Biochemical properties and potential endogenous substrates of polyphenoloxidase from chufa (Eleocharis tuberosa) corms

Polyphenoloxidase (PPO) was partially purified from chufa corms through ammonium sulphate precipitation and dialysis. Biochemical properties of chufa PPO were analysed using exogenous substrate catechol. Optimal pH and temperature for PPO activity were 5 and 45 °C. Ethylenediaminetetraacetic acid disodium salt and l-cysteine could not inhibit the PPO activity. However, sodium thiosulphate pentahydrate exhibited the strongest inhibiting effect, followed by ascorbic acid and anhydrous sodium sulphite. Except for K⁺, other metal ions such as Zn²⁺, Cu²⁺, Fe³⁺, Ca²⁺, Fe²⁺ and Na⁺ accelerated the enzymatic reaction between catechol and PPO. Kinetic analysis showed that the apparent K_m and V_max values were around 10.77 mM and 82 units/ml min. In addition, (−)-gallocatechin gallate, (−)-epicatechin gallate and (+)-catechin gallate isolated and identified from chufa corms were supposed to be the potential endogenous PPO substrates due to their ortho-diphenolic or pyrogallolic structures. These polyphenols might be catalysed by PPO, resulting in the browning of chufa corms after fresh-cut processing.     

Biochemical characteristics and thermal inhibition kinetics of polyphenol oxidase extracted from Thompson seedless grape

Polyphenol oxidase (PPO) was isolated from Thompson seedless grape (Vitis vinifera ‘Thompson Seedless’), and its biochemical characteristics were studied. The PPO showed activity to catechol and D, L-DOPA, but not towards monophenol l-Tyrosine, diphenols guaiacol and caffeic acid, and triphenols pyrogallic acid and gallic acid. Apparent Michaelis–Menten constant (K _m) and maximum velocity of the reaction (V _max) values were 45.0 ± 0.05 mM and 500.0 ± 15.3 OD_400 nm/min for catechol, and 34.6 ± 0.03 mM and 384.6 ± 11.7 OD_478 nm/min for D, L-DOPA, respectively. The obtained similar specificity values of V _max/K _m ratio of catechol and D, L-DOPA indicated their similar affinity to Thompson seedless PPO. The most effective inhibitor was l-cysteine, followed in decreasing order by ascorbic acid, sodium metabisulfite, EDTA, NaCl, and citric acid. It was discovered that metal ions of Mg²⁺ and Cu²⁺ increased, while Zn²⁺ and K⁺ reduced the PPO activity. Sugars showed inhibition on the PPO activity, with higher effect by sucrose and lower effect by fructose and glucose. Optimum pH and temperature for grape PPO activity were 6.0 and 25 °C with 10 mM catechol as substrate. The enzyme was heat stable between 10 and 25 °C, but showed significant activity loss at temperatures higher than 40 °C and completely inactivation at 70 °C for 10 min. Thermal inactivation of PPO showed a first-order kinetic with an activation energy (E _a) of 146.1 ± 10.8 kJ/mol at pH 6.0.     

Purification and characterisation of polyphenol oxidase from leaves of Cleome gynandra L.

A polyphenol oxidase was purified and characterised from leaves of the common spiderflower. Purification sequentially with ammonium sulphate, dialysis, DEAE-Sepharose ion-exchange chromatography and Sephadex G-75 gel filtration chromatography resulted in 37.8-fold enrichment in the specific activity and 44.3% recovery of the total activity. Purified PPO is a monomeric protein of 52.6 kDa revealed by Coomassie and active staining and Western blot. It was optimally active at pH 8.0 and 60 °C, and stable from pH 3.0 to 9.0 and below 60 °C. It displayed enzymatic activity towards monophenols, diphenols and triphenols, especially towards diphenols, and substrate specificity towards methylated and methoxylated substrates. Its activity was slightly increased by 0.1% SDS, heavily inhibited by Hg²⁺ and Pb²⁺, and completely inhibited by 1.0 mM of ascorbic acid, l-cysteine, β-mercaptoethanol, sodium diethyldithiocarbamate and thiourea, and by 10 mM of dithioerythritol, sodium metabisulphite and sodium sulphite.     

Purification and characterisation of polyphenol oxidase from red Swiss chard (Beta vulgaris subspecies cicla) leaves

We report purification and characterisation of a polyphenol oxidase from red Swiss chard (rcPPO). Our purification procedure resulted in a 39-fold enrichment in specific activity and 17% recovery of total enzyme activity. The purified rcPPO appeared as a monomeric protein of 41 kDa, with a specific conformation conserved in the Cu²⁺ combining region. It was optimally active at pH 7.5 and 45 °C. It had a diphenolase substrate preference towards l-DOPA, catechol and chlorogenic acid, but also exhibited weak monophenolase one toward 4-methoxyphenol and l-tyrosine. We also found that the enzyme was activated by K⁺, Na⁺, SDS and laurouyl sarcosine, but inhibited by divalent cations including Ca²⁺, Cu²⁺. Its activity was completely inhibited by ascorbic acid, cysteine, 1,4-dithiothreitol, β-mercaptoethanol, sodium diethyldithiocarbamate, sodium metabisulphite, sodium sulphite and thiourea. This first report on the purification and characterisation of red Swiss chard PPO provides a basis for understanding and use of this enzyme.     

Polyphenol oxidase activity,phenolic acid composition and browning in cashew apple (Anacardium occidentale, L.) after processing

This study describes the extraction and characterisation of cashew apple polyphenol oxidase (PPO) and the effect of wounding on cashew apple phenolic acid composition, PPO activity and fruit browning. Purification factor was 59 at 95% (NH₄)₂SO₄ saturation. For PPO activity, the optimal substrate was catechol and the optimum pH was 6.5. PPO K_m and V_max values were 18.8 mM and 13.6 U min⁻¹ ml⁻¹, respectively. Ascorbic acid, citric acid, sodium sulphite and sodium metabisulphite decreased PPO activity, while sodium chloride increased PPO activity. Wounding at 2 °C and 27 °C for 24 h increased PPO activity but storage at 40 °C reduced PPO activity. Gallic acid, protocatechuic acid and cinnamic acid (free and conjugate) were identified in cashew apple juice. Cutting and subsequent storage at 40 °C hydrolysed cinnamic acid. 5-Hydroxymethylfurfural content in cashew apple juice increased after injury and storage at higher temperatures, indicating non-enzymatic browning.     

Some properties of polyphenol oxidase from lily

A study of crude polyphenol oxidase (PPO) from lily bulbs was carried out to provide information useful for guiding food processing operations. Optimum pH for the enzyme activity in the presence of catechol, were 4.0 and 7.0 at room temperature(approximately 20 °C) and the enzyme was stable in the pH range from 5.0 to 6.5 at 4 °C for 10 h. Its optimum temperature was 40 °C and the heat inactivation of the enzyme followed first‐order kinetics. Lily PPO possessed a diphenolase activity toward catechol, catechin and gallic acid; catechin was the best substrate for the enzyme considering the V_max/K_m ratio. The most effective enzyme inhibitor was sodium sulphite, although ascorbic acid, l ‐cysteine and thiourea were also effective inhibitors at high concentration. But NaCl and citric acid were poor inhibitors of the enzyme. Data generated by this study might help to better prevent lily bulbs browning.     

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.     

Application of chemical and physical agents in model systems to controlling phenoloxidase enzymes

Several chemical and physical anti-browning agents are studied in different model systems in which caffeic acid (as substrate) and laccase from Trametes versicolor (LAC) and polyphenoloxidase from sunflower seeds (PPO) (as enzymes) are used to emulate the browning reaction. Temperature and low electric current were the tested physical agents, while acetic acid, sodium acetate, sodium chloride and, finally, sodium bisulfite were the chemical agents. Sunflower PPO was observed to be less heat sensitive than LAC that was fully inactivated after 1 and 3 min of exposure to 100 and 80 °C, respectively. Conversely, PPO required more than 3 min at 100 °C to be fully inactivated, and it still showed a significant activity (ca. 17%) after an exposure to 80 °C for 15 min. Both LAC and PPO were found to be active at frozen (−18 °C) and cool (+4 °C) temperature, and their activities were strengthened at 40 and 60 °C. As concerning chemical agents, inhibitory power of acetic acid on LAC was observed to be very weak. In the sodium acetate solution at the concentrations of 0.5, 1.0, 2.0 and 4.0%, LAC residual activity was equal to 81.5, 63.9, 61.1 and 35.2%, respectively. PPO is shown to be more sensitive to the NaCl than LAC and indifferent to the presence of NaHSO₃. A 28% residual activity of LAC was found in the solution with 200 mg L⁻¹ NaHSO₃. Finally, LAC activity was decreased to 72.3, 60.0, 16.7 and 8.4% after a low electric current (LEC) treatment of 30 s and 1, 3 and 6 min, respectively. Conversely, PPO activity was not affected under these conditions.     

Betacyanins in dragon fruit peels: the kinetic models of their degradation under different treatment conditions

In this study, after purification, the content of betacyanins in dragon fruit peels was 9.22 mg g⁻¹ dry sample. The stability of betacyanins under the effect of different concentration of metal cation (K⁺, Na⁺, Cu²⁺, Fe²⁺, Fe³⁺, Mg²⁺, Al³⁺) solutions and sugars (glucose, sucrose, fructose, maltose and lactose) solutions was observed. The results showed that 10% lactose and 0.08 mol L⁻¹ potassium chloride solution increased the stability of betacyanins and could be used as stabiliser agents. The degradation reaction under different temperatures (20, 30, 40, 50, 60, 70, 80 °C), pH (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0) and light conditions (light and dark) of mixed solution (betacyanins and stabiliser), including aqueous pigments solution (APS), lactose pigments solution (LPS) and potassium chloride pigments solution (PCPS), accorded with the first-order reaction kinetics. The t_1/2 values (the hours needed for 50.0% degradation of betacyanins) of LPS even up to 330.0 h at 20 °C and dark condition.     

Characterization of polyphenol oxidase from Uapaca kirkiana fruit

Polyphenol oxidase (PPO), the enzyme responsible for the postharvest spoilage of fruits, was extracted and purified from Uapaca kirkiana peel and pulp by ammonium sulfate precipitation and dialysis. Further purification of peel PPO was carried out by gel filtration chromatography. Optimum pH values were 7 and 8 for peel and pulp PPO, respectively. The optimum temperatures for peel and pulp PPO were 45 and 35 °C, respectively. Inhibition studies of the PPO enzyme were performed using citric acid, sodium azide, sodium metabisulfite and thiourea. The most effective inhibitors were sodium azide and citric acid for both peel and pulp PPO. V_max and K_m values were 13.63 units min^?1 and 4.923 mmol L^?1, respectively, for peel PPO and 14.03 units min^?1 and 5.43 mmol L^?1, respectively, for pulp PPO. Three isoenzymes of Uapaca kirkiana PPO were detected by polyacrylamide gel electrophoresis. One of the isoenzymes could be identified as having a molecular weight of 26 625 Da. Copyright © 2005 Society of Chemical Industry     

Influence of hot‐air treatment,superatmospheric O2 and elevated CO2 on bioactive compounds and storage properties of fresh‐cut pomegranate arils

The impact of heat treatment using hot air (HT 45 °C and 55 °C for 1 h) and two active modified atmosphere packaging (MAP) conditions of high oxygen atmosphere (HOA: 80 kPa O₂, 20 kPa N₂) and high CO₂ atmosphere (HCA: 20 kPa CO₂, 80 kPa N₂), individually or combined, on the antioxidant capacity, polyphenols, vitamin C content, total anthocyanins, polyphenoloxydase (PPO) activity and shelf life of fresh‐cut (FC) pomegranate arils stored for 14 days at 4 °C was studied. The results indicate that HT 45 °C along with HOA inhibited PPO activity and prevented loss of antioxidant capacity, vitamin C and phenolic compounds in arils, in comparison with control and HT 55 °C. All treatments reduced the accumulation of anthocyanins, but HCA‐treated arils lost more anthocyanins besides having worse a* colour parameter values. No significant differences in titrable acidity (TA) and total soluble solids (TSS) were observed between treatments. The combination of HOA and HT 45 °C enhanced the benefits of applying each treatment separately and could be useful to improve and extend postharvest life of pomegranate FC arils.     

Laundry detergent-stable lipase from Pacific white shrimp (Litopenaeus vannamei) hepatopancreas: Effect of extraction media and biochemical characterization

Extraction and biochemical properties of a new lipase from the hepatopancreas of Pacific white shrimp were studied. Recovery of the hepatopancreas powder with 50 mM Tris-HCl, pH 7.0 containing 0.2% (v/v) Brij35 gave a higher recovery of lipase activity than other extractants tested (p < 0.05). The optimal pH and temperature for lipase activity were 8.5 and 60°C, respectively, when p-nitrophenyl palmitate was used as a substrate. The enzyme was stable to heat treatment up to 40°C and over a pH range of 7.0–10.0 for 30–120 min. Lipase activities continuously decreased as the sodium deoxycholate (NaDC) concentration increased, but activities increased as NaCl concentration increased up to 3.0 M. Hydrolytic activity was enhanced by NaN₃, but strongly inhibited by Hg²⁺, Cu²⁺, Al³⁺, and phenylmethanesulfonyl fluoride. The lipase was evaluated as highly stable against surfactants (Tween 20, Tween 80, Triton X-100, and gum arabic). However, the enzyme was unstable against sodium dodecyl sulphate. Stability of the lipase with commercial liquid and solid detergents (Attack^®, Bres^®, Omo^®, and Pao^®) was also investigated. The lipase exhibited substantial stability and compatibility with tested commercial liquid and solid laundry detergents for 30–60 min. The overall properties of the lipase from Pacific white shrimp hepatopancreas, thus leading us to propose that it is an excellent candidate for use as biocatalysts for better detergent formulation.     

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.     

Characterization of polyphenoloxidase (PPO) and total phenolic contents in medlar (Mespilus germanica L.) fruit during ripening and over ripening

Characterization of polyphenoloxidase (PPO) enzyme and determination of total phenolic concentrations during fruit ripening and over ripening in medlar (Mespilus germanica L.) were determined. During ripening, PPO substrate specificity, optimum pH and temperature, optimum enzyme and substrate concentrations were determined. Among the five mono- and di-phenolic substrates examined ((p-hydroxyphenyl) propionic acid, l-3,4-dihydroxyphenylalanine, catechol, 4-methylcatechol and tyrosine), 4-methylcatechol was selected as the best substrate for all ripening stages. A range of pH 3.0–9.0 was also tested and the highest enzyme activity was at pH 7.0 throughout ripening. The optimum temperature for each ripening stage was determined by measuring the enzyme activity at various temperatures over the range of 10–70 °C with 10 °C increments. The optimum temperatures were found to be 30, 20 and 30 °C, respectively, for each ripening stage. Optimum enzyme and substrate concentrations were found to be 0.1 mg/ml and 40 mM, respectively. The V_max and K_m value of the reaction were determined during ripening and found to be 476 U/mg protein and 26 mM at 193 DAFB (days after full bloom) – stage 1, 256 U/mg protein and 12 mM at 207 DAFB – stage 2, 222 U/mg protein and 8 mM at 214 DAFB – stage 3. For all ripening stages sodium metabisulfite markedly inhibited PPO activity. For stage 1 of ripening, Cu²⁺, Hg²⁺ and Al³⁺, for stage 2, Cu²⁺ and Hg²⁺, and for stage 3, Cu²⁺, Hg²⁺, Al³⁺ and Ca²⁺ strongly inhibited diphenolase activity. Accordingly, it can be concluded that as medlar fruit ripen there is no significant changes in the optimum values of polyphenoloxidases, although their kinetic parametres change. As the fruit ripening progressed through ripe to over-ripe, in contrary to polyphenoloxidase activity, there was an apparent gradual decrease in total fruit phenolic concentrations, as determined by using the aqueous solvents and water extractions.     

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     

Polyphenol oxidase inactivation and vitamin C degradation kinetics of Fuji apple quarters by high humidity air impingement blanching

In this study, polyphenol oxidase (PPO) and vitamin C were used as the indicators of enzymes and nutrients to evaluate the apple quality during high humidity air impingement blanching (HHAIB) process. The PPO can be completely inactivated within 7 min at 90–120 °C and can retain relatively more vitamin C in the case of PPO fully inactivation. PPO inactivation followed zero‐order kinetics model at 90 and 100 °C, and followed first‐order fraction model at 110 and 120 °C. Activation energy (E_a) of PPO inactivation was between 11.61 and 13.66 kJ mol^?1 by Arrhenius equation. Vitamin C degradation under all processing temperatures was well described by first‐order model and its E_a value was 26.69 kJ mol^?1. Therefore, the HHAIB process was proved to be an effective pretreatment for Fuji apple quarters to inactivate PPO fast and meanwhile to maintain produce quality.     

Effect of rice bran protein extract on enzymatic browning inhibition in potato puree

Effect of rice bran protein extract (RBPE) on enzymatic browning inhibition in potato puree was studied by colour measurement and polyphenol oxidase (PPO) inhibition. RBPE inhibited browning in potato puree and showed a higher per cent potato PPO inhibition at pH 4.0 and 5.0 than those at pH 3.0, 6.0 and 7.0 (P ≤ 0.05). RBPE heated to 40 and 60 °C had browning inhibition in potato puree and per cent potato PPO inhibition at a similar extent to unheated RBPE (P > 0.05). Browning inhibition and per cent potato PPO inhibition of RBPE were decreased when it was heated to 80 °C (P ≤ 0.05). RBPE inhibited browning in potato puree higher than 5, 10 and 20 mm ascorbic acid and 5 and 10 mm citric acid (P ≤ 0.05). Regarding the kinetics study, RBPE exhibited a mixed‐type inhibition for potato PPO. Therefore, RBPE has a potential to be used as a natural antibrowning agent in the potato industry.     

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     

Investigations on tyrosinase activity in melanin-free ink from Sepia officinalis: potential for food proteins cross-linking

Protein modification via enzymatic cross-linking is an attractive way for altering food structure so as to create products with increased quality and nutritional value. In this study, enzymatic cross-linking of β-casein was performed by tyrosinase activity, from melanin-free ink from Sepia officinalis, which was monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) techniques. The melanin-free ink contains a strong tyrosinase activity with pH 7 and 58?°C as optima of pH and temperature, respectively. Such activity is stimulated by ferrous ions and strongly inhibited by Mn²⁺, EDTA, H₂O₂, arbutin, and p-coumaric acid. We also show that 2 Mercapto-ethanol (14?mM) quickly and completely inactivated sepia tyrosinase. The melanin-free ink exhibits a major protein on SDS–PAGE with an N-terminal sequence matching perfectly with an internal sequence of the sepia peroxidase. The zymogram confirmed the inactive state of this truncated protein and the presence of an active tyrosinase enzyme. Interestingly, this activity was able to cross-link the β-casein protein. The tyrosinase implication in reticulation was demonstrated by the addition of its inhibitors, with 2-mercaptoethanol being the most effective, followed by arbutin, p-coumaric acid, and hydrogen peroxide.     

Characterization of polyphenol oxidase from Napoleon grape

Polyphenol oxidase (PPO) from Napoleon grape was isolated using a two-phase partitioning approach with Triton X-114. The enzyme was purified in a latent form and could be optimally activated by the presence of 0.2% of sodium dodecyl sulphate (SDS) at pH 6.0. In the absence of SDS, the enzyme showed maximum activity at acid pH (3.0). The enzyme was kinetically characterized at pH 3.0 and pH 6.0 in the presence of 0.2% of SDS, using 4-tert-butylcatechol (TBC) as a substrate. The V_m/K_M ratio showed that Napoleon grape PPO presents greater affinity for TBC at acid pH (0.1 min⁻¹) that at pH 6.0 in the presence of SDS (0.02 min⁻¹). The enzyme was highly heat stable, 80% of activity remaining at 70 °C. Selected inhibitors were also studied, tropolone being the most active with a K_i value of 27 μM at acid pH and pH 6.0 in the presence of 0.2% SDS.