α-Amylase, glucoamylase and isopullulanase determine molecular weight of pullulan produced by Aureobasidium melanogenum P16.

A high molecular weight (Mw) pullulan has many potential applications in various fields. α-Amylase, glucoamylase and pullulanase were thought to play an important role in high Mw pullulan biosynthesis. However, there is no genetic evidence for this role. In this study, the genes encoding α-amylase, glucoamylase and pullulanase were cloned from Aureobasidium melanogenum P16, a high pullulan producing yeast and characterized. The proteins deduced from the cloned α-amylase gene, the glucoamylase gene and the isopullulanase gene, not a pullululanse gene had their corresponding conserved amino acid sequences, respectively. After the single gene of them was deleted, the Mw of the pullulan produced by the single disruptants greatly increased and the pullulan concentration decreased. It was found that the triple mutant DT15 grown at the flask level could produce 46.2 g/L of pullulan with a Mw of 3.02 × 106 Da and grown in the 10-L fermentor could yield 58.14 g/L of pullulan with the same Mw while its wild type strain P16 produced 65.5 ±â€¯3.5 g/L of pullulan with a Mw of 0.35 × 106 Da. After the genes were complemented, pullulan production, Mw of the produced pullulan and others were restored. All the results demonstrated that the α-amylase, glucoamylase and isopullulanase indeed could determine the Mw of the produced pullulan.

Improvement of heterologous protein production in Aspergillus oryzae by RNA interference with alpha-amylase genes.

Aspergillus oryzae RIB40 has three alpha-amylase genes (amyA, amyB, and amyC), and secretes alpha-amylase abundantly. However, large amounts of endogenous secretory proteins such as alpha-amylase can compete with heterologous protein in the secretory pathway and decrease its production yields. In this study, we examined the effects of suppression of alpha-amylase on heterologous protein production in A. oryzae, using the bovine chymosin (CHY) as a reporter heterologous protein. The three alpha-amylase genes in A. oryzae have nearly identical DNA sequences from those promoters to the coding regions. Hence we performed silencing of alpha-amylase genes by RNA interference (RNAi) in the A. oryzae CHY producing strain. The silenced strains exhibited a reduction in alpha-amylase activity and an increase in CHY production in the culture medium. This result suggests that suppression of alpha-amylase is effective in heterologous protein production in A. oryzae.

[Effects of administration of vitamin A, B 2 and B 15 to guinea pigs during the larval stage of ascariasis on alpha-amylase activity in the lungs and kidneys]. / Wplyw witamin A, B2 i B15 podawanych swinkom morskim w czasie glistnicy larwalnej na aktywnosc alfa-amylazy w plucach i nerkach.

The guinea pigs were administrated vit. A (400 i.u.), vit. B2 (1 mg) or vit. B15 (5 mg). On the 9-th day of the experiment part of them was infected with 5000 invasive eggs of Ascaris suum. The invasion lasted 6 days and was controlled by lungs and kidney weight, and number of larvae in the lungs. The activity of amylase was determined by saccharogenic method in both organs. In the lungs of infected animals the activity of alpha-amylase was about 3 times lower than in the control. The infection of guinea pigs which were given vitamins did not cause change of enzyme's activity. In the kidney directive tendency was the same, but the differences were smaller. The infection resulted in an increase of relative mass of lungs. This index and number of larvae was considerable smaller in guinea pigs with vitamins A and B15 administration. Any testing agent did not cause change of relative weight of kidney.

[Trypsin and alpha-amylase activity in the pancreas of guinea pigs. V. Effects of administration of vitamin A and B 2 during the larval stage of ascariasis]. / Aktywnosc trypsyny i alfa-amylazy w trzustce swinek morskich. V. Wplyw podawania witamin A i B2 w czasie askarydozy larwalnej.

The infection of guinea pigs with Ascaris suum larvae resulted in decrease of the activities of trypsin and alpha-amylase, and in increase of lipase activity in extracts from their pancreas. The activity of alpha-amylase, lipase and the relative weight of lungs of infected animals which were given vitamin A, did not differ from control animals. The activity of trypsin from pancreas these animals was higher than that measured in only infected guinea pigs but it was lower than in control animals. Application of vitamin B2 and the infection of guinea pigs with A. suum did not lead to the synonymous results.

Net absorption of starch in low birth weight infants.

Twelve 3-day metabolic balance studies were carried out in 12 low birth weight infants fed an infant formula providing 3.5 g of corn starch per kilogram body weight per day. The mean coefficients of net absorption were 88 +/- 6% for starch, 70 +/- 14% for fat and 90 +/- 4% for nitrogen. No relationship was found between starch absorption and nitrogen or fat absorption. There was no relationship between starch absorption and the duration of starch feeding. It is concluded that the ability of young infants to digest large quantities of starch is most likely limited resulting from low pancreatic alpha-amylase activity. Nevertheless, from a practical point of view, small amounts of starch in infant formulas can be considered not only as a thickener but also as a source of calories.