Optimization of production conditions of rice α-galactosidase II displayed on yeast cell surface.

The yeast surface displayed rice α-galactosidase II (YSD rice α-Gal II) was generated with the pYD1 vector. The expression and cultural conditions for the improvement of production of YSD rice α-Gal II were optimized. The results showed that several induction factors, which were the initial cell density, inoculation ratio, galactose (inducer) concentration, induction time and temperature, determined the activity and expression efficiency of YSD rice α-Gal II. Meanwhile, the medium composition also affected its activity and production. Moreover, the production of YSD rice α-Gal II was further improved by continuous feeding of galactose in the fermenter level. The highest production was obtained at an initial cell density of OD600 = 2.9, 2% inoculation ratio, and 2% galactose, with 0.6 g/L compound nitrogen source ((NH4)2SO4/urea = 2/1, w/w) and 5 g/L sucrose, followed by continuous feeding of galactose (20 g/L with flow rate of 1.5 mL/h). At such conditions, the enzyme activity and productivity reached to 676.2 U/g (DCW) and 1548.5 U/L, respectively, 26.4- and 63.7-fold to that before optimization. The results provided a basic and effective strategy for the industrial production of YSD rice α-Gal II.

Aspergillus niger production of pectinase and α-galactosidase for enzymatic soy processing.

Soybean is a most promising sustainable protein source for feed and food to help meet the protein demand of the rapidly rising global population. To enrich soy protein, the environment-friendly enzymatic processing requires multiple carbohydrases including cellulase, xylanase, pectinase, α-galactosidase and sucrase. Besides enriched protein, the processing adds value by generating monosaccharides that are ready feedstock for biofuel/bioproducts. Aspergillus could produce the required carbohydrases, but with deficient pectinase and α-galactosidase. Here we address this critical technological gap by focused evaluation of the suboptimal productivity of pectinase and α-galactosidase. A carbohydrases-productive strain A. niger (NRRL 322) was used with soybean hull as inducing substrate. Temperatures at 20 °C, 25 °C and 30 °C were found to affect cell growth on sucrose with an Arrhenius-law activation energy of 28.7 kcal/mol. The 30 °C promoted the fastest cell growth (doubling time = 2.1 h) and earliest enzyme production, but it gave lower final enzyme yield due to earlier carbon-source exhaustion. The 25 °C gave the highest enzyme yield. pH conditions also strongly affected enzyme production. Fermentations made with initial pH of 6 or 7 were most productive, e.g., giving 1.9- to 2.3-fold higher pectinase and 2.2- to 2.3-fold higher α-galactosidase after 72 h, compared to the fermentation with a constant pH 4. Further, pH must be kept above 2.6 to avoid limitation in pectinase production and, in the later substrate-limiting stage, kept below 5.5 to avoid pectinase degradation. α-Galactosidase production always followed the pectinase production with a 16-24 h lag; presumably, the former relied on pectin hydrolysis for inducers generation. Optimal enzyme production requires controlling the transient availability of inducers.

Microbial production and biotechnological applications of α-galactosidase.

α-Galactosidase, (E.C. 3.2.1.22) is an exoglycosidase that target galactooligosaccharides such as raffinose, melibiose, stachyose and branched polysaccharides like galactomannans and galacto-glucomannans by catalysing the hydrolysis of α-1,6 linked terminal galactose residues. The enzyme has been isolated and characterized from microbial, plant and animal sources. This ubiquitous enzyme possesses physiological significance and immense industrial potential. Optimization of the growth conditions and efficient purification strategies can lead to a significant increase in the enzyme production. To boost commercial productivity, cloning of novel α-galactosidase genes and their heterologous expression in suitable host has gained popularity. Enzyme immobilization leads to its greater reutilization, superior thermostability, pH tolerance and increased activity. The enzyme is well explored in food industry in the removal of raffinose family oligosaccharides (RFOs) in soymilk and sugar crystallization process. It also improves animal feed quality and biomass processing. Applications of the enzyme is in the area of biomedicine includes therapeutic advances in treatment of Fabry disease, blood group conversion and removal of α-gal type immunogenic epitopes in xenotransplantation. With considerable biotechnological applications, this enzyme has been vastly commercialized and holds greater future prospects.

Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides.

BACKGROUND: α-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of α-galactosidase of Saccharomyces cerevisiae. RESULTS: The MEL1 gene coding for the α-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-α-D-galactopyranoside than for natural substrates, followed by the natural glycosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with ß-mannosidase. Furthermore, this enzyme strongly resists proteases, and shows remarkable activation in their presence. Hydrolysis of galactose bonds linked to terminal non-reducing mannose residues of synthetic galactomannan-oligosaccharides confirms that ScAGal belongs to the first group of α-galactosidases, according to substrate specificity. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h. CONCLUSIONS: ScAGal characteristics and improvement in productivity that have been achieved contribute in making ScAGal a good candidate for application in the elimination of raffinose family oligosaccharides found in many products of the food industry.

Inhibition of Mitochondrial Complex I Impairs Release of α-Galactosidase by Jurkat Cells.

Fabry disease (FD) is caused by mutations in the GLA gene that encodes lysosomal α-galactosidase-A (α-gal-A). A number of pathogenic mechanisms have been proposed and these include loss of mitochondrial respiratory chain activity. For FD, gene therapy is beginning to be applied as a treatment. In view of the loss of mitochondrial function reported in FD, we have considered here the impact of loss of mitochondrial respiratory chain activity on the ability of a GLA lentiviral vector to increase cellular α-gal-A activity and participate in cross correction. Jurkat cells were used in this study and were exposed to increasing viral copies. Intracellular and extracellular enzyme activities were then determined; this in the presence or absence of the mitochondrial complex I inhibitor, rotenone. The ability of cells to take up released enzyme was also evaluated. Increasing transgene copies was associated with increasing intracellular α-gal-A activity but this was associated with an increase in Km. Release of enzyme and cellular uptake was also demonstrated. However, in the presence of rotenone, enzyme release was inhibited by 37%. Excessive enzyme generation may result in a protein with inferior kinetic properties and a background of compromised mitochondrial function may impair the cross correction process.

Production of Therapeutic Enzymes by Lentivirus Transgenesis.

Since ERT for several LSDs treatment has emerged at the beginning of the 1980s with Orphan Drug approval, patients' expectancy and life quality have been improved. Most LSDs treatment are based on the replaced of mutated or deficient protein with the natural or recombinant protein.One of the main ERT drawback is the high drug prices. Therefore, different strategies trying to optimize the global ERT biotherapeutic production have been proposed. LVs, a gene delivery tool, can be proposed as an alternative method to generate stable cell lines in manufacturing of recombinant proteins. Since LVs have been used in human gene therapy, clinical trials, safety testing assays and procedures have been developed. Moreover, one of the main advantages of LVs strategy to obtain manufacturing cell line is the short period required as well as the high protein levels achieved.In this chapter, we will focus on LVs as a recombinant protein production platform and we will present a case study that employs LVs to express in a manufacturing cell line, alpha-Galactosidase A (rhαGAL), which is used as ERT for Fabry disease treatment.

Improved Efficacy in a Fabry Disease Model Using a Systemic mRNA Liver Depot System as Compared to Enzyme Replacement Therapy.

Fabry disease is a lysosomal storage disorder caused by the deficiency of α-galactosidase A. Enzyme deficiency results in a progressive decline in renal and cardiac function, leading to cardiomyopathy and end-stage renal disease. Current treatments available, including enzyme replacement therapies, have provided significant benefit to patients; however, unmet medical needs remain. mRNA therapy, with drug-like properties, has the unique ability to produce therapeutic proteins endogenously. Here we describe the sustained delivery of therapeutic human α-galactosidase protein in vivo via nanoparticle-formulated mRNA in mouse and non-human primate, with a demonstration of efficacy through clinically relevant biomarker reduction in a mouse Fabry disease model. Multi-component nanoparticles formulated with lipids and lipid-like materials were developed for the delivery of mRNA encoding human α-galactosidase protein. Upon delivery of human GLA mRNA to mice, serum GLA protein levels reached as high as ∼1,330-fold over normal physiological values.

Priapism in a Fabry disease mouse model is associated with upregulated penile nNOS and eNOS expression.

Fabry disease is a glycosphingolipidosis caused by deficient activity of α-galactosidase A; it is one of a few diseases that are associated with priapism, an abnormal prolonged erection of the penis. The goal of this study was to investigate the pathogenesis of Fabry disease-associated priapism in a mouse model of the disease. We found that Fabry mice develop late-onset priapism. Neuronal nitric oxide synthase (nNOS), which was predominantly present as the 120-kDa N-terminus-truncated form, was significantly upregulated in the penis of 18-month-old Fabry mice compared to wild type controls (~fivefold). Endothelial NOS (eNOS) was also upregulated (~twofold). NO level in penile tissues of Fabry mice was significantly higher than wild type controls at 18 months. Gene transfer-mediated enzyme replacement therapy reversed abnormal nNOS expression in the Fabry mouse penis. The penile nNOS level was restored by antiandrogen treatment, suggesting that hyperactive androgen receptor signaling in Fabry mice may contribute to nNOS upregulation. However, the phosphodiesterase-5A expression level and the adenosine content in the penis, which are known to play roles in the development of priapism in other etiologies, were unchanged in Fabry mice. In conclusion, these data suggested that increased nNOS (and probably eNOS) content and the consequential elevated NO production and high arterial blood flow in the penis may be the underlying mechanism of priapism in Fabry mice. Furthermore, in combination with previous findings, this study suggested that regulation of NOS expression is susceptible to α-galactosidase A deficiency, and this may represent a general pathogenic mechanism of Fabry vasculopathy.

A Grapevine-Inducible Gene Vv-α-gal/SIP Confers Salt and Desiccation Tolerance in Escherichia coli and Tobacco at Germinative Stage.

Grapevine is an important fruit crop cultivated worldwide. Previously, we have reported the characterization of a salt stress-inducible gene Vv-α-gal/SIP isolated from the tolerant grapevine cultivar Razegui. In this study, we performed functional studies in both Escherichia coli and tobacco systems to gain more insights in the role of the Vv-α-gal/SIP gene. Our data revealed that the recombinant E. coli cells harboring the pET24b+ expression vector with the Vv-α-gal/SIP showed higher tolerance to desiccation and salinity compared to E. coli cells harboring the vector alone. In addition, the transgenic tobacco plants expressing the Vv-α-gal/SIP gene exhibited a higher percentage of seed germination and better growth under salt stress than the wild-type (WT) tobacco seedlings. This stress mitigation might be related to the putative function of this gene, which is thought to be involved in carbohydrate metabolism regulation. Collectively, these results suggest that Vv-α-gal/SIP is potentially a candidate gene for engineering drought and salt tolerance in cultivated plants.

Ionizing radiation reduces ADAM10 expression in brain microvascular endothelial cells undergoing stress-induced senescence.

Cellular senescence is associated with aging and is considered a potential contributor to age-associated neurodegenerative disease. Exposure to ionizing radiation increases the risk of developing premature neurovascular degeneration and dementia but also induces premature senescence. As cells of the cerebrovascular endothelium are particularly susceptible to radiation and play an important role in brain homeostasis, we investigated radiation-induced senescence in brain microvascular endothelial cells (EC). Using biotinylation to label surface proteins, streptavidin enrichment and proteomic analysis, we analyzed the surface proteome of stress-induced senescent EC in culture. An array of both recognized and novel senescence-associated proteins were identified. Most notably, we identified and validated the novel radiation-stimulated down-regulation of the protease, a disintegrin and metalloprotease 10 (ADAM10). ADAM10 is an important modulator of amyloid beta protein production, accumulation of which is central to the pathologies of Alzheimer's disease and cerebral amyloid angiopathy. Concurrently, we identified and validated increased surface expression of ADAM10 proteolytic targets with roles in neural proliferation and survival, inflammation and immune activation (L1CAM, NEO1, NEST, TLR2, DDX58). ADAM10 may be a key molecule linking radiation, senescence and endothelial dysfunction with increased risk of premature neurodegenerative diseases normally associated with aging.

Tetrahydrobiopterin deficiency in the pathogenesis of Fabry disease.

Fabry disease is caused by deficient activity of α-galactosidase A and subsequent accumulation of glycosphingolipids (mainly globotriaosylceramide, Gb3), leading to multisystem organ dysfunction. Oxidative stress and nitric oxide synthase (NOS) uncoupling are thought to contribute to Fabry cardiovascular diseases. We hypothesized that decreased tetrahydrobiopterin (BH4) plays a role in the pathogenesis of Fabry disease. We found that BH4 was decreased in the heart and kidney but not in the liver and aorta of Fabry mice. BH4 was also decreased in the plasma of female Fabry patients, which was not corrected by enzyme replacement therapy (ERT). Gb3 levels were inversely correlated with BH4 levels in animal tissues and cultured patient cells. To investigate the role of BH4 deficiency in disease phenotypes, 12-month-old Fabry mice were treated with gene transfer-mediated ERT or substrate reduction therapy (SRT) for 6 months. In the Fabry mice receiving SRT but not ERT, BH4 deficiency was restored, concomitant with ameliorated cardiac and renal hypertrophy. Additionally, glutathione levels were decreased in Fabry mouse tissues in a sex-dependent manner. Renal BH4 levels were closely correlated with glutathione levels and inversely correlated with cardiac and kidney weight. In conclusion, this study showed that BH4 deficiency occurs in Fabry disease and may contribute to the pathogenesis of the disease through oxidative stress associated with a reduced antioxidant capacity of cells and NOS uncoupling. This study also suggested dissimilar efficacy of ERT and SRT in correcting pre-existing pathologies in Fabry disease.

Improving the Secretory Expression of an -Galactosidase from Aspergillus niger in Pichia pastoris.

α-Galactosidases are broadly used in feed, food, chemical, pulp, and pharmaceutical industries. However, there lacks a satisfactory microbial cell factory that is able to produce α-galactosidases efficiently and cost-effectively to date, which prevents these important enzymes from greater application. In this study, the secretory expression of an Aspergillus niger α-galactosidase (AGA) in Pichia pastoris was systematically investigated. Through codon optimization, signal peptide replacement, comparative selection of host strain, and saturation mutagenesis of the P1' residue of Kex2 protease cleavage site for efficient signal peptide removal, a mutant P. pastoris KM71H (Muts) strain of AGA-I with the specific P1' site substitution (Glu to Ile) demonstrated remarkable extracellular α-galactosidase activity of 1299 U/ml upon a 72 h methanol induction in 2.0 L fermenter. The engineered yeast strain AGA-I demonstrated approximately 12-fold higher extracellular activity compared to the initial P. pastoris strain. To the best of our knowledge, this represents the highest yield and productivity of a secreted α-galactosidase in P. pastoris, thus holding great potential for industrial application.

Development of a model system for neuronal dysfunction in Fabry disease.

Fabry disease is a glycosphingolipid storage disorder that is caused by a genetic deficiency of the enzyme alpha-galactosidase A (AGA, EC 3.2.1.22). It is a multisystem disease that affects the vascular, cardiac, renal, and nervous systems. One of the hallmarks of this disorder is neuropathic pain and sympathetic and parasympathetic nervous dysfunction. The exact mechanism by which changes in AGA activity result in change in neuronal function is not clear, partly due to of a lack of relevant model systems. In this study, we report the development of an in vitro model system to study neuronal dysfunction in Fabry disease by using short-hairpin RNA to create a stable knock-down of AGA in the human cholinergic neuronal cell line, LA-N-2. We show that gene-silenced cells show specifically reduced AGA activity and store globotriaosylceramide. In gene-silenced cells, release of the neurotransmitter acetylcholine is significantly reduced, demonstrating that this model may be used to study specific neuronal functions such as neurotransmitter release in Fabry disease.

Kinetic properties and substrate inhibition of α-galactosidase from Aspergillus niger.

The recombinant AglB produced by Pichia pastoris exhibited substrate inhibition behavior for the hydrolysis of p-nitrophenyl α-galactoside, whereas it hydrolyzed the natural substrates, including galactomanno-oligosaccharides and raffinose family oligosaccharides, according to the Michaelian kinetics. These contrasting kinetic behaviors can be attributed to the difference in the dissociation constant of second substrate from the enzyme and/or to the ability of the leaving group of the substrates. The enzyme displays the grater kcat/Km values for hydrolysis of the branched α-galactoside in galactomanno-oligosaccharides than that of raffinose and stachyose. A sequence comparison suggested that AglB had a shallow active-site pocket, and it can allow to hydrolyze the branched α-galactosides, but not linear raffinose family oligosaccharides.

Production of a Highly Protease-Resistant Fungal α-Galactosidase in Transgenic Maize Seeds for Simplified Feed Processing.

Raffinose-family oligosaccharide (RFO) in soybeans is one of the major anti-nutritional factors for poultry and livestocks. α-Galactosidase is commonly supplemented into the animal feed to hydrolyze α-1,6-galactosidic bonds on the RFOs. To simplify the feed processing, a protease-resistant α-galactosidase encoding gene from Gibberella sp. strain F75, aga-F75, was modified by codon optimization and heterologously expressed in the embryos of transgentic maize driven by the embryo-specific promoter ZM-leg1A. The progenies were produced by backcrossing with the commercial inbred variety Zheng58. PCR, southern blot and western blot analysis confirmed the stable integration and tissue specific expression of the modified gene, aga-F75m, in seeds over four generations. The expression level of Aga-F75M reached up to 10,000 units per kilogram of maize seeds. In comparison with its counterpart produced in Pichia pastoris strain GS115, maize seed-derived Aga-F75M showed a lower temperature optimum (50 °C) and lower stability over alkaline pH range, but better thermal stability at 60 °C to 70 °C and resistance to feed pelleting inactivation (80 °C). This is the first report of producing α-galactosidase in transgenic plant. The study offers an effective and economic approach for direct utilization of α-galactosidase-producing maize without any purification or supplementation procedures in the feed processing.

Strategies for the production of difficult-to-express full-length eukaryotic proteins using microbial cell factories: production of human alpha-galactosidase A.

Obtaining high levels of pure proteins remains the main bottleneck of many scientific and biotechnological studies. Among all the available recombinant expression systems, Escherichia coli facilitates gene expression by its relative simplicity, inexpensive and fast cultivation, well-known genetics and the large number of tools available for its biotechnological application. However, recombinant expression in E. coli is not always a straightforward procedure and major obstacles are encountered when producing many eukaryotic proteins and especially membrane proteins, linked to missing posttranslational modifications, proteolysis and aggregation. In this context, many conventional and unconventional eukaryotic hosts are under exploration and development, but in some cases linked to complex culture media or processes. In this context, alternative bacterial systems able to overcome some of the limitations posed by E. coli keeping the simplicity of prokaryotic manipulation are currently emerging as convenient hosts for protein production. We have comparatively produced a "difficult-to-express" human protein, the lysosomal enzyme alpha-galactosidase A (hGLA) in E. coli and in the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 cells (P. haloplanktis TAC125). While in E. coli the production of active hGLA was unreachable due to proteolytic instability and/or protein misfolding, the expression of hGLA gene in P. haloplanktis TAC125 allows obtaining active enzyme. These results are discussed in the context of emerging bacterial systems for protein production that represent appealing alternatives to the regular use of E. coli and also of more complex eukaryotic systems.

Role of feed forward neural networks coupled with genetic algorithm in capitalizing of intracellular alpha-galactosidase production by Acinetobacter sp.

Alpha-galactosidase production in submerged fermentation by Acinetobacter sp. was optimized using feed forward neural networks and genetic algorithm (FFNN-GA). Six different parameters, pH, temperature, agitation speed, carbon source (raffinose), nitrogen source (tryptone), and K2HPO4, were chosen and used to construct 6-10-1 topology of feed forward neural network to study interactions between fermentation parameters and enzyme yield. The predicted values were further optimized by genetic algorithm (GA). The predictability of neural networks was further analysed by using mean squared error (MSE), root mean squared error (RMSE), mean absolute error (MAE), mean absolute percentage error (MAPE), and R2-value for training and testing data. Using hybrid neural networks and genetic algorithm, alpha-galactosidase production was improved from 7.5 U/mL to 10.2 U/mL.

A novel promising strain of Trichoderma evansii (WF-3) for extracellular α-galactosidase production by utilizing different carbon sources under optimized culture conditions.

A potential fungal strain of Trichoderma sp. (WF-3) was isolated and selected for the production of α-galactosidase. Optimum conditions for mycelial growth and enzyme induction were determined. Basal media selected for the growth of fungal isolate containing different carbon sources like guar gum (GG), soya bean meal (SM), and wheat straw (WS) and combinations of these carbon substrates with basic sugars like galactose and sucrose were used to monitor their effects on α-galactosidase production. The results of this study indicated that galactose and sucrose enhanced the enzyme activity in guar gum (GG) and wheat straw (WS). Maximum α-galactosidase production (213.63 U mL(-1)) was obtained when the basic medium containing GG is supplemented with galactose (5 mg/mL). However, the presence of galactose and sucrose alone in the growth media shows no effect. Soya meal alone was able to support T. evansii to produce maximum enzyme activity (170.36 U mL(-1)). The incubation time, temperature, and pH for the maximum enzyme synthesis were found to be 120 h (5 days), 28°C, and 4.5-5.5, respectively. All the carbon sources tested exhibited maximum enzyme production at 10 mg/mL concentration. Among the metal ions tested, Hg was found to be the strongest inhibitor of the enzyme. Among the chelators, EDTA acted as stronger inhibitor than succinic acid.

A new α-galactosidase from thermoacidophilic Alicyclobacillus sp. A4 with wide acceptor specificity for transglycosylation.

An α-galactosidase gene (gal36A4) of glycosyl hydrolase family 36 was identified in the genome of Alicyclobacillus sp. A4. It contains an ORF of 2,187 bp and encodes a polypeptide of 728 amino acids with a calculated molecular mass of 82.6 kDa. Deduced Gal36A4 shows the typical GH36 organization of three domains--the N-terminal ß-sheets, the catalytic (ß/α)8-barrels, and the C-terminal antiparallel ß-sheet. The gene product was produced in Escherichia coli and showed both hydrolysis and transglycosylation activities. The optimal pH for hydrolysis activity was 6.0, and a stable pH range of 5.0-11.0 was found. The enzyme had a temperature optimum of 60 °C. It is specific for α-1,6-glycosidic linkages and had a K m value of 1.45 mM toward pNPGal. When using melibiose as both donor and acceptor of galactose, Gal36A4 showed the transfer ratio of 23.25 % at 96 h. With respect to acceptor specificity, all tested monosaccharides, disaccharides, and oligosaccharides except for D-xylose and L-arabinose were good acceptors for transglycosylation. Thus, Gal36A4 may find diverse applications in industrial fields, especially in the food industry.

Production of α- and ß-galactosidases from Bifidobacterium longum subsp. longum RD47.

Approximately 50% of people in the world experience abdominal flatulence after the intake of foods containing galactosides such as lactose or soybean oligosaccharides. The galactoside hydrolyzing enzymes of α- and ß-galactosidases have been shown to reduce the levels of galactosides in both the food matrix and the human gastrointestinal tract. This study aimed to optimize the production of α- and ß-galactosidases of Bifidobacterium longum subsp. longum RD47 with a basal medium containing whey and corn steep liquor. The activities of both enzymes were determined after culturing at 37°C at pH 6.0 for 30 h. The optimal production of α- and ß-galactosidases was obtained with soybean oligosaccharides as a carbon source and proteose peptone no. 3 as a nitrogen source. The optimum pH for both α- and ß-galactosidases was 6.0. The optimum temperatures were 35°C for α-galactosidase and 37°C for ß- galactosidase. They showed temperature stability up to 37°C . At a 1 mM concentration of metal ions, CuSO4 inhibited the activities of α- and ß-galactosidases by 35% and 50%, respectively. On the basis of the results obtained in this study, B. longum RD47 may be used for the production of α- and ß-galactosidases, which may reduce the levels of flatulence factors.