Development of a stable continuous flow immobilized enzyme reactor for the hydrolysis of inulin

A 23.5-fold purified exoinulinase with a specific activity of 413 IU/mg and covalently immobilized on Duolite A568 has been used for the development of a continuous flow immobilized enzyme reactor for the hydrolysis of inulin. In a packed bed reactor containing 72 IU of exoinulinase from Kluyveromyces marxianus YS-1, inulin solution (5%, pH 5.5) with a flow rate of 4 mL/h was completely hydrolyzed at 55 °C. The reactor was run continuously for 75 days and its experimental half-life was 72 days under the optimized operational conditions. The volumetric productivity and fructose yield of the reactor were 44.5 g reducing sugars/L/h and 53.3 g/L, respectively. The hydrolyzed product was a mixture of fructose (95.8%) and glucose (4.2%) having an average fructose/glucose ratio of 24. An attempt has also been made to substitute pure inulin with raw Asparagus racemosus inulin to determine the operational stability of the developed reactor. The system remained operational only for 11 days, where 85.9% hydrolysis of raw inulin was achieved.

Improved skeletal muscle oxidative enzyme activity and restoration of PGC-1α and PPARß/δ gene expression upon rosiglitazone treatment in obese patients with type 2 diabetes mellitus

Objective: To examine whether rosiglitazone alters gene expression of some key genes involved in mitochondrial biogenesis and oxidative capacity in skeletal muscle of type 2 diabetic patients, and whether this is associated with alterations in skeletal muscle oxidative capacity and lipid content.

Design: Skeletal muscle gene expression, mitochondrial protein content, oxidative capacity and lipid accumulation were measured in muscle biopsies obtained from diabetic patients, before and after 8 weeks of rosiglitazone treatment, and matched controls. Furthermore, whole-body insulin sensitivity and substrate utilization were assessed.

Subjects: Ten obese type 2 diabetic patients and 10 obese normoglycemic controls matched for age and BMI.

Methods: Gene expression and mitochondrial protein content of complexes I–V of the respiratory chain were measured by quantitative polymerase chain reaction and Western blotting, respectively. Histochemical staining was used to quantify lipid accumulation and complex II succinate dehydrogenase (SDH) activity. Insulin sensitivity and substrate utilization were measured during a hyperinsulinemic–euglycemic clamp with indirect calorimetry.

Results: Skeletal-muscle mRNA of PGC-1a and PPARb/d – but not of other genes involved in glucose, fat and oxidative metabolism – was significantly lower in diabetic patients (Po0.01). Rosiglitazone significantly increased PGC-1a (B2.2-fold, Po0.01) and PPARb/d (B2.6-fold, Po0.01), in parallel with an increase in insulin sensitivity, SDH activity and metabolic flexibility (Po0.01). Surprisingly, none of the measured mitochondrial proteins was reduced in type 2 diabetic patients, nor affected by rosiglitazone treatment. No alterations were seen in muscular fat accumulation upon treatment.

Conclusion: These results suggest that the insulin-sensitizing effect of rosiglitazone may involve an effect on muscular oxidative capacity, via PGC-1a and PPARb/d, independent of mitochondrial protein content and/or changes in intramyocellular lipid.

Relationship between genotype and enzyme activity of glutathione S-transferases M1 and P1 in Chinese

Glutathione S-transferases (GSTs) are the major detoxifying Phase II enzyme for eliminating electrophilic compounds. Mutations in GSTM1, GSTP1 and GSTT1 in Caucasian and GSTA1 in Chinese have been found to reduce enzyme activity. However, data on the impact of common genetic polymorphisms of GSTM1 and GSTP1 on enzyme activity in Chinese is lacking. This study aimed to investigate the effect of common GSTP1 and GSTM1 polymorphisms on erythrocyte GST activity in healthy Chinese (n = 196). GSTM1 null mutation (GSTM1*0) was analyzed by a PCR-Multiplex procedure, whereas GSTP1 313A → G polymorphism (resulting in Ile105Val at codon 105) was analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis. Erythrocyte GST activity was measured using 1-chloro-2,4-dinitro-bezene (CDNB) as the model substrate. The frequency of GSTM1 null genotype was 54.3% and the frequency of GSTP1-Ile/Ile, -Ile/Val, and -Val/Val genotype was 60.7%, 35.2% and 4.1%, respectively, with a frequency of 21.7% for the 105 valine allele. Age, gender and smoking did not significantly affect the erythrocyte GST activities. The mean erythrocyte GST enzyme activity for GSTP1*-Ile/Val genotype group (3.53 ± 0.63 U/g Hb) was significantly lower than that for subjects with GSTP1-Ile/Ile genotype (4.25 ± 1.07 U/g Hb, P = 0.004), while subjects with the GSTP1-Val/Val genotype had the lowest enzyme activity (2.44 ± 0.67 U/g Hb). In addition, the GST activity in carriers of GSTM1*0/GSTP1-Ile/Ile was significantly higher than that of subjects inherited GSTM1*0/GSTP1-Ile/Val or GSTM1*0/GSTP1-Val/Val. However, there is no association between GSTM1 null mutation and reduced enzyme activity. GSTP1 codon 105 mutation led to reduced erythrocyte GST activity in Chinese. A combined GSTP1 and GSTM1 null mutations also resulted in significantly reduced GST activity. Further studies are needed to explore the clinical implications of GSTM1 and GSTP1 polymorphisms.

Immobilized enzyme technology for debittering citrus fruit juices

There has been increased interest in the use of immobilized enzymes in fruit juice industry for debittering of citrus fruit juices due to their high efficiency to remove bitter flavonoids. The structure of naringin, responsible for immediate bitterness, and of limonin, responsible for "delayed bitterness" has been discussed. This chapter also discusses various attempts that have been made to immobilize enzymes on an appropriate support so as to enable their use in debittering of citrus fruit juices. These include physicochemical and enzyme biotechnological approaches which makes the fruit juice more acceptable and cost effective to the consumer. Despite of high volume of production of citrus fruits and fruit juices, suitable processes to produce non-bitter citrus juice by immobilized enzymes technology has not yet commercialized globally.

Production of high fructose syrup from asparagus inulin using immobilized exoinulinase from Kluyveromyces marxianus YS-1

Extracellular exoinulinase from Kluyveromyces marxianus YS-1, which hydrolyzes inulin into fructose, was immobilized on Duolite A568 after partial puriWcation by ethanol precipitation and gel exclusion chromatography on Sephadex G-100. Optimum temperature of immobilized enzyme was 55 °C, which was 5 °C higher than the free enzyme and optimal pH was 5.5. Immobilized biocatalyst retained more than 90% of its original activity after incubation at 60 °C for 3 h, whereas in free form its activity was reduced to 10% under same conditions, showing a signiWcant improvement in the thermal stability of the biocatalyst after immobilization. Apparent Km values for inulin, raYnose and sucrose were found to be 3.75, 28.5 and 30.7 mM, respectively. Activation energy (Ea) of the immobilized biocatalyst was found to be 46.8 kJ/mol. Metal ions like Co2+ and Mn2+ enhanced the activity, whereas Hg2+ and Ag2+ were found to be potent inhibitors even at lower concentrations of 1 mM. Immobilized biocatalyst was eVectively used in batch preparation of high fructose syrup from Asparagus racemosus raw inulin and pure inulin, which
yielded 39.2 and 40.2 g/L of fructose in 4 h; it was 85.5 and 92.6% of total reducing sugars produced, respectively.

Upregulation of glutathione-related genes and enzyme activities in cultured human cells by sublethgal concentrations of inorganic arsenic

Inorganic arsenic (jAs), a known human carcinogen, acts as a tumor promoter in part by inducing a rapid burst of reactive oxygen species (ROS) in mammalian cells. This causes oxidative stress and a subsequent increase in the level of cellular glutathione (GSH). Glutathione, a ubiquitous reducing sulfhydryl tripeptide, is involved in ROS detoxification and its increase may be part of an adaptive response to the oxidative stress. Glutathione related enzymes including glutathione reductase (GR) and glutathione S-transferase (GST) also play key roles in these processes. In this study the regulatory effects of inorganic arsenite (As111) on the activities of GSH-related enzymes were investigated in cultured human keratinocytes. Substantial increases in GR enzyme activity and mRNA levels were shown in keratinocytes and other human cell lines after exposure to low, subtoxic, micromolar concentrations of As111 for 24 h. Upregulation of GSH synthesis paralleled the upregulation of GR as shown by increases in glutamatecysteine lyase (GeL) enzyme activity and mRNA levels, cystine uptake, and intracellular GSH levels. Glutathione S-transferase activity was also shown to increase slightly in keratinocytes, but not in fibroblasts or breast tumor cells. Overall the results show that sublethal arsenic induces a multicomponent response in human keratinocytes that involves upregulation of parts, but not all of the GSH system and counteracts the acute toxic effects of jAs. The upregulation of GR has not previously been shown to be an integral part of this response, although GR is critical for maintaining levels of reduced GSH.

Cell disruption optimization and covalent immobilization of beta-D-galactosidase from kluyveromyces marxianus YW-1 for lactose hydrolysis in milk

β-D-galactosidase (EC 3.2.1.23) from Kluyveromyces marxianus YW-1, an isolate from whey, has been studied in terms of cell disruption to liberate the useful enzyme. The enzyme produced in a bioreactor on a wheat bran medium has been successfully immobilized with a view to developing a commercially usable technology for lactose hydrolysis in the food industry. Three chemical and three physical methods of cell disruption were tested and a method of grinding with river sand was found to give highest enzyme activity (720 U). The enzyme was covalently immobilized on gelatin. Immobilized enzyme had optimum pH and temperature of 7.0 and 40 °C, respectively and was found to give 49% hydrolysis of lactose in milk after 4 h of incubation. The immobilized enzyme was used for eight hydrolysis batches without appreciable loss in activity. The retention of high catalytic activity compared with the losses experienced with several previously reported immobilized versions of the enzyme is significant. The method of immobilization is simple, effective, and can be used for the immobilization of other enzymes.

Covalent immobilization of naringinase for the transformation of a flavonoid

Naringinase (EC 3.2.1.40) from Penicillium sp was immobilized by covalent binding to woodchips to improve its catalytic activity. The immobilization of naringinase on glutaraldehyde-coated woodchips (600 mg woodchips, 10 U naringinase, 45 °C, pH 4.0 and 12h) through 1% glutaraldehyde cross-linking was optimized. The pH-activity curve of the immobilized enzyme shifted toward a lower pH compared with that of the soluble enzyme. The immobilization caused a marked increase in thermal stability of the enzyme. The immobilized naringinase was stable during storage at 4 °C. No loss of activity was observed when the immobilized enzyme was used for seven consecutive cycles of operations. The efficiency of immobilization was 120%, while soluble naringinase afforded 82% efficacy for the hydrolysis of standard naringin under optimal conditions. Its applicability for debittering kinnow mandarin juice afforded 76% debittering efficiency. 

Apparent in vivo Δ-6 desaturase activity, efficiency, and affinity are affected by total dietary C18 PUFA in the freshwater fish Murray cod

Dietary fatty acids are known to modulate fatty acid metabolism in fish. However, the innate capability of fish to bioconvert short chain fatty acids to health promoting long chain fatty acids (LCPUFA) is insufficient to compensate for a reduced dietary intake. While many studies have focused on the dietary regulation of the fatty acid bioconversion pathways, there is little known regarding the effects of the dietary levels of C18 polyunsaturated fatty acids (PUFA) on fatty acid metabolism. Here, we show a greater degree of apparent enzyme activity (Δ-6 desaturase) in fish fed a diet with higher amounts of dietary C18 PUFA. In particular, fish receiving high amounts of dietary C18 PUFA had a greater amount of Δ-6 desaturase activity acting on 18:3n-3 than 18:2n-6. However, with the gradual reduction of dietary C18 PUFA there was a shift in substrate preference of Δ-6 desaturase from 18:3n-3 to 18:2n-6. This information will provide valuable insight for the implementation of low fish oil diets, which permit the maintenance of n-3 LCPUFA levels in farmed Murray cod.

Antioxidant enzyme activities of iron-saturated bovine lactoferrin (Fe-bLf) in human gut epithelial cells under oxidative stress

Chemoprevention by dietary constituents in the form of functional food has emerged as a novel approach to control inflammatory diseases and cancers. Recently we reported for the first time that iron content is a critical determinant in the anti-tumour activity of bovine milk lactoferrin (bLf). We therefore wanted to evaluate the chemo-preventative efficacy of Apo-bLF and 100% iron-saturated bLF (Fe-bLF) on hydrogen peroxide (H2O 2)-induced colon carcinogenesis, and their influence on antioxidant enzyme activities within colon carcinogenesis. This was undertaken through observing how oxidative stress induced by H2O2 alters antioxidant enzyme activity within HT29 colon cancer cells, and then observing changes in this activity by treatments with the different antioxidants ascorbic acid (AA), Apo-bLF and Fe-bLF. All antioxidant enzymes (catalase, glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-s-transferase (GsT) and superoxide dismutase (SOD)) appeared to be increased within HT29 cells, even prior to H2O2 exposure, and all enzymes showed significant decreased activity when cells were treated with the antioxidants AA, Apo-bLF or Fe-bLF, with or without H2O2 exposure. The results indicate that all three antioxidants have the ability to scavenge ROS, lower antioxidant enzyme activities within already excited states, and possibly allow colon cancer cells to be overcome by oxidative stress that would normally be prevented, perhaps leading to damage and potential apoptosis of the cancer cells. In conclusion, the anti-oxidative effects of Apo-bLF and Fe-bLf studied for the first time, show dynamic changes that may allow for necessary protection from imbalanced oxidative conditions, and potential at reducing the ability of cancer cells to protect themselves from oxidative stress states.

Effect of selenium-saturated bovine lactoferrin (Se-bLF) on antioxidant enzyme activities in human gut epithelial cells under oxidative stress

Cancer and many chronic inflammatory diseases are associated with increased amounts of reactive oxygen species (ROS). The potential cellular and tissue damage created by ROS has significant impact on many disease and cancer states and natural therapeutics are becoming essential in regulating altered redox states. We have shown recently that iron content is a critical determinant in the antitumour activity of bovine milk lactoferrin (bLF). We found that 100% iron-saturated bLF (Fe-bLF) acts as a potent natural adjuvant and fortifying agent for augmenting cancer chemotherapy and thus has a broad utility in the treatment of cancer. Furthermore, we also studied the effects of iron saturated bLF's ability as an antioxidant in the human epithelial colon cancer cell line HT29, giving insights into the potential of bLF in its different states. Thus, metal saturated bLF could be implemented as anti-cancer neutraceutical. In this regard, we have recently been able to prepare a selenium (Se) saturated form of bLF, being up to 98% saturated. Therefore, the objectives of this study were to determine how oxidative stress induced by hydrogen peroxide (H₂O₂) alters antioxidant enzyme activity within HT29 epithelial colon cancer cells, and observe changes in this activity by treatments with different antioxidants ascorbic acid (AA), Apo (iron free)-bLF and selenium (Se)-bLF. The states of all antioxidant enzymes (glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-s-transferase (GsT), catalase and superoxide dismutase (SOD)) demonstrated high levels within untreated HT29 cells compared to the majority of other treatments being used, even prior to H₂O₂ exposure. All enzymes showed significant alterations in activity when cells were treated with antioxidants AA, Apo-bLF or Se-bLF, with and/or without H₂O₂ exposure. Obvious indications that the Se content of the bLF potentially interacted with the glutathione (GSH)/GPx/GR/GsT associated redox system could be observed immediately, showing capability of Se-bLF being highly beneficial in helping to maintain a balance between the oxidant/antioxidant systems within cells and tissues, especially in selenium deficient systems. In conclusion, the antioxidative defence activity of Se-bLf, investigated in this study for the first time, shows dynamic adaptations that may allow for essential protection from the imbalanced oxidative conditions. Because of its lack of toxicity and the availability of both selenium and bLF in whole milk, Se-bLF offers a promise for a prospective natural dietary supplement, in addition to being an immune system enhancement, or a potential chemopreventive agent for cancers.

Enzyme architectonics on graphene oxides.

The thesis focuses on use of carbon nanoparticles, namely graphene oxides for studying the effect of hydrophobicity on enzyme structure and how they it influences the enzyme activity at molecular level. It was observed that controlling the hydrophobicity of the nanoparticle is a key towards modulating enzyme activity.