Characterization of a recombinant thermostable xylanase from deep-sea thermophilic Geobacillus sp. MT-1 in East Pacific

A novel xylanase-producing thermophilic strain MT-1 was isolated from a deep-sea hydrothermal field in east Pacific. A xylanase gene encoding 331 amino-acid peptide from this isolate was cloned and expressed in Escherichia coli. The recombinant xylanase exhibited maximum activity at 70°C and had an optimum pH of 7.0. It was active up to 90°C and showed activity over a wide pH ranging from 5.5 to 10.0. The crude xylanase presented similar properties in temperature and pH to those of the recombinant xylanase. The recombinant xylanase was stable in 1 mM of enzyme inhibitors (PMSF, EDTA, 2-ME or DTT) and in 0.1% detergents (Tween 20, Chaps or Triton X-100), whereas, it was strongly inhibited by sodium dodecyl sulfate (SDS) (1 mM). In addition, its catalytic function was stable in the presence of Li⁺, Na⁺ or K⁺. However, it was strongly inhibited by Ni²⁺, Mn²⁺, Co²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺ and Al³⁺ (1 or 0.1 mM). The K _m and V _max of the recombinant xylanase for oat spelt xylan were calculated to be 1.579 mg/ml and 289 μmol/(min • mg), respectively. Our study, therefore, presented a rapid overexpression and purification of xylanase from deep-sea thermophile aimed at improving the enzyme yield for industrial applications and scientific research.     

Improved expression of a soluble single chain antibody fusion protein containing tumor necrosis factor in <Emphasis Type="Italic">Escherichia coli</Emphasis>

An epoxide hydrolase gene of about 0.8 kb was cloned from Rhodococcus opacus ML-0004, and the open reading frame (ORF) sequence predicted a protein of 253 amino acids with a molecular mass of about 28 kDa. An expression plasmid carrying the gene under the control of the tac promotor was introduced into Escherichia coli, and the epoxide hydrolase gene was successfully expressed in the recombinant strains. Some characteristics of purified recombinant epoxide hydrolase were also studied. Epoxide hydrolase showed a high stereospecificity for l(+)-tartaric acid, but not for d(+)-tartaric acid. The epoxide hydrolase activity could be assayed at the pH ranging from 3.5 to 10.0, and its maximum activity was obtained between pH 7.0 and 7.5. The enzyme was sensitive to heat, decreasing slowly between 30°C and 40°C, and significantly at 45°C. The enzyme activity was activated by Ca²⁺ and Fe²⁺, while strongly inhibited by Ag⁺ and Hg⁺, and slightly inhibited by Cu²⁺, Zn²⁺, Ba²⁺, Ni⁺, EDTA–Na₂ and fumarate.     

A thermophilic and acid stable family-10 xylanase from the acidophilic fungus Bispora sp. MEY-1

A complete gene, xyl10C, encoding a thermophilic endo-1,4-β-xylanase (XYL10C), was cloned from the acidophilic fungus Bispora sp. MEY-1 and expressed in Pichia pastoris. XYL10C shares highest nucleotide and amino acid sequence identities of 57.3 and 49.7%, respectively, with a putative xylanase from Aspergillus fumigatus Af293 of glycoside hydrolase family 10. A high expression level in P. pastoris (73,400 U ml⁻¹) was achieved in a 3.7–l fermenter. The purified recombinant XYL10C was thermophilic, exhibiting maximum activity at 85°C, which is higher than that reported from any fungal xylanase. The enzyme was also highly thermostable, exhibiting ~100% of the initial activity after incubation at 80°C for 60 min and >87% of activity at 90°C for 10 min. The half lives of XYL10C at 80 and 85°C were approximately 45 and 3 h, respectively. It had two activity peaks at pH 3.0 and 4.5–5.0 (maximum), respectively, and was very acid stable, retaining more than 80% activity after incubation at pH 1.5−6.0 for 1 h. The enzyme was resistant to Co²⁺, Mn²⁺, Cr³⁺ and Ag⁺. The specific activity of XYL10C for oat spelt xylan was 18,831 U mg⁻¹. It also had wide substrate specificity and produced simple products (65.1% xylose, 25.0% xylobiose and 9.9% xylan polymer) from oat spelt xylan.     

Effects of Ca2+ and Temperature on Potassium Uptake along Roots of Wheat, Rice and Cucumber

The differences in K⁺ uptake of different segments of excised roots of two thermophilic plants (rice, Oryza sativa L. cv. Dunghan Shali and cucumber, Cucumis sativus L. cv. Csemege Fürtös) and a non-thermophilic plant (wheat, Triticum aestivum L. cv. Aurora) were investigated in the presence and absence of Ca²⁺, at 0 and 25°C, using radiotracer K⁺(⁸⁶Rb⁺) technique. The K⁺ uptake exhibited different temperature- and Ca²⁺-dependent distributions along the root axis for the different species studied. In the case of rice and cucumber an extraordinarily large K⁺ uptake occurred in the apical root portion at 0°C if Ca²⁺ was omitted. The presence of Ca²⁺ diminished this anomaly. For wheat normal K⁺ uptake patterns were observed under similar conditions. At 25°C Ca²⁺-stimulated K⁺ uptake may appear in each root segment, depending upon species and composition of the uptake solution. The results indicate that there may be considerable differences in the compositions of the cell walls and membranes of root cells of thermophilic and non-thermophilic plants, and in their ion-exchange properties, especially in the apical region.     

De novo synthesis,constitutive expression of <Emphasis Type="Italic">Aspergillus sulphureus</Emphasis> β-xylanase gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and partial enzymic characterization

The endo-β-1, 4-xylanase gene xynA from Aspergillus sulphureus, encoded a lack-of-signal peptide protein of 184 amino acids, was de novo synthesized by splicing overlap extension polymerase chain reaction according to Pichia pastoris protein’s codon bias. The synthetic DNA, composed of 572 nucleotides, was ligated into the downstream sequence of an α-mating factor in a constitutive expression vector pGAPzαA and electrotransformed into the P. pastoris X-33 strain. The transformed yeast screened by Zeocin was able to constitutively secrete the xylanase in yeast–peptone–dextrose liquid medium. The heterogenous DNA was stabilized in the strain by 20-times passage culture. The recombinant enzyme was expressed with a yield of 120 units/mL under the flask culture at 28°C for 3 days. The enzyme showed optimal activity at 50°C and pH 2.4–3.4. Residual activity of the raw recombinant xylanase was not less than 70% when fermentation broth was directly heated at 80°C for 30 min. However, the dialyzed xylanase supernatant completely lost the catalytic activity after being heated at 60°C for 30 min. The recombinant xylanase showed no obvious activity alteration by being pretreated with Na₂HPO₄-citric acid buffer of pH 2.4 for 2 h. The xylanase also showed resistance to certain metal ions (Na⁺, Mg²⁺, Ca²⁺, K⁺, Ba²⁺, Zn²⁺, Fe²⁺, and Mn²⁺) and EDTA. These biochemical characteristics suggest that the recombinant xylanase has a prospective application in feed industry as an additive.     

Molecular cloning,purification, and characterization of a novel thermostable cinnamoyl esterase from Lactobacillus helveticus KCCM 11223

A gene encoding cinnamoyl esterase (CE), which breaks down chlorogenic acid (ChA) into caffeic and quinic acids, was cloned from Lactobacillus helveticus KCCM 11223. The gene with an open reading frame of 759 nucleotides was expressed in Escherichia coli, which resulted in a 51.6-fold increase in specific activity compared to L. helveticus KCCM 11223. The recombinant CE exists as a monomeric enzyme having a molecular weight of 27.4?kDa. Although the highest activity was observed at pH 7, the enzyme showed stable activity at pH 4.0–10.0. Its optimum temperature was 65°C, and it also possessed a thermophilic activity: the half-life of CE was 24.4?min at 65°C. The half-life of CE was 145.5, 80.5, and 24.4?min at 60, 62, and 65°C, respectively. The K_m and V_max values for ChA were 0.153?mM and 559.6?µM/min, respectively. Moreover, the CE showed the highest substrate specificity with methyl caffeate among other methyl esters of hydroxycinnamic acids such as methyl ferulate, methyl sinapinate, methyl p-coumarate, and methyl caffeate. Ca²⁺, Cu²⁺, and Fe²⁺ significantly reduced the relative activity on ChA up to 70%. This is the first report on a thermostable CE from lactic acid bacteria that can be useful to hydrolyze ChA from plant cell walls.     

Purification and characterization of a thermostable λ-carrageenase from a hot spring bacterium,Bacillus sp.

Purpose of work The purpose of this study is to report a thermostable λ-carrageenase that can degrade λ-carrageenan yielding neo-λ-carrabiose at 75 °C. A thermophilic strain Lc50-1 producing λ-carrageenase was isolated from a hot spring in Indonesia and identified as a Bacillus sp. The λ-carrageenase, Cga-L50, with an apparent molecular weight of 37 kDa and a specific activity of 105 U/mg was purified from the culture supernatant. The optimum pH and temperature of Cga-L50 were 8.0 and 75 °C, respectively. The enzyme was stable from pH 6–9 and retained ~50 % activity after holding at 85 °C for 10 min. Significant activation of Cga-L50 was observed with K⁺, Ca²⁺, Co²⁺, and Na⁺; whereas, the enzyme activity was inhibited by Sr²⁺, Mn²⁺, Fe²⁺, Cu²⁺,Cd²⁺, Mg²⁺, and EDTA. Cga-L50 is an endo-type λ-carrageenase that hydrolyzes β-1,4-linkages of λ-carrageenan, yielding neo-λ-carrabiose as the main product. This study is the first to present evidence of thermostable λ-carrageenase from hot spring bacteria.     

深海嗜热噬菌体GVE2头尾连接蛋白EV8的原核表达和功能分析

为了解头尾连接蛋白在噬菌体感染和装配中起的作用,对高温噬茵体GVE2(virulent bacteriophage of Geobacillus sp.E263)的头尾连接蛋白EV8进行了原核表达和功能鉴定.将EV8编码序列克隆入pGEX4T-2原核表达载体,并转染大肠埃希氏菌BL21.诱导表达后用SDS-PAGE进行鉴定,显示获得相对分子质量约36kD的谷胱甘肽硫转移酶(GST-EV8)融合蛋白.Western blot检测发现EV8在GVE2感染后2h开始表达,说明该蛋白为噬菌体晚期表达蛋白.免疫电镜定位表明头尾连接蛋白位于噬菌体头尾的连接处,为进一步研究高温噬菌体的组装和表达调控奠定了基础.     

The ATPase activity of the <Emphasis Type="Italic">G2alt</Emphasis> gene encoding an aluminium tolerance protein from <Emphasis Type="Italic">Anoxybacillus gonensis</Emphasis> G2

The G2ALT gene was cloned and sequenced from the thermophilic bacterium Anoxybacillus gonensis G2. The gene is 666 bp long and encodes a protein 221 amino acids in length. The gene was overexpressed in E. coli and purified to homogeneity and biochemically characterized. The enzyme has a molecular mass of 24.5 kDa and it could be classified as a member of the family of bacterial aluminium resistance proteins based on homology searches. When this fragment was expressed in E. coli, it endowed E. coli with Al tolerance to 500 μM. The purified G2ALT protein is active at a broad pH range (pH 4.0–10.0) and temperature range (25°C–80°C) with optima of 6.0 and the apparent optimal temperature of 73°C respectively. Under optimal conditions, G2ALT exhibited a low ATPase activity with K _m ⁻ and V _max ⁻ values of 10±0.55 μM and 26.81±0.13 mg Pi released/min/mg enzyme, respectively. The ATPase activity of G2ALT requires Mg²⁺ and Na⁺ ions, while Zn²⁺ and Al³⁺ stimulate the activity. Cd²⁺ and Ag⁺ reduced the activity and Li⁺, Cu²⁺, and Co²⁺ inhibited the activity. Known inhibitors of most ATPases, like such as β-mercaptoethanol and ouabain, also inhibited the activity of the G2ALT. These biochemical characterizations suggested that G2ALT belongs to the PP-loop ATPase superfamily and it can be responsible for aluminium tolerance in A. gonensis G2.     

Constitutive expression,purification and characterization of bovine prochymosin in <Emphasis Type="Italic">Pichia pastoris</Emphasis> GS115

Chymosin can specifically break down the Phe105–Met106 peptide bond of milk κ-casein to form insoluble para-κ-casein, resulting in milk coagulation, a process that is used in making cheese. In this study, in order to obtain an alternative milk coagulant which is safe and efficient, and simultaneously can produce cheese with a good taste, bovine prochymosin B was chosen and constitutively expressed to a high level in Pichia pastoris. The recombinant chymosin was expressed mainly as a secretory form, and it exhibited milk-clotting activity. It was purified by ammonium sulfate fractionation, anion exchange, followed by cation exchange chromatography. A final yield of 24.2% was obtained for the purified enzyme, which appeared as a single band in SDS–PAGE having a molecular mass of approximate 36 kDa. Proteolysis assay showed that it specifically hydrolyzed κ-casein. It was stable at 25–50°C and had optimal activity at 37°C and pH 4.0. The activity of the recombinant chymosin was activated by cations such as Mn²⁺, Fe³⁺, Mg²⁺ and Na⁺, but inhibited by K⁺, Co²⁺, Zn²⁺, Ni²⁺, and to a lesser extent by Cu²⁺. These results suggested that recombinant bovine chymosin is an acid milk coagulant, and it could be considered as a safe and efficient enzyme suitable for use in cheese production.     

Cloning,Expression, Purification,and Characterization of Cold-Adapted α-Amylase from <Emphasis Type="Italic">Pseudoalteromonas arctica</Emphasis> GS230

A cold-adapted α-amylase (ParAmy) gene from Pseudoalteromonas arctica GS230 was cloned, sequenced, and expressed as an N-terminus His-tag fusion protein in E. coli. A recombinant protein was produced and purified with DEAE-sepherose ion exchange chromatography and Ni affinity chromatography. The molecular weight of ParAmy was estimated to be 55 KDa with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). With an optimum temperature for activity 30 °C, ParAmy showed 34.5% of maximum activity at 0 °C and its activity decreased sharply at above 40 °C. ParAmy was stable in the range of pH 7–8.5 at 30 °C for 1 h. ParAmy was activated by Mn²⁺, K⁺ and Na⁺, and inhibited by Hg²⁺, Cu²⁺, and Fe³⁺. N-Bromosuccinimid showed a significant repressive effect on enzyme activity. The K _m and V _max values of the α-amylase for soluble starch were 7.28 mg/mL and 13.07 mg/mL min, respectively. This research suggests that Paramy has a good potential to be a cold-stable and alkalitolerant amylase in detergent industry.     

Expression, purification and characterization of flavin reductase from Citrobacter freundii A1

Flavin reductase plays an important biological role in catalyzing the reduction of flavin by NAD(P)H oxidation. The gene that codes for flavin reductase from Citrobacter freundii A1 was cloned and expressed in Escherichia coli BL21(DE3)pLysS. In this study, we aimed to characterize the purified recombinant flavin reductase of C. freundii A1. The recombinant enzyme was purified to homogeneity and the biochemical profiles, including the effect of pH, temperature, metal ions and anions on flavin reductase activity and stability, were determined. This enzyme exhibited optimum activity at 45 °C in a 10-min reaction at pH 7.5 and was stable at temperatures up to 30 °C. At 0.1 mM concentration of metal ions, flavin reductase activity was stimulated by divalent cations including Mn²⁺, Sr²⁺, Ni²⁺, Sn²⁺, Ba²⁺, Co²⁺, Mg²⁺, Ca²⁺ and Pb²⁺. Ag⁺ was noticeably the strongest inhibitor of recombinant flavin reductase of C. freundii A1. This enzyme should not be defined as a standard flavoprotein. This is the first attempt to characterize flavin reductase of C. freundii origin.     

Purification and Properties of Phage 95-induced Lytic Enzyme of Pseudomonas aeruginosa

A lytic enzyme was isolated from the lysate of Ps. aeruginosa infected with a new strain of bacteriophage, phage 95. The enzyme, LE95, was purified by chromatography in twice on IRC50 column and by gel filtration in twice on Sephadex G–75 column. The molecular weight was estimated as 21,000. The optimal condition for the hydrolysis of acetone-dried cells of Ps. aeruginosa was determined to be following: the optimal pH was between 6.5 and 7.0, the temperature about 70°C and the concentration of phosphate buffer about 5 mm. The enzyme was strongly inhibited by Ag⁺, Hg²⁺, Ni², Fe²⁺ and Cu²⁺ ions. When peptideglycan obtained from Ps. aeruginosa was digested by LE95, free amino groups were liberated without release of reducing sugars. The enzyme was suggested to be amidase or peptidase.     

Gene cloning,expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacterium Psychrobacter sp. C18

A psychrophilic bacterium Psychrobacter sp. C18 previously isolated from the Southern Okinawa Trough deep-sea sediments showed extracellular lipolytic activity towards tributyrin. A genomic DNA library was constructed and screened to obtain the corresponding lipase gene. The sequenced DNA fragment contains an open reading frame of 945 bp, which was denoted as the lipX gene, from which a protein sequence LipX was deduced of 315 amino acid residues with a molecular mass of 35,028 Da. This protein contained the bacterial lipase GNSMG (GxSxG, x represents any amino acid residue) and HG consensus motifs. The recombinant pET28a(+)/lipX gene was overexpressed in heterologous host Escherichia coli BL21 (DE3) cells to overproduce the lipase protein LipX_His with a 6× histidine tag at its C-terminus. Nickel affinity chromatography was used for purification of the expressed recombinant lipase. The maximum lipolytic activity of the purified recombinant lipase was obtained at temperature of 30°C and pH 8.0 with p-nitrophenyl myristate (C14) as a substrate. Thermostability assay indicated that the recombinant LipX_His is a cold-adapted lipase, which was active in 10% methanol, ethanol, acetone and 30% glycol, and inhibited partially by Zn²⁺, Co²⁺, Mn²⁺, Fe³⁺ and EDTA. Most non-ionic detergents, such as DMSO, Triton X-100, Tween 60 and Tween 80 enhanced the lipase activity but 1% SDS completely inhibited the enzyme activity. Additionally, the highest lipolytic rate of the recombinant LipX_His lipase was achieved when p-nitrophenyl myristate was used as a substrate, among all the p-nitrophenyl esters tested.     

Cloning,expression, and characterization of Aureobasidium melanogenum lipase in Pichia pastoris

cDNA of Aureobasidium melanogenum lipase comprises 1254 bp encoding 417 amino acids, whereas genomic DNA of lipase comprises 1311 bp with one intron (57 bp). The lipase gene contains a putative signal peptide encoding 26 amino acids. The A. melanogenum lipase gene was successfully expressed in Pichia pastoris. Recombinant lipase in an inducible expression system showed the highest lipase activity of 3.8 U/mL after six days of 2% v/v methanol induction. The molecular mass of purified recombinant lipase was estimated as 39 kDa using SDS-PAGE. Optimal lipase activity was observed at 35–37 °C and pH 7.0 using p-nitrophenyl laurate as the substrate. Lipase activity was enhanced by Mg²⁺, Mn²⁺, Li⁺, Ca²⁺, Ni²⁺, CHAPS, DTT, and EDTA and inhibited by Hg²⁺, Ag⁺, SDS, Tween 20, and Triton X-100. The addition of 10% v/v acetone, DMSO, p-xylene, and octanol increased lipase activity, whereas that of propanol and butanol strongly inhibited it.     

Purification and Some Properties of a New Levanase from Bacillus sp. No. 71

A levanase from Bacillus sp. was purified to a homogeneous state. The enzyme had a molecular weight of 135,000 and an isoelectric point of pH 4.7. The enzyme was most active at pH 6.0 and 40°C, stable from pH 6.0 to 10.0 for 20 hr of incubation at 4°C and up to 30°C for 30 min of incubation at pH 6.0. The enzyme activity was inhibited by Ag ⁺, Hg^{2 +}, Cu^{2 +}, Fe^{3 +}, Pb²⁺, and p-chloromercuribenzoic acid. The enzyme hydrolyzed levan and phlein endowise to produce levanheptaose as a main product. The limit of hydrolysis of levan and phlein were 71% and 96%, respectively.     

Cloning,purification, and characterization of β-galactosidase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> DSM 13

The gene encoding homodimeric β-galactosidase (lacA) from Bacillus licheniformis DSM 13 was cloned and overexpressed in Escherichia coli, and the resulting recombinant enzyme was characterized in detail. The optimum temperature and pH of the enzyme, for both o-nitrophenyl-β-d-galactoside (oNPG) and lactose hydrolysis, were 50°C and 6.5, respectively. The recombinant enzyme is stable in the range of pH 5 to 9 at 37°C and over a wide range of temperatures (4–42°C) at pH 6.5 for up to 1 month. The K _m values of LacA for lactose and oNPG are 169 and 13.7 mM, respectively, and it is strongly inhibited by the hydrolysis products, i.e., glucose and galactose. The monovalent ions Na⁺ and K⁺ in the concentration range of 1–100 mM as well as the divalent metal cations Mg²⁺, Mn²⁺, and Ca²⁺ at a concentration of 1 mM slightly activate enzyme activity. This enzyme can be beneficial for application in lactose hydrolysis especially at elevated temperatures due to its pronounced temperature stability; however, the transgalactosylation potential of this enzyme for the production of galacto-oligosaccharides (GOS) from lactose was low, with only 12% GOS (w/w) of total sugars obtained when the initial lactose concentration was 200 g/L.     

Purification and Characterisation of an F16L Mutant of a Thermostable Lipase

A mutant of the lipase from Geobacillus sp. strain T1 with a phenylalanine to leucine substitution at position 16 was overexpressed in Escherichia coli strain BL21(De3)pLysS. The crude enzyme was purified by two-step affinity chromatography with a final recovery and specific activity of 47.4 and 6,315.8 U/mg, respectively. The molecular weight of the purified F16L lipase was approximately 43 kDa by 12% SDS-PAGE analysis. The F16L lipase was demonstrated to be a thermophilic enzyme due its optimum temperature at 70 °C and showed stability over a temperature range of 40–60 °C. The enzyme exhibited an optimum pH 7 in phosphate buffer and was relatively stable at an alkaline pH 8–9. Metal ions such as Ca²⁺, Mn²⁺, Na⁺, and K⁺ enhanced the lipase activity, but Mg²⁺, Zn²⁺, and Fe²⁺ inhibited the lipase. All surfactants tested, including Tween 20, 40, 60, 80, Triton X-100, and SDS, significantly inhibited the lipolytic action of the lipase. A high hydrolytic rate was observed on long-chain natural oils and triglycerides, with a notable preference for olive oil (C18:1; natural oil) and triolein (C18:1; triglyceride). The F16L lipase was deduced to be a metalloenzyme because it was strongly inhibited by 5 mM EDTA. Moderate inhibition was observed in the presence of PMSF at a similar concentration, indicating that serine residues are involved in its catalytic action. Further, the activity was not impaired by water-miscible solvents, including methanol, ethanol, and acetone.     

Comparison of the Cathepsin D from Mackerel (Scomber australasicus) and Milkfish (Chanos chanos) Muscle

Muscle proteases from mackerel and milkfish were purified to electrophoretical homogeneity by concanavalin A-Sepharose and Sephadex G-100 chromatographies. Both proteases appear to be an aspartic protease, cathepsin D (EC 3.4.23.5). The molecular weights of the purified cathepsin D’s from mackerel and milkfish were 51,000 and 54,000, estimated by Sephadex G-100, and 59,000 and 61,000 by SDS–PAGE, respectively. Both cathepsin D’s were completely inhibited by pepstatin, but not affected by leupeptin, N-ethylmaleimide, dithiothreitol, or glutathione. ß-Mercaptoethanol, iodoacetic acid, p-chloromercuri-benzoate, phenylmethylsulfonyl fluoride, and sodium dodecyl sulfate partially or completely inhibited both cathepsin D’s. Na⁺ and K⁺ partially activated the cathepsin D from milkfish. Both cathepsin D’s were inhibited by Mg²⁺, Sr²⁺, Fe²⁺, and H²⁺, but activated by Ca²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, and Cd²⁺. The pI and optimal temperature of the cathepsin D’s from mackerel and milkfish were 5.04 and 4.91, 45°, and 50°C, respectively. The temperatures for inactivating 50% activity of the cathepsin D’s from mackerel and milkfish during 20 min of incubation were 53° and 48°C, respectively. Both cathepsin D’s had similar optimal pHs near 3. The activity of that from milkfish markedly decreased when the pH was higher than 4, and was almost completely lost at pH above 6, while that from mackerel still had at least 40% activity at pH 6.