Miguel Angel Duarte-Vázquez

Biotechnological applications of peroxidases

Peroxidases are widely distributed in nature. Reduction of peroxides at the expense of electron donating substrates, make peroxidases useful in a number of biotechnological applications. Enzymes such as lignin peroxidase and manganese peroxidase, both associated with lignin degradation, may be successfully used for biopulping and biobleaching in the paper industry, and can produce oxidative breakdown of synthetic azo dyes. Oxidative polymerization of phenols and aromatic amines conducted by horseradish peroxidase (HRP) in water and water-miscible organic solvents, may lead to new types of aromatic polymers. Site directed mutagenesis of HRP has been used to improve the enantioselectivity of arylmethylsulfide oxidations. Peroxidase has a potential for soil detoxification, while HRP as well as soybean and turnip peroxidases have been applied for the bioremediation of wastewater contaminated with phenols, cresols, and chlorinated phenols. Peroxidase based biosensors have found use in analytical systems for determination of hydrogen peroxide and organic hydroperoxides, while co-immobilized with a hydrogen peroxide producing enzyme, they can be used for determination of glucose, alcohols, glutamate and choline. Peroxidase has also been used for practical analytical applications in diagnostic kits, such as quantitation of uric acid, glucose, cholesterol, lactose, and so on. Enzyme linked immunorbent assay (ELISA) tests on which peroxidase is probably the most common enzyme used for labeling an antibody, are a simple and reliable way of detecting toxins, pathogens, cancer risk in bladder and prostate, and many other analytes. Directed evolution methods, appear to be a valuable alternative to engineer new catalyst forms of plant peroxidases from different sources to overcome problems of stability and to increase thermal resistance.

Monosaccharide composition and properties of a deglycosylated turnip peroxidase isozyme

A neutral peroxidase isozyme (TP) purified from turnip (Brassica napus L. var. purple top white globe) was partially deglycosylated, using chemical and enzymatic treatment. A 32% carbohydrate removal was achieved by exposing TP to a mixture of PNGase F, O-glycosidase, NANase, GALase III and HEXase I, while m-periodate treatment removed about 88% of TP carbohydrate moiety. The glycoprotein fraction of the TP contained a relatively high mannose and fucose content (37 and 31%, w/w, respectively), 16% (w/w) galactose, and 15% (w/w) GlcNAc. Thus, the carbohydrate moiety was classified as a hybrid type. Partially deglycosylated TP had reduced activity (by 50-85%), was more susceptible to proteolysis, and showed a slight decrease in thermostability compared to the native enzyme. Circular dichroism studies strongly suggested that although the carbohydrate moiety of TP did not influence the conformation of the polypeptide backbone, its presence considerably enhanced protein conformational stability toward heat. Removal of oligosaccharide chains from TP caused a decrease in K(m) and V(max) for hydrogen peroxide. Native and chemically deglycosylated TP were similarly immunodetected by rabbit polyclonal antibodies raised against TP. The results suggest that the carbohydrate moiety of TP is important for peroxidase activity and stability.

Production of Cow’s Milk Free from Beta-Casein A1 and Its Application in the Manufacturing of Specialized Foods for Early Infant Nutrition

Beta-casein (BC) is frequently expressed as BC A2 and BC A1 in cow’s milk. Gastrointestinal digestion of BC A1 results in the release of the opioid peptide beta-casomorphin 7 (BCM7) which is less likely to occur from BC A2. This work was aimed to produce milk containing BC A2 with no BC A1 (BC A2 milk) using genetically selected CSN2 A2A2 Jersey cows. Additionally, we aimed to develop an infant formula (IF) suitable for healthy full-term infants during the first six months of life based on BC A2 milk. The concentration of BCM7 released from BC A2 IF, from commercially available IFs as well as from human milk and raw cow’s milk was evaluated after simulated gastrointestinal digestion (SGID). BC A2 IF presented the lowest mean relative abundance of BC A1 (IF 1 = 0.136 ± 0.010), compared with three commercially available IFs (IF 2 = 0.597 ± 0.020; IF 3 = 0.441 ± 0.014; IF 4 = 0.503 ± 0.011). Accordingly, SGID of whole casein fraction from BC A2 IF resulted in a significantly lower release of BCM7 (IF 1 = 0.860 ± 0.014 µg/100 mL) compared to commercially available IFs (IF 2 = 2.625 ± 0.042 µg/100 mL; IF 3 = 1.693 ± 0.012 µg/100 mL; IF 4 = 1.962 ± 0.067 µg/100 mL). Nevertheless, BCM7 levels from BC A2 IF were significantly higher than those found in SGID hydrolysates of BC A2 raw milk (0.742 ± 0.008 µg/100 mL). Interestingly, results showed that BCM7 was also present in human milk in significantly lower amounts (0.697 ± 0.007 µg/100 mL) than those observed in IF 1 and BC A2 milk. This work demonstrates that using BC A2 milk in IF formulation significantly reduces BCM7 formation during SGID. Clinical implications of BC A2 IF on early infant health and development need further investigations.

Use of urea-polyacrylamide electrophoresis for discrimination of A1 and A2 beta casein variants in raw cow’s milk

Abstract Beta-casein (BC) in cow’s milk occurs in several genetic variants, where BC A1 (BCA1) and BC A2 (BCA2) are the most frequent. This work deals with a method based on modified polyacrylamide gel electrophoresis using urea PAGE to discriminate BCA1 and BCA2 variants from Holstein Friesian (HF) and genetically selected Jersey A2/A2 (JA2) cow’s milk. Two well defined bands were obtained from BC fraction of HF milk, while that of JA2 showed a single band. Proteins from these bands were sequenced by HPLC-quadrupole linear ion trap/mass spectrometry, resulting in BCA1 and BCA2 separation from the BC fraction of HF milk, whereas BCA2 was the only constituent of JA2 fraction. This method represents a feasible and useful tool to on site phenotyping of BC fraction of cow’s milk for pharmaceutical and food industries applications.

A Double-Blind, Active-Controlled Clinical Trial of Sodium Bicarbonate and Calcium Gluconate in the Treatment of Bilateral Osteoarthritis of the Knee

Objective: To evaluate the effect of intra-articular injections of sodium bicarbonate with a single (SBCG1) or double dose (SBCG2) of calcium gluconate administered monthly compared with methylprednisolone (MP) for treatment of knee osteoarthritis. Methods: A 3-month, randomized, double-blind clinical trial with patients diagnosed with knee osteoarthritis (OA). The outcome variables were the Western Ontario-McMaster University Osteoarthritis Index (WOMAC) and the Lequesne functional index. Results: After 3 months, all treatments significantly improved in overall WOMAC and Lequesne scores. Mean changes (95% confidence interval) in WOMAC total score and the Lequesne index, respectively, for SBCG1 (−12.5 [−14.3, −10.7]; −9.0 [−11.4, −6.7]) and SBCG2 (−12.3 [−14.3, −10.4]; −8.9 [−10.4, −7.4]) were significantly greater than for MP (−5.0 [−7.2, −2.8]; −3.2 [−4.9, −1.5]) (P < .001). Conclusions: Intra-articular injections of sodium bicarbonate and calcium gluconate are useful for short-term relief of OA symptoms in patients with bilateral knee osteoarthritis. Both treatments are more effective than MP injections in the reduction of knee OA symptoms. Trial Registration: Clinicaltrials.gov NCT00977444

Lower Protein Intake Supports Normal Growth of Full-Term Infants Fed Formula: A Randomized Controlled Trial

Infant formulas have been conventionally prepared with an excess of total protein in order to provide sufficient amounts of essential amino acids to the rapidly growing infant. However, this practice leads to higher than necessary protein intake during early infant development, inducing accelerated growth patterns correlated with the development of chronic diseases later in life. This study was aimed at assessing the safety of an infant formula enriched with bovine alpha-lactalbumin containing a total protein concentration very close to that of human milk, and determining its efficacy in the support of healthy infant growth from the first month to the fourth month of age. Healthy full-term infants ≤40 days of age were randomized in this controlled single blind trial to one of the following infant formulas: IF 1 (containing 1.0 g protein/dL; n = 30), IF 2 (containing 1.3 g protein/dL; n = 24), and IF 3 (containing 1.5 g protein/dL; n = 42). A control group consisting of exclusively breastfed infants (HM; n = 212) was included in the study. Anthropometric measurements and Z-scores were evaluated at baseline, at 1 month of age, and at 4 months of age. Weight gain (g/day) was similar in the IF 1 and the HM groups (p = 0.644), and it was significantly greater in the IF 2 and IF 3 groups than in the HM group. Growth patterns in both breastfed or IF-fed infants were in accordance with the World Health Organization (WHO) growth standards. At four months of age, the mean weight-for-age Z-score (WAZ) adjusted for initial value in the IF 1 group was similar to that of the HM group and significantly lower than that of the IF 2 and IF 3 groups (p = 0.031 and p = 0.014 for IF 2 and IF 3, respectively). Length-for-age (LAZ) adjusted for initial value was similar among all groups at four months of age. From 1 to 4 months of life, IF 1 containing 1.0 g protein/dL promotes growth and weight gain similar to those observed in exclusively breastfed infants. As this is a first approach to studying an IF containing total protein in a level below that recommended by international committees on nutrition, further investigations are needed to support these findings evaluating infant’s metabolic profile and growth in the long term.

Effect of a β-Hydroxyphosphonate Analogue of L-Carnitine on Insulin-Sensitive and Insulin-Resistant 3T3-L1 Adipocytes

This study investigated the effect of a β-hydroxyphosphonate analog of L-carnitine (L-CA) (CAS number: 1220955-20-3, Component: 1221068-91-2, C12H29NO4PI), (3-Hexanaminium, 1-(dimethoxyphosphinyl)-2-hydroxy-N,N,N,5-tetramethy-iodide (1:1), (2R, 3S)) on parameters related with type-2 diabetes in an in vitro model. Nontoxic concentrations of L-CA were assayed and compared to commercial L-carnitine effects. L-CA did not affect adipogenesis in normal cells, but an increment of TG accumulation was observed on insulin-resistant adipocytes (80%) when compared with resistant control. L-CA also stimulated glucose analog 2-NBDG uptakes on insulin-resistant adipocytes in a similar way as insulin when compared to insulin-resistant cells. Our results show that the L-CA promoted insulin-like responses on insulin-resistant adipocytes without appreciable pro-adipogenic effect in sensitive adipocytes.