Production Of Alpha-galactosidase Research Articles

The inflammatory pathogenetic pathways of Fabry nephropathy

The high variability in clinical features and outcomes observed in monogenic diseases like Fabry disease suggests the presence of additional pathogenetic pathways beyond the lysosomal deposition of Gb3 and Lyso-GB3. Research indicates that the deposition of Gb3 and Lyso-Gb3 can stimulate the inflammatory processes. Mononuclear immune-competent cells exposed to Gb3 deposition exhibit surface adhesion molecules and release pro-inflammatory and fibrotic cytokines such as IL β, TNFα, and TGFβ, culminating in the activation of inflammatory cascades associated with oxidative stress, apoptotic mechanisms maintained by renal residents and infiltrating cells, leading to chronic inflammation and tissue fibrosis. Furthermore, in another avenue of inquiry (termed Agalopathy), the mutated galactosidase alpha gene can result in the production of an altered alpha-galactosidase A enzyme, inducing endoplasmic reticulum stress and triggering the unfolded protein response (UPR) in an effort to prevent the production of altered proteins. The UPR, in turn, instigates the release of pro-inflammatory cytokines, thereby contributing to the inflammatory milieu. Experimental findings have demonstrated that the pathogenetic mechanisms activated by Gb3 and Lyso Gb3 deposition can become independent from the initial stimulus and may exhibit limited responsiveness to therapy. Cellular pathway alterations can persist post-therapy or gene correction. Moreover, biochemical and histological lesions characteristic of Fabry disease manifest in the absence of Gb3 in the Zebrafish experimental model. This review endeavors to describe the role of these processes in Fabry nephropathy and aims to synthesize the available evidence on the pathogenesis of renal damage.

The role of podocyte injury in the pathogenesis of Fabry disease nephropathy.

Renal involvement is one of the most severe morbidities of Fabry disease (FD), a multisystemic lysosomal storage disease with an X-linked inheritance pattern. It results from pathogenic variants in the GLA gene (Xq22.2), which encodes the production of alpha-galactosidase A (α-Gal), responsible for glycosphingolipid metabolism. Insufficient activity of this lysosomal enzyme generates deposits of unprocessed intermediate substrates, especially globotriaosylceramide (Gb3) and derivatives, triggering cellular injury and subsequently, multiple organ dysfunction, including chronic nephropathy. Kidney injury in FD is classically attributed to Gb3 deposits in renal cells, with podocytes being the main target of the pathological process, in which structural and functional alterations are established early and severely. This configures a typical hereditary metabolic podocytopathy, whose clinical manifestations are proteinuria and progressive renal failure. Although late clinical outcomes and morphological changes are well established in this nephropathy, the molecular mechanisms that trigger and accelerate podocyte injury have not yet been fully elucidated. Podocytes are highly specialized and differentiated cells that cover the outer surface of glomerular capillaries, playing a crucial role in preserving the structure and function of the glomerular filtration barrier. They are frequent targets of injury in many nephropathies. Furthermore, dysfunction and depletion of glomerular podocytes are essential events implicated in the pathogenesis of chronic kidney disease progression. We will review the biology of podocytes and their crucial role in regulating the glomerular filtration barrier, analyzing the main pathogenic pathways involved in podocyte injury, especially related to FD nephropathy.

Enzymatic Reduction of Anti-nutritional Factors in Fermenting Soybeans by Lactobacillus plantarum Isolates from Fermenting Cereals

Soybean is rich in dietary protein but contains some anti-nutritional factors (ANFs), including phytates, tannins, trypsin inhibitors and oligosaccharides. It is used with cereals in weaning foods to improve the protein content and supply essential amino acids. The objective of this work, therefore, was to use microorganisms, specifically Lactobacillus plantarum and the enzymes it produces to reduce anti-nutritional factors and improve the nutritional composition of such food blends. Nine strains of Lactobacillus plantarum isolated from spontaneously fermenting cereals, identified and characterised, were selected based on the abundant production of alpha-galactosidase for the fermentation of the legume. Samples were subjected to fermentation for 5 days and the reduction of anti-nutritional factors was monitored. Anti-nutritional factors and alpha-galactosidase were determined by UV-spectrophotometry. Data were analysed using ANOVA at p = 0.05. Fermentation reduced the tannin content in the raw samples from 1.93 to 0.12 mg/g. Phytate content reduced from 1.16 to 0.04 mg/g. The trypsin inhibitor and protease inhibitor also reduced from 1.20 to 0.010 and 1.2 to 0.020 respectively. The production of alpha-galactosidase by L. plantarum (1.8 unit/ml) enhanced the reduction while the nutritional composition of the food blend in which the soybean was added improved significantly. The use of alpha-galactosidase enzyme by Lactobacillus plantarum from local food sources is thus shown to reduce anti-nutritional factors in soybeans, which can be of benefit in enhancing the nutritional quality of cereal-legume gruels.

Construction of the industrial ethanol-producing strain of Saccharomyces cerevisiae able to ferment cellobiose and melibiose

The gene mel1, encoding alpha-galactosidase in Schizosaccharomyces pombe, and the gene bgl2, encoding and beta-glucosidase in Trichoderma reesei, were isolated and co-expressed in the industrial ethanol-producing strain of Saccharomyces cerevisiae. The resulting strains were able to grow on cellobiose and melibiose through simultaneous production of sufficient extracellular alpha-galactosidase and beta-glucosidase activity. Under aerobic conditions, the growth rate of the recombinant strain GC 1 co-expressing 2 genes could achieve 0.29 OD600 h(-1) and a biomass yield up to 7.8 g l(-1) dry cell weight on medium containing 10.0 g l(-1) cellobiose and 10.0 g l(-1) melibiose as sole carbohydrate source. Meanwhile, the new strain of S. cerevisiae CG 1 demonstrated the ability to directly produce ethanol from microcrystalline cellulose during simultaneous saccharification and fermentation process. Approximately 36.5 g l(-1) ethanol was produced from 100 g of cellulose supplied with 5 g l(-1) melibose within 60 h. The yield (g of ethanol produced/g of carbohydrate consumed) was 0.44 g/g, which corresponds to 88.0% of the theoretical yield.

α-Galactosidase from Bacillus megaterium VHM1 and Its Application in Removal of Flatulence-Causing Factors from Soymilk

A bacterial strain capable of producing extracellular alpha-galactosidase was isolated from sugar cane industrial waste soil sample. Microbiological, physiological, and biochemical studies revealed that isolate belonged to Bacillus sp,. Furthermore, 16S rDNA sequence analysis of new isolates was identified as Bacillus megaterium VHM1. The production of alpha-galactosidase was optimized by various physical culture conditions. Guar gum and yeast extract acted as the best carbon and nitrogen source, respectively for the production of alpha-galactosidase. The enzyme showed an optimum pH at 7.5 and was stable over a pH between 5 and 9. The enzyme was optimally active in 55degreesC and the enzyme was thermostable with half life of 120 minutes at 55 degrees C and lost their 90%, residual activity in 120 minutes at 60 degrees C. alpha-Galactosidase was strongly inhibited by Ag2, Cu2, and Hg2+ at 1mM concentration. The metal ions Fe2, Mn2+, and Mg2+ had no effect on alpha-galactosidase activity, Zn2+,Ni2+, and Ca2+ reduced the enzyme activity slightly. The B megaterium VHM1 enzyme treatment completely hydrolyzed flatulence-causing sugars of soymilk within one and half hour.

High-Yield Production of Alpha-Galactosidase Excreted from Penicillium Chrysogenum and Aspergillus Niger

ABSTRACTα-Galactosidase production by the fungal strains Penicillium chrysogenum LN33 and Aspergillus niger M47 cultivated in shaking and stationary conditions using soya meal extract as nutrient medium was investigated. Submerged shake flask fermentation was preferred over stationary cultivation for P. chrysogenum LN33 as it gave a high α-galactosidase yield (4200U/l). Because of pellet formation the semicontinuous shake-flask fermentation was carried out for P. chrysogenum LN33 α-galactosidase production during 32 days. The maximum α-galactosidase activity by A. niger M47 (7000U/l) was obtained in stationary conditions of cultivation which is 27–28% greater as compared with the enzyme titer in shaking cultures. Air-lift laboratory bioreactor cultivation of both P. chrysogenum LN33 and A. niger M47 resulted in high-yield production of α-galactosidase (8700U/l and 11200U/l, respectively), which is of particular interest for biotechnological application in food processing.

Investigation on α-Galactosidase Production by Streptomyces griseoloalbus in a Forcefully Aerated Packed-Bed Bioreactor Operating in Solid-State Fermentation Condition

Solid-state fermentation (SSF) was carried out for the production of extracellular alpha-galactosidase by Streptomyces griseoloalbus. Soybean flour was the best solid substrate for alpha-galactosidase production. Packed-bed bioreactor performed well in enhancing the enzyme yield and resulted in a highest yield of 197.2 +/- 0.02 U/gds with a forced aeration of 2 vvm, which was approximately twofold the yield obtained in flasks. The alpha-galactosidase production was growth-associated, and the highest enzyme yield was obtained after 96 h of incubation. It is significant that this is the first report on alpha-galactosidase production by a filamentous bacterium in SSF using packed-bed bioreactor.

Evaluation of α-galactosidase biosynthesis by Streptomyces griseoloalbus in solid-state fermentation using response surface methodology

The present study was aimed at evaluating the effects of the three crucial factors, galactose concentration, inoculum size and moisture content, on alpha-galactosidase production by the filamentous actinobacterium Streptomyces griseoloalbus in solid-state fermentation. Central Composite design was adopted to derive a statistical model for the optimization of fermentation conditions. Maximum alpha-galactosidase yield (117 U g(-1) of dry fermented substrate) was obtained when soya bean flour supplemented with 1.5% galactose and with initial moisture content of 40% was inoculated with 1.9 x 10(6) CFU g(-1) initial dry substrate. The model was valid and could result in considerably enhanced enzyme yield. The results indicated a cost effective method for the production of alpha-galactosidase using soya bean flour. This is the first report on exploitation of the potential of filamentous bacterium for the production of alpha-galactosidase, an enzyme having versatile applications.

Effects of cultivation medium and conditions on the production of alpha-galactosidase by Aspergillus awamore & A/ carbonarius

The α-gal production by Aspergillus awamori and A. carbonarius (local isolates) as affected by several culture conditions was studied showing that 0.5 % corn bran and 2.0 % cane baggase were used for the production of α-gal by Aspergillus awamori & Aspergillus carbonarius respectively; the best pH value, incubation temperature and incubation period for the production of α-gal were 4.8, 30º C and 6 days for both experimental fungi; raffinose at the concentration 1 % supplemented with 0.5 % corn bran and acacia gum at concentration 1 % supplemented with 2 % cane baggase were the best synthetic carbon source for the production of α-gal for Aspergillus awamori and Aspergillus carbonarius respectively; L-Asparagine (0.262 %) was the best concentration of nitrogen source for Aspergillus awamori whereas sodium nitrate (0.4 %) was the best concentration of nitrogen source for Aspergillus carbonarius and the best ratio of surface area to volume of medium was 0.525 for Aspergillus carbonarius and 2.260 for Aspergillus carbonarius. Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 60-69

Response surface methodology for optimizing the fermentation medium of alpha-galactosidase in solid-state fermentation

Alpha-galactosidase is applied in food and feed industries for hydrolysing raffinose series oligosaccharides (RO) that are the factors primarily responsible for flatulence upon ingestion of soybean-derived products. The objective of the current work was to develop an optimal culture medium for the production of alpha-galactosidase in solid-state fermentation (SSF) by a mutant strain Aspergillus foetidus. Response surface methodology (RSM) was applied to evaluate the effects of variables, namely the concentrations of wheat bran, soybean meal, KH(2)PO(4), MnSO(4).H(2)O and CuSO(4).5H(2)O on alpha-galactosidase production in the solid substrate. A fractional factorial design (FFD) was firstly used to isolate the main factors that affected the production of alpha-galactosidase and the central composite experimental design (CCD) was then adopted to derive a statistical model for optimizing the composition of the fermentation medium. The experimental results showed that the optimum fermentation medium for alpha-galactosidase production by Aspergillus foetidus ZU-G1 was composed of 8.2137 g wheat bran, 1.7843 g soybean meal, 0.001 g MnSO(4).H(2)O and 0.001 g CuSO(4).5H(2)O in 10 g dry matter fermentation medium. After incubating 96 h in the optimum fermentation medium, alpha-galactosidase activity was predicted to be 2210.76 U g(-1) dry matter in 250 ml shake flask. In the present study, alpha-galactosidase activity reached 2207.19 U g(-1) dry matter. Optimization of the solid substrate was a very important measure to increase enzyme activity and realize industrial production of alpha-galactosidase. The process of alpha-galactosidase production in laboratory scale may have the potential to scale-up.

Optimization of the Fermentation Medium for Alpha‐Galactosidase Production from Aspergillus foetidus ZU‐G1 Using Response Surface Methodology

The optimization of fermentation medium for alpha-galactosidase production by Aspergillus foetidus ZU-G1 was investigated in shaker flask fermentation. A one-factor-at-a-time experiment was used to screen the preferable nutriment (carbon sources, nitrogen sources, and essential elements) for alpha-galactosidase production. A fractional factorial design was used to screen the main 5 factors, soybean meal, wheat bran, KH2PO4, FeSO4 x 7 H2O, and the medium initial pH, that affected the production of alpha-galactosidase. The central composite experimental design was further adopted to derive a statistical model for optimizing the composition of the fermentation medium. The experimental results showed that the optimum fermentation medium for alpha-galactosidase production by Aspergillus foetidus ZU-G1 was composed of 3.2% soybean meal (w/v), 2% wheat bran (w/v), 0.1% KH2PO4 (w/v), and 0.05% FeSO4 x 7 H2O (w/v); initial medium pH was 6.31. The results further predicted that alpha-galactosidase activity reached 64.75 U/mL after 96-h incubation in this medium, which was approximately 7 times higher than that incubated in the nonoptimized medium. The time course of alpha-galactosidase production in the optimized medium composition was also carried out to validate the model.

Production of a Thermoresistant Alpha-galactosidase from Thermus sp. Strain T2 for Food Processing

The gene of a thermostable alpha-galactosidase, aglA, from Thermus sp. strain T2 was sequenced, cloned and overexpressed in Escherichia coli (strain MC 2508. The purified enzyme proved to be quite thermostable, retaining high levels of activity even after incubation at 70°C. The optimal T was 65°C at pH 7. The highest activity was achieved at acid pH values, although good activity could be obtained in a broad pH range. Enzyme stability depends on the enzyme concentration at alkaline pH values, suggesting that under these conditions the subunit dissociation may be the first step in the inactivation of the enzyme. However, at pH 5 the enzyme stability becomes independent of the enzyme concentration. The enzyme also exhibited a quite broad specificity, although it shows the best activity with alpha derivatives of galactose, and was able to recognize very different substrates (alpha derivatives of mannose, xylose and maltose, and even beta galactose derivatives). There was no detectable activity against glucose derivatives.

Adenovirus-Transduced Lung as a Portal for Delivering α-Galactosidase A into Systemic Circulation for Fabry Disease

Gene therapy efforts have focused primarily on the use of either the liver or skeletal muscle as depot organs for the production of a variety of therapeutic proteins that act systemically. Here we examined the lung to determine whether it could function as yet another portal for the secretion of proteins into the circulation. Fabry disease is caused by a deficiency of the lysosomal hydrolase alpha-galactosidase A, resulting in the abnormal deposition of the glycosphingolipid globotriaosylceramide (GL-3) in vascular lysosomes. Pulmonary instillation of a recombinant adenoviral vector (Ad2/CMVHI-alpha(gal)) encoding human alpha-galactosidase A into Fabry mice resulted in high-level transduction and expression of the enzyme in the lung. Importantly, enzymatic activity was also detected in the plasma, liver, spleen, heart, and kidneys of the Fabry mice. The detection of enzymatic activity outside of the lung, along with the finding that viral DNA was limited to the lung, indicates that the enzyme crossed the air/blood barrier, entered the systemic circulation, and was internalized by the distal visceral organs. The levels of alpha-galactosidase A attained in these tissues were sufficient to reduce GL-3 to basal levels in the lung, liver, and spleen and to approximately 50% of untreated levels in the heart. Together, these results suggest that the lung may be a viable alternate depot organ for the production and systemic secretion of alpha-galactosidase A for Fabry disease.

Expression and characterization of glycosylated and catalytically active recombinant human alpha-galactosidase A produced in Pichia pastoris.

Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the lysosomal enzyme alpha-galactosidase A. This enzyme is responsible for the hydrolysis of terminal alpha-galactoside linkages in various glycolipids. An improved method of production of recombinant alpha-galactosidase A for use in humans is needed in order to develop new approaches for enzyme therapy. Human alpha-galactosidase A for use in enzyme therapy has previously been obtained from human sources and from recombinant clones derived from human cells, CHO cells, and insect cells. In this report we describe the construction of clones of the methylotrophic yeast Pichia pastoris that produce recombinant human alpha-galactosidase A. Recombinant human alpha-galactosidase A is secreted by these Pichia clones and the level of production is more than 30-fold greater than that of previously used methods. Production was optimized using variations in temperature, pH, cDNA copy number, and other variables using shake flasks and a bioreactor. Expression of the human enzyme increased with increasing cDNA copy number at 25 degrees C, but not at the standard growth temperature of 30 degrees C. The recombinant alpha-galactosidase A was purified to homogeneity using ion exchange (POROS 20 CM, POROS 20 HQ) and hydrophobic (Toso-ether, Toso-butyl) chromatography with a BioCAD HPLC Workstation. Purified recombinant alpha-galactosidase A was taken up by fibroblasts derived from Fabry disease patients and normal enzyme levels could be restored under these conditions. Analysis of the carbohydrate present on the recombinant enzyme indicated the predominant presence of N-linked high-mannose structures rather than complex carbohydrates.

Use of Plackett-Burman design for rapid screening of several nitrogen sources, growth/product promoters, minerals and enzyme inducers for the production of alpha-galactosidase by Aspergillus niger MRSS 234 in solid state fermentation system

Five sources of nitrogen, six minerals, six enzyme inducers and one each of growth as well as product promotors were screened by Plackett-Burman design, consisting of a total of 20 experiments for the above 19 sources/categories of medium ingredients, for their effect on the production of alpha-galactosidase by Aspergillus niger MRSS 234 in solid state fermentation system. The enzyme production was recorded from 2 to 5 days of fermentation. Data on enzyme titres was analysed by compatible analysis to obtain regression coefficients and t-ratios. Among the nitrogen sources, urea contributed positively to enzyme production and its effect increased with the fermentation time, in contrast to negative effect of all the ammonium salts used. Corn steep liquor, citric acid and legume seed flours showed significantly positive effects on enzyme production, though lactose showed negative effect upto 3 days of fermentation and then turned positive but not significantly. Calcium chloride and ferrous sulphate showed considerable negative effect, in contrast to mixed effect by other mineral salts studied. Among the legume seed flours, guar and French bean flours showed larger positive effects. The studies allowed the selection of urea, corn steep liquor, guar flour, soy bean flour and citric acid as most promising sources/categories for further optimization studies based on the effects as well as their trend with fermentation time. The use of Plackett-Burman design for rapid screening of large number of nutrients, in a very small number of experiments, for reliable short-listing of a few of most effective sources/categories for further optimization, has been scarce in submerged fermentation and never attempted earlier in solid state fermentation system.

Use of Plackett–Burman design for rapid screening of several nitrogen sources, growth/product promoters, minerals and enzyme inducers for the production of alpha-galactosidase by Aspergillus niger MRSS 234 in solid state fermentation system

Use of Plackett–Burman design for rapid screening of several nitrogen sources, growth/product promoters, minerals and enzyme inducers for the production of alpha-galactosidase by Aspergillus niger MRSS 234 in solid state fermentation system

Comparative study on the production of guar alpha-galactosidase by Saccharomyces cerevisiae SU50B and Hansenula polymorpha 8/2 in continuous cultures

Saccharomyces cerevisiae SU50B and Hansenula polymorpha 8/2, both carrying a multicopy integrated guar alpha-galactosidase, have been cultivated in continuous cultures, using various mixtures of carbon sources and cultivation conditions. Both S. cerevisiae SU50B and H. polymorpha 8/2 are stable and produce high levels of extracellular alpha-galactosidase in continuous cultures for more than 500 h. For these expression systems the strong inducible promoter systems GAL7 and methanol oxidase, respectively, were used. The induction of alpha-galactosidase synthesis by galactose in SU50B is limited by the low galactose uptake. Apart from that, at high dilution rates, the glucose repression is substantial, and a maximal expression level of 28.6 mg of extracellular alpha-galactosidase.g (dry weight) of biomass-1 could be obtained. In H. polymorpha, the induction of alpha-galactosidase synthesis, in addition to methanol oxidase synthesis using formaldehyde, is very effective up to 42 mg of extracellular alpha-galactosidase.g (dry weight) of biomass-1. Productivities in terms of specific production rate enable a good comparison with those of other heterologous expression systems in the literature. The productivities found with S. cerevisiae SU50B and H. polymorpha, 3.25 and 5.5 mg of alpha-galactosidase.g of biomass-1.liter-1.h-1, respectively, rank among the highest reported in the literature. Enzyme production and secretion in H. polymorpha are more complex. A two-peaked optimum is found in enzyme production. No clear explanation of this phenomenon can be given.

Regulation of basal and induced levels of the MEL1 transcript in Saccharomyces cerevisiae.

The MEL1 gene in Saccharomyces cerevisiae is required for the production of alpha-galactosidase and for the catabolism of melibiose. Production of alpha-galactosidase is induced by galactose or melibiose and repressed by glucose. Inducibility is controlled by the positive and negative regulatory proteins GAL4 and GAL80, respectively. We have cloned the MEL1 gene to study its transcriptional expression and regulation. Evidence is presented that the MEL1 gene encodes alpha-galactosidase and that mel0 is a naturally occurring allele which lacks the alpha-galactosidase-coding sequences. RNAs prepared from wild-type cells and from cells carrying either the noninducible gal4-2 or GAL80S-100 allele grown on three different carbon sources were examined by Northern hybridization analyses. In wild-type cells under noninducing conditions, such as growth on glycerol-lactic acid, the MEL1 transcript was detected at a basal level which was 1 to 2% of the fully induced level. The basal level of expression was diminished in cells carrying the gal4-2 mutant allele but not in cells carrying the GAL80S-100 allele. The basal and induced RNA levels are repressed by glucose. Size determinations of the MEL1 transcripts detected in glycerol-lactic acid- and galactose-grown cells provided no evidence for two distinct transcripts.

Regulation of basal and induced levels of the MEL1 transcript in Saccharomyces cerevisiae.

The MEL1 gene in Saccharomyces cerevisiae is required for the production of alpha-galactosidase and for the catabolism of melibiose. Production of alpha-galactosidase is induced by galactose or melibiose and repressed by glucose. Inducibility is controlled by the positive and negative regulatory proteins GAL4 and GAL80, respectively. We have cloned the MEL1 gene to study its transcriptional expression and regulation. Evidence is presented that the MEL1 gene encodes alpha-galactosidase and that mel0 is a naturally occurring allele which lacks the alpha-galactosidase-coding sequences. RNAs prepared from wild-type cells and from cells carrying either the noninducible gal4-2 or GAL80S-100 allele grown on three different carbon sources were examined by Northern hybridization analyses. In wild-type cells under noninducing conditions, such as growth on glycerol-lactic acid, the MEL1 transcript was detected at a basal level which was 1 to 2% of the fully induced level. The basal level of expression was diminished in cells carrying the gal4-2 mutant allele but not in cells carrying the GAL80S-100 allele. The basal and induced RNA levels are repressed by glucose. Size determinations of the MEL1 transcripts detected in glycerol-lactic acid- and galactose-grown cells provided no evidence for two distinct transcripts.

Genetic co-regulation of galactose and melibiose utilization in Saccharomyces.

The gal3 mutation of Saccharomyces, which is associated with an impairment in the utilization of galactose, has been shown to be pleiotropic, causing similar impairments in the utilization of melibiose and maltose. Milibiose utilization and alpha-galactosidase production are directly controlled by the galactose regulatory elements i, c, and GAL4. The fermentation of maltose and the induction of alpha-glucosidase are regulated independently of the i, c, GAL4 system. The production of alpha-galactosidase and galactose-1-phosphate uridyl transferase is coordinate in galactokinaseless strains. Galactose serves as a nonmetabolized, gratuitous inducer of alpha-galactosidase in strains lacking the genes for one or more of the Leloir pathway enzymes.