Purification and characteristics of trypsins from cold-zone fish,Pacific cod (Gadus macrocephalus) and saffron cod (Eleginus gracilis)

Trypsins from the pyloric ceca of Pacific cod (Gadus macrocephalus) (GM-T) and saffron cod (Eleginus gracilis) (EG-T) were purified by gel filtration on Sephacryl S-200 and Sephadex G-50. The final enzyme preparations were nearly homogeneous on SDS–PAGE and the molecular weights of both enzymes were estimated to be approximately 24 kDa by SDS–PAGE. The specific trypsin inhibitors, soybean trypsin inhibitor and TLCK, strongly inhibited the activities of GM-T and EG-T. The optimum pH and optimum temperature of both trypsins were around pH 8.0 and 50 °C, respectively, using N^α-p-tosyl-l-arginine methyl ester as substrate. The GM-T and EG-T were unstable above 30 °C and below pH 5.0, and they were stabilised by calcium ion. The N-terminal amino acid sequences of GM-T (IVGGYECTRHSQAHQVSLNS) and EG-T (IVGGYECPRHSQAHQVSLNS) were found. The percentage of hydrophobic amino acid in the N-terminal 20 amino acids sequences of these cold-zone fish trypsins was lower (28%) than those of temperate-zone fish trypsins (34%), tropical-zone fish trypsins (37%) and mammalian trypsins (34%). Whereas the content of charged amino acids in the GM-T and EG-T was relatively higher than those of trypsins from temperate-zone fish, tropical-zone fish and mammals. Moreover, the GM-T catalyzed synthesis of N^α-(tert-butoxycarbonyl)-l-alanyl-l-alanine-p-nitroanilide (N^α-Boc-l-Ala-l-Ala-pNA) has been studied by using N^α-(tert-butoxycarbonyl)-l-alanine-p-guanidinophenyl ester [N^α-Boc-l-Ala-OpGu (inverse substrate)] as acyl donor and l-alanine-p-nitroanilide (l-Ala-pNA) as acyl acceptor, respectively.     

A heat-stable trypsin from the hepatopancreas of the cuttlefish (Sepia officinalis): Purification and characterisation

Thermostable trypsin from the hepatopancreas of Sepia officinalis was purified by fractionation with ammonium sulphate, Sephadex G-100 gel filtration, DEAE-cellulose an ion-exchange chromatography, Sephadex G-75 gel filtration and Q-Sepharose anion-exchange chromatography, with a 26.7-fold increase in specific activity and 21.8% recovery. The molecular weight of the purified enzyme was estimated to be 24,000 Da by SDS-PAGE and size exclusion chromatography. The purified enzyme showed esterase specific activity on Nα -benzoyl-L-arginine ethyl ester (BAEE) and amidase activity on Nα -benzoyl-DL-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the enzyme activity were pH 8.0 and 70 °C, respectively, using BAPNA as a substrate. The enzyme was extremely stable in the pH range 6.0–10.0 and highly stable up to 50 °C after 1 h of incubation. The purified enzyme was inhibited by soybean trypsin inhibitor (SBTI) and phenylmethylsulphonyl fluoride (PMSF), a serine-protease inhibitor. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGKESSPYNQ. S. officinalis trypsin, which showed high homology with trypsins from marine vertebrates and invertebrates, had a charged Lys residue at position 5 and a Ser residue at position 7, where Tyr and Cys are common in all marine vertebrates and mammalian trypsins. Further, the enzyme had an Asn at position 11, not found in any other trypsins.     

Purification and characterization of trypsin from the pyloric caeca of brownstripe red snapper (Lutjanus vitta)

Trypsin was purified from the pyloric caeca of brownstripe red snapper (Lutjanus vitta) by ammonium sulphate (40–60% saturation) precipitation, soybean trypsin inhibitor (SBTI)-Sepharose 4B column and DEAE-Sephacel column chromatography. Purified trypsin showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) and native-PAGE. A yield of 4.9% with the purification-fold of 20 was obtained. Trypsin had an apparent molecular weight of 23 kDa. SBTI and N-ρ-tosyl-l-lysine-chloromethylketone (TLCK) showed a strong inhibitory effect on the purified trypsin, while other protease inhibitors exhibited negligible inhibition. Trypsin had maximal activity at pH 8.5 and 60 °C for the hydrolysis of α-N-benzoyl-dl-arginine-ρ-nitroanilide (BAPNA). It was stable within the temperature range of 25–55 °C and pH range of 7.0–10.0. Purified trypsin had a Michaelis–Menten constant (K_m) and catalytic constant (k_cat) of 0.507 mM and 4.71 s⁻¹, respectively, when BAPNA was used as the substrate. For the hydrolysis of α-N-ρ-tosyl-l-arginine methyl ester (TAME), K_m and k_cat were 0.328 mM and 112 s⁻¹, respectively.     

A novel aspartic protease from the viscera of Sardinelle (Sardinella aurita): Purification and characterisation

A novel aspartic protease was extracted from the defatted viscera of sardinelle (Sardinella aurita) and purified, with a 9.5-fold increase in specific activity and 23.3% recovery. The molecular weight of the purified enzyme was estimated to be 17 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The purified enzyme appeared as a single band on native-PAGE. The optimum pH and temperature for protease activity were around 3.0 and 40 °C, respectively. The enzyme showed pH stability between 2.0 and 5.0 and retained more than 50% of its activity after heating for 30 min at 50 °C. The enzyme lost 90% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride. Its K_m value was determined to be 0.73 × 10⁻⁴ M using haemoglobin as a substrate. The N-terminal 12 amino acid sequence of the purified acidic protease was R V I I E D X D Q F C T. This sequence showed low homology with aspartic peptidases of several other species of fish, suggesting that the enzyme is a new aspartic protease.     

New alkaline trypsin from the intestine of Grey triggerfish (Balistes capriscus) with high activity at low temperature: Purification and characterisation

A highly alkaline trypsin from the intestine of Grey triggerfish (Balistes capriscus), with high activity at low temperature, was purified and characterised. The enzyme was purified to homogeneity using acetone precipitation, Sephadex G-100 gel filtration and Mono Q-Sepharose anion-exchange chromatography, with a 13.9-fold increase in specific activity and 41.3% recovery. The molecular weight of the purified alkaline trypsin was estimated to be 23.2 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and size exclusion chromatography. Purified trypsin appeared as a single band on native–PAGE. Interestingly, the enzyme was highly active over a wide range of pH, from 9.0 to 11.5, with an optimum at pH 10.5, using Nα-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as a substrate. The relative activities at pH 9.0, 11.5 and 12.0 were 86.5%, 92.6% and 52.4%, respectively. The enzyme was extremely stable in the pH range 7.0–12.0. In addition, the enzyme had high activity at low and moderate temperatures with an optimum at around 40 °C and had more than 80% of its maximum activity at 20 °C. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI) and phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor. The enzyme showed extreme stability towards oxidising agents, retaining about 87% and 80% of its initial activity after 1 h incubation at 40 °C in the presence of 1% sodium perborate and 1% H₂O₂, respectively. In addition, the enzyme showed excellent stability and compatibility with some commercial solid detergents.     

Purification and characterisation of two enzymes related to endogenous formaldehyde in Lentinula edodes

In this study, γ-glutamyl transpeptidase (GGT) and l-cysteine sulphoxide lyase (C-S lyase) were purified from the fruiting body of Lentinula edodes in three steps and then characterised. We found that GGT together with C-S lyase caused the generation of endogenous formaldehyde in L. edodes. GGT was composed of a large subunit of 41 kDa and a small subunit of 25 kDa, and C-S lyase was composed of two identical subunits of 46 kDa, as determined by SDS–PAGE. GGT was stable at pH 8.0–10.0 with an optimum pH of 8.8, and was stable at 20–50 °C with an optimum activity at 37 °C. C-S lyase was stable at pH 8.0–9.0 with an optimum pH of 8.5, and was stable at 20–60 °C with an optimum activity at 40 °C. The present work supports the study of the mechanism of endogenous formaldehyde in L. edodes.     

Purification and characterisation of exo- and endo-inulinase from Aspergillus ficuum JNSP5-06

Three exoinulinases (Exo-I, Exo-II, and Exo-III) and two endoinulinases (Endo-I and Endo-II) were purified from the culture broth of Aspergillus ficuum JNSP5-06 by ammonium sulphate precipitation, DEAE-cellulose column chromatography, Sepharose CL-6B column chromatography and preparative electrophoresis. The molecular weights of Exo-I, Exo-II, Exo-III, Endo-I, and Endo-II were determined to be 70 kDa, 40 kDa, 46 kDa, 34 kDa, and 31 kDa, respectively. Using inulin as the substrate, their K_m values were 43.1 mg/ml, 31.5 mg/ml, 25.3 mg/ml, 14.8 mg/ml, and 25.6 mg/ml, respectively. These five inulinases were stable below 50 °C with optimum activity at 45 °C, and were stable at a pH range of 4–8 with an optimum pH at 4.5 for exoinulinase and at 5.0 for endoinulinase. The inulinase activity was completely inhibited by Ag⁺ and strongly inhibited by Fe²⁺ and Al³⁺, whereas K⁺, Ca²⁺, Li²⁺, EDTA and urea had no significant influence on the inulinase activity.     

Purification and characterization of a novel glucoamylase from Fusarium solani

Thermostable enzymes are currently being investigated to improve industrial processes of starch saccharification. A novel glucoamylase was purified to electrophoretic homogeneity from the culture supernatant of Fusarium solani on a fast protein liquid chromatographic system (FPLC). The recovery of glucoamylase after gel filtration on FPLC was 31.8% with 26.2-fold increase in specific activity. The enzyme had a molecular mass of 40 kDa by SDS-PAGE and 41 kDa by gel filtration. The glucoamylase exhibited optimum activity at pH 4.5. The K_cat and K_m were 441/min and 1.9 mg/ml, respectively, for soluble starch, specificity constant (K_cat/K_m) was 232. The enzyme was thermally stable at 50 °C and retained 79% activity after 60 min at this temperature. The half-life of the enzyme was 26 min at 60°C. The enzyme was slightly stimulated by Cu²⁺ and Mg²⁺ and strongly inhibited by Hg²⁺, Pb²⁺, Zn²⁺, Ni²⁺ and Fe³⁺.     

Artocarpus integer leaf protease: Purification and characterisation

The presence of a protease in Artocarpus integer leaves, which are traditionally used as a meat tenderiser, was verified by the presence of a band at 69 kDa, using caseinolytic zymography. Purification by temperature phase partitioning with Triton X-114, ammonium sulphate precipitation and gel filtration chromatography yielded a preparation with a 12-fold increase in enzyme purity and a final specific activity of 76.67 U/mg. The cysteinic nature of this enzyme was confirmed through inhibition of enzyme activity by E-64 and iodoacetamide and enhancement of activity by cysteine and 2-mercaptoethanol. The protease retained 70% of its activity over a broad pH range (pH 6–12), with optimal activity recorded at pH 10 and 40 °C. The enzyme was stable at temperatures up to 70 °C, with 80% of its activity intact. Addition of 5 mM Ca²⁺ stimulated enzyme activity and a kinetic study of the enzyme yielded K_m and V_max values of 0.304 mg/mL and 0.735 mg/mL/min, respectively.     

Purification and characterisation of trypsins from the spleen of skipjack tuna (Katsuwonus pelamis)

Three trypsin isoforms, trypsins A, B and C, from the spleen of skipjack tuna (Katsuwonus pelamis) were purified by a series of chromatographies including Sephacryl S-200, Sephadex G-50 and diethylaminoethyl-cellulose to obtain a single band on native-PAGE and SDS–PAGE. The molecular mass of all the trypsin isoforms was estimated to be 24 kDa by size exclusion chromatography and SDS–PAGE. The optimum pH and temperature of the three isoforms for the hydrolysis of N-p-tosyl-l-arginine methyl ester hydrochloride were 8.5 and 60 °C, respectively. Trypsins were stable to heat treatment up to 50 °C, and over a pH range of 6.0–11.0. All isoforms were stabilised by calcium ions. The trypsin activities were effectively inhibited by soybean trypsin inhibitor, TLCK and partially inhibited by ethylenediaminetetraacetic acid, while E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A showed no inhibitory effect. Activities decreased continuously as NaCl concentration (0–30%) increased. Trypsins A, B and C showed K_m of 0.11–0.29 mM and K_cat of 57.1–114 s⁻¹. The N-terminal amino acid sequence of 20 residues of three trypsin isoforms was IVGGYECQAHSQPHQVSLNS and had high homology to those of other fish trypsins.     

Purification and characterization of trypsin from the viscera of sardine (Sardina pilchardus)

Trypsin from the viscera of Sardina pilchardus was purified by fractionation with ammonium sulphate, heat treatment and Sephadex G-100 gel filtration with a ninefold increase in specific activity and 9% recovery. The molecular weight of the enzyme was estimated to be 25,000 Da on SDS–PAGE. This enzyme showed esterase-specific activity on Nα-benzoyl-l-arginine ethyl ester (BAEE). The purified enzyme was inhibited by benzamidine, a synthetic trypsin inhibitor, and phenylmethylsulphonyl fluoride (PMSF) a serine-protease inhibitor, but was not inhibited by the β-mercaptoethanol. The optimum pH and temperature for the enzyme activity were pH 8.0 and 60 °C, respectively. The relative activity at pH 9.0 was 95.5% and the enzyme showed pH stability between 6.0 and 9.0. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECQKYSQ. S. pilchardus trypsin, which showed high homology to other fish trypsins, had a charged Lys residue at position 9, where Pro or Ala are common in fish trypsins. The enzyme was strongly inhibited by Zn²⁺ and Cu²⁺.     

Purification and characterization of chymotrypsins from the hepatopancreas of crucian carp (Carassius auratus)

Two chymotrypsins (chymotrypsin A and B) have been purified to homogeneity from the hepatopancreas of crucian carp (Carassius auratus) by ammonium sulphate fractionation and chromatographies on DEAE-Sepharose, Sephacryl S-200 HR, Phenyl-Sepharose and SP-Sepharose. The molecular masses of chymotrypsin A and B were approximately 28 and 27 kDa, respectively, by SDS–PAGE. Purified chymotrypsins also revealed single bands by native-PAGE. Optimum temperatures of chymotrypsin A and B were 40 and 50 °C, and optimal pHs were 7.5 and 8.0 using Suc-Leu-Leu-Val-Tyr-AMC as substrate. Both enzymes were effectively inhibited by serine proteinase inhibitors and slightly activated by metal ions such as Ca²⁺ and Mg²⁺, while inactivated by Mn²⁺, Cd²⁺, Cu²⁺, Fe²⁺ to different degrees. Apparent K_ms of chymotrypsin A and B were 1.4 and 0.5 μM, and K_cats were 2.7 S⁻¹ and 3.4 S⁻¹, respectively. Immunoblotting analysis using anti-chymotrypsin B weakly cross reacted with chymotrypsin A.     

Purification and characterisation of two extracellular acid proteinases from Monascus pilosus

Genus Monascus is one of the most important microorganisms in the fermentation industry in Asia. However, only a little attention has been paid to the proteinases produced by this fungus and their role in the fermentation process. The main objective of this study was to purify and characterise acid proteinases produced by Monascus pilosus. Two acid proteinases (MpiAP1 and MpiAP2) were purified to homogeneity. Both purified enzymes, MpiAP1 and MpiAP2, were monomeric structures with molecular masses of around 43 and 58 kDa, respectively. The former was an acidic non-glycoprotein, whereas the latter was an acidic glycoprotein with 27% carbohydrate content. Although amino-terminal amino acid sequence analysis of both enzymes (MpiAP1 and MpiAP2) of 20 amino acid length showed over 90% similarity, their amino-terminal amino acids were different from each other. Both enzymes were optimally active at 55 °C and at pH 2.5–3.0 against casein or human haemoglobin. The T_1/2 values of MpiAP1 and MpiAP2 were 65 and 70 °C, respectively. Both of the enzymes were completely inhibited by pepstatin A, and markedly by SDS. MoO₃ also showed a partial inhibition of both enzymes. Milk casein and haemoglobin were good substrates for these enzymes. Eleven cleavages were detected using the oxidised insulin B-chain as a peptide for the proteolytic specificity test of MpiAP1, while seven cleavages were detected for MpiAP2.     

Purification and partial characterization of polyphenol oxidase from the flower buds of Lonicera japonica Thunb.

The purification and partial enzymology characteristics of polyphenol oxidase from Lonicera japonica (LjPPO) were studied in this paper. The crude enzyme solution was purified in turn by ammonium sulfate, dialysis, and DEAE-cellulose ion-exchange chromatography after preliminary treatments. Purification resulted in 31-fold enrichment and its molecular weight was estimated to be ∼49 kDa exhibited on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The pH for optimal conditions of LjPPO was 7.5, and the temperature was 25 °C, in addition, the inhibitive effects of inhibitors were enhanced positively with increasing of the concentration. Moreover, crude enzyme solution showed diphenolase activity toward catechol, l-dopa and chlorogenic acid rather than monophenolase and triphenolase activity, and the best substrate was catechol because of the highest V_max/K_m value. However, the oxidation of diphenol related to browning significantly, so the data obtained in this research provided theoretical basis for the prevention of enzymatic browning of L. japonica during processing.     

Structural characterisation and bioactivities of hybrid carrageenan-like sulphated galactan from red alga Furcellaria lumbricalis

The red alga, Furcellaria lumbricalis from the coast of the Prince Edward Island (PEI) in Atlantic Canada, was extracted with hot water and fractionated with 0.125 M KCl to obtain a carrageenan-like polysaccharide. The polysaccharide was further purified on ion-exchange and gel-permeation chromatography to yield a fraction (FB1) of uniform size and charge, with an average molecular weight of 428 kD. Oligosaccharides generated with acid hydrolysis of FB1 were sequenced using the electrospray ionisation collision induced dissociation tandem mass spectrometry (ES-CID-MS/MS) technique. On the basis of chemical and spectroscopic analysis, FB1 was characterised to be composed of 1,4-linked 3,6-anhydro-galactose (40%), 1,3-linked 4-sulphated-galactose (30%), 1,3-linked galactose (20%), 1,4-linked galactose (8%) and 1,4-linked 3-O-methyl-galactose (2%), which makes it be a novel sulphated galactan hybrid. The β-secretase (BACE) inhibition and immunomodulation activities of FB1-derived oligosaccharides were evaluated in vitro.     

Purification and characterization of transglutaminase from a newly isolated Streptomyces hygroscopicus

Transglutaminase (TGase, EC 2.3.2.13) from a Streptomyces hygroscopicus strain isolated from soil was purified from culture broth by ethanol precipitation, followed by successive chromatographies on CM-cellulose and Sephadex G-75 columns with a yield and purification-fold of 21.1% and 30%, respectively. The enzyme’s molecular weight was estimated as 38,000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified microbial transglutaminase (MTG) exhibited optimum activity at 37–45 °C and in a range of pH 6.0–7.0 for hydroxamate formation from N-carboxybenzoyl-l-glutaminyl-glycine and hydroxylamine. The enzyme was not stable above 50 °C and was stable within a pH range of 5.0–8.0 at lower temperature. The MTG was not inhibited by Ca²⁺ and ethylenediaminetetraacetic acid, suggesting it was calcium-independent. Purified MTG was strongly inactivated by 5,5′-dithiobis (2-nitrobenzoic acid), Cu²⁺, Zn²⁺, Pb²⁺, and Hg²⁺, suggesting that this enzyme could possess a thiol group at the active site. The MTG stability was strongly affected by ethanol concentration. The enzyme activity was slightly elevated at a lower concentration of ethanol at 25 °C.     

Isolation and characterisation of acid-solubilised collagen from the skin of Nile tilapia (Oreochromis niloticus)

Acid-solubilised collagen (ASC) was extracted from the skin of Nile tilapia (Oreochromis niloticus) and characterisation was studied. The results indicated that the yield of ASC was 39.4% on the basis of dry weight. This ASC was rich in glycine (35.6%). The amount of imino acids, proline and hydroxyproline, in ASC was 210 residues per 1000 residues. The ultraviolet (UV) absorption spectrum of ASC showed that the distinct absorption was at 220 nm. ASC showed transition curve at maximum temperature (T_max) of 32.0 °C in 0.05 M acetic acid, about 12 °C lower than that of calf skin collagen. Maximum solubility (0.75 mg/ml) in 0.5 M acetic acid was observed at pH 3. Solubility reached the minimum at pH 7. A sharp decrease in solubility was observed in 2% (w/v) NaCl or above. Biochemical studies indicated that ASC was composed of the α1α2α3 heterotrimers.     

Purification and characterisation of Saccharomyces cerevisiae external invertase isoforms

Four external invertase isoforms (EINV1, EINV2, EINV3 and EINV4) from Saccharomyces cerevisiae were highly purified by isoelectric precipitation, ethanol precipitation, ion-exchange on QAE-Sephadex and gel filtration using Sephacryl S-200. Unlike previously published procedures for external invertase purification, a specially designed step elution was applied on QAE-Sephadex which enabled the separation of four isoforms. The isoforms have the same molecular mass and catalytic properties: K_m for sucrose (25.6 mM), pH optimum (3.5–5.0) and temperature optimum (60 °C), but they exhibit significant difference in pI values, thermal stability and chemical reactivity. Deglycosylation studies showed that the observed differences between isoforms arise from posttranslational modifications. Results showed that external invertase is a mixture of at least four isoforms, but in order to improve the efficiency of food industry processes, only the most stable isoform (EINV1) should be purified and utilised. Substantially different chemical reactivity of the isoforms could be used to improve the yield of covalent immobilization procedures.     

Purification and characterization of soluble invertases from suspension-cultured bamboo (Bambusa edulis) cells

An alkaline invertase (IT I) and an acid invertase (IT II) were purified from the soluble fraction of suspension cultured bamboo cells. Both purified invertases were homogeneous as examined by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and were identified as β-fructofuranosidases able to attack the β-fructofuranoside from the fructose end. With respect to sucrose hydrolysis, the optimal pHs were 7.0 and 4.5 for IT I and IT II, respectively. The Km’s were 10.9 and 3.7 mM. The IT I and IT II molecular masses were 240 and 68 kDa, respectively, as estimated by gel filtration. The isoelectric points were 4.8 and 7.4. IT I was a homotetrameric enzyme activated by bovine serum albumin (BSA). IT II was a monomeric enzyme activated by BSA, concanavalin A (ConA) and urease. Both isoforms were significantly inhibited by heavy metal ions Ag⁺ (5 mM) and Hg²⁺ (1 mM), and mercaptide forming agent ρ-chloromercuribenzoic acid (PCMB; 0.5 mM).