Production of thermostable α-galactosidase from thermophilic fungusHumicola sp

The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular -galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu²⁺, Ni²⁺ and Hg²⁺ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml^–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg²⁺ strongly inhibited enzyme activity. TheK _m andV _max forp-nitrophenyl--d-galactopyranoside were 60M and 33.6 mol min^–1 mg^–1, respectively, while for raffinose those values were 10.52 mM and 1.8 mol min^–1 mg^–1, respectively.     

Redirecting Carbon Flux in Torulopsis glabrata from Pyruvate to α-Ketoglutaric Acid by Changing Metabolic Co-factors

The nutrition conditions needed to redirect the carbon flux in Torulopsis glabrata, a pyruvate hyper-production yeast, from pyruvate to α-ketoglutaric acid (KG) were investigated in a stirred fermentor. A minor amount of KG (1.3 gl⁻¹) was produced when NaOH was used to control the pH, while 12 g KG l⁻¹ was produced when CaCO₃ was used instead. When thiamine and biotin were included in the medium, 13 g KG l⁻¹ and 68 g pyruvate l⁻¹ were produced after 48 h when glucose was nearly consumed (approximately 5 gl⁻¹). With fermentation continuing for a further 16 h, the concentration of pyruvate decreased to 31 gl⁻¹, and KG increased to 30 gl⁻¹. KG thus accumulated at the expense of pyruvate consumption. Received 2 June 2005; Revisions requested 30 June 2005 and 1 September 2005; Revisions received 1 September 2005 and 28 October 2005; Accepted 28 October 2005     

The Mode of Action of Endo α-1,4 Polygalactosaminidase from Pseudomonas sp. 881 on Galactosaminooligosaccharides

The mode of action of the endo (α-1,4 polygalactosaminidase from Pseudomonas sp. 881 on galactosaminooligosaccharides (GOSs) was studied. The enzyme could hydrolyze (α-1,4 polygalactosamine to GOSs by the endo-split manner. Tetraose and longer GOSs were hydrolyzed to galactosaminobiose and galactosaminotriose as the final products. Galactosaminomonomer (galactosamine) could not be produced as an enzymatic product. From the dependency of kinetic parameters on the chain lengths of the substrates, it was suggested that the enzyme has 8 subsites. A catalytic site of the enzyme is located between the third and the fourth sites from the non-reducing end, since the main product from GOSs was galactosaminotriose, and galactosaminotetraitol remained in the hydrolyzate of galactosaminoheptaitol digestion. The enzyme showed transglycosylating activity on GOS4.     

Production of Maltohexaose by α-Amylase from Bacillus circulans G-6

An α-amylase which produces maltohexaose as the main product from strach was found in the culture filtrate of Bacillus circulans G-6 which was isolated from soil and identified by the author.

The enzyme was purified by means of ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephadex G-200 column chromatography. The purified enzyme was homogeneous on disc electrophoresis. The optimum pH and temperature of the enzyme were around pH 8.0 and around 60°C, respectively. The enzyme was stable in the range of pH 5–10. Metal ions such as Hg²⁺, Cu²⁺, Zn²⁺, Fe²⁺ and Co²⁺ inhibited the enzyme activity. The molecular weight was about 76,000. The yield of maltohexaose from soluble starch of DE (dextrose equivalent*) 1.8-12.6 was about 30%, and the combined action of the enzyme and pullulanase or isoamylase increased the yield of maltohexaose.     

Characterization of α-maltotetraohydrolase produced by Pseudomonas sp. MS300 isolated from the deepest site of the Mariana Trench

We have isolated a Pseudomonas-like amylase producer, the strain MS300, which displayed a large halo on starch medium, from the deepest site of the Mariana Trench. The strain MS300 produced two major and two minor α-maltotetraohydrolases (G4-amylase). The two major G4-amylases share the same molecular weight of 55 000 but had different pI values, 5.0 and 4.7, respectively. The optimum temperature for activity of both major G4-amylases is 40°C, and the optimum pH is 6.8 for one and 8.9 for the other. MS300 produced more amylase under high hydrostatic pressure than under atmospheric pressure. Strain MS300 may be active in the deep sea at a depth of 10 897 m. Received: December 11, 1997 / Accepted: April 16, 1998     

Monoamine Transamination Catalyzed by ω-Amino Acid; Pyruvate Aminotransferase of Pseudomonas sp. F-126

ω-Amino acid: pyruvate aminotransferase of Pseudomonas sp. F-126 catalyzes stoichiometricalJy a transamination between various amines and pyruvate. Most of alkyl and aromatic monoamines served as an amino donor. The enzyme activity was affected by carbon number of straight-chain alkylmonoamines with a maximum activity at 5-carbon unit, n-amylamine. Michaelis constants for n-butylamine and pyruvate were calculated to be 66.6 mm and 5.5 mm respectively. The enzyme was active in the alkaline range with a maximum at pH 10.5 ~ 11.0, though not any activity was observed at the pH below 8.0. The optimum temperature for the reaction was at 60°C.     

Purification and Some Properties of the Endo α-1,4 Polygalactosaminidase from Pseudomonas sp.

The endo α-1,4 polygalactosaminidase from Pseudomonas sp. 881 was purified from the culture nitrate by ethanol precipitation and sequential column chromatographies on CM-Sephadex C-25, Sephadex G-50 and Phenyl-Sepharose CL-4B. The purified enzyme was electrophoretically homogeneous and its molecular weight and isoelectric point were 31,000 and 6.7, respectively. The optimum pH and temperature for hydrolysis of polygalactosamine were 5.0 and 55°C, respectively. The enzyme was stable up to 45°C for 15min and from pH 4.0 to 7.6 at 37°C for 1 hr.

The Km value was 0.05% α-1,4 polygalactosamine and the V was 0.154μmol reducing sugar (galactosamine)/min/μg protein. This polygalactosaminidase was inhibited by Sn²⁺ , Fe²⁺ , Fe³⁺ , Hg²⁺, Cu²⁺ ions and SDS. The enzyme did not hydrolyze oligo galactosamines (n < tetramer) or N-acetyl-polygalactosamines. It acted only on oligo galactosamine (n > trimer) and polygalactosamine endogeneously so far tested.     

Production of β-galactosidase from thermophilic fungus Rhizomucor sp

A thermostable β-galactosidase was produced extracellularly by a thermophilic Rhizomucor sp, with maximum enzyme activity (0.21 U mg⁻¹) after 4 days under submerged fermentation condition (SmF). Solid state fermentation (SSF) resulted in a nine-fold increase in enzyme activity (2.04 U mg⁻¹). The temperature range for production of the enzyme was 38–55°C with maximum activity at 45°C. The optimum pH and temperature for the partially purified enzyme was 4.5 and 60°C, respectively. The enzyme retained its original activity on incubation at 60°C up to 1 h. Divalent cations like Co²⁺, Mn²⁺, Fe²⁺ and Zn²⁺ had strong inhibitory effects on the enzyme activity. The K _m and V _max for p-nitrophenyl-β- _D-galactopyranoside and o-nitrophenyl-β - _D-galactopyranoside were 0.39 mM, 0.785 mM and 232.1 mmol min⁻¹ mg⁻¹ respectively. The K _m and V _max for the natural substrate lactose were 66.66 μM and 0.20 μ mol min⁻¹ mg⁻¹. Received 10 March 1997/ Accepted in revised form 17 July 1997     

Enzymatic Synthesis of L-Pipecolic Acid by Δ1-Piperideine-2-carboxylate Reductase from Pseudomonas putida

L-Pipecolic acid is a chiral pharmaceutical intermediate. An enzymatic system for the synthesis of L-pipecolic acid from L-lysine by commercial L-lysine α-oxidase from Trichoderma viride and an extract of recombinant Escherichia coli cells coexpressing Δ¹-piperideine-2-carboxylate reductase from Pseudomonas putida and glucose dehydrogenase from Bacillus subtilis is described. A laboratory-scale process provided 27 g/l of L-pipecolic acid in 99.7% e.e.     

Characterization of α-Glucosyltransferase from Pseudomonas mesoacidophila MX-45

α-Glucosyltransferase was purified from Pseudomonas mesoacidophila MX-45. The molecular weight was estimated to be 63,000 by SDS–PAGE, and the isoelectric point was pi 5.4. For enzyme activity based on sucrose decomposition, the optimum pH and the optimum temperature were pH 5.8 and 40°C, respectively. The ranges of stable pH and temperature were pH 5.1–6.7 and below 40°C, respectively. The purified enzyme of MX-45 converted sucrose into trehalulose (1-O-α-d-glucopyranosyl-d-fructose) and isomaltulose (palatinose, 6–O-α-d-glucopyranosyl-d-fructose) simultaneously, and the ratio of trehalulose to isomaltulose increased at lower reaction temperatures. Therefore, optimum conditions for trehalulose production were pH 5.5–6.5 at 20°C. The yield of trehalulose from sucrose (20–40% solution) was 91%. The K_m for sucrose was 19.2 ± 3.3 mm estimated by the Hanes–Woolf plot. Product inhibition was observed, and the product inhibition constant was 0.17 m. Hg²⁺, Fe³⁺, Cu²⁺, Mg²⁺, Ag⁺, Pb²⁺, glucono-1,5-lactone, and Tris(hydroxymethyl)aminomethane inhibited the reaction.     

Diamine Transamination Catalyzed by ω-Amino Acid : Pyruvate Aminotransferase of Pseudomonas sp. F–126

ω-Amino acid: pyruvate aminotransferase of Pseudomonas sp. F–126 catalyzes a transamination between various diamines and pyruvate, an exclusive amino acceptor. Based on a stoichiometric studies it was shown that one of the two amino groups of 1,2-diaminoethane, putrescine and cadaverine transaminated to pyruvate. The transamination between putrescine and pyruvate seemed to proceed by a ping-pong bi bi mechanism. Michaelis constants for putrescine and pyruvate were calculated to be 76.9 and 6.25 mm, respectively.     

Production of N-acetyl-β-d-glucosamine from chitin by Aeromonas sp. GJ-18 crude enzyme

A bacterium, GJ-18, having strong chitinolytic activity was isolated from coastal soil. The isolated strain was identified as Aeromonas sp. by morphological and biochemical properties along with 16S rRNA gene sequence. The crude chitinolytic activity of culture supernatants was maximal on the 5th day of culture. Below 45°C, chitin was effectively hydrolyzed to N-acetyl--d-glucosamine (GlcNAc) by Aeromonas sp. GJ-18 crude enzymes, but hydrolysis decreased markedly above 50°C. The optimum pH for enzyme activity was 5.0. TLC and HPLC analysis revealed that, below 45°C, the major reaction product was GlcNAc with a small amount of (GlcNAc)₂ and (GlcNAc)₃, whereas above 50°C the major product was (GlcNAc)₂. When swollen chitin (100 mg) was incubated with crude enzyme preparations (10 U) at 40°C, chitin was hydrolyzed to 83.0 and 94.9% yield of GlcNAc within 5 and 9 days, respectively.     

Production,Purification, and Characterization of α-Amylase from Solventogenic Clostridium sp. BOH3

A solventogenic strain of Clostridium sp. BOH3 produces extracellular α-amylase (7.15 U/mg protein) in reinforced clostridial medium supplemented with sugarcane bagasse hydrolysate (1 % w/v) and a small amount of starch (0.1 % w/v), which is essential for the expression of α-amylase. In the presence of α-amylase, BOH3 utilizes starch directly without any pretreatment and produces butanol almost equivalent (～90 %) to the production of butanol from glucose. α-Amylase can be purified from culture supernatant by using one-step weak anion exchange chromatography with a yield of 43 %. In peptide fingerprinting analysis, this enzyme shows homology with α-amylase produced by Clostridium acetobutylicum ATCC824. However, the molecular weight is 54 kDa, which is smaller than α-amylase of ATCC824 (84 kDa). This enzyme has optimum temperature at 45–50 °C and optimum pH at 4.5–5.5. Under this condition, the enzyme activity is 91.32 U/mg protein, and its K _m and V _max values are 1.71?±?0.02 mg/ml and 96.13?±?0.15 μmol/min/mg protein, respectively. Activity of this α-amylase can be enhanced (>1.5 times) by addition of Ca²⁺ and Co²⁺ and its activity can be maintained at an acidic pH (pH 3–5) for about 24 h. These unique characteristics suggest that this enzyme can be used for saccharification of starch for production of biofuel in one pot.     

The release of oligosaccharides from glycoproteins by endo-β-N-acetylglucosaminidase of Flavobacterium sp.

Endo-β-N-acetylglucosaminidase, purified to homogenicity from the cultural filtrate of Flavobacterium sp., liberated oligosaccharides from various glycoproteins. The enzyme could liberate the carbohydrate chain from native ovalbumin. The release of oligosaccharides from ribonuclease B, yeast carboxypeptidase and a Ricinus lectin was also observed. These glycoproteins contain neutral oligosaccharides that are attached to the protein through glycosyl asparagine bonds. The treatment of glycoprotein with SDS and boiling was more effective for removal of oligosaccharides by the enzyme. The enzyme hydrolyzed all five heterogeneous ovalbumin glycopeptides, although the rate of hydrolysis decreased as the size of the sugar moiety increased. Removal of the neutral oligosaccharides did not appear to effect the enzymatic properties of the hemagglutination ability of these glycoproteins.