Alterations in Tyrosine Hydroxylase and Monoamine Oxidase Activity in Blood Vessels

THE enzyme tyrosine hydroxylase¹ (TH), which has been reported as the rate limiting step in noradrenaline biosynthesis, can be modified by nerve stimulation, cold^2,3, exercise⁴, reser-pine, phenoxybenzamine and monoamine oxidase inhibitors^5–7. These treatments affect not only the enzyme in vitro but also catecholamine synthesis in vivo. Much of this information has come from studies with heart, brain, adrenals and spleen, but we found that blood vessels contain appreciable concentrations of noradrenaline⁸ and synthesize it in vivo from its precursor tyrosine. We now report that blood vessels have higher tyrosine hydroxylase activity than the heart and that this activity can be modified by reserpine and L-dihydroxyphenylalanine (L-dopa). Furthermore, the activity of tyrosine hydroxylase in the blood vessels of a spontaneously hypertensive rat differs from that in its normotensive control. We also found that the activity of the enzyme monoamine oxidase in the vasculature was affected by drugs and changes in blood pressure.     

Gene <Emphasis Type="Italic">dilp6</Emphasis> regulates octopamine metabolism in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The effect of strong hypomorphic mutation of the insulin-like protein gene (dilp6) on metabolism of octopamine (one of the main biogenic amines in insects) was studied in Drosophila melanogaster males and females. The activity of tyrosine decarboxylase (the key enzyme of octopamine synthesis) and the activity of octopamine-dependent N-acetyltransferase (the enzyme of its degradation) were measured. It was demonstrated that the activity of both studied enzymes is decreased under normal conditions in the dilp6⁴¹ mutants (as we previously demonstrated, this is correlated with an increased level of octopamine). It was also found that hypomorphic mutation of the dilp6 gene decreases the intensity of tyrosine decarboxylase response to heat stress. Thus, it was demonstrated for the first time that insulin-like DILP6 protein in drosophila influences the level of octopamine (regulating the activity of the enzyme degrading octopamine).     

Daily activity rhythm of desert omni-seasonal Tenebrionid beetles, <Emphasis Type="Italic">Trigonoscelis gigas</Emphasis> and <Emphasis Type="Italic">T. sublaevicollis</Emphasis> (Coleoptera,Tenebrionidae) in constant darkness and under alternating 1-hour pulses of light and darkness

In the Karakum Desert (Turkmenistan) the beetles Trigonoscelis gigas are only active in the morning and evening while T. sublaevicollis are strictly nocturnal, regardless of the season and weather. The daily activity rhythm of T. gigas and T. sublaevicollis was studied in the laboratory according to the following pattern: 5 days under a light-darkness cycle of 15: 9 h (LD 15: 9), then 10 days in constant darkness (DD), and then 10 more days under alternating 1-h pulses of light and darkness (LD 1: 1). The temperature was 25°C in all the modes. At LD 15: 9, beetles of both species maintained a 24-h period and a natural pattern of the activity rhythm. In DD, the circadian rhythm ran with a period of 23.5 ± 0.3 h (n = 40) in T. gigas and 23.6 ± 0.4 h (n = 40) in T. sublaevicollis. At DD, the morning and evening activity peaks of T. gigas merged to form a rhythm with only one peak. Under LD 1: 1, both T. gigas and T. sublaevicollis recovered a 24-h period of the rhythm, while the rhythm of T. gigas regained the two-peak structure. Our research confirmed the assumption of Tshernyshev (1980) about the 24-h period of the free-running endogenous rhythm and the distorting effect of constant conditions on this rhythm.     

Possible role of a peroxidative system in melanin synthesis inVerticillium

Manometric tests demonstrated phenolase activity in potato and mushroom extracts but little in extracts from a microsclerotial isolate ofVerticillium albo-atrum. The purpurogallin test indicated the presence of peroxidase activity in theseVerticillium extracts. An assay for an enzyme system which produced dark pigment from catechol was developed. Mn⁺⁺ stimulated pigment synthesis about twice as much as Mg⁺⁺ or Ca⁺⁺. Other cations, Co⁺⁺, Ni⁺⁺, Zn⁺⁺, Cu⁺⁺ and Fe⁺⁺ had less effect. The cell-free enzyme system containing H₂O₂ and Mn⁺⁺ produced dark-colored products from catechol, dopa, andp-phenylenediamine. Pyrogallol yielded a bright yellow color. Chemicals which did not yield colored products as a result of enzyme action included aniline, ascorbic acid, chlorogenic acid,p-cresol, gallic acid, hydroquinone, phenol, phenylalanine, protocatechuic acid, resorcinol, shikimic acid, and tyrosine. In view of these results and the failure of others to demonstrate more than weak phenolase activity inVerticillium, we conclude that a peroxidation probably initiates most melanin synthesis inVerticillium.     

Molecular mechanisms of action of natural amino acids and serotonin on infusorian adenylyl cyclase and guanylyl cyclase

Earlier, it has been shown that some amino acids and their derivatives are able to regulate activities of adenylyl cyclase (AC) and guanylyl cyclase (GC) in free-living infusoria Dileptus anser and Tetrahymena pyriformis. The goal of this work consisted in studying the molecular mechanisms of action of methionine, tyrosine, alanine, and neurohormone serotonin on the activity of enzyme-cyclases and in identification of their specific receptors in D. anser and T. pyriformis. Methionine and serotonin significantly increased the basal AC activity in both infusoria; the effect of serotonin on AC in T. pyriformis took place with participation of the Ca²⁺-dependent form of AC and of the heterotrimeric G-proteins. The AC-stimulating effect of tyrosine and alanine was expressed weakly and was revealed only in D. anser. Serotonin in both infusoria and alanine in D. anser stimulated GC activity, whereas methionine and tyrosine did not affect GC. Methionine and serotonin were bound with a high affinity to the surface receptors of infusoria. The K_D for [methyl-³H]methionine binding to D. anser and T. pyriformis were equal to 7.5 and 35.6 nM, and for [³H]serotonin binding, they were 2.7 and 4.7 nM, respectively. Alanine and tyrosine were bound to infusoria with low affinity. Thus, in the infusoria D. anser and T. pyriformis, there are chemosignal systems regulated by amino acids and their derivatives, including enzymes with cyclase activity. These systems are suggested to be similar to the hormonal signal systems of the higher eukaryotes and to be their predecessors.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

Regulation of Multimeric Structure of NO-synthase as a New Factor of Organogenesis

The effect of NO on organogenesis in Drosophila is discussed. A new model of regulation of the activity of NO-producing enzyme, NO synthase is described, which takes into account endogenous synthesis of its reduced isoform. The reduced isoform of NO synthase is capable of suppressing the enzymatic activity of full-sized NO synthase during formation of a heterodimer in vivo and in vitro. The reduced form of this enzyme inhibits the antiproliferative effect of the full-sized NO synthase isoform during formation of eye structure in Drosophila by affecting the pathways of cell cycle regulation. The reduced form of NO synthase is an endogenous dominant-negative factor of regulation of the NO synthase activity in development of Drosophila.     

Chromosome Replication in Toluenized <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Cells

BIOCHEMICAL studies of chromosome replication have been hampered by the unavailability of an adequate in vitro system with the basic features of in vivo DNA replication. The criteria for such a system are: (1) semiconservative replication; (2) normal biological activity of newly synthesized DNA; (3) normal advancement of the original replication fork; (4) rate of DNA replication equivalent to in vivo; and (5) expected phenotypic behaviour of temperature-sensitive dna mutants. Systems in Escherichia coli, a membrane-DNA fraction¹, an agar-embedded cell lysate² and toluene-treated cells³ have met two or three of the requirements. Several laboratories have also reported the expected behaviour of ts-dna E. coli mutants in toluenized cells^3–5.     

Effect of light and temperature on the daily rhythm of adult eclosion in the blood-sucking mosquitoes <Emphasis Type="Italic">Aedes communis</Emphasis> De Geer and <Emphasis Type="Italic">Culex pipiens</Emphasis> L. (Diptera,Culicidae)

A comparative study on the effect of light and temperature on the daily rhythm of adult eclosion was carried out on the blood-sucking mosquitoes Aedes (Ochlerotatus) communis De Geer and Culex pipiens pipiens L. Two forms of C. p. pipiens were investigated: the autogenous form molestus occurring in the underground habitats (the urban mosquito), and the anautogenous form pipiens inhabiting mostly the above-ground biotopes. The study included both field observations and laboratory experiments. Under natural conditions (southern Karelia, 62°N) at optimal temperatures A. communis and C. p. p. f. pipiens demonstrated a bimodal eclosion rhythm with morning and evening peaks, whereas in C. p. p. f. molestus only an evening maximum was observed. The fraction of adults eclosing in the middle of the day increased at lower temperatures. In all the mosquitoes studied, the daily eclosion dynamics in the nature did not differ significantly between the variants with different levels of illumination, suggesting that temperature was the main factor regulating the eclosion rhythm. However, experiments with constant temperature showed that light also could influence the timing of eclosion. The responsiveness to light was different in two studied forms of C. p. pipiens. Under a constant temperature and light-dark cycles the rhythm became weak or disappeared completely in the mosquitoes from above-ground biotopes (A. communis and C. p. p. f. pipiens) but persisted in the urban mosquito. The effect of gradual twilight transitions was more prominent than that of switching the light on or off abruptly. The observed differences in the light and temperature dependence of the eclosion rhythm are discussed in relation to the ecological conditions in different habitats.     

Carbenicillin as Inhibitor of β-Lactamase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

CARBENICILLIN was produced as a new, semi-synthetic penicillin with antibacterial activity against Pseudomonas aeruginosa and some other microorganisms¹, but this compound was known to be destroyed by staphylococcal penicillinase². Newsom et al.³ described the substrate profile of a constitutive β-lactamase from one strain of Pseudomonas aeruginosa and reported the hydrolysis of carbenicillin at a rate higher than benzylpenicillin. When compared with the inducible enzyme described by Sabath et al.⁴, it differed both in the substrate profile and the ability to hydrolyse carbenicillin. Lack of activity of the inducible enzyme on carbenicillin was also reported by Garber and Friedman⁵ when studying eight strains of Pseudomonas aeruginosa. Sykes and Richmond⁶ were able to identify three types of β-lactamases among fifty-six strains of Pseudomonas aeruginosa according to induci-bility, substrate profile and activity on carbenicillin. Type I (Sabath et al.⁴) was inducible, highly active on cephaloridine and showed no activity on carbenicillin. Types II (Sykes and Richmond⁷) and III (Newsom et al.³) were constitutive and inactivated carbenicillin at different rates. Only the constitutive enzymes conferred resistance towards carbenicillin. We have investigated the activity on carbenicillin of β-lactamases from strains of Pseudomonas aeruginosa isolated from clinical specimens. Activity on benzylpenicillin and cephaloridine was also studied.     

Stereochemistry of Intercalation: Interaction of Daunomycin with DNA

DAUNOMYCIN^1–3, a glycosidic anthracycline antibiotic from Streptomyces peucetius⁴, is being used in the treatment of acute leukaemia and solid tumours in man^5,6. The biological activity seems to be due to complex formation with the DNA of deoxyribonucleoprotein⁴. In vivo, daunomycin inhibits both RNA and DNA synthesis^7,8 and, in vitro, DNA-dependent RNA polymerase and DNA polymerase^7–9.     

Single point mutations enhance activity of <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase

Objectives

To enhance activity of cis-epoxysuccinate hydrolase from Klebsiella sp. BK-58 for converting cis-epoxysuccinate to tartrate.

Results

By semi-saturation mutagenesis, all the mutants of the six important conserved residues almost completely lost activity. Then random mutation by error-prone PCR and high throughput screening were further performed to screen higher activity enzyme. We obtained a positive mutant F10D after screening 6000 mutations. Saturation mutagenesis on residues Phe10 showed that most of mutants exhibited higher activity than the wild-type, and the highest mutant was F10Q with activity of 812 U mg^?1 (k _cat /K _m , 9.8 ± 0.1 mM^?1 s^?1), which was 230 % higher than that of wild-type enzyme 355 U mg^?1 (k _cat /K _m , 5.3 ± 0.1 mM^?1 s^?1). However, the thermostability of the mutant F10Q slightly decreased.

Conclusions

The catalytic activity of a cis-epoxysuccinate hydrolase was efficient improved by a single mutation F10Q and Phe10 might play an important role in the catalysis.

     

Overexpression and characterization of a novel cold-adapted and salt-tolerant GH1 β-glucosidase from the marine bacterium <Emphasis Type="Italic">Alteromonas</Emphasis> sp. L82

A novel gene (bgl) encoding a cold-adapted β-glucosidase was cloned from the marine bacterium Alteromonas sp. L82. Based on sequence analysis and its putative catalytic conserved region, Bgl belonged to the glycoside hydrolase family 1. Bgl was overexpressed in E. coli and purified by Ni²⁺ affinity chromatography. The purified recombinant β-glucosidase showed maximum activity at temperatures between 25°C to 45°C and over the pH range 6 to 8. The enzyme lost activity quickly after incubation at 40°C. Therefore, recombinant β-glucosidase appears to be a cold-adapted enzyme. The addition of reducing agent doubled its activity and 2 M NaCl did not influence its activity. Recombinant β-glucosidase was also tolerant of 700 mM glucose and some organic solvents. Bgl had a K_m of 0.55 mM, a V_max of 83.6 U/mg, a k_cat of 74.3 s^-1 and k_cat/K_m of 135.1 at 40°C, pH 7 with 4-nitrophenyl-β-D-glucopyranoside as a substrate. These properties indicate Bgl may be an interesting candidate for biotechnological and industrial applications.     

Mutant tRNA<Superscript>His</Superscript> Ineffective in Repression and Lacking Two Pseudouridine Modifications

TRANSFER RNA has been implicated in the regulation of a number of amino-acid biosynthetic operons^1–4. Histidyl-tRNA^His has been shown to be involved in regulation of the histidine operon by analysis of six genes (hisO, hisR, hisS, hisT, hisU, hisW), mutation of which causes derepression of the enzymes of the histidine biosynthetic pathway in Salmonella typhimurium^5–7. A class of derepressed mutants (hisR) has only about 55% as much tRNA^His as the wild type⁴ and in the one example sequenced, contains tRNA^HIS with a structure identical to that of the wild type⁸. Studies of mutants of the gene for histidyl-tRNA synthetase (hisS) indicated that the derepressed phenotype was associated with defects in the charging of tRNA^HISin vitro². The amounts of charged and uncharged tRNA^His present in vivo during physiological derepression of the wild type and in the six classes of regulatory mutants, have been determined⁹. This work has shown that repression of the histidine operon is correlated directly with the concentration of charged histidyl-tRNA^Hisin vivo and not with the ratio of charged to uncharged or the absolute amount of uncharged tRNA^His. The derepression observed in mutants, of hisS (the gene for histidyl-tRNA synthetase), hisR (the presumed structural gene for the single species of tRNA^His) and hisU and hisW (genes presumably involved in tRNA modification) may be explained by the lower cellular concentration of charged tRNA^His which these mutants contain.     

Antirestriction and antimodification activities of T7 Ocr: Effects of amino acid substitutions in the interface

The T7 antirestriction protein Ocr, encoded by 0.3 (ocr), specifically inhibits ATP-dependent type I restriction-modification systems. T7 0.3 (ocr) was cloned in pUC18. Ocr inhibited both restriction and modification activities of the type I restriction-modification system (EcoKI) in Escherichia coli K12. The Ocr F53D A57E mutant was obtained and proved to inhibit only restriction activity of EcoKI. The 0.3 (ocr) and Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the P _ltetO-1 promoter, strongly controlled by the TetR repressor. The bioluminescence intensity and luciferase content varied up to 5000-fold in E. coli K12 MG1655Z1 tetR⁺ (pZE21-luxCDABE) cells, depending on the environmental concentration of the inductor anhydrotetracycline. The antirestriction activity of Ocr and Ocr F53D A57E was studied as a function of their concentration in the cell. The dissociation constant K _d, characterizing the binding with EcoKI, differed 1000-fold between Ocr and Ocr F53D A57E (10^?10 M versus 10^?7 M).     

Two distinct <Emphasis Type="Italic">waxy</Emphasis> alleles impact the granule-bound starch synthase in sorghum

The granule-bound starch synthase (GBSS) is the enzyme responsible for amylose synthesis in starch granules. Loss of GBSS activity results in starch granules containing mostly amylopectin and little or no amylose, a phenotype described as waxy. Previously, two phenotypic classes of waxy alleles were identified in sorghum (Sorghum bicolor L. Moench) characterized by the absence (waxy ^a ; wx ^a ) or presence (waxy ^b ; wx ^b ) of the GBSS protein in the endosperm. To characterize these alleles, we examined endosperm architecture using scanning electron microscopy (SEM), assayed GBSS enzymatic activities, and identified DNA lesions associated with the mutations in the GBSS (Sb10g002140) gene. wx ^a , the allele present in B Tx630 and R Tx2907, contained a large insertion in the third exon, which was consistent with the absence of the GBSS protein previously observed. wx ^b , the allele present in B 9307 and B TxARG1, contained a missense mutation that resulted in conversion of glutamine 268 to histidine in a conserved domain in starch synthases. In wx ^b , GBSS activity was less than 25% that of the non-waxy line B Wheatland, and GBSS activity was not detected in wx ^a . SEM showed that endosperm architecture was very similar in both wx ^a and wx ^b alleles, but altered in comparison to non-waxy lines R Tx430 and B Wheatland. Both alleles may have a range of potential applications in grain sorghum because of low amylose content in their starch and the presence or absence of the GBSS protein. PCR based markers were developed for both the wx ^a and the wx ^b alleles to aid in molecular breeding of low amylose sorghum.     

Over-expression of tobacco <Emphasis Type="Italic">UBC1</Emphasis> encoding a ubiquitin-conjugating enzyme increases cadmium tolerance by activating the 20S/26S proteasome and by decreasing Cd accumulation and oxidative stress in tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis>)

Ubiquitin (Ub)-conjugating enzyme (UBC, E2) receives Ub from Ub-activating enzyme (E1) and transfers it to target proteins, thereby playing a key role in Ub/26S proteasome-dependent proteolysis. UBC has been reported to be involved in tolerating abiotic stress in plants, including drought, salt, osmotic and water stresses. To isolate the genes involved in Cd tolerance, we transformed WT (wild-type) yeast Y800 with a tobacco cDNA expression library and isolated a tobacco cDNA, NtUBC1 (Ub-conjugating enzyme), that enhances cadmium tolerance. When NtUBC1 was over-expressed in tobacco, cadmium tolerance was enhanced, but the Cd level was decreased. Interestingly, 20S proteasome activity was increased and ubiquitinated protein levels were diminished in response to cadmium in NtUBC1 tobacco. By contrast, proteasome activity was decreased and ubiquitinated protein levels were slightly enhanced by Cd treatment in control tobacco, which is sensitive to Cd. Moreover, the oxidative stress level was induced to a lesser extent by Cd in NtUBC1 tobacco compared with control plants, which is ascribed to the higher activity of antioxidant enzymes in NtUBC1 tobacco. In addition, NtUBC1 tobacco displayed a reduced accumulation of Cd compared with the control, likely due to the higher expression of CAX3 (Ca²⁺/H⁺ exchanger) and the lower expression of IRT1 (iron-responsive transporter 1) and HMA-A and -B (heavy metal ATPase). In contrast, atubc1 and atubc1atubc2 Arabidopsis exhibited lower Cd tolerance and proteasome activity than WT. In conclusion, NtUBC1 expression promotes cadmium tolerance likely by removing cadmium-damaged proteins via Ub/26S proteasome-dependent proteolysis or the Ub-independent 20S proteasome and by diminishing oxidative stress through the activation of antioxidant enzymes and decreasing Cd accumulation due to higher CAX3 and lower IRT1 and HMA-A/B expression in response to 50 µM Cd challenge for 3 weeks.     

Effect of cold adaptation of α-and β-adrenergic responses of blood pressure in hindlimb and small intestine in situ and systemic blood pressure in rabbits

Changes in the main parameters of α-and β-adrenergic responses, sensitivity to agonists (EC ₅₀) and maximum response (P _m) of hindlimb and small intestinal blood pressure in situ and systemic blood pressure were studied in rabbits adapted to cold for 1–30 days (daily exposures to ?10°C for 6 h). The responses to phenylephrine, noradrenaline, adrenaline, clonidine (α-agonists), and isopropylnoradrenaline (β-agonist) corresponded to the equation p = (P _m A ⁿ )/(EC ₅₀ ⁿ + A ⁿ ) (1) with n = 1 and n = 2, respectively. Cold adaptation induced reciprocal changes in the response of both EC ₅₀ and P _m to α-agonists and in the response of P _m alone to isopropylnoradrenaline. The significant differences of the parameters from control observed during the first 5 days of adaptation gradually decreased by day 30. After 10 days of adaptation, the efficiency (E = P _m/2EC ₅₀) of response to α-and β-agonists of adrenoceptors significantly increased.     

<Emphasis Type="Italic">Synechocystis</Emphasis> PCC 6803 cells heterologously expressing bacterial tyrosine ammonia lyase can use exogenous tyrosine for <Emphasis Type="Italic">p</Emphasis>-coumaric acid production

Phototrophic cyanobacteria may be considered as an alternative host for producing numerous bioactive compounds. We demonstrate that the Synechocystis PCC 6803 expressing tyrosine ammonia-lyase from Rhodobacter sphaeroides under Ptrc1O promoter produce p-coumaric acid at a rate three times higher than that under Ptrc1Ocore promoter, accounting for 18.4 ± 1.5 μg of p-coumaric acid per 10⁸ cells (0.36 mg L^?1). Additionally, our study is the first report to show the biotransformation of tyrosine to p-coumaric acid reaching a maximum 2.4-fold increase when 0.5 mM tyrosine was supplemented to the growth medium. Liquid chromatography-mass spectrometry analysis revealed the occurrence of diverse patterns of metabolites under different concentrations of supplemented tyrosine, suggesting that it is used in additional metabolic pathways.     

Deciphering the mode of action,structural and biochemical analysis of heparinase II/III (<Emphasis Type="Italic">Ps</Emphasis>PL12a) a new member of family 12 polysaccharide lyase from <Emphasis Type="Italic">Pseudopedobacter saltans</Emphasis>

Heparinases are widely used for production of clinically and therapeutically important bioactive oligosaccharides and in analyzing the polydisperse, heterogeneous, and complex structures of heparin/heparan sulfate. In the present study, the gene (1911 bp) encoding heparinase II/III of family 12 polysaccharide lyase (PsPL12a) from Pseudopedobacter saltans was cloned, expressed, and biochemically and functionally characterized. The purified enzyme PsPL12a of molecular size approximately 76 kDa exhibited maximum activity in the temperature range 45–50 °C and at pH 6.0. PsPL12a gave maximum activity at 1% (w/v) heparin under optimum conditions. The kinetic parameters, K _m and V_max, for PsPL12a were 4.6?±?0.5 mg/ml and 70?±?2 U/mg, respectively. Ten millimolars of each Mg²⁺ and Mn²⁺ ions enhanced PsPL12a activity by 80%, whereas Ni²⁺ inhibited by 75% and Co²⁺ by 10%, and EDTA completely inactivated the enzyme. Protein melting curve of PsPL12a gave a single peak at 55 °C and 10 mM Mg²⁺ ions and shifted the peak to 60 °C. The secondary structure analysis of PsPL12a by CD showed 65.12% α-helix, 11.84% β-strand, and 23.04% random coil. The degradation products of heparin by PsPL12a analyzed by ESI-MS spectra displayed peaks corresponding to heparin di-, tetra-, penta-, and hexa-saccharides revealing the endolytic mode of enzyme action. Heparinase II/III (PsPL12a) from P. saltans can be used for production of low molecular weight heparin oligosaccharides for their utilization as anticoagulants. This is the first report on heparinase cloned from P. saltans.