Purification and properties of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase expressed in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH₄)₂SO₄ fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5 and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature stability was up to 45 °C. Metal ions, such as, Mn²⁺ and K⁺ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg²⁺ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu²⁺ and Ag²⁺) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K _m of 120 and 330 μM and V _max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively.     

Optimized expression of an acid xylanase from <Emphasis Type="Italic">Aspergillus usamii</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its biochemical characterization

The xylanase gene xyn II from Aspergillus usamii E001 was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPIC9K and integrated into the genome of a methylotrophic yeast, P. pastoris GS115, by electroporation. His⁺ transformants were screened for on the basis of their resistance to G418 and activity assay. A transformant, P. pastoris GSC12, which showed resistance to over 6 mg G418/ml and highest xylanase activity was selected. Recombinant xylanase was secreted by P. pastoris GSC12 24 h after methanol induction of shake-flask cultures, and reached a final yield of 3139. About 68 U/mg 120 h after the induction. The molecular mass of this xylanase was estimated to be 21 kDa by SDS-PAGE. The optimum pH and temperature were 4.2 and 50 °C, respectively. Xylanase was stable below 50 °C and within pH 3.0–7.0. Its activity was increased by EDTA and Co²⁺ ion and strongly inhibited by Mn²⁺, Li⁺ and Ag⁺ ions. The K _m and V _max values with birchwood xylan as the substrate were found to be 5.56 mg/ml and 216 μmol/mg/min, respectively. This is the first report on expression and characterization of xylanase from A. usamii in P. pastoris. The hydrolysis products consisted of xylooligosaccharides together with a small amount of xylose. This property made the enzyme attractive for industrial purposes, as relatively pure xylooligosaccharides could be obtained.     

A Novel NAD<Superscript>+</Superscript>-dependent aldehyde dehydrogenase encoded by the <Emphasis Type="Italic">puuC</Emphasis> gene of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> DSM 2026 that utilizes 3-hydroxypropionaldehyde as a substrate

3-Hydroxypropionic acid (3-HP), a versatile and valuable platform chemical, has diverse industrial applications; but its biological production from glycerol is often limited by the capability of the enzyme aldehyde dehydrogenase (ALDH) to convert an intermediary compound, 3-hydroxypropionaldehyde (3-HPA), to 3-HP. In this study, we report a new ALDH, PuuC, from Klebsiella pneumoniae DSM 2026, that efficiently converts 3-HPA to 3-HP. The identified gene puuC was cloned, expressed in Escherichia coli, purified, and characterized for its properties. The recombinant enzyme with a molecular weight of 53.8 kDa exhibited broad substrate specificity for various aliphatic aldehydes, especially C₂–C₅ aldehydes. NAD⁺ was the preferred coenzyme for the oxidation of most aliphatic and aromatic aldehydes tested. The optimum pH and temperature for PuuC activity were pH 8.0 and 45°C. The K _m values for 3-HPA and NAD⁺ were 0.48 and 0.09 mM, respectively. The activity of PuuC was enhanced in the presence of reducing agents such as 2-mercaptoethanol or dithiothreitol, while several metal ions, particularly Hg²⁺, Ag⁺, and Cu²⁺ inhibited its activity. The predicted structure of PuuC indicated the presence of K191 and E194 in close proximity to the glycine motif, suggesting that PuuC belongs to class 2 ALDHs.     

Purification and characterization of chitinases from <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">variotii</Emphasis> DG-3 parasitizing on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> eggs

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag⁺ and Hg²⁺ while Chi46 by Hg²⁺ and Pb²⁺ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co²⁺. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) _n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.     

Benzyladenine Promotes Flowering in <Emphasis Type="Italic">Doritaenopsis</Emphasis> and <Emphasis Type="Italic">Phalaenopsis</Emphasis> Orchids

Two experiments were performed to determine how application of the cytokinin benzyladenine (BA) influenced flowering in Doritaenopsis and Phalaenopsis orchid clones. In the first experiment, two vegetative orchid clones growing in 15-cm pots were transferred from a 28°C greenhouse that inhibited flowering to a 23°C greenhouse for flower induction (day 0). A foliar spray (0.2 L m⁻²) containing BA at 100, 200, or 400 mg L⁻¹ or 25, 50, or 100 mg L⁻¹ each of BA and gibberellins A₄ + A₇ (BA+GA) was applied on days 0, 7, and 14. Plants treated with BA alone at 200 or 400 mg L⁻¹ had a visible inflorescence 3–9 days earlier and had a mean of 0.7–3.5 more inflorescences and 3–8 more flowers per plant than nontreated plants. The application of BA+GA had no effect on inflorescence number and total flower number at the rates tested. In the second experiment, three orchid clones received a single foliar spray of BA at 200 mg L⁻¹ at six time points relative to time of transfer from 29°C to 23°C (−1, 0, +1, +2, +4, or +6 weeks). A separate group of plants received a BA application at week 0 but was maintained at 29°C. Inflorescence number was greatest in all three orchid clones when plants were treated with BA 1 week after the temperature transfer. Plants that were sprayed with BA and maintained at 29°C did not initiate inflorescences. The promotion of flowering by the application of BA suggests that cytokinins at least partially regulate inflorescence initiation of Doritaenopsis and Phalaenopsis, but its promotion is conditional and BA application cannot completely substitute for an inductive low temperature.     

Expression,purification and characterization of pectate lyase A from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Pectate lyase A (PelA) of Aspergillus nidulans was successfully expressed in Escherichia coli and effectively purified using a Ni²⁺-nitrilotriacetate-agarose column. Enzyme activity of the recombinant PelA could reach 360 U ml⁻¹ medium. The expressed PelA exhibited its optimum level of activity over the range of pH 7.5–10 at 50°C. Mn²⁺, Ca²⁺, Fe²⁺, Mg²⁺ and Fe³⁺ ions stimulated the pectate lyase activity, but Cu²⁺ and Zn²⁺ inhibited it. The recombinant PelA had a V _max of 77 μmol min⁻¹ mg⁻¹ and an apparent K _m of 0.50 mg ml⁻¹ for polygalacturonic acid. Low-esterified pectin was the optimum substrate for the PelA, whereas higher-esterified pectin was hardly cleaved by it. PelA efficiently macerated mung bean hypocotyls and potato tuber tissues into single cells.     

Purification and Properties of an <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase from <Emphasis Type="Italic">Streptomyces cerradoensis</Emphasis>

An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The K_m and V_max values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg⁻¹ protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity was strongly inhibited by Cu²⁺ and Hg²⁺ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose.     

Characterization of an extracellular alkaline serine protease from marine <Emphasis Type="Italic">Engyodontium album</Emphasis> BTMFS10

An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications. Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated as 28.6 kDa. Isoelectric focusing yielded pI of 3–4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin confirmed the serine protease nature of the enzyme. K _m, V _max, and K _cat of the enzyme were 4.727 × 10⁻² mg/ml, 394.68 U, and 4.2175 × 10⁻² s⁻¹, respectively. Enzyme was noted to be active over a broad range of pH (6–12) and temperature (15–65°C), with maximum activity at pH 11 and 60°C. CaCl₂ (1 mM), starch (1%), and sucrose (1%) imparted thermal stability at 65°C. Hg²⁺, Cu²⁺, Fe³⁺, Zn²⁺, Cd⁺, and Al³⁺ inhibited enzyme activity, while 1 mM Co²⁺ enhanced enzyme activity. Reducing agents enhanced enzyme activity at lower concentrations. The enzyme showed considerable storage stability, and retained its activity in the presence of hydrocarbons, natural oils, surfactants, and most of the organic solvents tested. Results indicate that the marine protease holds potential for use in the detergent industry and for varied applications.     

Purification and some properties of an extracellular acid protease from <Emphasis Type="Italic">Aspergillus</Emphasis><Emphasis Type="Italic">clavatus</Emphasis>

Acid proteases represent an important group of enzymes, widely used in food, beverage and pharmaceutical industries. For most of these applications the enzymatic preparation must be at least partially purified and free of substances that could change the characteristics of the product or the process. Fungal proteases have replaced other sources because they are easily obtained mainly from Mucor, Rhizopus, Penicillium and Aspergillus species. A strain of Aspergillus clavatus was selected by producing high level of acid protease activity. An extracellular aspartatic protease from this strain was purified 37.2 times with 37% recovery using (NH₄)₂SO₄ fractionation and ion-exchange chromatography. The enzyme was found to be monomeric having a molecular mass of 30.4 kDa. The purified enzyme is an acid protease with optimum pH of 5.5 and temperature for optimum activity of 50 °C. Its high pH stability was verified in the range of 3.5–6.5. The acid protease was strongly inhibited by Hg⁺² and partially inhibited by Cu⁺², Zn⁺² and Mn⁺². The enzyme was sensitive to denaturing agent SDS and activated by thiol-containing reducing agent dithiotreitol (DTT). The protease activity was not influenced by iodoacetic acid, E-64 and PMSF, while it was lightly actived by EDTA and totally inhibited by pepstatin, with a K_i of 7.8 μM, indicating that is an aspartic protease. A. clavatus acid protease presents interesting characteristics for biotechnological process, such as cheese and flavor manufacture and dietary supplements, in which activity and stability in acid pH are required.     

Purification and biochemical characterization of thermostable alkaline phosphatases produced by <Emphasis Type="Italic">Rhizopus microsporus</Emphasis> var. <Emphasis Type="Italic">rhizopodiformis</Emphasis>

The biochemical properties of the alkaline phosphatases (AlPs) produced by Rhizopus microsporus are described. High enzymic levels were produced within 1–2 d in agitated cultures with 1 % wheat bran. Intra- and extracellular AlPs were purified 5.0 and 9.3×, respectively, by DEAE-cellulose and ConA-sepharose chromatography. Molar mass of 118 and 120 kDa was estimated by gel filtration for both forms of phosphatases. SDS-PAGE indicated dimeric structures of 57 kDa for both forms. Mn²⁺, Na⁺ and Mg²⁺ stimulated the activity, while Al³⁺ and Zn²⁺ activated only the extracellular form. Optimum temperature and pH for both phosphatases were 65 °C and pH 8.0, respectively. The enzymes were stable at 50 °C for at least 15 min. Hydrolysis of 4-nitrophenyl phosphate exhibited a K _m 0.28 and 0.22 mmol/L, with υ _lim 5.89 and 4.84 U/mg, for intra- and extracellular phosphatases, respectively. The properties of the reported AlPs may be suitable for biotechnological application.     

Expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> of the recombinant <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> metalloprotease and its purification and characterization

The full length empA gene encoding Vibrio anguillarum metalloprotease was amplified by PCR and fused to the expression vector pBAD24. The carboxy-terminal 6xHis-tagged recombinant metalloprotein (rEmpA) was expressed from plasmid pBAD-VAP6his in E. coli TOP10 and purified with affinity chromatography using a Ni-NTA column. SDS-PAGE analysis and Western blotting revealed a molecular mass of the mature rEmpA predicted to be 36 kDa. The optimal temperature and pH for the purified rEmpA were 37°C and 8.0, respectively. The enzyme was stable below 30°C and between pH 5.0 and 8.0, respectively. The results show that Ca²⁺, Na⁺ and Mg²⁺ had an activating effect on the enzyme while Zn²⁺ and Cu²⁺ acted as inhibitors of the enzyme. The purified rEmpA was characterized as a zinc metalloprotease as it was inhibited by zinc- and metal-specific inhibitors, such as 1,10-phenanthroline, EDTA and EGTA. The results indicate that some characteristics of EmpA from marine V. anguillarum had been modified after expression and processing in the engineered E. coli. The purified rEmpA showed degradation activity towards various kinds of proteins, indicating its potential role in pathogenesis.     

Characterization of <Emphasis Type="SmallCaps">d</Emphasis>-tagatose-3-epimerase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> that converts <Emphasis Type="SmallCaps">d</Emphasis>-fructose into <Emphasis Type="SmallCaps">d-</Emphasis>psicose

A non-characterized gene, previously proposed as the d-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with d-fructose and decreased for other substrates in the order: d-tagatose, d-psicose, d-ribulose, d-xylulose and d-sorbose. Its activity was maximal at pH 9 and 40°C while being enhanced by Mn²⁺. At pH 9 and 40°C, 118 g d-psicose l⁻¹ was produced from 700 g d-fructose l⁻¹ after 3 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Secretory Expression of Nattokinase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> YF38 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant strain was induced by 0.7 mmol l⁻¹ isopropyl-β-D- thiogalactopyranoside (IPTG) at 20°C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.     

Protective action of reactivating factor of <Emphasis Type="Italic">Luteococcus japonicus</Emphasis> subsp. <Emphasis Type="Italic">casei</Emphasis> toward cells of <Emphasis Type="Italic">Escherichia coli</Emphasis> reparation mutants inactivated with UV-light

Reactivating factor (RF) from Luteococcus japonicus subsp. casei had a protective action on UV-irradiated cells of Escherichia coli AB1157 with a native reparation system and on cells of isogenic reparation mutants of E. coli UvrA⁻, RecA⁻, and PolA⁻: the effect resulted in multifold increase of survivability. Defense action of L. casei exometabolite is not connected with stimulating reparation systems in E. coli, and, probably, it is mediated by involvement of the exometabolite in the mechanism of cell division. RF did not provoke the reactivation of E. coli cells inactivated by UV-light.     

High expression of recombinant <Emphasis Type="Italic">Streptomyces</Emphasis> sp. S38 xylanase in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by codon optimization and analysis of its biochemical properties

In recent years, the biotechnological use of xylanases has grown remarkably. To efficiently produce xylanase for food processing and other industry, a codon-optimized recombinant xylanase gene from Streptomyces sp. S38 was synthesized and extracellularly expressed in Pichia pastoris under the control of AOX1 promoter. SDS-PAGE and activity assay demonstrated that the molecular mass of the recombinant xylanase was estimated to be 25 kDa, the optimum pH and optimum temperature were 5.5 and 50°C, respectively. In shake flask culture, the specific activity of the xylanase activity was 5098.28 U/mg. The K _m and V _max values of recombinant xylanase were 11.0 mg/ml and 10000 μmol min⁻¹ mg⁻¹, respectively. In the presence of metal ions such as Ca²⁺, Cu²⁺, Cr³⁺ and K⁺, the activity of the enzyme increased. However, strong inhibition of the enzyme activity was observed in the presence of Hg²⁺. This is the first report on the expression properties of a recombinant xylanase gene from the Streptomyces sp. S38 using Pichia pastoris. The attractive biochemical properties of the recombinant xylanase suggest that it may be a useful candidate for variety of commercial applications.     

Quantitative Structure-Activity Relationships for the Pre-Steady State of <Emphasis Type="Italic">Pseudomonas Species</Emphasis> Lipase Inhibitions by <Emphasis Type="Italic">p</Emphasis>-Nirophenyl-<Emphasis Type="Italic">N</Emphasis>-Substituted Carbamates

The pre-steady states of Pseudomonas species lipase inhibitions by p-nitrophenyl-N-substituted carbamates (1–6) are composed of two steps: (1) formation of the non-covalent enzyme–inhibitor complex (E:I) from the inhibitor and the enzyme and (2) formation of the tetrahedral enzyme–inhibitor adduct (E–I) from the E:I complex. From a stopped-flow apparatus, the dissociation constant for the E:I complex, K_S, and the rate constant for formation of the tetrahedral E–I adduct from the E:I complex, k₂ are obtained from the non-linear least-squares of curve fittings of first-order rate constant (k_obs) versus inhibition concentration ([I]) plot against k_obs=k₂+k₂[I]/(K_S+[I]). Values of pK_S, and log k₂ are linearly correlated with the σ^* values with the ρ^* values of −2.0 and 0.36, respectively. Therefore, the E:I complexes are more positive charges than the inhibitors due to the ρ^* value of −2.0. The tetrahedral E–I adducts on the other hand are more negative charges than the E:I complexes due to the ρ^* value of 0.36. Formation of the E:I complex from the inhibitor and the enzyme are further divided into two steps: (1) the pre-equilibrium protonation of the inhibitor and (2) formation of the E:I complex from the protonated inhibitor and the enzyme.     

Purification and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-tagatose

l-arabinose isomerase (EC5.3.1.4. AI) mediates the isomerization of d-galactose into d-tagatose as well as the conversion of l-arabinose into l-ribulose. The AI from Lactobacillus plantarum SK-2 was purified to an apparent homogeneity giving a single band on SDS–PAGE with a molecular mass of 59.6 kDa. Optimum activity was observed at 50°C and pH 7.0. The enzyme was stable at 50°C for 2 h and held between pH 4.5 and 8.5 for 1 h. AI activity was stimulated by Mn²⁺, Fe³⁺, Fe²⁺, Ca²⁺ and inhibited by Cu²⁺, Ag⁺, Hg²⁺, Pb²⁺. d-galactose and l-arabinose as substrates were isomerized with high activity. l-arabitol was the strongest competitive inhibitor of AI. The apparent Michaelis–Menten constant (K _m), for galactose, was 119 mM. The first ten N-terminal amino acids of the enzyme were determined as MLSVPDYEFW, which is identical to L. plantarum (Q88S84). Using the purified AI, 390 mg tagatose could be converted from 1,000 mg galactose in 96 h, and this production corresponds to a 39% equilibrium.