Effective production of a thermostable alpha-glucosidase from Sulfolobus solfataricus in Escherichia coli exploiting a microfiltration bioreactor.

A microfiltration (MF) membrane bioreactor was developed for an efficient production of a recombinant thermostable alpha-glucosidase (rSsGA) from Sulfolobus solfataricus MT-4. The aim of the membrane bioreactor was to improve the control of the concentration of key components in the growth of genetic engineered microorganisms, such as Escherichia coli. The influence of medium composition was studied in relation to cell growth and alpha-glucosidase production. The addition of components such as yeast extract and tryptone resulted in a higher enzyme production. High cell density cultivation of E. coli BL21(DE3) on semidefined medium, exploiting a microfiltration bioreactor, was studied in order to optimize rSsGA production. In addition to medium composition, the inducer employed (either isopropyl beta-D-thiogalactopyranoside or lactose), the induction duration, and the cultivation mode influenced both the final biomass and the enzyme yield. The MF bioreactor allowed a cell concentration of 50 g/L dry weight and a corresponding alpha-glucosidase production of 11,500 U/L. The improvement obtained in the enzyme production combining genetic engineering and the microfiltration strategy was estimated to be 2,000-fold the wild-type strain.

A microfiltration bioreactor to achieve high cell density in Sulfolobus solfataricus fermentation.

A novel technique is proposed to achieve higher cell yield in extremophile fermentation. Because the accumulation of toxic compounds is thought to be responsible for low biomass yields, a bioreactor has been designed based on a microfiltration hollow-fiber module located inside the traditional fermentation vessel. Using the cultivation of the thermoacidophilic archeon Sulfolobus solfataricus theta as a model, a biomass of 35gl(-1) dry weight was obtained which proved greater than that of 2gl(-1) obtained in batch fermentation. The bioreactor was characterized by running several fermentation experiments to check the high stability of the membrane module to sterilization cycles, high temperatures, and acidic pHs, even for prolonged periods of time. It was shown that the exhaust medium is unable to sustain growth for the presence of toxic compounds, and ultrafiltration and ion-exchange techniques were used in all the attempts to regenerate it. The results demonstrated the ability of the method to lower inhibitor concentrations and prolong the growth phase, thus achieving high cell density. Furthermore, they indicated that the toxic compounds are ionic species of less than 1kDa.

Identification of a high-molecular-weight cadmium-binding protein in copper-resistant Bacillus acidocaldarius cells.

Bacillus acidocaldarius grown in the presence of Cu++ was capable of accumulating the metal in the form of a protease-sensitive high molecular weight (HMW) moiety whose formation was inhibited by actinomycin D. Only cells preadapted in Cu++ were able to grow in a Cd(++)-containing medium. A cell-free extract from cadmium-stressed cells was fractionated by gel-permeation chromatography. The majority of cadmium was found associated with a HMW protein fraction which was further purified by anion exchange chromatography and high-performance liquid chromatography. The molecular weight of the purified protein was estimated to be 23,000 by SDS-PAGE. Amino acid analysis showed a low cysteine content and an abundance of aspartate and glutamate. It is likely that the cadmium-binding protein is an essential component of the mechanism mediating recovery from heavy metal toxicity.