Applied Biochemistry and Biotechnology (v.177, #5)

Partial Nitrification and Denitrifying Phosphorus Removal in a Pilot-Scale ABR/MBR Combined Process by Peng Wu; Lezhong Xu; Jianfang Wang; Zhenxing Huang; Jiachao Zhang; Yaoliang Shen (1003-1012).
A pilot-scale combined process consisting of an anaerobic baffled reactor (ABR) and an aerobic membrane bioreactor (MBR) for the purpose of achieving easy management, low energy demands, and high efficiencies on nutrient removal from municipal wastewater was investigated. The process operated at room temperature with hydraulic retention time (HRT) of 7.5 h, recycle ratio 1 of 200 %, recycle ratio 2 of 100 %, and dissolved oxygen (DO) of 1 mg/L and achieved good effluent quality with chemical oxygen demand (COD) of 25 mg/L, NH4 +-N of 4 mg/L, total nitrogen (TN) of 11 mg/L, and total phosphorus (TP) of 0.7 mg/L. The MBR achieved partial nitrification, and NO2 -N has been accumulated (4 mg/L). Efficient short-cut denitrification was occurred in the ABR with a TN removal efficiency of 51 %, while the role of denitrification and phosphorus removal removed partial TN (14 %). Furthermore, nitrogen was further removed (11 %) by simultaneous nitrification and denitrification in the MBR. In addition, phosphorus accumulating organisms in the MBR sufficiently uptake phosphorus; thus, effluent TP further reduced with a TP removal efficiency of 84 %. Analysis of fluorescence in situ hybridization (FISH) showed that ammonia oxidizing bacteria (AOB) and phosphorus accumulating organisms (PAOs) were enriched in the process. In addition, the accumulation of NO2 -N was contributed to the inhibition on the activities of the NOB rather than its elimination.
Keywords: Anaerobic processes; Aerobic processes; Membrane bioreactors; Municipal wastewater; Nutrient removal

Advanced Glycation-Modified Human Serum Albumin Evokes Alterations in Membrane and Eryptosis in Erythrocytes by Saurabh Awasthi; S. K. Gayathiri; R. Ramya; R. Duraichelvan; A. Dhason; N. T. Saraswathi (1013-1024).
Increased burden of advanced glycation end-products (AGEs) in case of hyperglycemic conditions leads to the development of retinopathy, nephropathy, and cardiovascular and neurological disorders such as Alzheimer’s disease. AGEs are considered as pro-oxidants, and their accumulation increases the oxidative stress. The prolonged exposure to these AGEs is the fundamental cause of chronic oxidative stress. Abnormal morphology of red blood cells (RBCs) and excessive eryptosis has been observed in diabetes, glomerulonephritis, dyslipidemia, and obesity, but yet the contribution of extracellular AGEs remains undefined. In this study, we investigated the effect of AGEs on erythrocytes to determine their impact on the occurrence of different pathological forms of these blood cells. Specifically, carboxymethyllysine (CML), carboxyethyllysine (CEL), and Arg-pyrimidine (Arg-P) which have been reported to be the most pre-dominant AGEs formed under in vivo conditions were used in this study. Results suggested the eryptotic properties of CML, CEL, and Arg-P for RBCs, which were evident from the highly damaged cell membrane and occurrence of abnormal morphologies. Methylglyoxal-modified albumin showed more severe effects, which can be attributed to the high reactivity and pro-oxidant nature of glycation end products. These findings suggest the possible role of AGE-modified albumin towards the morphological changes in erythrocyte’s membrane associated with diabetic conditions.
Keywords: Advanced glycation end products; Albumin; Erythrocytes; Carboxymethyllysine; Carboxyethyllysine; Arg-pyrimidine; Eryptosis

Fe(II)-Co(II) Double Salt Incorporated Magnetic Hydrophobic Microparticles for Invertase Adsorption by Kazım Köse; Kadir Erol; Asiye Aslı Emniyet; Dursun Ali Köse; Gülçin Alp Avcı; Lokman Uzun (1025-1039).
Invertase (β-fructofuranoside fructohydrolase, EC is an enzyme widely used in the food industry. Its main function is the formation of glucose and fructose through hydrolysis of sucrose. For the separation and purification of this commercially important enzyme from aqueous solutions, magnetic poly(2-hydroxyethyl methacrylate-N-methacryloyl-L-tryptophan), m-poly(HEMA-MATrp) microparticles were developed. Magnetic properties of microparticles are provided using ferromagnetic Fe(II)-Co(II) double salt. Characterization studies of magnetic microparticles were conducted via vibrating sample magnetometer (VSM), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) analysis. Specific surface area of magnetic microparticles is 6.75 m2/g. Because of all experiments performed in this study, the adsorption capability of magnetic microparticles was optimized by variation of different conditions (pH, interaction time, initial invertase concentration, temperature, and ionic strength) and maximum adsorption capacity was determined as 992.64 mg invertase/g magnetic microparticles.
Keywords: Double salt; Magnetic separation; Hydrophobic microparticles; Invertase; Adsorption

Characterization and Differentiation of Stem Cells Isolated from Human Newborn Foreskin Tissue by Özge Sezin Somuncu; Pakize Neslihan Taşlı; Hatice Burcu Şişli; Salih Somuncu; Fikrettin Şahin (1040-1054).
Circumcision is described as a cultural, medical, and religious process which states surgical removal of the foreskin either partly or fully. Cells isolated from the circumcised tissues are referred as foreskin cells. They have been thought as feeder cell lines for embryonic stem cells. Their fibroblastic properties were also utilized for several experiments. The waste tissues that remain after the circumcision thought to have stem cell properties. Therefore, there have been very few attempts to expose their stem cell properties without turning them into induced pluripotent stem cells. Although stem cell isolation from prepuce and their mesenchymal multilineage differentiation potential have been presented many times in the literature, the current study explored hematopoietical phenotype of newborn foreskin stem cells for the first time. According to the results, human newborn foreskin stem cells (hnFSSCs) were identified by their capability to turn into all three germ layer cell types under in vitro conditions. In addition, these cells have exhibited a stable phenotype and have remained as a monolayer in vitro. hnFSSCs suggested to carry different treatment potentials for bone damages, cartilage problems, nerve damages, lesion formations, and other diseases that are derive from mesodermal, endodermal, and ectodermal origins. Owing to the location of the tissue in the body and differentiation capabilities of hnFSSCs, these cells can be considered as easily obtainable and utilizable even better than the other stem cell sources. In addition, hnFSSCs offers a great potential for tissue engineering approaches due to exhibiting embryonic stem cell-like characteristics, not having any ethical issues, and teratoma induction as in embryonic stem cell applications.
Keywords: Neurogenesis; Osteogenesis; Adipogenesis; Chondrogenesis; Human newborn foreskin stem cell

Spermine synthase (SPMS, EC, enzyme of spermine (Spm) biosynthesis, has been shown to be related to stress response. In this study, attempts were made to clone and characterize a gene encoding SPMS from tea plant (Camellia sinensis). The effect of exogenous application of Spm in C. sinensis subjected to low-temperature stress was also investigated. A full-length SPMS complementary DNA (cDNA) (CsSPMS) with an open reading frame of 1113 bp was cloned using reverse transcription-PCR and rapid amplification of cDNA ends (RACE) techniques from cultivar “Yingshuang”. The CsSPMS gene, which encoded a 371 amino acid polypeptide, in four cultivars is highly homologous. Quantitative real-time PCR indicated that the CsSPMS gene shows tissue-specific expression, mainly in the leaf and root of tea plant. The expression analysis demonstrated that the CsSPMS gene is quickly induced by cold stress and had similar trends in four cultivars. Spm-supplemented “Baicha” cultivar contains higher endogenous polyamines compared to the control, coupling with higher expression levels of ADC and SPMS. In addition, activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), as well as free proline content in the Spm-supplemented samples were higher than the control during the experiment course or at a given time point, indicating that Spm exerted a positive effect on antioxidant systems. Moreover, Agrobacterium-mediated expression of CsSPMS in tobacco leaves showed relatively higher cold tolerance. Taken together, these findings will enhance the understanding of the relationships among CsSPMS gene regulatory, polyamines accumulation, and cold tolerance in tea plant.
Keywords: Cold tolerance; CsSPMS ; Gene expression; Polyamine; Tea plant

Comparative Study of Biosafety, DNA, and Chromosome Damage of Different-Materials-Modified Fe3O4 in Rats by Dong-Lin Xia; Yan-Pei Chen; Chao Chen; Yu-Fei Wang; Xiao-dong Li; Hong He; Hai-Ying Gu (1069-1082).
The increasing use of modified Fe3O4 magnetic microparticles has raised safety concerns regarding their use and effect on human health. This study assessed the in vivo biosafety, DNA, and chromosome damage of modified Fe3O4 microparticles such as Au@Fe3O4, Ag@Fe3O4, Cs@Fe3O4, Pt@Fe3O4, and CdS@Fe3O4, using spleen-deficient rats. Spleen-deficient rats treated with naked and modified (Au, Cs, Pt) Fe3O4 microparticles (5000 mg/kg) displayed low toxicity. Only treatment with Cds@Fe3O4 resulted in elevated toxicity and death in rats. Au-, Ag-, and Pt-modified Fe3O4 increased the rate of hemolysis in rats relative to treatment with naked Fe3O4. Despite this, Au- and Pt-modified Fe3O4 increased the biocompatibility and reduced DNA and chromosome damage in rats relative to naked Fe3O4. While Cs@Fe3O4 microparticles displayed a higher biocompatibility than naked Fe3O4, they displayed no significant reduction in DNA and chromosome damage. In summary, Au and Pt surface-modified Fe3O4 microparticles display elevated in vivo biosafety compared to unmodified particles. The precious metal material, with good biological compatibility, surface modification of Fe3O4 is an effective strategy to improve the overall safety and potential therapeutic utility of these magnetic materials.
Keywords: Biosafety; DNA damage; Chromosome damage; Fe3O4 ; Modified; SD rats

Oleaginous yeast Trichosporon cutaneum CGMCC 2.1374 was found to utilize inulin directly for microbial lipid fermentation without a hydrolysis step. The potential inulinase-like enzyme(s) in T. cutaneum CGMCC 2.1374 were characterized and compared with other inulinase enzymes produced by varied yeast strains. The consolidated bioprocessing (CBP) for lipid accumulated using inulin was optimized with 4.79 g/L of lipid produced from 50 g/L inulin with the lipid content of 33.6 % in dry cells. The molecular weight of the enzyme was measured which was close to invertase in Saccharomyces cerevisiae. The study provided information for inulin hydrolyzing enzyme(s) in oleaginous yeasts, as well as a preliminary CBP process for lipid production from inulin feedstock.
Keywords: Inulin hydrolyzing enzyme; Trichosporon cutaneum ; Dry cell weight; Lipid production; Consolidated bioprocessing

Lactic Acid and Biosurfactants Production from Residual Cellulose Films by Oscar Manuel Portilla Rivera; Guillermo Arzate Martínez; Lorenzo Jarquín Enríquez; Pedro Alberto Vázquez Landaverde; José Manuel Domínguez González (1099-1114).
The increasing amounts of residual cellulose films generated as wastes all over the world represent a big scale problem for the meat industry regarding to environmental and economic issues. The use of residual cellulose films as a feedstock of glucose-containing solutions by acid hydrolysis and further fermentation into lactic acid and biosurfactants was evaluated as a method to diminish and revalorize these wastes. Under a treatment consisting in sulfuric acid 6 % (v/v); reaction time 2 h; solid liquid ratio 9 g of film/100 mL of acid solution, and temperature 130 °C, 35 g/L of glucose and 49 % of solubilized film was obtained. From five lactic acid strains, Lactobacillus plantarum was the most suitable for metabolizing the glucose generated. The process was scaled up under optimized conditions in a 2-L bioreactor, producing 3.4 g/L of biomass, 18 g/L of lactic acid, and 15 units of surface tension reduction of a buffer phosphate solution. Around 50 % of the cellulose was degraded by the treatment applied, and the liqueurs generated were useful for an efficient production of lactic acid and biosurfactants using L. plantarum. Lactobacillus bacteria can efficiently utilize glucose from cellulose films hydrolysis without the need of clarification of the liqueurs.
Keywords: Residual cellulose films; Acid hydrolysis; Glucose; Lactic acid; Biosurfactants

An Injectable PEG-BSA-Coumarin-GOx Hydrogel for Fluorescence Turn-on Glucose Detection by Gayathri Srinivasan; Jun Chen; Joseph Parisi; Christian Brückner; Xudong Yao; Yu Lei (1115-1126).
Diabetes mellitus is a chronic metabolic disorder, requiring vigilant monitoring of blood glucose levels. In this study, an injectable fluorescent enzymatic hydrogel was designed for rapid glucose detection. The leakage-free glucose-responsive hydrogel was constructed by the covalent linkage of a multi-arm poly-(ethylene glycol) (PEG), bovine serum albumin (BSA), glucose oxidase (GOx), and 4-(aminomethyl)-6,7-dimethoxycoumarin (Coumarin-NH2). The GOx serves as glucose-recognition element and the pH-sensitive Coumarin-NH2 as a fluorescence turn-on reporter. The material properties of the fluorescent hydrogel were systematically characterized which show high elasticity with good mechanical strength. Upon the addition of glucose, the as-developed fluorescent hydrogel shows a fast response time, good sensitivity, and good reproducibility at physiological pH and ambient temperature. The glucose-sensing mechanism is based on the oxidation of the glucose by GOx that generates protons to change the local pH. Consequently, protonation of the covalently immobilized and pH-sensitive Coumarin-NH2 turns on the fluorescence of the coumarin. The fluorescence hydrogel developed holds great promise as an injectable, implantable glucose-sensing biomaterials for in vivo continuous glucose monitoring.
Keywords: Fluorescence; Hydrogel; Glucose; Glucose oxidase; Glucose sensing; Switch-on fluorescent chemosensors

While bacteria have been primarily studied for phosphorus (P) removal in wastewater treatment, fungi and their ability to accumulate intracellular polyphosphate are less investigated. P-accumulating fungal strains were screened from soybean plants and surrounding soil by flask cultivation with potato dextrose broth and KH2PO4 in this study. Mucor circinelloides was selected for its high efficiency in P removal efficiency and high cellular P content. Neisser staining and growth-curve analysis confirmed that M. circinelloides stored polyphosphate intracellularly by luxury phosphate uptake. The effect of culture medium compositions on P removal efficiency and cellular P content was also investigated. Monosaccharides (such as glucose and fructose) and organic nitrogen (N, such as urea, and peptone) promoted fungi growth and P accumulation. M. circinelloides also preferred organic phosphates. When glucose, urea, and phytic acid sodium salt were used as the carbon, N, and P source, respectively, the maximum utilization efficiency was 40.1 % for P and 7.08 % for cellular P content. In addition, the potential of M. circinelloides for P removal from waste streams was investigated. Compared with the non-inoculated control culture, inoculation with M. circinelloides improved the soluble P removal in treating wastewater centrate, screened manure, and digested manure.
Keywords: Phosphorus-accumulating fungi; Mucor circinelloides ; Polyphosphate; Phosphorus removal; Waste streams

Postsynthetic Domain Assembly with NpuDnaE and SspDnaB Split Inteins by Daniel Demonte; Naiyi Li; Sheldon Park (1137-1151).
Inteins are protein segments embedded in frame within a precursor sequence that catalyze a self-excision reaction and ligate the flanking sequences with a standard peptide bond. Split inteins are expressed as two separate polypeptide fragments and trans-splice upon subunit association. Split inteins have found use in biotechnology applications but their use in postsynthetic domain assembly in vivo has been limited to the ligation of two protein domains. Alternatively, they have been used to splice three domains and fragments in vitro. To further develop split intein-based applications in vivo, we have designed a cell-based assay for the postsynthetic splicing of three protein domains using orthogonal split inteins. Using naturally and artificially split inteins, NpuDnaE and SspDnaB, we show that a multidomain protein of 128 kDa can be assembled in Escherichia coli from individually expressed domains. In the current system, the main bottleneck in achieving high yield of tandem trans-spliced product appears to be the limited solubility of the SspDnaB precursors. Optimizing protein solubility should be important to achieve efficient combinatorial synthesis of protein domains in the cell.
Keywords: Split intein; Protein trans-splicing; NpuDnaE; SspDnaB; Domain assembly; Protein ligation

The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate. It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method. In this paper, we have compared the DNS and NS reducing sugar assays using a range of xylan-type substrates and accurately compared the molar response factors for xylose and a range of xylo-oligosaccharides. Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity. Unlike the DNS assay, the NS assay gave the equivalent colour response with equimolar amounts of xylose, xylobiose, xylotriose and xylotetraose demonstrating that it accurately measures the quantity of glycosidic bonds cleaved by the endo-xylanase. The authors strongly recommend cessation of the use of the DNS assay for measurement of endo-xylanase due to the fact that the values obtained are grossly overestimated due to secondary reactions in colour development.
Keywords: endo-xylanase; Beechwood xylan; Wheat arabinoxylan; Nelson-Somogyi; 3,5-Dinitrosalicylic acid; Xylo-oligosaccharides; Reducing sugars

Interferon (IFN)-λ, also known as IL-28A, IL-28B, or IL-29, is a new type III IFN, which shares many functional characteristics with type I IFN (α/β). Currently, IFN-α is used in the treatment of certain forms of cancer with severe adverse effects. Some researches had stated that IFN-λs induced a similar but restricted growth inhibition of tumor cells relative to IFN-α; moreover, mutations of IFN-λs could strongly impact its biological properties. In this study, three hIL-29 mutants (K33R, R35K, and K33R/R35K) were generated by site-directed mutagenesis and efficiently expressed in Pichia pastoris GS115, which have considerable abilities to inhibit the growth of BEL-7402, HCT-8, and SGC-7901 tumor cells in vitro. The results showed that these mutants (K33R, R35K, and K33R/R35K) exhibited a significantly enhanced anti-proliferation activity against these tumor cells, compared with native hIL-29 in vitro. Further assay in vitro indicated that superior to K33R and R35K, K33R/R35K had a significant increase in anti-tumor activity compared with IFN-α2b, which suggested that the K33R/R35K could make improvement for the effectiveness of native hIL-29 in clinic and could be used as a potentially powerful candidate for cancer immunotherapy.
Keywords: Human interleukin-29; Site-directed mutagenesis; Interferon-α; Anti-proliferation activity; Tumor cell

Non-edible oils are preferred raw materials for biodiesel production. However, the properties of raw materials significantly affect the synthesis process, leading to difficulties to design one process suitable for any kind of raw material. In this study, the composition of five typical non-edible oils was analyzed. The major difference was the content of free fatty acids, reflected from their acid values. The influence of different oils was investigated by using lipase from Candida sp. 99–125. At low lipase dosage and low water content, the conversion was found proportional to the acid value. However, by increasing the water content or lipase dosage, we observed that the conversions for all kinds of oils used in this study could exceed 80 %. Time course analysis indicates that the lipase used in this study catalyzed hydrolysis followed by esterification, rather than direct transesterification. Accumulation of free fatty acids at the very beginning was necessary. A high water content facilitated the hydrolysis of oils with low acid value. This lipase showed capability to transform all the oils by controlling the water content.
Keywords: Acid value; Biodiesel; Lipase; Non-edible oils; Raw materials

Seventeen bacterial isolates were screened for their cellulase activity by carboxymethyl cellulose (CMC) plate assay. The bacterial strain K1 showed the largest depolymerized region in CMC plate assay and was further studied for quantitative cellulase activity. On the basis of 16S rDNA sequence analysis, the strain K1 was found to be Bacillus sp. This strain produced the maximum CMCase at pH 6 and 50 °C in the presence of peptone (1 %) as a source of nitrogen. The CMCase activity was stimulated by Ca2+ (2 mM) by 20 % over the control. The CMCase activity of this Bacillus sp. K1 was highly induced when lactose was used as a source of carbon during fermentation.
Keywords: Cellulase; Bacillus ; Optimization

Erratum to: Nonclassically Secreted Proteins as Possible Antigens for Vaccine Development: A Reverse Vaccinology Approach by Mauricio de Alvarenga Mudadu; Viviane Carvalho; Sophie Yvette Leclercq (1199-1199).