Niklas von Weymarn

Extreme Halophiles Synthesize Betaine from Glycine by Methylation

Glycine betaine is a compatible solute, which is able to restore and maintain osmotic balance of living cells. It is synthesized and accumulated in response to abiotic stress. Betaine acts also as a methyl group donor and has a number of important applications including its use as a feed additive. The known biosynthetic pathways of betaine are universal and very well characterized. A number of enzymes catalyzing the two-step oxidation of choline to betaine have been isolated. In this work we have studied a novel betaine biosynthetic pathway in two phylogenically distant extreme halophiles,Actinopolyspora halophila and Ectothiorhodospira halochloris. We have identified a three-step series of methylation reactions from glycine to betaine, which is catalyzed by two methyltransferases, glycine sarcosine methyltransferase and sarcosine dimethylglycine methyltransferase, with partially overlapping substrate specificity. The methyltransferases from the two organisms show high sequence homology. E. halochlorismethyltransferase genes were successfully expressed inEscherichia coli, and betaine accumulation and improved salt tolerance were demonstrated.

Influence of pH on the production of xylanases by Trichoderma reesei Rut C-30

Trichoderma reesei Rut C-30 was cultivated in bioreactors at different pH on a medium with lactose as the main carbon source. Compared to an earlier study, in which T. reesei Rut C-30 was cultivated using polysaccharides (cellulose or xylan) as the main carbon sources, we now report a slightly lower pH value for maximal xylanase levels. The highest xylanase activity (IU/ml) on the lactose-based medium was observed at pH 6.0 compared to pH 7.0 on the polysaccharide-based media. When the pattern of different xylanases was analyzed by isoelectric focusing and activity zymogram, we observed that a low pH (4.0) favoured the production of xylanase I, whilst a high pH (6.0) favoured the production of xylanase III. Xylanase II was clearly produced at both pH values. The results at pH 4 and 6 correlate with the pH activity profiles of xylanase I, II and III. Hence, the different T. reesei xylanases were produced according to which enzyme is most active in that particular environment.

Phytase production by high cell density culture of recombinant Bacillus subtilis

Bacillus subtilis BD170, harboring a plasmid pGT44[phyC] carrying the phytase gene (phyC) and a phosphate-depletion inducible pst-promoter, was grown in a 2 l bioreactor. Using a controlled feeding of glucose, high cell densities of 32 and 56 g dry cell weight l−1 were achieved with peptone and yeast extract, respectively, as the complex nitrogen sources in a semi-defined growth medium. The fed-batch protocol was applied to production of recombinant phytase and a high extracellular phytase activity (48 U ml−1) was reached with peptone. Although the yeast extract feeding resulted in a higher cell density, it was unsuitable as a medium component for phytase expression due to its relatively high phosphate content.

Production of recombinant HIV-1 Nef (negative factor) protein using Pichia pastoris and a low-temperature fed-batch strategy.

In the present paper we describe the cloning and extracellular expression of the HIV‐1 Nef (negative factor) protein utilizing the yeast Pichia pastoris, as well as the successful use of a low‐temperature fed‐batch strategy for decreasing end‐product degradation by proteases. The nef gene in a pPICZαA vector was integrated into the genome of three different P. pastoris strains, namely X‐33, GS115 and KM71H. On the basis of its efficient growth and production characteristics the wild‐type strain (X‐33) was found to be the best choice. The decreased end‐product degradation at low temperatures was not due to lower amounts of proteases but due to their diminished activity. The yield of biomass from methanol was improved 1.44‐fold utilizing the low‐temperature strategy compared with the standard fermentation. Purification of histidine‐tagged Nef was performed in one step using a Ni2+‐nitrilotriacetate–Sepharose column. The purified product was characterized by SDS/PAGE, Western blotting, matrix‐assisted laser‐desorption ionization–time‐of‐flight MS, reversed‐phase HPLC and N‐terminal‐sequence analysis.

Comparison of pulp species in IONCELL-P: Selective hemicellulose extraction method with ionic liquids

Abstract In our recent studies, it was demonstrated that the IONCELL-P process selectively dissolves hemicelluloses from bleached birch kraft pulp in a mixture of 1-ethyl-3-methylimidazolium acetate ([emim][OAc]) and water as a solvent system. The IONCELL-P method refines paper-grade pulp to dissolving pulp with <5% hemicelluloses and allows isolation of polymeric xylan without yield losses or polymer degradation. This paper is a comparative study where paper-grade pine, birch, and eucalyptus pulps are subjected to the IONCELL-P process with two [emim]-based ionic liquids (ILs), i.e. [emim]acetate and [emim]dimethylphosphate. Also, the effect of an endoglucanase pretreatment was investigated to check whether 1) the pulp viscosity could be adjusted for the following process steps before the hemicellulose extraction and 2) the decreasing pulp viscosity would open the fiber structure and thus enhance the extraction. Under optimum conditions, the birch xylan content could be reduced from 25.4% down to 1.3% and for eucalyptus from 16.6% to 2.4%. Pine pulp xylan and glucomannan were decreased from 8.1% and 7.1% to 0.9% and 2.2%, respectively. The residual hemicellulose contents of the pine pulp could be further decreased with a hemicellulase pretreatment. The selectivity of the dissolution towards hemicelluloses was better for hardwoods. Adjusting the pulp viscosity by endoglucanase prior to the IONCELL-P process reduced the selectivity as short-chain cellulose molecules were extracted along with the hemicelluloses.

Production of recombinant HIV-1 nef protein using different expression host systems: A techno-economical comparison

Three popular expression host systems Escherichia coli, Pichia pastoris and Drosophila S2 were analyzed techno‐economically using HIV‐1 Nef protein as the model product. On scale of 100 mg protein, the labor costs corresponded to 52–83% of the manufacturing costs. When analyzing the cost impact of the different phases (strain/cell line construction, bioreactor production, and primary purification), we found that with the microbial host systems the strain construction phase was most significant generating 56% (E. coli) and 72% (P. pastoris) of the manufacturing costs, whereas with the Drosophila S2 system the cell line construction and bioreactor production phases were equally significant (46 and 47% of the total costs, respectively). With different titers and production goal of 100 mg of Nef protein, the costs of P. pastoris and Drosophila S2 systems were about two and four times higher than the respective costs of the E. coli system. When equal titers and bioreactor working volumes (10 L) were assumed for all three systems, the manufacturing costs of the bioreactor production of the P. pastoris and Drosophila S2 systems were about two and 2.5 times higher than the respective costs of the E. coli system.