Xin Gen Lei

Selenium-dependent cellular glutathione peroxidase protects mice against a pro-oxidant-induced oxidation of NADPH, NADH, lipids, and protein

Since our prior work indicated that Se‐dependent cellular glutathione peroxidase (GPX1) was necessary for protection against paraquat lethality, the present studies were to elucidate the biochemical mechanisms related to that protection. Four groups of mice [Se‐deficient or ‐adequate GPX1 knockout and wild‐type (WT)] were injected (i.p.) with 50 mg paraquat/kg body weight and tissues were collected 0, 0.5, 1, 2, 3, or 4 h after the injection. Whereas the ratios of NADPH/NADP and NADH/NAD in lung were reduced by 50–70% only 0.5 h after the injection in all groups, these two ratios in liver of the Se‐adequate WT were significantly higher than those of the three GPX1 knockout or deficient groups 2–4 h after the injection. The paraquat‐induced pulmonary lipid peroxidation and hepatic protein oxidation, measured as F2‐isoprostanes and carbonyl contents, respectively, peaked at 1 h in these three groups. No such oxidative events were shown in any tissue of the Se‐adequate WT throughout the time course. Whereas the F2‐isoprostane formation was accelerated by both GPX1 knockout and Se deficiency in liver, it was not significantly elevated by the paraquat treatment in brain of any group. The paraquat injection also resulted in temporal changes in lung GPX activity and GPX1 protein in the Se‐adequate WT, and significant reductions in lung total SOD activity in the GPX1 knockout or deficient groups. In conclusion, GPX1 plays a critical role in maintaining the redox status of mice under acute oxidative stress, and protects against paraquat‐induced oxidative destruction of lipids and protein in vivo. These protections of GPX1 seem to be inducible and coordinated with those of other antioxidant enzymes.—Cheng, W.‐H., Fu, Y. X., Porres, J. M., Ross, D. A., Lei, X. G. Selenium‐dependent cellular glutathione peroxidase protects mice against a pro‐oxidant‐induced oxidation of NADPH, NADH, lipids, and protein. FASEB J. 13, 1467–1475 (1999)

Nutritional benefits of phytase and dietary determinants of its efficacy.

Abstract Lei, X.G. and Stahl, C.H. 2000. Nutritional benefits of phytase and dietary determinants of its efficacy. J. Appl. Anim. Res., 17: 97–112. Over 60% of the total P in cereal grains and oil seeds and their by-products is found as phytate, myo-inositol hexakisphosphate. Phytases are a group of enzymes that initiate the phosphate hydrolysis from phytate and produce myo-inositol and inorganic phosphate by catalyzing the stepwise removal of the phosphate groups. Microbial phytases effectively improve dietary phytate-P utilization and may partially or completely replace inorganic P supplementation in swine and poultry diets. Replacing inorganic P with phytase in these diets can reduce P excretion by up to 50% and, enhance bioavailabilities of Ca, Zn and Fe. The improvement in nitrogen retention and protein and amino acid utilization by dietary phytase supplementation is relatively small and highly variable. The efficacy of supplemental phytase is affected by at least three dietary factors: Ca:P ratio, cholecalciferol and organic adds.

An improved method for a rapid determination of phytase activity in animal feed

The current direct colorimetric assay for phytase activity in feeds has interference from high P background and other factors. Our objective was to develop a rapid and reliable spin column method to accurately determine phytase activity in feed ingredients or complete diets. After the feed sample was extracted by stirring in 0.2 M citrate buffer, pH 5.5, for 30 min at room temperature, the oily layer of the supernatant fraction was removed by passing through an acrodisc syringe filter (0.45-microm HT Tuffryn membrane, Gelman Laboratory, Ann Arbor, MI). The filtrate was then loaded onto a spin column (MW cutoff 30,000, Millipore, Bedford, MA) to remove free phosphate before the phytase activity assay. Compared with the direct assay, this new procedure improved both accuracy and reproducibility. When diets contained phytase at 0 to 1,500 U/kg (as fed), the CV for multiple assays of the same samples (n = 6) by the new method ranged from 1 to 6% compared with 28 to 39% by the direct method. A linear relationship was found between the added phytase activity in practical diets and the analyzed activity by the new method (r2 = 0.99; P < 0.01). In conclusion, the spin column method is an improved assay for phytase activity in animal feed, and may be used for quality control of phytase supplementation.

Comparison of extracellular Escherichia coli AppA phytases expressed in Streptomyces lividans and Pichia pastoris

A phytase gene (appA) from Escherichia coli was cloned into Streptomyces lividans and expressed as an extracellular protein which was then compared with the same enzyme expressed in Pichia pastoris. The phytase expressed in S.xa0lividans was not glycosylated and had a molecular mass of 45xa0kDa. Compared with the glycosylated phytase expressed in P.xa0pastoris, this non-glycosylated phytase was 25–50% less active (p<0.05) at pHxa02 to 3.5 or at 45 and 55xa0°C, but 50% more active (p<0.05) at 75xa0°C. The thermo-tolerance of the non-glycosylated phytase was 26 and 48% higher (p<0.05) than that of the glycosylated phytase at 45 and 55xa0°C, but was 80 and 94% lower (p<0.05) at 65xa0° and 75xa0°C, respectively.

Expression of Escherichia coli AppA2 phytase in four yeast systems

To develop an effective fermentation system for producing Escherichia coliphytase AppA2, we expressed the enzyme in three inducible yeast systems: Saccharomyces cerevisiae (pYES2), Schizosaccharomyces pombe (pDS472a), and Pichia pastoris (pPICZ αA), and one constitutive system: P. pastoris (pGAPZαA). All four systems produced an extracellular functional AppA2 phytase with apparent molecular masses ranging from 51.5 to 56xa0kDa. During 8-day batch fermentation in shaking flasks, the inducible Pichia system produced the highest activity (272 units ml−1 medium), whereas the Schizo. pombesystem produced the lowest activity (2.8 units ml−1). The AppA2 phytase expressed in Schizo. pombehad 60–75% lower Kmfor sodium phytate and 28% higher heat-stability at 65 °C than that expressed in other three systems. However, all four recombinant AppA2 phytases had pH optimum at 3.5 and temperature optimum at 55 °C and similar efficacy in hydrolyzing phytate–phosphate from soybean meal.

Phytase: An Enzyme to Improve Soybean Nutrition

Soybean (Glycine max) serves as a major human food and animal feed component due to its nutritional and health values. As an important dietary source of protein, fat, fiber, minerals and vitamins, soybean also provides many bioactive components such as phytoestrogens with potential benefits for human health (Messina, 1999). Meanwhile, other components present in soybean like trypsin inhibitors and phytate can act as anti-nutritional factors that interfere with protein digestion or chelate nutritionally essential elements including Ca, Zn and Fe (Liener, 1994; Hurrell, 2003). While trypsin inhibitors are heat labile and are usually inactivated in the production of soybean meal or soy protein isolate, phytate is heat stable and needs phytases for its hydrolysis. Phytases are phosphohydrolytic enzymes that initiate the stepwise removal of phosphates from inositol hexaphosphate (Lei & Porres, 2007). Phytase supplementation has become an efficient tool to improve bioavailability of P present in feedstuffs and to reduce the amount of phytate-derived P excreted to the environment by animals. Phytase-mediated hydrolysis of phytate also releases several other essential minerals (Lei et al., 1993a,b,c). Soybean meal is a common ingredient to be mixed with corn and other cereals for the swine and poultry ration. Various sources of plant and microbial phytases, along with other feed supplements such as citric acid, vitamin D, and strontium, have been tested to enhance utilization of P and other nutrients in the cornsoybean meal based diets (Han et al., 1998; Snow et al., 2004; Pagano et al., 2007b). Findings from these experiments have been used to improve performance and health of commercial herds and spare costly non-renewable sources of inorganic P. Soybean serves as an important component of many dishes oriented to human nutrition. It is consumed as cooked, sprouted, and processed into soy milk, tofu, miso, tempeh or natto. Industrial processing of soybean is derived not only by its nutritional properties, but also by its chemical characteristics. Soy proteins contain lipophilic, polar, non-polar, and negatively and positively charged groups that enable them to be associated with many different types of compounds (Endres, 2001). Representing the major industrial products, soy oil and soybean meal are produced through a solvent extraction process. Crude soybean oil is further processed in to a variety of products, whereas soybean meal can be further processed to protein concentrates, protein isolates, or textured protein products for