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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.94, #3)

Conversion of sodium lactate to lactic acid with water-splitting electrodialysis by Anna Persson; Arvid Garde; Ann-Sofi Jönsson; Gunnar Jonsson; Guido Zacchi (pp. 197-211).

The conversion of sodium lactate to lactic acid with water-splitting electrodialysis was investigated. One way of reducing the power consumption is to add a conductive layer to the acid compartment. Doing this reduced the power consumption by almost 50% in a two-compartment cell, whereas the electric current efficiency was not affected at all. Three different solutions were treated in the electrodialysis unit: a model solution with 70 g/L of sodium lactate and a fermentation broth that had been prefiltered two different ways. The fermentation broth was either filtered in an open ultrafiltration membrane (cut-off of 100,000 Dalton) in order to remove the microorganisms or first filtered in the open ultrafiltration membrane and then in an ultrafiltration membrane with a cut-off of 2000 Dalton to remove most of the proteins. The concentration of sodium lactate in the fermentation broth was 70 g/L, as well. Organic molecules present in the broth (peptides and similar organic material) fouled the membranes and, therefore, increased power consumption. Power consumption increased more when permeate from the more open ultrafiltration membrane was treated in the electrodialysis unit than when permeate from the membrane with the lower cut-off was treated, since there was a higher amount of foulants in the former permeate. However, the electrodialysis membranes could be cleaned efficiently with a 0.1 M sodium hydroxide solution.

Keywords: Lactic acid; electrodialysis; bipolar membranes; fermentation broth; wheat

Substrate selectivity of Gluconobacter oxydans for production of 2,5-diketo-d-gluconic acid and synthesis of 2-keto-l-gulonic acid in a multienzyme system by Aiguo Ji; Peiji Gao (pp. 213-223).

Substrate selectivity of Gluconobacter oxydans (ATCC 9937) for 2,5-diketo-d-gluconic acid (2,5-DKG) production was investigated with glucose, gluconic acid, and gluconolactone in different concentrations using a resting-cell system. The results show that gluconic acid was utilized favorably by G. oxydans as substrate to produce 2,5-DKG. The strain was coupled with glucose dehydrogenase (GDH) and 2,5-DKG reductase for synthesis of 2-keto-l-gulonic acid (2-KLG), a direct precursor of l-ascorbic acid, from glucose. NADP and NADPH were regenerated between GDH and 2,5-DKG reductase. The mole yield of 2-KLG of this multienzyme system was 16.8%. There are three advantages for using the resting cells of G. oxydans to connect GDH with 2,5-DKG reductase for production of 2-KLG: gluconate produced by GDH may immediately be transformed into 2,5-DKG so that a series of problems generally caused by the accumulation of gluconate would be avoided; 2,5-DKG is supplied directly and continuously for 2,5-DKG reductase, so it is unnecessary to take special measures to deal with this unstable substrate as it was in Sonoyama’s tandem fermentation process; and NADP(H) was regenerated within the system without any other components or systems.

Keywords: 2-Keto-l-gulonic acid; Gluconobacter oxydans ; glucose dehydrogenase; 2,5-diketo-d-gluconic acid reductase; 2,5-diketo-d-gluconic acid; l-ascorbic acid; glucose; gluconic acid; NADP(H) regeneration

Polysaccharide hydrolases from leaves of Boscia senegalensis by Mamoudou H. Dicko; Marjo J. F. Searle-van Leeuwen; Alfred S. Traore; Riet Hilhorst; Gerrit Beldman (pp. 225-241).

The leaves of Boscia senegalensis are traditionally used in West Africa in cereal protection against pathogens, pharmacologic applications, and food processing. Activities of α-amylase, β-amylase, exo-(1→3, 1→4)-β-d-glucanase, and endo-(1→3)-β-d-glucanase were detected in these leaves. The endo-(1→3)-β-d-glucanase (EC3.2.1.39) was purified 203-fold with 57% yield. The purified enzyme is a nonglycosylated monomeric protein with a molecular mass of 36 kDa and pI≥10.3. Its optimal activity occurred at pH 4.5 and 50°C. Kinetic analysis gave V _max, k cat, and K m values of 659 U/mg, 395 s⁻¹, and 0.42 mg/mL, respectively, for laminarin as substrate. The use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry and high-performance liquid chromatography revealed that the enzyme hydrolyzes not only soluble but also insoluble (1→3)-β-glucan chains in an endo fashion. This property is unusual for endo-acting (1→3)-β-d-glucanase from plants. The involvement of the enzyme in plant defense against pathogenic microorganisms such as fungi is discussed.

Keywords: α-Amylase; β-amylase; β-glucanase; disodium 2,2′-bichinconitate; yeast glucan; high-performance liquid chromatography; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Cloning and expression of thermostable β-glycosidase gene from thermus nonproteolyticus HG102 and characterization of recombinant enzyme by Xiangyuan He; Shuzheng Zhang; Shoujun Yang (pp. 243-255).

The gene coding for β-glycosidase (EC 3.2.1.21) from Thermus nonproteolyticus HG102 was cloned and expressed in Escherichia coli. The gene open reading frame was 1311 bp, and it codes for 437 amino acids. The deduced amino acid sequence of the enzyme showed identity with members of the glycosyl hydrolase family I. The enzyme had high content of Arg and Pro. The recombinant enzyme was purified to homogeneity with heat precipitation, ammonium sulfate precipitation, DEAE-cellulose (DE52) chromatography, and prepared slab polyacrylamide gel electrophoresis. The enzyme was monomeric and had a molecular mass of 48,900 Daltons and a pI of 5.2. The enzyme showed optimum activity at pH 5.6 and 90°C. It catalyzed the hydrolysis of β-d-glucoside, β-d-galactoside, β-d-fucoside, and β-d-mannoside. Lineweaver-Burk plots showed that the k cat/K m ratio for β-d-glucoside and β-d-fucoside was higher than for β-d-mannoside and β-d-galactoside. The enzyme was extremely thermostable, with a half-life of 2.5 h at 90°C, and was stable over a wide range of pH. It also had transglycosidic activity at high temperature.

Keywords: Thermus nonproteolyticus HG102; thermostable β-glycosidase; gene cloning and expression

Optimization of inulinase production by Kluyveromyces marxianus using factorial design by Susana J. Kalil; Rodrigo Suzan; Francisco Maugeri; Maria I. Rodrigues (pp. 257-264).

Factorial design and response surface techniques were used to optimize the culture medium for the production of inulinase by Kluyveromyces marxianus. Sucrose was used as the carbon source instead of inulin. Initially, a fractional factorial design (2^5–1) was used in order to determine the most relevant variables for enzyme production. Five parameters were studied (sucrose, peptone, yeast extract, pH, and K₂HPO₄), and all were shown to be significant. Sucrose concentration and pH had negative effects on inulinase production, whereas peptone, yeast extract, and K₂HPO₄ had positive ones. The pH was shown to be the most significant variable and should be preferentially maintained at 3.5. According to the results from the first factorial design, sucrose, peptone, and yeast extract concentrations were selected to be utilized in a full factorial design. The optimum conditions for a higher enzymatic activity were then determined: 14 g/L of sucrose, 10 g/L of yeast extract, 20 g/L of peptone, 1 g/L of K₂HPO₄. The enzymatic activity in the culture conditions was 127 U/mL, about six times higher than before the optimization.

Keywords: Inulinase; Kluyveromyces marxianus ; optimization; factorial design and response surface analysis

Use of reversible denaturation for adsorptive immobilization of urease by Fereshteh Azari; Saman Hosseinkhani; Mohsen Nemat-Gorgani (pp. 265-277).

Urease was chosen as a model multimeric protein to investigate the utility of reversible denaturation for immobilization to a hydrophobic support. Of the various procedures investigated, acidic denaturation provided the highest degree of immobilization and enzymatic activity with lowering of K m (apparent). Exposure of hydrophobic clusters in the protein molecule induced by the acidic pH environment was confirmed by fluorescence studies using 8-anilino-1-naphtalene-sulfonate as a hydrophobic-reporter probe. The catalytic potential of the enzyme at low pH values was dramatically improved with significant heat and pH stability enhancement on immobilization. Furthermore, the immobilized preparation was used successfully in continuous catalytic transformations. Based on the results presented in this article and a recent report involving a relatively more simple monomeric protein, it is suggested that reversible denaturation may be of general utility for immobilization of proteins, which are not normally adsorbed on hydrophobic supports.

Keywords: Adsorptive immobilization; urease; molten globule; 8-anilino-1-naphthalene-sulfonate; hydrophobic matrix

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.94, #3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.94, #3)