24 kDa Trypsin: A predominant protease purified from the viscera of hybrid catfish (Clarias macrocephalus × Clarias gariepinus)

Trypsin was purified to homogeneity from the viscera of hybrid catfish (Clarias macrocephalus × Clarias gariepinus) through ammonium sulphate fractionation and a series of chromatographies including Sephacryl S-200, Sephadex G-50 and DEAE-cellulose. It was purified to 47.6-fold with a yield of 12.7%. Based on native-PAGE, the purified trypsin showed a single band. The molecular weight of purified trypsin was estimated as 24 kDa by size exclusion chromatography and SDS–PAGE. The optimum pH and temperature for N^α-p-tosyl-l-arginine methyl ester hydrochloride (TAME) hydrolysis were 8.0 and 60 °C, respectively. Trypsin was stable to heat treatment up to 50 °C, and over a pH range of 6.0–11.0. Trypsin was stabilized by calcium ion. The trypsin activity was strongly inhibited by soybean trypsin inhibitor and N-p-tosyl-l-lysine chloromethyl ketone and partially inhibited by ethylenediaminetetraacetic acid. Activity decreased continuously as NaCl concentration (0–30%) increased. Apparent K_m value of trypsin was 0.3 mM and K_cat value was 92.1 S⁻¹ for TAME. The N-terminal amino acid sequence of 20 residues of trypsin was IVGGYECQAHSQPPTVSLNA, which is highly homologous with trypsins from other species of fish.     

Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.)

Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification – by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose – was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0–11.0 and an optimal temperature of approximately 55–60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis–Menten constant (K_m) and catalytic constant (K_cat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s⁻¹, respectively. The catalytic efficiency (K_cat/K_m) was 238 s⁻¹ mM⁻¹.     

Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: Its purification and characterization

Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5 kDa according to SDS–PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40 °C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe²⁺ > Hg²⁺ > Mn²⁺ > K⁺ > Mg²⁺ > Li⁺ > Cu²⁺). Trypsin activity decreased continuously as NaCl concentration increased (0–30%). K_m and k_cat values were 0.13 mM and 1.46 s⁻¹, respectively. Results suggest the enzyme have a potential application where room processing temperatures (25–35 °C) or high salt (30%) concentration are needed, such as in fish sauce production.     

Biochemical properties of two isoforms of trypsin purified from the Intestine of skipjack tuna (Katsuwonus pelamis)

Two trypsins (A and B) from the intestine of skipjack tuna (Katsuwonus pelamis) were purified by Sephacryl S-200, Sephadex G-50 and DEAE-cellulose with a 177- and 257-fold increase in specific activity and 23% and 21% recovery for trypsin A and B, respectively. Purified trypsins revealed a single band on native-PAGE. The molecular weights of both trypsins were 24 kDa as estimated by size exclusion chromatography and SDS–PAGE. Trypsin A and B exhibited the maximal activity at 55 °C and 60 °C, respectively, and had the same optimal pH at 9.0. Both trypsins were stable up to 50 °C and in the pH range from 6.0 to 11.0. Both trypsin A and B were stabilised by calcium ion. Activity of both trypsins continuously decreased with increasing NaCl concentration (0–30%) and were inhibited by the specific trypsin inhibitors – soybean trypsin inhibitor and N-p-tosyl-l-lysine chloromethyl ketone. Apparent K_m and K_cat of trypsin A and B were 0.22–0.31 mM and 69.5–82.5 S⁻¹, respectively. The N-terminal amino acid sequences of the first 20 amino acids of trypsin A and B were IVGGYECQAHSQPPQVSLNA and IVGGYECQAHSQPPQVSLNS, respectively.     

Characterisation of trypsin purified from the viscera of Tunisian barbel (Barbus callensis) and its application for recovery of carotenoproteins from shrimp wastes

Trypsin was purified from the viscera of barbel by precipitation using ammonium sulphate (0-80%), Sephadex G-100, and Mono Q-Sepharose ion exchange chromatography. The trypsin was purified 27-fold, with 79 U/mg specific activity and 31% recovery. The enzyme had a molecular weight of 24 kDa; purified trypsin appeared as a single band on native-PAGE. The optimum pH and temperature for enzyme activity were pH 10.0 and 55 °C with BAPNA used as a substrate. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECTPYSQ. The Michaelis-Menten constant (K_m) and catalytic constant (k_cat) values of the enzyme were 0.018 mM and 1.21 s⁻¹, respectively. The study also investigated the effects of purified trypsin on the recovery of carotenoproteins from shrimp (Parapenaeus longirostris) shells through hydrolysis using 1.0 U barbel trypsin/g shrimp shells for 1 h at 30 °C. The freeze-dried carotenoproteins recovered contained 71.09% protein, 16.47% lipid, 7.78% ash, and 1.79% chitin.     

Trypsin from zebra blenny (Salaria basilisca) viscera: Purification,characterisation and potential application as a detergent additive

An alkaline trypsin was purified from the viscera of zebra blenny (Salaria basilisca) by ammonium sulphate (40?80% saturation) precipitation, Sephadex G-100, Mono Q-Sepharose and ultrafiltration. A yield of 12% with a purification-fold of 4.2 was obtained. The trypsin had an apparent molecular weight of 27 kDa. Soybean trypsin inhibitor and phenylmethylsulfonyl fluoride showed a strong inhibitory effect on the purified trypsin. Trypsin had maximal activity at pH 9.5 and 60 °C for the hydrolysis of Nα-benzoyl-dl-arginine-p-nitroanilide (BAPNA). It was stable at low temperatures and in the pH range of 7.0?12.0. The N-terminal amino acid sequence of the first 12 amino acids of the purified protease was IVGGRECTEPSQ. S. basilisca trypsin, which showed high homology with other fish trypsins, had a charged Arg residue at position 5, where Tyr is common in marine vertebrates and mammalian trypsins. The trypsin kinetic constants, K_m and k_cat for BAPNA, were 0.6 mM and 1.38 s^?1, respectively.     

Characteristics of trypsin from the pyloric ceca of walleye pollock (Theragra chalcogramma)

Trypsin was purified from the pyloric ceca of walleye pollock (Theragra chalcogramma) by gel filtration on Sephacryl S-200 and Sephadex G-50. The final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the molecular mass of the enzyme was estimated to be 24 kDa by SDS–PAGE. Trypsin activity was effectively inhibited by serine protease inhibitors, such as soybean trypsin inhibitor and TLCK. Trypsin had maximal activities at around pH 8.0 and 50 °C for the hydrolysis of N^α-p-tosyl-l-arginine methyl ester hydrochloride. Trypsin was unstable above 30 °C and below pH 5.0, and was stabilized by calcium ions. Walleye pollock trypsin was more thermally unstable than trypsin from the Temperate Zone fish and Tropical Zone fish. The N-terminal amino acid sequence of the trypsin, IVGGYECTKHSQAHQVSLNS, was found, and the sequential identity between the walleye pollock trypsin and Frigid Zone fish trypsin was higher (85–100%) than with Temperate Zone fish trypsin (75–90%), Tropical Zone fish trypsin (75–85%), or mammalian trypsin (60–65%).     

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 characterisation of trypsins from the pyloric caeca of mandarin fish (Siniperca chuatsi)

Two trypsins of anionic form (trypsin A) and cationic form (trypsin B) from the pyloric caeca of mandarin fish (Siniperca chuatsi) were highly purified by a series of chromatographies, including DEAE-Sephacel, Sephacryl S-200 HR, Q-Sepharose or SP-Sepharose. Purified trypsins revealed a single band on native-PAGE. The molecular weights of trypsin A and B were 21 kDa and 21.5 kDa, respectively, as estimated by SDS–PAGE, both under reducing and non-reducing conditions. Zymography analysis showed that both trypsins were active in degrading casein. Trypsin A and B exhibited maximal activity at 35 °C and 40 °C, respectively, and shared the same optimal pH of 8.5, using Boc-Phe-Ser-Arg-MCA as substrate. The two trypsins were stable up to 45 °C and in the pH range from 4.5 to 11.0. Trypsin inhibitors are effective on these two enzymes and their susceptibilities were similar. Both trypsins were activated by metal ions such as Ca²⁺ and Mg²⁺ and inactivated by Fe²⁺, Zn²⁺, Mn²⁺, Cu²⁺, Al³⁺, Ba²⁺ and Co²⁺ to different degrees. Apparent K_m values of trypsin A and B were 2.18 μM and 1.88 μM, and K_cat values were 81.6 S⁻¹ and 111.3 S⁻¹ for Boc-Phe-Ser-Arg-MCA, respectively. Immunoblotting analysis using anti-common carp trypsin A positively cross-reacted with the two enzymes, suggesting their similarity. The N-terminal amino acid sequence of trypsin B was determined as IVGGYECEAH, which is highly homologous with trypsins from other species of fish.     

Giant Amazonian fish pirarucu (Arapaima gigas): Its viscera as a source of thermostable trypsin

A trypsin was purified from pyloric caeca of pirarucu (Arapaima gigas). The effect of metal ions and protease inhibitors on its activity and its physicochemical and kinetic properties, as well its N-terminal sequence, were determined. A single band (28.0 kDa) was observed by SDS–PAGE. Optimum pH and temperature were 9.0 and 65 °C, respectively. The enzyme was stable after incubation for 30 min in a wide pH range (6.0–11.5) and at 55 °C. The kinetic parameters K_m, k_cat and k_cat/K_m were 0.47 ± 0.042 mM, 1.33 s⁻¹ and 2.82 s⁻¹ mM⁻¹, respectively, using BApNA as substrate. This activity was shown to be very sensitive to some metal ions, such as Fe²⁺, Hg²⁺, Zn²⁺, Al³⁺, Pb²⁺, and was highly inhibited by trypsin inhibitors. The trypsin N-terminal sequence IVGGYECPRNSVPYQ was found. The features of this alkaline peptidase suggest that it may have potential for industrial applications (e.g. food and detergent industries).     

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.     

Isolation and characterisation of trypsin from sardinelle (Sardinella aurita) viscera

BACKGROUND: In Tunisia, sardinelle (Sardinella aurita) catches totalled about 13 300 t in 2002. During processing, solid wastes including heads and viscera are generated, representing about 30% of the original raw material. Viscera, one of the most important by‐products of the fishing industry, are recognised as a potential source of digestive enzymes, especially proteases with high activity over a wide range of pH and temperature conditions. This paper describes the purification procedure and some biochemical characterisation of trypsin from S. aurita viscera. RESULTS: Trypsin from the viscera of sardinelle (S. aurita) was purified by fractionation with ammonium sulphate, Sephadex G‐75 gel filtration, Sepharose mono Q anion exchange chromatography, ultrafiltration and a second Sephadex G‐75 gel filtration, resulting in a 5.42‐fold increase in specific activity and 6.1% recovery. The molecular weight of the purified enzyme was estimated to be 24 kDa using size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme showed esterase‐specific activity on N‐α‐benzoyl‐L ‐arginine ethyl ester (BAEE) that was four times greater than its amidase‐specific activity on N‐α‐benzoyl‐DL ‐arginine‐p‐nitroanilide (BAPNA). The optimal pH and temperature for enzyme activity were pH 8 and 55 °C respectively using BAEE as a substrate. The trypsin kinetic constants K_m and k_cat on BAPNA were 1.67 mmol L^?1 and 3.87 s^?1 respectively, while the catalytic efficiency k_cat/K_m was 2.31 s^?1 L mmol^?1. CONCLUSION: Trypsin was purified from sardinelle (S. aurita) viscera. Biochemical characterisation of S. aurita trypsin showed that this enzyme can be used as a possible biotechnological tool in the fish‐processing and food industries. Copyright © 2008 Society of Chemical Industry     

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.     

Isolation and properties of 5′-nucleotidase isolated from jumbo squid (Dosidicus gigas) mantle muscle from the Gulf of California,Mexico

The enzyme 5′-nucleotidase of jumbo squid (Dosidicus gigas) mantle was purified and its SDS–PAGE showed a single band of 33 kDa, whereas a protein with a molecular mass of 107 kDa was detected by gel filtration suggesting a homotrimeric nature of this enzyme. Subunits of the named enzyme were not linked by covalent bonds. Isoelectric focusing of this enzyme showed a pI of 3.6–3.8 and presented a hyperbolic kinetics with V_max of 1.16 μM/min/mg of protein, K_m of 1.49 mM, K_cat of 3.48 μM of P_ι s⁻¹ and K_cat/K_m relation of 356.52 ((mol/L)⁻¹ s⁻¹). Purified enzyme preferred AMP as substrate (by 6.7-folds) than IMP, showing a K_m of 6.34 mM, V_max of 0.19 μM/min/mg of protein a K_cat of 0.3388 mol of P_ι s⁻¹ and K_cat/K_m relation of 53.44 ((mol/L)⁻¹ s⁻¹). The low K_m in relation to purified AMP deaminase of the same organism suggested a high contribution of 5′-nucleotidase in AMP degradation in jumbo squid mantle.     

The trypsin inhibitors present in seed of different grain legume species and cultivar

Trypsin inhibitors in a selection of grain legume seeds from different species and cultivars were studied. The results showed that trypsin inhibition content ranged from negligible in Lupinus spp. to very high in Glycine max. Although there is variation among cultivars, generally the highest TIU mg⁻¹ sample values occured in soybean (43–84) and common bean (21–25). Inhibitor content of different Lathyrus cultivars, ranged from 19–30 TIU mg⁻¹ sample. This was higher than the contents in chickpea (15–19 TIU mg⁻¹ sample) and pea (6–15 TIU mg⁻¹ sample). Lentil and faba bean had low values in most cvs (3–8, and 5–10 TIU mg⁻¹ sample, respectively). Trypsin inhibitor isoform analyses showed that the amount of TI detected, varied with legume species and variety.     

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⁺.     

Use of pyloric caeca extract from bigeye snapper (Priacanthus macracanthus) for the production of gelatin hydrolysate with antioxidative activity

Proteolytic activity of pyloric caeca extract (PCE) from bigeye snapper (Priacanthus macracanthus) was studied. The highest activity was observed at 55 °C and pH 8.0 when casein, N^α-Benzoyl-dl-arginine-p-nitroanilide (BAPNA) and N^α-p-Tosyl-l-arginine methyl ester hydrochloride (TAME) were used as the substrates. The activity was inhibited markedly by 1 mg/ml soybean trypsin inhibitor, whereas E-64, pepstatin A and EDTA exhibited a negligible effect on activity. The results suggested that a trypsin-like enzyme was most likely the major proteinase in PCE. As determined by activity staining, two proteolytic activity bands with apparent molecular weights of 55 and 24 kDa were found. Gelatin hydrolysate from bigeye snapper skin prepared using PCE exhibited the increases in 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities and ferric reducing antioxidative power (FRAP) as degrees of hydrolysis (DHs) increased (P < 0.05). Hydrolysates derived from gelatin using Alcalase combined with PCE showed the highest ABTS radical scavenging activity (P < 0.05). Gelatin hydrolysate prepared using Alcalase in combination with Neutrase or PCE at 500 and 1000 ppm, respectively, retarded the oxidation in both linoleic acid oxidation and lecithin liposome oxidation systems. The antioxidative peptide of gelatin hydrolysate had a molecular weight of 1.7 kDa.     

Polyacrylamide–gelatine carrier system used for invertase immobilization

Invertase was immobilized into polyacrylamide–gelatin carrier system by chemical cross-linking with chromium (III) acetate, chromium (III) sulphate, and potassium chromium (III) sulphate. The optimum conditions, namely substrate concentration, temperature, and pH were determined. The effect of polyacrylamide–gelatin ratio and cross-linker concentration on immobilized enzyme activity were analysed. Maximum immobilized enzyme activities were obtained with chromium (III) acetate (0.01 mol dm⁻³), chromium (III) sulphate (0.004 mol dm⁻³) and potassium chromium (III) sulphate (0.001 mol dm⁻³) for 0.177 (w/w) polyacrylamide–gelatin carrier ratio as 79%, 72% and 79%, respectively. The K_m values were 86 and 166 mM for free and immobilized enzyme, respectively. All immobilized samples were used 20 times over a period of 2 months without a considerable loss of activity.     

Identification,quantification and antioxidant activity of acylated flavonol glycosides from sea buckthorn (Hippophae rhamnoides ssp. sinensis)

A novel acylated flavonol glycoside: isorhamnetin (3-O-[(6-O-E-sinapoyl)-β-d-glucopyranosyl-(1 → 2)]-β-d-glucopyranosyl-7-O-α-l-rhamnopyranoside) (1), together with two known acylated flavonol glycosides: quercetin (3-O-[(6-O-E-sinapoyl)-β-d-glucopyranosyl-(1 → 2)]-β-d-glucopyranosyl-7-O-α-l-rhamnopyranoside) (2) and kaempferol (3-O-[(6-O-E-sinapoyl)-β-d-glucopyranosyl-(1 → 2)]-β-d-glucopyranosyl-7-O-α-l-rhamnopyranoside) (3) were isolated from the n-butanol fraction of sea buckthorn (Hippophae rhamnoides ssp. sinensis) berries for the first time by chromatographic methods, and their structures were elucidated using UV, MS, ¹H and ¹³C NMR, and 2D NMR. Compounds 1–3 showed good scavenging activities, with respective IC₅₀ values of 8.91, 4.26 and 30.90 μM toward the 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical; respective Trolox equivalent antioxidant capacities of 2.89, 4.04 and 2.44 μM μM⁻¹ toward 2,2′-azino-bis-3-ethyl-benzothiazoline-6-sulphonate (ABTS) radical. The quantitative analysis of the isolated acylated flavonol glycosides was performed by HPLC–DAD method. The contents of compounds 1–3 were in the range of 12.2–31.4, 4.0–25.3, 7.5–59.7 mg/100 g dried berries and 9.1–34.5, 75.1–182.1, 29.2–113.4 mg/100 g dried leaves, respectively.     

Substrate specificity of a recombinant d-lyxose isomerase from Providencia stuartii for monosaccharides

The specific activity and catalytic efficiency (k_cat/K_m) of the recombinant putative protein from Providencia stuartii was the highest for d-lyxose among the aldose substrates, indicating that it is a d-lyxose isomerase. Gel filtration analysis suggested that the native enzyme is a dimer with a molecular mass of 44 kDa. The maximal activity for d-lyxose isomerization was observed at pH 7.5 and 45 °C in the presence of 1 mM Mn²⁺. The enzyme exhibited high isomerization activity for aldose substrates with the C2 and C3 hydroxyl groups in the left-hand configuration, such as d-lyxose, d-mannose, l-ribose, d-talose, and l-allose (listed in decreasing order of activity). The enzyme exhibited the highest activity for d-xylulose among all pentoses and hexoses. Thus, d-lyxose was produced at 288 g/l from 500 g/l d-xylulose by d-lyxose isomerase at pH 7.5 and 45 °C for 2 h, with a conversion yield of 58 % and a volumetric productivity of 144 g l^− 1 h^− 1. The observed k_cat/K_m (920 mM^− 1 s^− 1) of P. stuartiid-lyxose isomerase for d-xylulose is higher than any of the k_cat/K_m values previously reported for sugar and sugar phosphate isomerases with monosaccharide substrates. These results suggest that the enzyme will be useful as an industrial producer of d-lyxose.