulfonyl‐fluoride (PMSF) and 2‐mercaptoethanol (2‐ME) while Mn²⁺ and ethylenediaminetetraacetic acid (EDTA) had no effect. The purified enzyme had a K_m of 18 mg·mL^?1 and a V_max of 11.1 μmol · mL^?1 · min^?1 with casein as substrate.     

Purification and characterisation of β‐mannanase from Lactobacillus plantarum (M24) and its applications in some fruit juices

β‐Mannanase was purified 2619.05‐fold from the Lactobacillus plantarum (M24) bacterium by ammonium sulphate precipitation and ion exchange chromatography (DEAE‐Sephadex). The purified enzyme gave two protein bands at a level of approximately 36.4 and 55.3 kDa in the SDS‐PAGE. The purified mannanase enzyme has shown its maximum activity at 50 °C and pH 8, and it has been also determined that the enzyme was stable at 5–11 pH range and over 50 °C. The V_max and K_m values have been identified as 82 mg mannan mL^?1 and 0.178 mm , respectively. The effects of some metal ions such as Fe²⁺, Ca²⁺, Co²⁺, Ni²⁺, Mn²⁺, Cu²⁺ and Zn²⁺ on the mannanase enzyme have been also investigated, and it has been determined that all metal ions had significant effects on the activation of the mannanase enzyme. In addition, the effectiveness of the purified mannanase enzyme on the clarification of some fruit juices such as orange, apricot, grape and apple has been investigated. During the clarification processes, the enzyme was more effective than crude extracts on the clarification of the peach juice with a ratio of 223.1% at most.     

An extra-cellular alkaline metallolipase from Bacillus licheniformisMTCC 6824: Purification and biochemical characterization

An extra-cellular lipase produced by Bacillus licheniformis MTCC 6824 was purified to homogeneity by ammonium sulphate fractionation, ethanol/ether precipitation, dialysis, followed by anion-exchange chromatography on Amberlite IRA 410 (Cl⁻ form) and gel exclusion chromatography on Sephadex G 100 using Tris–HCl buffer (pH 8.0). The crude lipase extract had an activity of 41.7 LU/ml of culture medium when the bacterium was cultured for 48 h at 37 °C and pH 8.0 with nutrient broth supplemented with sardine oil as carbon source. The enzyme was purified 208-fold with 8.36% recovery and a specific activity of 520 LU/mg after gel exclusion chromatography. The pure enzyme is a monomeric protein and has an apparent molecular mass of 74.8 kDa. The lipase had a V_max and K_m of 0.64 mM/mg/min and 29 mM, respectively, with 4-nitro phenylpalmitate as a substrate, as calculated from the Lineweaver–Burk plot. The lipase exhibited optimum activity at 45 °C and pH 8.0, respectively. The enzyme had half-lives (T_1/2) of 82 min at 45 °C, and 48 min at 55 °C. The catalytic activity was enhanced by Ca²⁺ (18%) and Mg²⁺ (12%) at 30 mM. The lipase was inhibited by Co²⁺, Cu²⁺, Zn²⁺, Fe² even at low concentration (10 mM). EDTA, at 70 mM concentration, significantly inhibited the activity of lipase. Phenyl methyl sulfonyl fluoride (PMSF, 70 mM) completely inactivated the original lipase. A combination of Ca²⁺ and sorbitol induced a synergistic effect on the activity of lipase with a significantly high residual activity (100%), even after 45 min, as compared to 91.5% when incubated with Ca²⁺ alone. The lipase was found to be hydrolytically resistant toward triacylglycerols with more double bonds.     

Polyphenol oxidase properties,anti-urease,and anti-acetylcholinesterase activity of Diospyros lotus L. (Plum Persimmon)

Diospyros lotus fruit polyphenol oxidase was purified using affinity chromatography, resulting in a 15-fold enrichment in specific activity. The purified enzyme, having 16.5 kDa molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibited the highest activity toward 4-methylcatechol. Maximum diphenolase activity was reached at pH 7.0 and 60°C in the presence of 4-methylcatechol. K_m and V_max values were calculated as 3.8 mM and 1250 U/mg protein, respectively. Ascorbic acid was a promising inhibitor with an IC₅₀ value of 0.121 µM. The activity of the purified enzyme was stimulated by Fe²⁺, Sr²⁺, Zn²⁺, and K⁺ and deeply inhibited by Hg²⁺, at 1 mM final concentration. Aqueous extract of Diospyros lotus L. fruit showed strong substantial urease and acetylcholinesterase inhibition, with IC₅₀ values of 1.55 ± 0.05 and 16.75 ± 0.11 mg/mL, respectively.     

Alpha-Amylase from Persimmon Honey: Purification and Characterization

The α-amylase was extracted from pure persimmon honey and purified by DEAE-Toyopearl 650M, CM-Toyopearl 650M, and Toyopearl HW-55F column chromatographies. Molecular weight of purified enzyme was estimated to be about 58 kDa by Toyopearl HW-55F gel chromatography and SDS-PAGE, respectively suggested that the purified enzyme was a monomer. Optimum pH of the enzyme was 6.0?7.0 and optimum temperature 40°C. The enzyme was extremely inactivated at pH was higher than 7.0 or lower than 5.0. Heat inactivation occurred at 40°C. This enzyme activated by Ca ²⁺ , Mn²⁺, PCMB, and DTNB, but inhibited by Ba²⁺, Fe³⁺, Hg²⁺, Mg²⁺, and iodoacetic acid. The purified enzyme was of α?-type by TLC analysis. The relative rate of hydrolysis of the polymeric substance decreased with decreasing percentage of α?-1,4-linkages and with increasing percentage of α?-1,6-linkages in substrate similar to the results from commercially available honey.     

Purification and properties of polyphenol oxidase in head lettuce (Lactuca sativa)

Polyphenol oxidase (EC 1.10.3.1) in head lettuce (Lactuca sativa L) was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56 000 amu by Sephadex G-100 gel filtration. The purified enzyme quickly oxidised chlorogenic acid (5-caffeoyl quinic acid) and (—)-epicatechin. The K^m values for the enzyme, using chlorogenic acid (pH 4·5, 30°C) and (—)-epicatechin (pH 7·0, 30°C) as substrate, were 0·67 mM and 0·91 mM, respectively. The optimal pH of chlorogenic acid oxidase and (—)-epicatechin oxidase activities were 4·5 and 7·8, respectively, and both activities were stable in the pH range 6–8 at 5°C for 20 h. Potassium cyanide and sodium diethyldithiocarbamate markedly inhibited both activities of the purified enzyme. The inhibitory effect of metallic ions such as Ca²⁺, Mn²⁺, Co²⁺ and Ni²⁺ for chlorogenic acid oxidase activity was stronger than that for (—)-epicatechin oxidase activity.     

PURIFICATION AND CHARACTERIZATION OF A SERINE PROTEINASE FROM THE TUNA PYLORIC CAECA

A 15.0 kDa serine proteinase with collagenase activity from pyloric caeca of tuna, Thunnus thynnus, was purified in four steps; acetone precipitation, gel filtration chromatography on a Sephadex G‐100, ion‐exchange chromatography on a DEAE‐Sephadex α‐50 and gel filtration chromatography on a Sephadex G‐75 column. The purification and yield were 30.5‐fold and 0.023%, respectively, as compared with those in the starting crude extract. The optimum pH and temperature for the purified collagenolytic enzyme were around pH 7.5 and 55C, respectively. The purified proteinase was strongly inhibited by metal ions (Hg²⁺ and Zn²⁺) and serine proteinase inhibitors (PMSF, TLCK and soybean trypsin inhibitor) suggesting it is a serine protease. The K_m and V_max of the purified enzyme for collagen type I were approximately 3.82 mM and 851.5 U, respectively.     

PURIFICATION AND PROPERTIES OF CYCLODEXTRIN GLUCANOTRANSFERASE FROM AN ALKALOPHILIC BACILLUS sp. 7-12

Cyclodextrin glucanotransferases (EC 2.4.1.19) (CGTase) are industrially important enzymes for production of cyclodextrin (CD) from starch. γ‐CD yield of CGTase from alkalophilic Bacillus species is usually much lower than β‐CD, while from alkalophilic Bacillus sp. 7‐12. γ‐CD yield is close to β‐CD. A CGTase from alkalophilic Bacillus sp. 7‐12 was purified and characterized. When purified by ammonium sulfate fractionation, DEAE‐cellulose column chromatography and Sepharose CL‐6B column chromatography, the enzyme obtained consisted of a single band that did not dissociate into subunits by SDS polyacrylamide gel electrophoresis. Molecular weight of the purified enzyme was determined to be 69,000 Da by SDS‐PAGE. The enzyme showed a K_mof 1.24 mg/mL and V_max0.101 µM/min when potato starch was used as substrate. The enzyme was stable below 70C with an optimum activity at 60C, and stable at pH range 6–10 with an optimum pH at 8.5. The enzyme activity was strongly inhibited by Ag⁺, Cu²⁺, Mg²⁺, Al³⁺, Co²⁺, Zn²⁺, Fe²⁺and slightly inhibited by Sn²⁺, Mn²⁺. The ions Ca²⁺and K⁺, EDTA and DTT had no influence on the enzyme activity.     

Purification and characterization of an alkaline pectin lyase produced by a newly isolated Brevibacillus borstelensis (P35) and its applications in fruit juice and oil extraction

An alkaline pectin lyase (PNL) (EC 4.2.2.10) secreted by Brevibacillus borstelensis P35 (GenBank Number: FJ417406) was purified using ammonium sulfate fractionation, anion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-150. The pH and temperature optima of the enzyme were found to be 8.0 and 60 °C. The enzyme does not loose activity up to 60 °C if exposed for 1 h. The values of K _m and V _max of the enzyme were 0.625 mg/mL and 126.32 s^?1, respectively. The molecular weight was found to be 36 ± 01 kDa. The presence of 10 mM concentration of Ca²⁺, Cu²⁺, Mn²⁺, Mg²⁺, Zn²⁺, Hg²⁺, Fe²⁺ and EDTA, l-cystein, ascorbic acid significantly enhanced the PNL of the purified enzyme. In the course of the laboratory trials, it was demonstrated that PNL from B. borstelensis (P35) could be successfully applied to the production and clarification of fruit juice and oil extraction.     

Purification and partial characterization of β-glucanase produced by <Emphasis Type="Italic">Trichoderma viride</Emphasis> TP09 isolated from sewage of beer-making

As an initial investigation to improve the insoluble yeast β-1, 3-glucan solubility, a novel β-glucanase from Trichoderma viride TP09 was purified in the culture supernatant and partially characterized. By 70% saturation ammonium sulfate and chromatography on DEAE-Sepharose CL-6B column, β-glucanase was purified 28.7-fold, with recovery of 45.2% of the initial activity. The molecular weight of this enzyme was estimated to be 54.6 KD by SDS-PAGE. The optimum pH and the optimum temperature for the enzyme were 5.0 and 50 °C, respectively. The enzyme showed high stability within the range of pH 3.0–5.0 and thermostability between 30 and 70 °C. The enzyme activity was inhibited by Fe³⁺, Mg²⁺, Mn²⁺, Cu²⁺, and stimulated by Zn²⁺, Ca²⁺, Fe²⁺. Substrate specificity studies revealed the enzyme to be a β-1, 3–1, 4-glucanase. The β-glucanase showed preference for β-1, 3 linkage and β-1, 4 linkage, but had no activity on α-1, 4 and α-1, 6 linkage. The above results indicated that the enzyme extracted from T. viride TP09 of the beer-making sewage could be used as a potential predominant tool to enhance solubility of the insoluble yeast β-1, 3-glucan. These findings may lead to an enhanced solubility and expedite the progress of application in immunotherapy.     

Purification and some properties of a cysteine proteinase from sorghum malt variety SK5912

A cysteine proteinase from sorghum malt variety SK5912 was purified by a combination of 4 M sucrose fractionation, ion‐exchange chromatography on Q‐ and S‐Sepharose (fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 8.4‐fold to give a 13.4% yield relative to the total activity in the crude extract and a final specific activity of 2057.1 U mg^?1 protein. SDS—PAGE revealed two migrating protein bands corresponding to apparent relative molecular masses of 55 and 62 kDa, respectively. The enzyme was optimally active at pH 6.0 and 50 °C, not influenced across a relatively broad pH range of 5.0–8.0 and retained over 60% activity at 70 °C after 30‐min incubation. It was highly significantly (P < 0.001) inhibited by Hg²⁺, appreciably (P < 0.01) inhibited by Ag⁺, Ba²⁺ and Pb²⁺ but highly significantly (P < 0.001) activated by Co²⁺, Mn²⁺ and Sr²⁺. The proteinase was equally highly significantly (P < 0.001) inhibited by both iodoacetate and p‐chloromercuribenzoate and hydrolysed casein to give the following kinetic constants: K_m = 0.33 mg ml^?1; V_max = 0.08 µmol ml^?1 min^?1. Copyright © 2004 Society of Chemical Industry     

Purification and characterisation of thermo-alkaline pectinase enzyme from Hylocereus polyrhizus

The thermo-alkaline pectinase enzyme from Hylocereus polyrhizus was purified 232.3-fold with a 73.3 % recovery through ammonium sulphate precipitation, gel filtration, and ion exchange chromatography. Ion exchange chromatography combined with sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) revealed that the enzyme was monomeric with a molecular weight of 34.2 kDa. The pectinase exhibited broad specificity towards polygalacturonic acid, arabinan, oat spelt xylan, and pNP-α-glucopyranoside. The optimum pH and temperature were 8.0 and 75 °C, respectively. This enzyme was stable over a wide pH range (3.0–11.0) and at relatively high temperature (85 °C for 1 h). The K_m and V_max values of pectinase towards polygalacturonic acid were 2.7 mg/ml and 34.30 U/mg proteins, respectively. In addition, the enzyme activity was inhibited by Ni²⁺, Al³⁺, and Fe²⁺ and was increased in the presence of Ca²⁺ and Mg²⁺ by 120 and 112 %, respectively. The purified pectinase demonstrated robust stability in response to surfactants and oxidising agents. EDTA, which is a powerful chelating agent, did not exert any significant effect on the enzyme stability. Thus, enzymes with these unique properties may be widely used in different types of industries and biotechnological applications.     

Purification and characterization of a new endo-β-1,3-glucanase exhibiting a high specificity for curdlan for production of β-1,3-glucan oligosaccharides

Endo-β-1,3-glucanase (Endo23) was purified from a Trichoderma reesei GIMCC 3.498 fermentation broth using anion exchange and 2-stage size exclusion chromatography. Purification of 44.5× and a 12% recovery yield of enzyme activity were achieved. The Mw and isoelectric point were estimated to be 24 kDa and 3.85 using SDS-PAGE and IEF, respectively. The highest substrate specificity was observed for water-insoluble curdlan. The optimal conditions for hydrolyzing curdlan were pH 5.0 and 50°C. The main hydrolytic products were glucobiose and glucotriose. Minor amounts of glucose and glucotetraose were detected. Hg²⁺, Fe²⁺, Fe³⁺, and Sn²⁺ inhibited the hydrolysis activity of Endo23 at 5 and 50 mM. K⁺ slightly promoted Endo23 activity. Endo23 belongs to the category EC3.2.1.39. The peptide sequences of Endo23 showed identity with conserved sequences that typically exist in β-1,3-glucanases of the glycoside hydrolase family. The Endo23 sequence was partially similar to a hypothetical lignocellulase from Penicillium oxalicum 114-2.     

Partial characterization of β‐ d‐xylosidase from wheat malts

Arabinoxylans (AXs) from wheat malts potentially affect beer quality and production. β‐ d ‐Xylosidase is a key enzyme that degrades the main chains of AXs to produce xylose. This study performed a partial characterization of β‐ d ‐xylosidase from wheat malts. The optimal temperature was 70 °C and the enzyme exhibited excellent thermostability, that is, residual activities were 92.6% at 60 °C for 1 h. The enzyme was stable over a pH range of 3.0–6.0 and showed optimum activity at pH 3.5 and 4.5. Kinetic parameters K_m and V_max of wheat malt β‐ d ‐xylosidase against p‐nitrophenyl‐xyloside were 1.74 mmol L⁻¹ and 0.76 m m min⁻¹, respectively. The enzyme activity was severely inhibited by Cu²⁺, moderately inhibited by Mn²⁺, Mg²⁺, Al³⁺, Ca²⁺, Ba²⁺ and Na⁺ and mildly inhibited by Fe³⁺ and Fe²⁺. The partial enzymatic characterization achieved in this study can be used as a theoretical basis for purifying β‐ d ‐xylosidase from wheat malts. Copyright © 2015 The Institute of Brewing & Distilling     

Production, purification, and characterization of a potential thermostable galactosidase for milk lactose hydrolysis from Bacillus stearothermophilus

β-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 β-galactosidase gene bgaB from Bacillus stearothermophilus was cloned and expressed in B. sub-tilis WB600. The recombinant enzyme was purified by a combination of heat treatment, ammonium sulfate fractionation, ion exchange, and gel filtration chromatography techniques. The purified β-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 β-galactosidase activity were 70°C and pH 7.0, respectively. Kinetics of thermal inactivation and half-life times for this thermostable enzyme at 65 and 70°C were 50 and 9 h, respectively, and the K_m and V_max values were 2.96 mM and 6.62 μmol/min per mg. Metal cations and EDTA could not activate this thermostable enzyme, and some divalent metal ions, namely, Fe²⁺, Zn²⁺, Cu²⁺, Pb²⁺, and Sn²⁺, 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.     

Purification and some properties of a novel raw starch‐digesting amylase from Aspergillus carbonarius

A medium was developed to obtain the maximum yield of raw starch‐digesting amylase from Aspergillus carbonarius (Bainier) Thom IMI 366159 in submerged culture with raw starch as the sole carbon source. The amylase was purified to apparent homogeneity by sucrose concentration and ion exchange chromatography on S‐ and Q‐Sepharose (fast flow) columns. SDS‐PAGE revealed two migrating protein bands corresponding to relative molecular masses of 31.6 and 32 KDa. The enzyme was optimally active at pH 6.0–7.0 and 40 °C, was uninfluenced across a relatively broad pH range of 3.0–9.0 and retained over 85% activity between 30 and 80 °C after 20 min incubation. The enzyme was strongly activated by Co²⁺ and only slightly by Fe²⁺, while Ca²⁺, Hg²⁺, EDTA and N‐bromosuccinamide elicited significant repression of the enzyme activity. The enzyme hydrolysed amylopectin (K_m 0.194 mg ml ⁻¹), glycogen (K_m 0.215 mg ml ⁻¹), pullulan (K_m 0.238 mg ml ⁻¹), amylose (K_m 0.256 mg ml ⁻¹) and raw potato starch (K_m 0.260 mg ml ⁻¹), forming predominantly maltose and relatively smaller amounts of glucose. © 2000 Society of Chemical Industry