Screening and mutagenesis of Aspergillus niger for the improvement of glucose 6-phosphate dehydrogenase production

The strain of Aspergillus niger ZBY-7 was selected as the original strain of glucose 6-phosphate dehydrogenase production. After mutagenesis of the strain using UV irradiation and nitrosoguanidine, mutants of Aspergillus niger resistant to certain metabolic inhibitor were obtained. Five of the mutants showed increased glucose 6-phosphate dehydrogenase production. The mutant resistant to antimycin A (Aspergillus niger AM-23) produced the highest level of glucose 6-phosphate dehydrogenase (695.9% of that from the original strain).     

Selection of biochemical mutants of Aspergillus niger with enhanced extracellular ribonuclease production

Combined with u.v. irradiation and the nitrosoguanidine method, selection of biochemical mutants resistant to metabolic inhibitors (2-deoxy-D-glucose, antimycin A, sodium orthovanadate and sodium azide) was a very efficient method for improvement of ribonuclease production by Aspergillus niger. Resistance to sodium azide produced highest RNase production, greatest frequency of positive mutation and shortest sporulation time. The most active strain, Aspergillus niger SA-13-20 resistant to sodium azide, was obtained, which had a 433% increase in RNase production in comparison with the parent strain and had good subculturing stability. Its dynamic characters were similar to those of the parent strain.     

Sugar and phosphate metabolism and alkaloid production phases in submerged cultures of two Claviceps strains

Summary In submerged cultures of Claviceps sp. CP II, elymoclavine was synthesized only by the growing mycelium (phase P1), whereas cultures of C. purpurea strain 129 produced agroclavine after vegetative growth had also ceased (phase P2). In strain CP II, the peak of activity of malate dehydrogenase, glucose-6-phosphate dehydrogenase and phosphatases was related to the time of maximum growth rate and alkaloid production. Citrate synthase activity paralleled the course of alkaloid synthesis. Strain 129 exhibited a further activity peak of the same magnitude during phase P2. ATP levels in both cultures corresponded to the pattern of change in enzyme activities. Strain CP II contained roughly twice as much orthophosphate and ATP in its cells as strain 129 and exhibited higher average activity of glucose-6-phosphate dehydrogenase. It follows from these results that alkaloid synthesis requires the processes of primary metabolism, even when it occurs after active growth of the culture has ceased. Cultures producing alkaloids oxidized at C-8 exhibit higher glucose-6-phosphate dehydrogenase activity, probably because of a higher NADPH consumption.     

Strain improvement and metabolic flux modeling of wild-type and mutant <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. NX-3 for synthesis of exopolysaccharide welan gum

Low-energy nitrogen ion beam implantation technique was used for the strain improvement of Alcaligenes sp. NX-3 for the production of exopolysaccharide welan gum. A high welan gum producing mutant, Alcaligenes sp. NX-3-1, was obtained through 20 keV N⁺ ion beam irradiation. Starting at a concentration of 50 g/L of glucose, mutant NX-3-1 produced 25.0 g/L of welan gum after 66 h of cultivation in a 7.5 L bioreactor, which was 34.4% higher than that produced by the wild-type strain. The results of metabolic flux analysis showed that the glucose-6-phosphate and acetyl coenzyme A nodes were the principle and flexible nodes, respectively. At the glucose-6-phosphate node, the fraction of carbon measured from glucose-6-phosphate to glucose-1-phosphate was enhanced after mutagenesis, which indicated that more flux was used to synthesize welan gum in the mutant. By analyzing the activities of related enzymes in the biosynthetic pathway of sugar nucleotides essential for welan gum production, we found that the specific activities of phosphoglucomutase, UDP-glucose pyrophosphorylase, UDP-glucose dehydrogenase, and dTDP-glucose pyrophosphorylase in the mutant strain were higher than those in the wild-type strain. These improvements in enzyme activities could be due to the affected of ion beam implantation.     

Screening, mutagenesis and protoplast fusion of Aspergillus niger for the enhancement of extracellular glucose oxidase production

Various strains of Aspergillus niger were screened for extracellular glucose oxidase (GOD) activity. The most effective producer, strain FS-3 (15.9 U mL^–1), was mutagenized using UV-irradiation or ethyl methane sulfonate. Of the 400 mutants obtained, 32 were found to be resistant to 2-deoxy d-glucose, and 17 of these exhibited higher GOD activities (from 114.5 to 332.1%) than the original FS-3 strain. Following determination of antifungal resistance of the highest producing mutants, four mutants were selected and used in protoplast fusions in three different intraspecific crosses. All fusants showed higher activities (from 285.5 to 394.2%) than the original strain. Moreover, of the 30 fusants isolated, 19 showed higher GOD activity than their corresponding higher-producing parent strain.     

Selection of Escherichia coli mutants lacking glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase

Glucose is metabolized in Escherichia coli chiefly via the phosphoglucose isomerase reaction; mutants lacking that enzyme grow slowly on glucose by using the hexose monophosphate shunt. When such a strain is further mutated so as to yield strains unable to grow at all on glucose or on glucose-6-phosphate, the secondary strains are found to lack also activity of glucose-6-phosphate dehydrogenase. The double mutants can be transduced back to glucose positivity; one class of transductants has normal phosphoglucose isomerase activity but no glucose-6-phosphate dehydrogenase. An analogous scheme has been used to select mutants lacking gluconate-6-phosphate dehydrogenase. Here the primary mutant lacks gluconate-6-phosphate dehydrase (an enzyme of the Enter-Doudoroff pathway) and grows slowly on gluconate; gluconate-negative mutants are selected from it. These mutants, lacking the nicotinamide dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase, grow on glucose at rates similar to the wild type. Thus, these enzymes are not essential for glucose metabolism in E. coli.     

Comparative study of fungal cell disruption—scope and limitations of the methods

Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems.     

Strain improvement of Aspergillus niger for the enhanced production of asperenone

The enhancement of production of asperenone (Fig. 1), an inhibitor of lipoxygenase and human platelet aggregation from Aspergillus niger CFTRI 1105, was achieved by UV and nitrous acid mutagenesis. Nitrous acid mutants exhibited increased inhibitor production when compared with UV irradiated mutants. I N 41 a first-generation nitrous acid mutant produced 5.1 fold increased asperenone over parent strain. Mutant II N 31 obtained by second-generation nitrous acid treatment produced 60.3 mg asperenone/g biomass, which was 131 fold increase when compared to first generated mutant I N 41 and 670 fold increase over the parent strain. This mutant was stable for several generations on production medium.     

Glucose oxidase biosynthesis in relation to biochemical mutations in Aspergillus niger

The production of extracellular glucose oxidase in a submerged culture by a number of auxotrophic, 2-deoxy-D-glucose resistant and protease-less mutants of Aspergillus niger was evaluated. Among the auxotrophic strains, no evident dependence was found between the kind of the nutritional requirements and the level of the glucose oxidase activity. However, the majority of auxotrophs, requiring serine or niacin, showed a higher enzyme activity (from 16 to 680%) than the parent strain. The dynamics of the glucose oxidase synthesis by the free and immobilized mycelium of the most active niacin^? mutant of A. niger was also investigated.     

Production of mold lactase

A process for production of mold lactase was developed. Tests were carried out in pilot and industrial scale with an Aspergillus niger strain selected after screening a number of molds.A computer coupled autoanalyzer system was used for monitoring enzyme formation in the pilot fermentor. Lactase production was investigated using different pH- and temperature-profiles. A. niger lactase has an acid pH optimum, a high temperature optimum and good stability. It does not require any metal ions. It is suitable for immobilization for hydrolysis of lactose in acid whey.Three-fold enhancement of lactase production was obtained by mutagenizing A. niger using NTG as mutagenic agent.The lactases produced by the mutants have the same pH and temperature optima and stability but the growth properties of the mutants were different from those of the original strain.Sufficient specific activity of the enzyme preparation for immobilization was obtained by purifying the enzyme by selective adsorption on Na-Ca-silicate.     

The cytochemical assay of NAD+ kinase activity in the follicular cells of guinea-pig thyroid gland

Summary A quantitative cytochemical assay for NAD⁺ kinase-like activity in the guinea-pig thyroid gland is described. The NADP⁺ produced by the activity of the kinase was used to drive the NADP⁺-dependent enzyme glucose-6-phosphate dehydrogenase which is endogenous to the tissue. The activity of glucose-6-phosphate dehydrogenase is greatly in excess of that of the kinase and was unaffected by the constituents of the kinase incubation medium (ATP, Mg²⁺ and NAD⁺) either alone or in combination. Kinase activity was dependent both on ATP and Mg²⁺, with maximal activity seen when the Mg-ATP ratio was between 1:1 and 4:1. Free ATP inhibited the activity of the enzyme. Enzyme activity was exhibited over a broad pH range (7–9) with a peak at pH 8.2. The sulphhydryl-blocking agents,p-chloromercuribenzoate, iodoacetate and iodoacetamide (at 1 mM), completely abolished kinase activity but were without effect on glucose-6-phosphate dehydrogenase activity.N-ethylmaleimide and citrate (both at 1 mM) had no effect on either kinase or glucose-6-phosphate dehydrogenase activities.     

Characterization of a mutation that causes overproduction of inositol in Neurospora crassa

Summary Slow-growing (inl ^+/-) spontaneous mutants have been isolated from an inositol requiring (inl) strain of Neurospora crassa that produces defective myo-inositol-1-phosphate synthase (MIPS), the enzyme responsible for the production of inositol-1-phosphate from glucose-6-phosphate. The defective enzyme has some residual activity. In the inl ^+/- strain the synthesis of the defective enzyme is enhanced, which enables the strain to grow slowly on minimal medium. The mutation (opi1) responsible for the partial inositol independence segregates independently from the inositol locus, and suppresses the inositolless character by overproduction of defective MIPS. opi1 acting upon the wild type (inl ⁺) allele increases MIPS production and causes inositol excretion.     

Hemoglobin and red cell enzyme variation in some populations of the Republic of Vietnam with comments on the malria hypothesis

The blood of Vietnamese, Khmer, Cham, Rhade, Sedang and Stieng populations of the Republic of Vietnam was studied for hemoglobins, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase and adenylate kinase by starch gel electrophoresis. Plasmodium falciparum malaria parasite rates were obtained in all groups but the Stieng. The prevalence of glucose-6-phosphate dehydrogenase deficiency was lowest in the Sedang (0.004), the Vietnamese (0.014) and the Rhade (0.023). The highest prevalence of glucose-6-phosphate dehydrogenase deficiency was found in the Stieng (0.153). The lowest frequencies of hemoglobin^E were in the Vietnamese (0.025) and the Sedang (0.029). All other groups had high frequencies of hemoglobin^E, the highest being the Stieng (0.365). The prevalence of phosphogluconate dehydrogenase^B ranged from 0.000 in the Stieng to 0.054 in the Vietnamese. The Vietnamese were not differentiated from the Sedang at the glucose-6-phosphate dehydrogenase or the hemoglobin loci but were differentiated at the phosphogluconate dehydrogenase locus. Using the three markers most of the populations studied in South Vietnam were distinguishable one from the other. There was variable correlation between the frequency of hemoglobin^E and the prevalence of glucose-6-phosphate dehydrogenase deficiency. A correlation of the endemicity of falciparum malaria and the frequency of glucose-6-phosphate dehydrogenase deficiency and of hemoglobin^E was inconclusive. The frequency of the adenylate kinase² allele was low to absent. Adenylate kinase³ was found in the Khmer and in the Stieng.     

Changes of some enzymatic activities in iodoacetate-treated microconidiating cultures of Neurospora crassa

In iodoacetate-treated microconidiating cultures of Neurospora crassa, mycelial yield, sucrose consumption and ethanol production are reduced. The specific activity of glyceraldehyde-3-phosphate dehydrogenase is sharply decreased while the specific activities of glucose-6-phosphate dehydrogenase and of 6-phosphogluconate dehydrogenase are stimulated. A polyphenoloxidase is induced in the microconidiating cultures.     

Presence of Nonoxidative Enzymes of the Pentose Phosphate Shunt in Tetrahymena*

SYNOPSIS. Tetrahymena pyriformis, strain HSM, do not have glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase, but contain transaldolase, transketolase, ribose 5-phosphate isomerase, ribulose-5-phosphate 3-epimerase, and ribokinase. The nonoxidative enzymes of the pentose phosphate shunt function in metabolism as indicated by the incorporation of label from [1-¹⁴C]ribose into CO₂ and glycogen and by the increase in total glycogen content of cultures supplemented with ribose.     

Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L

In the tricarboxylic acid (TCA) cycle, NADP⁺-specific isocitrate dehydrogenase (NADP⁺-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form α-ketoglutaric acid with NADP⁺ as a cofactor. We constructed an NADP⁺-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP⁺-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP⁺-ICDH activity. Therefore, NADP⁺-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering.     

Reduction of acetophenone to R(+)-phenylethanol by a new alcohol dehydrogenase from Lactobacillus kefir

Summary A new alcohol dehydrogenase catalysing the enantioselective reduction of acetophenone to R(+)-phenylethanol was found in a strain of Lactobacillus kefir. A 70-fold enrichment of the enzyme with an overall yield of 76% was obtained in two steps. The addition of Mg²⁺ ions was found to be necessary to prevent rapid deactivation. The enzyme depends essentially on NADPH and was inactive when supplied with NADH as the coenzyme. Important enzymological data of the dehydrogenase are: K _m (acetophenone) 0.6 mM, K _m (NADPH) 0.14 mM, and a pH optimum for acetophenone reduction at 7.0. Addition of EDTA leads to complete deactivation of the enzyme activity. Added iodoacetamide or p-hydroxymercuribenzoate cause only slight inhibition, revealing that the active centre of the enzyme contains no essential SH-group. Besides acetophenone several other aromatic and long-chain aliphatic secondary ketones are substrates for this enzyme. Batch production of phenylethanol was examined using three different methods for the regeneration of NADPH: glucose/glucose dehydrogenase, glucose-6-phosphate/glucose-6-phosphate dehydrogenase, and isopropanol.     

Characterization of a zwf mutant of Synechococcus sp. strain PCC 7942.

A mutant of the cyanobacterium Synechococcus sp. strain PCC 7942 carrying a disrupted gene encoding glucose-6-phosphate dehydrogenase (zwf) produced no detectable glucose-6-phosphate dehydrogenase as assessed by enzyme assay and Western blot (immunoblot) analysis. This mutant exhibited significantly impaired dark viability.