Applied Biochemistry and Biotechnology (v.126, #1)

Methylotrophic yeast Pichia pastoris is convenient for the expression of eukaryotic foreign proteins owing to its potential for posttranslational modifications, protein folding, and facile culturing. In this work, human interleukin (hIL)-2 was successfully produced as a secreted fusion form in recombinant P. pastoris. By employing green fluorescent protein (GFP) as a monitoring fusion partner, clear identification of fusion protein expression and quantification of intracellular hIL-2 were possible even though there was no correlation between culture supernatant fluorescence and secreted hIL-2 owing to high media interference. Importantly, by the addition of casamino acids in basal medium, we were able to enhance threefold amount of secreted hIL-2, which was present both as a fusion and as a clipped fragment.
Keywords: Human interleukin-2; green fluorescent protein; Pichia pastoris ; fusion protein; secretion

7-Aminocephalosporanic acid (7-ACA), the starting material for the production of a number of clinically used semisynthetic cephalosporins, is produced by deacylation of cephalosporin-C. The production of 7-ACA was studied in various modes, at the optimal conditions using free and immobilized whole cells of Pseudomonas diminuta.
Keywords: 7-ACA; CPC acylase; GL-7-ACA; GL-7-ACA acylase; immobilization

Bacterial polyhydroxyalkanoates (PHAs) are perceived to be a suitable alternative to petrochemical plastics because they have similar material properties, are environmentally degradable, and are produced from renewable resources. In this study, the in situ degradation of medium-chain-length PHA (PHAMCL) films in tropical forest and mangrove soils was assessed. The PHAMCL was produced by Pseudomonas putida PGA1 using saponified palm kernel oil (SPKO) as the carbon source. After 112 d of burial, there was 16.7% reduction in gross weight of the films buried in acidic forest soil (FS), 3.0% in the ones buried in alkaline forest soil by the side of a stream (FSst) and 4.5% in those buried in mangrove soil (MS). There was a slight decrease in molecular weight for the films buried in FS but not for the films buried in FSst and in MS. However, no changes were observed for the melting temperature, glass transition temperature, monomer compositions, structure, and functional group analyses of the films from any of the burial sites during the test period. This means that the integral properties of the films were maintained during that period and degradation was by surface erosion. Scanning electron microscopy of the films from the three sites revealed holes on the film surfaces which could be attributed to attack by microorganisms and bigger organisms such as detritivores. For comparison purposes, films of polyhydroxybutyrate (PHB), a short-chain-length PHA, and polyethylene (PE) were buried together with the PHAMCL films in all three sites. The PHB films disintegrated completely in MS and lost 73.5% of their initial weight in FSst, but only 4.6% in FS suggesting that water movement played a major role in breaking up the brittle PHB films. The PE films did not register any weight loss in any of the test sites.
Keywords: Medium-chain-length polyhydroxyalkanoate; degradation; tropical forest soils; mangrove soil

Macrophage-stimulating activity of exo-biopolymer from cultured rice bran with Monascus pilosus by K. W. Yu; Y. S. Kim; K. S. Shin; J. M. Kim; H. J. Suh (35-48).
To find a new use of rice bran, five fungi were examined for the production of exo-biopolymer with macrophage-stimulating activity from rice bran. Among the exo-biopolymers produced from the cultures, Monascus pilosus had the most potent macrophage stimulating activity in a liquid culture rather than in a solid culture. In order to improve the yield of exo-biopolymer with macrophage-stimulating activity, a suitable medium for exo-biopolymer was tested in submerged culture of M. pilosus. The highest amount of exo-biopolymer (13.9 mg/mL) was obtained in a medium containing rice bran as an only carbon source followed by media with additional maltose and sucrose (13.8 and 13.7 mg/mL, respectively). The addition of peptone resulted in the production of high amount of exo-biopolymer (15.1 mg/mL), meanwhile the addition of ammonium chloride resulted in 264.0 µg/mL of glucosamine content. Among eight different kinds of inorganic salts tested, potassium phosphate (0.1%) was the most effective inorganic salt for the mycelial growth and exo-biopolymer production. Therefore the optimal medium composition was as follows (g/L): 20 g of rice bran, 5 g of peptone, and 1 g of KH2PO4. The optimal culture pH and time for mycelial growth and exo-biopolymer production was pH 5.0 and 25°C, respectively. The maximum exo-biopolymer (20.1 mg/mL) was observed at the fourth day of cultivation. Exo-biopolymer, a crude polysaccharide fraction, mainly contained neutral sugar (81.8%) with considerable amounts of uronic acid (18.2%). Component sugar analysis showed that the active fraction consisted mainly of arabinose, galactose, glucose, which was digested from starch of rice bran during cultivation, and uronic acid (molar ratio; 0.8:1.0:0.7:0.8).
Keywords: Macrophage-stimulating activity; Monascus pilosus ; rice bran; submerged culture

Enzymatic kinetic of cellulose hydrolysis by Rui M. F. Bezerra; Albino A. Dias (49-59).
The ethanol effect on the Trichoderma reesei cellulases was studied to quantify and clarify this inhibition type. To determine inhibition parameters of crude cellulase and purified exoglucanase Cel7A, integrated Michaelis-Menten equations were used assuming the presence of two inhibitors: cellobiose as the reaction product and ethanol as a possible bioproduct of cellulose fermentation.It was found that hydrolysis of cellulose by crude enzyme follows a model that considers noncompetitive inhibition by ethanol, whereas Cel7A is very slightly competitively inhibited. Crude cellulase is much more inhibited (K iul=K icl=151.9 mM) than exoglucanase Cel7A (K icl=1.6 × 1015 mM). Also, calculated inhibition constants showed that cellobiose inhibition is more potent than ethanol inhibition both for the crude enzyme as well as exoglucanase Cel7A.
Keywords: Cellulase kinetics; ethanol inhibition; exoglucanase Cel7A; integrated Michaelis-Menten equations; kinetic with two inhibitors

Production of poplar xyloglucan endotransglycosylase using the methylotrophic yeast Pichia pastoris by Monika Bollok; Hongbin Henriksson; Åsa Kallas; Mehmedalija Jahic; Tuula T. Teeri; Sven-Olof Enfors (61-77).
The gene XET16A encoding the enzyme xyloglucan endotransglycosylase (XET) from hybrid aspen (Populus tremula xtremuloides Mich) was transformed into Pichia pastoris GS115 and the enzyme was secreted to the medium. The influence of process conditions on the XET production, activity, and proteolytic degradation were examined. Inactivation of XET occurred in the foam, but could be decreased significantly by using an efficient antifoam. Rich medium (yeast extract plus peptone) was needed for product accumulation, but not for growth. The proteolytic degradation of the enzyme in the medium was substantially decreased by also adding yeast extract and peptone to the glycerol medium before induction with methanol. Decreasing the fermentation pH from 5.0 to 4.0 further reduced the proteolysis. The specific activity was further improved by production at 15°C instead of 22°C. In this way a XET production of 54 mg/L active enzyme could be achieved in the process with a specific activity of 18 Unit/mg protein after a downstream process including centrifugation, micro- and ultrafiltration, and ion exchange chromatography.
Keywords: Pichia pastoris ; xyloglucan endotransglycosylase; foaming; proteolysis