Torbjörn Linden

Fermentation of lignocellulose hydrolysates with yeasts and xylose isomerase

Untreated spent sulfite liquor (SSL) was fermented with five yeasts, Candida tropicalis, Pichia stipitis, Pachysolen tannophilus, Schizosaccharomyces pombe, Saccharomyces cerevisiae, and a co-culture of P. tannophilus and S. cerevisiae, in the presence of commercial xylose (glucose) isomerases and 4.6 mM azide. The highest yield of ethanol, 0.41 g g− total sugar, was obtained with S. cerevisiae. The yield based on consumed sugars and per gram cell dry weight was also highest with this yeast.C. tropicalis and P. tannophilus produced considerable amounts of polyoles, mainly xylitol. With P. stipitis sugar uptake was rapidly inhibited in untreated SSL. The presence of azide contributed to the yield by about 0.04, mainly due to the fermentation of stored carbohydrates. The fermentation of hydrogen fluoride-pretreated and acid-hydrolysed wheat straw with S. cerevisiae, xylose isomerase, and azide gave a yield of 0.40 g ethanol g−1 total sugar. In this substrate the xylose utilization was 84% compared with 51% in SSL, which is discussed in relation to the salt sensitivity of xylose isomerases.

Ethanolic fermentation of pentoses in lignocellulose hydrolysates.

In the fermentation of lignocellulose hydrolysates to ethanol, two major problems are encountered: the fermentation of the pentose sugar xylose, and the presence of microbial inhibitors. Xylose can be directly fermented with yeasts, such as Pachysolen tannophilus, Candida shehatae, and Pichia stipis, or by isomerization of xylose to xylulose with the enzyme glucose (xylose) isomerase (XI; EC 5.3.1.5), and subsequent fermentation with bakers’ yeast, Saccharomyces cerevisiae. The direct fermentation requires low, carefully controlled oxygenation, as well as the removal of inhibitors. Also, the xylose-fermenting yeasts have a limited ethanol tolerance. The combined isomerization and fermentation with XI and S. cerevisiae gives yields and productivities comparable to those obtained in hexose fermentations without oxygenation and removal of inhibitors. However, the enzyme is not very stable in a lignocellulose hydrolysate, and S. cerevisiae has a poorly developed pentose phosphate shunt. Different strategies involving strain adaptation, and protein and genetic engineering adopted to overcome these different obstacles, are discussed.

3,4-DGE is important for side effects in peritoneal dialysis what about its role in diabetes.

Breakdown of glucose under physiological conditions gives rise to glucose degradation products (GDPs). GDPs are also formed during heat sterilization of glucose-containing peritoneal dialysis fluids (PD-fluids). In PD-fluids GDPs have been shown in many different in vitro assays to be responsible for adverse effects such as growth inhibition, and impaired leukocyte function and impaired wound healing of peritoneal mesothelial cells. They have been linked to changes in the peritoneal membrane as well as to the decline in residual renal function of PD-patients. In diabetes one of the GDPs, 3-deoxyglucosone (3-DG), has been proposed as responsible for side-effects rather the glucose itself. 3,4-dideoxyglucosone-3-ene (3,4-DGE) was recently identified as the most bio-reactive GDP in PD-fluids. It exists in equilibrium with a pool of precursors, consisting of 3-DG but also of other hitherto unidentified GDPs. In PD-fluids the concentration of GDPs in this pool is 10-20 times as high as that of 3,4-DGE. In vitro 3,4-DGE induces caspase-dependent apoptosis of neutrophils and peripheral blood mononuclear cells. Such induction may explain immunosuppressive properties of 3,4-DGE and contribute to an impaired peritoneal antibacterial defense. 3,4-DGE also induces renal cell apoptosis. This may explain the better preservation of residual renal function in PD patients not exposed to GDPs. The concentration of 3-DG increases with worsening glycemic control and has been implicated in the genesis of diabetic microangiopathy. As 3,4-DGE is much more bio-reactive than 3-DG and as it may be easily recruited from the pool, it seems probable that 3,4-DGE is the molecule involved in the diabetic lesions rather than 3-DG itself. Thus, 3,4-DGE might contribute to diabetic nephropathy and to the impaired antibacterial defenses in diabetics. Unraveling of the pool dynamics of the GDPs and the molecular mechanisms of GDP-mediated cell injury may provide new therapeutic insights in PD and diabetes.

A rapid chromatographic method for the production of preparative amounts of xylulose

Abstract High-purity d -xylulose > 97% was prepared from d -xylose in gram quantities using a simple three-step procedure; enzymatic isomerization with commercial glucose isomerase (xylose isomerase EC. 5.3.1.5), ethanol precipitation, and ion-exchange chromatography on an anionic resin in sulfite form. In the chromatographic step, 0.7–1.4 g xylulose was produced within 10–15 h. Impurities in the preparation are of sugar origin and may consist of glucose, ribulose, and xylose. Fractions of high purity, > 99%, but in small amounts, could be produced in the chromatographic step. When the procedure was simplified by performing the precipitation in a saturated isomerization solution, almost identical results were obtained. If the precipitation step was excluded, it resulted in poorer separation in the chromatographic step, but xylulose could still be produced with a purity with a purity of 96–97%, though in smaller amounts, 0.35–0.74g.

HPLC determination of xylulose formed by enzymatic xylose isomerization in lignocellulose hydrolysates

In the concentration range appropriate for enzymatic xylose isomerization, xylulose was measured in a lignocellulose hydrolysate using HPLC with two hydrogen loaded ion exchange columns in series. Spent sulphite liquour (SSL) was used as a model for lignocellulose hydrolysates. In buffer the separation took 22 minutes and in SSL the analysis time was 47 minutes due to the presence of ethanol. The enzymatic isomerization of xylose to xylulose was followed directly in SSL, providing a method for the direct determination of xylose isomerase activity in lignocellulose hydrolysates.

Infusion fluids contain harmful glucose degradation products

PurposeGlucose degradation products (GDPs) are precursors of advanced glycation end products (AGEs) that cause cellular damage and inflammation. We examined the content of GDPs in commercially available glucose-containing infusion fluids and investigated whether GDPs are found in patients’ blood.MethodsThe content of GDPs was examined in infusion fluids by high-performance liquid chromatography (HPLC) analysis. To investigate whether GDPs also are found in patients, we included 11 patients who received glucose fluids (standard group) during and after their surgery and 11 control patients receiving buffered saline (control group). Blood samples were analyzed for GDP content and carboxymethyllysine (CML), as a measure of AGE formation. The influence of heat-sterilized fluids on cell viability and cell function upon infection was investigated.ResultsAll investigated fluids contained high concentrations of GDPs, such as 3-deoxyglucosone (3-DG). Serum concentration of 3-DG increased rapidly by a factor of eight in patients receiving standard therapy. Serum CML levels increased significantly and showed linear correlation with the amount of infused 3-DG. There was no increase in serum 3-DG or CML concentrations in the control group. The concentration of GDPs in most of the tested fluids damaged neutrophils, reducing their cytokine secretion, and inhibited microbial killing.ConclusionsThese findings indicate that normal standard fluid therapy involves unwanted infusion of GDPs. Reduction of the content of GDPs in commonly used infusion fluids may improve cell function, and possibly also organ function, in intensive-care patients.

Activity and stability of xylose isomerase preparations from whole cells of Lactobacillus brevis in spent sulfite liquor

Three differently treated xylose isomerase preparations of Lactobacillus brevis DSM 20054 whole cells were compared with a commercial immobilized preparation, Maxazyme GI-immob., with respect to activity and stability. All isomerizations were performed in spent sulfite liquor. The activity (∼300 mg xylulose g−1 dry weight biocatalyst h−1) of one preparation, heat-, ethanol-, and glutaraldehyde-treated whole cells of L. brevis, was 30 to 40 times higher, at pH 5 and 5.5, respectively, than that of the commercial enzyme at pH 5.5. The commercial enzyme was not active at pH 5. During four repeated 24-h isomerizations, this L. brevis preparation retained 70% of the activity at pH 5.5 and 30% at pH 5, as compared with 66 and 0%, respectively, for the commercial preparation.