Cloning and heterologous expression of xylanase from Pichia stipitis in Escherichia coli.

AIMS: The main goal of this study was to characterize the xylanase (xynA) gene from Pichia stipitis NRRL Y-11543. METHODS AND RESULTS: The xylanase gene was cloned into pUC19 in Escherichia coli DH5alphaF' and selected by growth on RBB-xylan. All functional clones contained a recombinant plasmid with an insert of 2.4 kbp, as determined by restriction mapping. The nucleotide sequence of the P. stipitis xylanase gene consisted of 1146 bp and encoded a protein of 381 amino acids with a molecular weight of 43 649 Da. The sequence contained a putative 20-amino acid N-terminal signal sequence and four N-linked glycosylation sites. The Km values for non-glycosylated and glycosylated xylanases were 1.4 mg ml-1 and 4.2 mg ml-1, respectively, and Vmax values were 0.8 and 0.082 micromol min-1 mg-1 protein, respectively. CONCLUSION: Xylanase, a rarely found enzyme in yeast species, has been characterized in detail. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study can be used to develop better xylanase-utilizing yeast strains.

Purification and characterization of acetyl esterase from Candida guilliermondii.

An extracellular acetyl esterase (EC 3.1.1.6) from Candida guilliermondii NRRL Y-17257 was purified to homogeneity by acetone precipitation and QAE sepharose anion-exchange chromatography. The enzyme was a monomer with an apparent molecular weight of 67 kDa and a pI of 7.6. It had maximum activity at pH 7.5 and at 50-60 degrees C. It was relatively stable over a pH range of 5.8-8.0 and exhibited thermal stability up to 60 degrees C. The Km and Vmax values on alpha-naphthylacetate were 2.63 mM and 213.3 micromol alpha- naphthol min-1 mg-1 protein, respectively.

Glucosyltransferase activity of commercial juice-processing enzyme preparations.

Of various commercial enzyme preparations examined, Cytolase M102 was found to contain the highest glucosyltransferase activity (55 U ml-1). It rapidly converted maltose to panose (Glc alpha-->6 Glc alpha l-->4 Glc) with a Vmax value of 5.8 mmol l-1 min-1 at 50 degrees C in 0.05 mol l-1 sodium acetate buffer (pH 4.4). The Km value of the enzyme for maltose was 750 mmol l-1. Yields of panose and glucose after 45 min of reaction, for example, were 47.2% and 52.8%, respectively, on the basis of the amount of maltose consumed.

Transfructosylating enzyme activity of Penicillium roquefortii.

The blue cheese mold, Penicillium roquefortii NRRL 844, was found to produce a transfructosylating enzyme that exhibited optimal activity at 50 degrees C in the pH range of 5.0-6.0. The enzyme rapidly catalysed the conversion of sucrose to a syrup containing nearly 60% fructo-oligosaccharides of the total sugars present.

Effect of inulin on yeast inulinase production in sauerkraut brine.

Kluyveromyces marxianus NRRL Y-1196 produced the highest inulinase activity (38 U/mg protein) of six yeasts examined after 24 h growth in sauerkraut brine in shaking flasks at 30°C with 0.3% inulin as an enzyme inducer. The enzyme was recovered by acetone fractionation, with a yield of 81%. It had maximum activity at pH 4.4 and 55°C with K m values for inulin and sucrose of 3.92 mM and 11.9 mM, respectively. The yeast raised the pH from 3.4 to above 7.0, using all the lactic acid in the brine. Growth of K. marxianus in sauerkraut brine with a small amount of inulin may usefully decrease the BOD and concomitantly produce inulinase.

Kluyveromyces marxianus: a potential source of diacetyl reductase.

Kluyveromyces marxianus had a higher specific activity of diacetyl reductase (EC 1.1.1.5) than all other organisms previously reported. The enzyme was NADH-dependent and irreversibly catalysed the conversion of diacetyl to acetoin with an optimum pH of 7.0. It was stable at 40°C but lost 50% of its activity at 50°C in 30 min. The K m and V max values for diacetyl were 1.8 mM and 0.053 mM/min, respectively.

Amylolytic activity of Byssochlamys fulva.

Byssochlamys fulva was found to produce a glucoamylase (EC 3.2.1.3) that exhibited its maximal activity at 50°C and had a broad optimum pH range of 4.0-5.2. The Km and Vmax values of the crude enzyme for amylopectin were 0.15% and 17.9 mg glucose l-1 min--1 , respectively. The molecular weight of the enzyme as estimated by the gel-filtration method was 34 kDa.

[Clinical analysis of senile and non-senile affective psychosis].

This article analysed 101 cases of affective psychosis in accordance with CCMD-2 diagnosis criteria. There were 42 cases over and 59 under 60 in age. The results indicated that the patients of the senile affective psychotics accounted for 30.47% of the hospitalized psychotics in the same time. 78.57% of the old group were psychically and somatically precipitated. The proportion was much higher than the non-senile. The degree of the senile group was much higher than the non-senile in manic attack, while it was much lower in depressive attack. The dosages and the plasma level of Li in the former were markedly decreased in comparison to the latter group.

Production and characterization of polygalacturonase fromGeotrichum candidum.

An extracellular polygalacturonase (EC 3.2.1.15) fromGeotrichum candidum ATCC 34614 grown onsauerkraut brine was produced and characterized. Polygalacturonic acid markedly increased the enzyme yield in the brine. The fungus produced the highest activity (290 U/l) in brine with 0.3% (w/v) polygalacturonic acid. The pH and temperature optima of the enzymes were 4.5 to 5.0 and 30°C, respectively. It was stable from pH 4.0 to 5.8 and at 30°C but lost its activity at higher temperatures. The Km and Vmax values for polygalacturonic acid were 4.2 mg/ml and 0.19MM galacturonic acid/min, respectively. The enzyme was not substrate inhibited.

Amylolytic enzyme production byRhizopus oryzae grown on agricultural commodities.

The amylolytic enzyme production byRhizopus oryzae NRRL 395 grown on different agricultural commodities was datermined. The mould produced much higher enzyme activity from barley, corn, bats, and rice than from cassava. The optimal temperature for enzyme production was 30°C. Neutralization with CaCO3 greatly enhanced the rate of enzyme production. Nitrogen supplementation of cassava resulted in higher enzyme yields.

Toxic action of citric acid onGeotrichum candidum.

Citric acid had a toxic effect on the mycelial growth ofGeotrichum candidum. At equal pH, citric acid was more toxic than HCl but its effect was minimized by adding CaCl2. The results indicate that the toxic effect of citric acid is due to the undissociated component and its ability to sequester the essential cations present in the growth medium.

Lipase production byGeotrichum candidum from sauerkraut brine.

Sauerkraut brine could serve as a substrate for the production of extracellular lipase (EC 3.1.1.3) byGeotrichum candidum ATCC 34614. The fungus produced the highest specific activity (more than 140 units/mg protein) when cultivated in shake flasks for 72 h at 30°C in the brine, with a small amount of olive oil (0.3%, v/v) as an enzyme inducer. Lipase activity was recovered from the culture filtrate by fractionation with acetone and the yield was as high as 59%.

Production of alcohol from apple pomace.

Production of ethyl alcohol from apple pomace with a Montrachet strain of Saccharomyces cerevisiae is described. More than 43 g of the ethyl alcohol could be produced per kg of apple pomace fermented at 30 degrees C in 24 h. The fermentation efficiency of this process was approximately 89%.

Assimilation of lemonade-processing wastewater by yeasts.

Of the five species of yeasts examined in shake-flask studies at 30 degrees C, only Candida utilis and Saccharomyces fragilis gave higher cell yields (>2.5 g /liter) in 32 h and reduced approximately 87% of the biological oxygen demand in lemonade-processing wastewater fortified with 0.05% ammonium sulfate and 0.01% potassium phosphate. The latter, however, settled more rapidly than the former.

Continuous Removal of Lactic Acid from Wastewater by Candida utilis.

Under steady-state conditions, the maximum rate of lactic acid removal was found to be 588 mg/h per liter of continuous culture.

Baked-bean waste: a potential substrate for producing fungal amylases.

Baked-bean waste was found to be a favorable substrate for amylase production by Aspergillus foetidus NRRL 337. Under optimum conditions, the yields of alpha-amylase (EC 3.2.1.1) and glucoamylase (EC 3.2.1.3) were 47 and 226 U, respectively, per ml of the waste fermented.

Utilization of brewery spent grain liquor by Aspergillus niger.

Aspergillus niger was found capable of rapidly converting about 97% of the sugar from brewery spent grain liquor to fungal mass. The yield of dry mycelium, based on the sugar consumed, was approximately 57%. This fungus produced 1.10% titratable acid calculated as citric acid and reduced the biochemical oxygen demand by 96%.

Propagation of Geotrichum candidum in acid brine.

Geotrichum candidum completely neutralized the acid brine and reduced its biochemical oxygen demand (BOD) by 88%. Yield of dry mycelium was 62 g per 100 g of BOD utilized.

Production of yeast invertase from sauerkraut waste.

Sauerkraut waste was found to be a favorable medium for the production of invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) by Candida utilis.