Applied Biochemistry and Biotechnology (v.178, #5)

Potential Application of Biohydrogen Production Liquid Waste as Phosphate Solubilizing Agent—A Study Using Soybean Plants by Saurabh Jyoti Sarma; Satinder Kaur Brar; Yann LeBihan; Gerardo Buelna (865-875).
With CO2 free emission and a gravimetric energy density higher than gasoline, diesel, biodiesel, and bioethanol, biohydrogen is a promising green renewable energy carrier. During fermentative hydrogen production, 60–70 % of the feedstock is converted to different by-products, dominated by organic acids. In the present investigation, a simple approach for value addition of hydrogen production liquid waste (HPLW) containing these compounds has been demonstrated. In soil, organic acids produced by phosphate solubilizing bacteria chelate the cations of insoluble inorganic phosphates (e.g., Ca3 (PO4)2) and make the phosphorus available to the plants. Organic acid-rich HPLW, therefore, has been evaluated as soil phosphate solubilizer. Application of HPLW as soil phosphate solubilizer was found to improve the phosphorus uptake of soybean plants by 2.18- to 2.74-folds. Additionally, 33–100 % increase in seed germination rate was also observed. Therefore, HPLW has the potential to be an alternative for phosphate solubilizing biofertilizers available in the market. Moreover, the strategy can be useful for phytoremediation of phosphorus-rich soil.
Keywords: Biohydrogen; Bioremediation; Organic acids; Phosphate solubilization

A laboratory study was delineated to ascertain the impact and the extent of Dolichos yellow mosaic virus (DYMV) on biochemical constituents and various enzyme levels in the leaves of hyacinth bean. DYMV-infected leaves of all the genotypes used in the study revealed significant and consistent changes in activities of CAT, APX, PPO, DHAR, and MDHAR paralleled with a compelling hike in proline levels. Unlike that in non-infected leaves of the genotypes VRSEM-301 and VRSEM-749, VRSEM-894 and VRSEM-855, the enzyme level did not alter much which extended equally with increased phenolics, suggesting a well-coordinated generation of free radicals thereby suppressing oxidative stress in the latter. The genotypes were also evaluated at molecular level for elucidating the presence of the virus by using five sets of primer pairs. Two primers viz., DAC1 and DAC2 witnessed the presence of the virus in both non-infected and infected leaves. The difference in the appearance and/or disappearance of bands according to non-infected to infect reverberates the variation between genotypes in defense against infection.
Keywords: Dolichos yellow mosaic virus (DYMV); Lablab perpureus ; Antioxidant enzymes; Photosynthetic pigments

A Novel, Poly(Ethyl Ethylene Ether) Inhibitor to Trypsin from Marine Cyanobacteria, Lyngbya confervoides by Ambika Devi; Shankar Prasanth; Easwaran Murugesh; Karickal R. Haridas; Abdulhameed Sabu; Madhathilkovilakathu Haridas (891-899).
A novel, poly(ethyl ethylene ether) inhibitor to trypsin was purified from marine cyanobacteria, Lyngbya confervoides from the coastal areas of Thalassery, North Kerala. The kinetics and the thermodynamic parameters of its interactions with the enzyme were also studied. It was demonstrated that the substrate binding, catalytic triad of the enzyme could be blocked by the inhibitor, as expressed by molecular simulation studies. The study also showed that the cyanobacterial group could prove to be a potential source of novel enzyme inhibitors for various applications.
Keywords: Cyanobacteria; Lyngbya sp; Trypsin inhibitors; Poly(ethyl ethylene ether)

A Comprehensive Review on l-Asparaginase and Its Applications by Tahira Batool; Essam A. Makky; Muna Jalal; Mashitah M. Yusoff (900-923).
l-asparaginase (LA) catalyzes the degradation of asparagine, an essential amino acid for leukemic cells, into ammonia and aspartate. Owing to its ability to inhibit protein biosynthesis in lymphoblasts, LA is used to treat acute lymphoblastic leukemia (ALL). Different isozymes of this enzyme have been isolated from a wide range of organisms, including plants and terrestrial and marine microorganisms. Pieces of information about the three-dimensional structure of l-asparaginase from Escherichia coli and Erwinia sp. have identified residues that are essential for catalytic activity. This review catalogues the major sources of l-asparaginase, the methods of its production through the solid state (SSF) and submerged (SmF) fermentation, purification, and characterization as well as its biological roles. In the same breath, this article explores both the past and present applications of this important enzyme and discusses its future prospects.
Keywords: l-asparaginase; Lymphoblasts; Acute lymphoblastic leukemia; Solid state fermentation; Submerged fermentation

Abnormal concentrations of amino acids in blood and urine can be indicative of several diseases, including cancer and diabetes. Therefore, analyses that examine amino acid concentrations are useful for the diagnosis of such diseases. In this study, we developed an enzyme-immobilized, small reactor column for flow analysis of amino acid concentrations. For the recognition of asparagine and lysine, asparaginyl-tRNA synthetase and lysyl-tRNA synthase were immobilized onto microparticles, respectively, and coupled with coloration reagents for spectrophotometric detection. This assay has some advantages in the analytical field, such as the ability to detect small amounts of analyte, allowing for the use of a small reaction volume, and ensuring a rapid and efficient reaction rate. This approach provided selective quantitation of up to 480 μM of asparagine and lysine in 200 mM Tris–HCl buffer (pH 8.0).
Keywords: Amino acid; Aminoacyl-tRNA synthetase; Biosensing; Enzyme reaction; Flow analysis

Dry Co-Digestion of Poultry Manure with Agriculture Wastes by Fatma Abouelenien; Yuzaburo Namba; Naomichi Nishio; Yutaka Nakashimada (932-946).
This study tested the effect on thermophilic and mesophilic digestion of poultry manure (PM) or treated poultry manure (TPM) by the addition of agriculture wastes (AWS) as a co-substrate under dry conditions. PM was co-digested with a mixture of AWS consisting of coconut waste, cassava waste, and coffee grounds. Results were increased methane content in biogas, with decreased ammonia accumulation and volatile acids. The highest performance occurred under mesophilic conditions, with a 63 and 41.3 % increase in methane production from addition of AWS to TPM (562 vs. 344 mL g VS−1 from control) and PM (406 vs. 287 mL g VS−1 from control), respectively. Thermophilic conditions showed lower performance than mesophilic conditions. Addition of AWS increased methane production by 150 and 69.6 % from PM (323.4 vs. 129 mL g VS−1 from control) and TPM (297.6 vs. 175.5 mL g VS−1 from control), respectively. In all experiments, 100 % acetate produced was degraded to methane. Maximum ammonia accumulation was lowered to 43.7 % by mixing of AWS (range 5.35–8.55 vs. 7.81–12.28 g N kg−1 bed). The pH was held at 7.3–8.8, a range suitable for methanogenesis.
Keywords: Dry co-digestion; Methane production; Poultry manure; Coffee waste; Cassava waste; Coconut waste

Characteristics of a Novel Aerobic Denitrifying Bacterium, Enterobacter cloacae Strain HNR by Long-Jie Guo; Bin Zhao; Qiang An; Meng Tian (947-959).
A novel aerobic denitrifier strain HNR, isolated from activated sludge, was identified as Enterobacter cloacae by16S rRNA sequencing analysis. Glucose was considered as the most favorable C-source for strain HNR. The logistic equation well described the bacterial growth, yielding a maximum growth rate (μmax) of 0.283 h−1 with an initial NO3 -N concentration of 110 mg/L. Almost all NO3 -N was removed aerobically within 30 h with an average removal rate of 4.58 mg N L−1 h−1. Nitrogen balance analysis revealed that proximately 70.8 % of NO3 -N was removed as gas products and only 20.7 % was transformed into biomass. GC-MS result indicates that N2 was the end product of aerobic denitrification. The enzyme activities of nitrate reductase and nitrite reductase, which are related to the process of aerobic denitrification, were 0.0688 and 0.0054 U/mg protein, respectively. Thus, the aerobic denitrification of reducing NO3 to N2 by strain HNR was demonstrated. The optimal conditions for nitrate removal were C/N ratio 13, pH value 8, shaking speed 127 rpm and temperature 30 °C. These findings show that E. cloacae strain HNR has a potential application on wastewater treatment to achieve nitrate removal under aerobic conditions.
Keywords: Aerobic denitrification; Nitrate removal; Nitrogen balance; Nitrate reductase; Nitrite reductase

Community-Level Physiological Profiling of Microbial Communities in Constructed Wetlands: Effects of Sample Preparation by Mark Button; Kela Weber; Jaime Nivala; Thomas Aubron; Roland Arno Müller (960-973).
Community-level physiological profiling (CLPP) using BIOLOG® EcoPlates™ has become a popular method for characterizing and comparing the functional diversity, functional potential, and metabolic activity of heterotrophic microbial communities. The method was originally developed for profiling soil communities; however, its usage has expanded into the fields of ecotoxicology, agronomy, and the monitoring and profiling of microbial communities in various wastewater treatment systems, including constructed wetlands for water pollution control. When performing CLPP on aqueous samples from constructed wetlands, a wide variety of sample characteristics can be encountered and challenges may arise due to excessive solids, color, or turbidity. The aim of this study was to investigate the impacts of different sample preparation methods on CLPP performed on a variety of aqueous samples covering a broad range of physical and chemical characteristics. The results show that using filter paper, centrifugation, or settling helped clarify samples for subsequent CLPP analysis, however did not do so as effectively as dilution for the darkest samples. Dilution was able to provide suitable clarity for the darkest samples; however, 100-fold dilution significantly affected the carbon source utilization patterns (CSUPs), particularly with samples that were already partially or fully clear. Ten-fold dilution also had some effect on the CSUPs of samples which were originally clear; however, the effect was minimal. Based on these findings, for this specific set of samples, a 10-fold dilution provided a good balance between ease of use, sufficient clarity (for dark samples), and limited effect on CSUPs. The process and findings outlined here can hopefully serve future studies looking to utilize CLPP for functional analysis of microbial communities and also assist in comparing data from studies where different sample preparation methods were utilized.
Keywords: CLPP; Domestic wastewater; Horizontal flow; Microbial activity; Treatment wetland

Surface modification of multiwalled carbon nanotubes (MWCNTs) through functionalization could improve the characteristics of these nanomaterials as support for enzymes. Carboxylation of MWCNTs (MWCNT-COOH) has been carried out in this study using the dielectric barrier discharge (DBD) plasma reactor through humidified air. The chemical method was also used for further functionalization of the MWCNT-COOH through which the amidation of the surfaces with either butylamine (MWCNT-BA) or octadecylamine (MWCNT-OA) was performed. By immobilization of Candida antarctica B lipase (CALB) on these nanoparticles, performance of the immobilized enzyme in catalyzing methanolysis of rapeseed oil was evaluated. The CALB loading on the MWCNT-BA and MWCNT-COOH was 20 mg protein/g, while the value for MWCNT-OA was 11 mg protein/g. The yield of biodiesel was determined as percentage of mass of fatty acid methyl ester (FAME) produced per initial mass of the oil, and the yield value for the two of these three supports namely, MWCNT-COOH and MWCNT-BA used for the CALB immobilization was similar at about 92 %, while 86 % was the yield for the reaction catalyzed by the lipase immobilized on MWCNT-OA. Thermal stability of the immobilized CALB and the catalytic ability of the enzyme in the repeated batch experiments have also been determined.
Keywords: MWCNTs; DBD reactor; Plasma functionalized MWCNTs; Immobilized CALB; Rapeseed oil; Biodiesel

Properties of Polyhydroxyalkanoate Granules and Bioemulsifiers from Pseudomonas sp. and Burkholderia sp. Isolates Growing on Glucose by Laís Postai Sacco; Tereza Cristina Luque Castellane; Erica Mendes Lopes; Eliana Gertrudes de Macedo Lemos; Lúcia Maria Carareto Alves (990-1001).
A Burkholderia and Pseudomonas species designated as AB4 and AS1, respectively, were isolated from soil containing decomposing straw or sugar cane bagasse collected from Brazil. This study sought to evaluate the capacities of culture media, cell-free medium, and crude lysate preparations (containing PHB inclusion bodies) from bacterial cell cultures to stabilize emulsions with several hydrophobic compounds. Four conditions showed good production of bioemulsifiers (E24 ≥ 50 %), headed by substantially cell-free media from bacterial cell cultures in which bacterial isolates from Burkholderia sp. strain AB4 and Pseudomonas sp. strain AS1 were grown. Our results revealed that the both isolates (AB4 and AS1 strains) exhibited high emulsification indices (indicating usefulness in bioremediation) and good stabilities.
Keywords: Bioemulsifier; Biopolymers; Burkholderia sp.; Emulsifying activity; Polyhydroxybutyrate; Pseudomonas sp.

The present investigation aimed to evaluate the degree and pattern of DNA methylation using methylation-sensitive AFLP (MS-AFLP) markers in genetically stable in vitro regenerates of Jatropha curcas L.. The genetically stable in vitro regenerates were raised through direct organogenesis via enhanced axillary shoot bud proliferation (Protocol-1) and in vitro-derived leaf regeneration (Protocol-2). Ten selective combinations of MS-AFLP primers produced 462 and 477 MS-AFLP bands in Protocol-1 (P-1) and Protocol-2 (P-2) regenerates, respectively. In P-1 regenerates, 15.8–31.17 % DNA was found methylated with an average of 25.24 %. In P-2 regenerates, 15.93–32.7 % DNA was found methylated with an average of 24.11 %. Using MS-AFLP in P-1 and P-2 regenerates, 11.52–25.53 % and 13.33–25.47 % polymorphism in methylated DNA was reported, respectively. Compared to the mother plant, P-1 regenerates showed hyper-methylation while P-2 showed hypo-methylation. The results clearly indicated alternation in degree and pattern of DNA methylation; hence, epigenetic instability in the genetically stable in vitro regenerates of J. curcas, developed so far using two different regeneration systems and explants of two different origins. The homologous nucleotide fragments in genomes of P-1 and P-2 regenerates showing methylation re-patterning might be involved in immediate adaptive responses and developmental processes through differential regulation of transcriptome under in vitro conditions.
Keywords: Epigenetic; In vitro; Jatropha ; Methylation polymorphism and MS-AFLP

AGEs Induce Apoptosis in Rat Osteoblast Cells by Activating the Caspase-3 Signaling Pathway Under a High-Glucose Environment In Vitro by Jiaqiang Liu; Jing Mao; Yi Jiang; Lunguo Xia; Lixia Mao; Yong Wu; Pan Ma; Bing Fang (1015-1027).
Advanced glycation end products (AGEs) accumulate under high-glucose conditions and affect the healing of bone damage through various pathways; however, the detail mechanisms underlying these changes are unknown. In this study, we investigated the effects of AGEs on the apoptosis of in vitro-cultured rat osteoblasts under high-glucose conditions and explored the underlying mechanisms of these effects. First, we cultured rat osteoblasts and determined the accumulation of AGEs in the culture medium under high-glucose conditions. Then, we cultured rat osteoblasts under a high glucose concentration (35 mM), a normal glucose concentration (5.5 mM), and a normal glucose concentration (5.5 mM) in the presence of AGEs. We examined the effects of high glucose and AGEs on the apoptosis of rat osteoblasts at different time points and further analyzed the activity and changes in the levels of procaspase-3, caspase-3, and the caspase-3 substrate poly ADP-ribose polymerase (PARP). Finally, we added sRAGE (soluble RAGE) (an AGE inhibitor) or DEVD (a caspase-3 inhibitor) to each culture group and examined apoptosis under each culture condition and the changes in the levels of procaspase-3, caspase-3, and its substrate PARP. The results showed that the high-glucose condition and the addition of AGEs increased the apoptosis of rat osteoblast cells and simultaneously increased the activity and quantity of caspase-3. These increases could be inhibited by the AGE inhibitor sRAGE or the caspase-3 inhibitor DEVD. The above results demonstrate that high-glucose conditions lead to the accumulation of AGEs and activation of the caspase-3 signaling pathway, resulting in the increased apoptosis of cultured rat osteoblast cells.
Keywords: High glucose; AGEs; Osteoblasts; Apoptosis; Caspase-3

The Induction of Growth Inhibition and Apoptosis in HeLa and MCF-7 Cells by Teucrium sandrasicum, Having Effective Antioxidant Properties by Leman Tarhan; Mahmure Nakipoğlu; Berna Kavakcıoğlu; Burcu Tongul; Ayşe Nalbantsoy (1028-1041).
The hidromethanolic (Met/W), ethyl acetate (EA(EA/W)), and water (W(EA/W)) extracts from Teucrium sandrasicum leaves (L) and flowers (F) were investigated for antioxidant properties and antiproliferative effects on HeLa, MCF-7, and L929. The highest DPPH scavenging, metal chelating capacities, and total phenolic and flavonoid contents were observed in Met/WL. The highest hydroxyl scavenging and reducing power capacities were found in EA(EA/W)L. Met/WL, EA(EA/W)L and EA(EA/W)F inhibited cancer cell growths, while they did not show significant cytotoxicity on L929. While the reactive oxygen species (ROS) levels were generally close to controls in HeLa, they were induced in MCF-7 with the treatment of Met/WL, EA(EA/W)L, and EA(EA/W)F and acted as antioxidant for L929. The highest apoptosis inductions were observed in Met/WL-treated HeLa and EA(EA/W)L-treated MCF-7, which were supported with the changes in mitochondrial membrane potentials. The highest caspase-9 activities were found in Met/WL-treated HeLa and EA(EA/W)F-treated MCF-7. Caspase-3 activity was only induced in EA(EA/W)F-treated HeLa.
Keywords: Teucrium sandrasicum ; HeLa; MCF-7; Antioxidant; Intracellular ROS levels; Apoptotic cell death

SSR Markers Associated with Proline in Drought Tolerant Wheat Germplasm by Muhammad Javid Iqbal; Yasir Maqsood; Zain Ul Abdin; Atif Manzoor; Muhammad Hassan; Amer Jamil (1042-1052).
Water stress causes major agricultural loss throughout the world as survival of the crops remained under stress and loss in yield. Plants respond to drought stress by means of different adaptive mechanisms such as accumulation of osmoprotectants to counteract the water stress. Amino acid proline is known to occur widely in higher plants and normally accumulates in large quantities as an osmolyte in response to environmental stresses. Biochemical estimation of proline was done in the drought-affected wheat genotypes by spectrophotometric method. Proline promoted a positive effect as root/shoot ratio was enhanced in wheat germplasm under drought stress. SSR primer pairs (45) were tested for polymorphism among selected wheat genotypes. The dendrogram results have shown the wheat genotype association with the levels of proline during induced drought stress. The relationship between pattern of drought responsive biochemical attributes and DNA markers in the selected wheat genotypes was recognized to select drought tolerant genotypes for sowing in drought affected areas of the country.
Keywords: Wheat; Proline; Stress; Markers; Drought

The battle against the prevalence of hospital-acquired infections has underscored the importance of identifying and maintaining the cleanliness of possible infection transmission sources in the patient’s environment. One of the most crucial lines of defense for mitigating the spread of pathogens in a healthcare facility is the removal of microorganisms from the environment by air filtration systems. After removing the pathogenic microorganisms, the filters used in these systems can serve as reservoirs for the pathogens and pose a risk for secondary infection. This threat, combined with the ever-growing prevalence of drug-resistant bacterial strains, substantiates the need for an effective bactericidal air filter. To this end, a broad-spectrum bactericidal polyurethane incorporating immobilized quaternary ammonium groups was developed for use as an air filter coating. In this study, the bactericidal activity of the polymer coating on high-efficiency particulate air (HEPA) filter samples was quantified against eight bacterial strains commonly responsible for nosocomial infection—including drug-resistant strains, and confirmed when applied as a filter coating in conditions mimicking those of its intended application. The coated HEPA filter samples exhibited high bactericidal activity against all eight strains, and the polyurethane was concluded to be an effective coating in rendering HEPA filters bactericidal.
Keywords: Pathogens; Antibacterial; Air filtration system; Drug-resistant strains; Coating