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BACKGROUNDUrinary excretion of the tetrasaccharide 6-α-D-glucopyranosyl-maltotriose (Glc4) is increased in various clinical conditions associated with increased turnover or storage of glycogen, making Glc4 a potential biomarker for glycogen storage diseases (GSD). We developed an ultraperformance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) assay to detect Glc4 in urine without interference of the Glc4 isomer maltotetraose (M4).METHODSUrine samples, diluted in 0.1% ammonium hydroxide containing the internal standard acarbose, were filtered, and the filtrate was analyzed by UPLC-MS/MS.RESULTSWe separated and quantified acarbose, M4, and Glc4 using the ion pairs m/z 644/161, 665/161, and 665/179, respectively. Response of Glc4 was linear up to 1500 μmol/L and the limit of quantification was 2.8 μmol/L. Intra- and interassay CVs were 18.0% and 18.4% (10 μmol/L Glc4), and 10.5% and 16.2% (200 μmol/L Glc4). Glc4 in control individuals (n = 116) decreased with increasing age from a mean value of 8.9 mmol/mol to 1.0 mmol/mol creatinine. M4 was present in 5% of urine samples. Mean Glc4 concentrations per age group in untreated patients with Pompe disease (GSD type II) (n = 66) were significantly higher, ranging from 39.4 to 10.3 mmol/mol creatinine (P < 0.001–0.005). The diagnostic sensitivity of Glc4 for GSD-II was 98.5% and the diagnostic specificity 92%. Urine Glc4 was also increased in GSD-III (8 of 9), GSD-IV (2 of 3) and GSD-IX (6 of 10) patients.CONCLUSIONSThe UPLC-MS/MS assay of Glc4 in urine was discriminative between Glc4 and M4 and confirmed the diagnosis in >98% of GSD-II cases.

We report the progress of a multi-disciplinary research project on solid-state fermentation (SSF) of the filamentous fungus Aspergillus oryzae. The molecular and physiological aspects of the fungus in submerged fermentation (SmF) and SSF are compared and we observe a number of differences correlated with the different growth conditions. First, the aerial hyphae which occur only in SSFs are mainly responsible for oxygen uptake. Second, SSF is characterised by gradients in temperature, water activity and nutrient concentration, and inside the hyphae different polyols are accumulating. Third, pelleted growth in SmF and mycelial growth in SSF show different gene expression and protein secretion patterns. With this approach we aim to expand our knowledge of mechanisms of fungal growth on solid substrates and to exploit the biotechnological applications.

Using a combination of dye adsorption and affinity elution we purified Aspergillus niger citrate synthase to homogeneity using a single column and characterised the enzyme. An A. niger citrate synthase cDNA was isolated by immunological screening and used to clone the corresponding citA gene. The deduced amino acid sequence showed high similarity to other fungal citrate synthases. After processing upon mitochondrial import, the calculated Mr of A. niger citrate synthase is 48 501, which agrees well with the estimated molecular mass of the purified protein (48 kDa). In addition to an N-terminal mitochondrial import signal, a peroxisomal target sequence (AKL) was found at the C-terminus of the protein. Whether both signals are functional in vivo is not clear. Strains overexpressing citA were made by transformation and cultured under citric acid-producing conditions. Up to 11-fold overproduction of citrate synthase did not increase the rate of citric acid production by the fungus, suggesting that citrate synthase contributes little to flux control in the pathway involved in citric acid biosynthesis by a non-commercial strain.

In our paper, we analysed the effects of overproduction of citrate synthase on citric acid production by Aspergillus niger in addition to some descriptive work on the enzyme itself and the citA gene. Dr Ratledge argues that our attempt to increase productivity by amplifying citrate synthase was 'a complete waste of time and effort’. He states that we could have known beforehand that this was not going to work by looking at biochemical data. I oppose his reasoning and will explain my view. Dr Ratledge performs a rough calculation to show that, in an unmodified strain, citrate synthase is well in excess of the amount required to sustain the metabolic flux observed during fermentation. A comparison between the maximal …