Applied Biochemistry and Biotechnology (v.186, #2)

Improving of Anticancer Activity and Solubility of Cisplatin by Methylglycine and Methyl Amine Ligands Against Human Breast Adenocarcinoma Cell Line by Fatemeh Safa Shams Abyaneh; Mahboube Eslami Moghadam; Adeleh Divsalar; Davood Ajloo; Moyaed Hosaini Sadr (271-291).
Methylglycine, also known sarcosine, is dramatically used in drug molecules and its metal complexes can interact to DNA and also do cleavage. Hence, to study the influence of methylglycine ligand on biological behavior of metal complexes, two water-soluble platinum (II) complexes with the formula cis-[Pt(NH3)2(CH3-gly)]NO3 and cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 (where CH3-gly is methylglycine) have been synthesized and characterized by spectroscopic methods, molar conductivity measurements, and elemental analyzes. The anticancer activity of synthesized complexes was tested against human breast adenocarcinoma cell line of MCF7 using MTT assay and results showed excellent anticancer activity with Cc50 values of 126 and 292 μM after 24 h incubation time, for both complexes of cis-[Pt(NH3)2(CH3gly)]NO3 and cis-[Pt(NH2-CH3)2(CH3gly)]NO3, respectively. Also, the interaction between Pt(II) complexes with calf thymus DNA was extensively studied by means of absorption spectroscopy, fluorescence titration spectra displacement with ethidium bromide (EtBr), and circular dichroism studied in Tris-buffer. The obtained spectroscopic results revealed that two complexes can bind to highly polymerized calf thymus DNA cooperatively and denature at micromolar concentrations. The fluorescence data indicate that quenching effect for cis-[Pt(NH3)2(CH3gly)]NO3 (K sv = 9.48 mM−1) was higher than that of cis-[Pt(NH2-CH3)2(CH3gly)]NO3 (K sv = 1.98 mM−1). These results were also confirmed by circular dichrosim spectra. Consequently, docking data showed that cis-[Pt(NH3)2(CH3gly)]NO3 with more interaction energy binds on DNA via groove binding which is more compatible with experimental results. Graphical AbstractᅟTwo anticancer Pt(II) complexes, cis-[Pt(NH3)2(CH3gly)]NO3 and cis-[Pt(NH2−CH3)2(CH3gly)]NO3, have been synthesized and interacted with calf thymus DNA. Improving solubility of these compounds reduce side effects and increase anticancer activity against human breast cell line. Modes of binding have been studied by electronic absorption, fluorescence, and CD measurements. Results show that both Pt(II) complexes can interact to DNA via groove binding.
Keywords: Cisplatin analog; Anticancer drug; Cytotoxicity; Methylglycine; DNA interaction; Thermodynamic parameters; Molecular docking

Synthesis of Lactosucrose Using a Recombinant Levansucrase from Brenneria goodwinii by Wei Xu; Qian Liu; Shuhuai Yu; Tao Zhang; Wanmeng Mu (292-305).
Lactosucrose is a kind of trisaccharide that functions as a significant prebiotic in the maintenance of gastrointestinal homeostasis for human. In this study, a levansucrase from Brenneria goodwinii was further used for the lactosucrose production. The recombinant levansucrase showed efficiency in the lactosucrose production by transfructosylation from sucrose and lactose, and no other oligosaccharide or polysaccharide was detected in the reaction mixture. The transfructosylation product by this recombinant enzyme was structurally determined to be lactosucrose by FT-IR and NMR. The production condition was optimized as pH at 6.0, temperature at 35 °C, 5 U mL−1 enzyme, 180 g L−1 sucrose, and 180 g L−1 lactose. Under the optimal condition, the enzyme could approximately produce 100 g L−1 lactosucrose when the reaction reached equilibrium. The recombinant levansucrase could effectively and exclusively catalyze the formation of lactosucrose, which might expand the enzymatic choice for further preparation of lactosucrose.
Keywords: Lactosucrose; Levansucrase; Transfructosylation; Optimized

An Overview of the Genetics of Plant Response to Salt Stress: Present Status and the Way Forward by Fawad Kaleem; Ghulam Shabir; Kashif Aslam; Sumaira Rasul; Hamid Manzoor; Shahid Masood Shah; Abdul Rehman Khan (306-334).
Salinity is one of the major threats faced by the modern agriculture today. It causes multidimensional effects on plants. These effects depend upon the plant growth stage, intensity, and duration of the stress. All these lead to stunted growth and reduced yield, ultimately inducing economic loss to the farming community in particular and to the country in general. The soil conditions of agricultural land are deteriorating at an alarming rate. Plants assess the stress conditions, transmit the specific stress signals, and then initiate the response against that stress. A more complete understanding of plant response mechanisms and their practical incorporation in crop improvement is an essential step towards achieving the goal of sustainable agricultural development. Literature survey shows that investigations of plant stresses response mechanism are the focus area of research for plant scientists. Although these efforts lead to reveal different plant response mechanisms against salt stress, yet many questions still need to be answered to get a clear picture of plant strategy to cope with salt stress. Moreover, these studies have indicated the presence of a complicated network of different integrated pathways. In order to work in a progressive way, a review of current knowledge is critical. Therefore, this review aims to provide an overview of our understanding of plant response to salt stress and to indicate some important yet unexplored dynamics to improve our knowledge that could ultimately lead towards crop improvement.
Keywords: ABA pathway; Plant signaling; Plant stress response mechanisms; Salinity; SOS pathway

A Chimeric Two-Component Regulatory System-Based Escherichia coli Biosensor Engineered to Detect Glutamate by Sambandam Ravikumar; Yokimiko David; Si Jae Park; Jong-il Choi (335-349).
In this study, we constructed amino acid biosensors that can be used as a high-throughput system to screen microorganisms that produce glutamate. The biosensors are based on two-component regulatory systems (TCRSs) combined with green fluorescent protein (GFP) as a reporter. A chimeric DegS/EnvZ (DegSZ) TCRS was constructed by fusing the N-terminal domain of the sensor kinase DegS from Planococcus sp. PAMC21323 with the catalytic domain of the osmosensor EnvZ from Escherichia coli to control expression of gfp in response to glutamate. gfp was controlled by the ompC promoter through the activated response regulator OmpR-P. The chimeric TCRS-based biosensors showed a 4-fold increase in the fluorescent signal after adding glutamate. A linear correlation was observed between fluorescence intensity and exogenously added glutamate concentration. The chimeric TCRS-based biosensor was used to determine glutamate concentration at the single-cell level by fluorescence-activated cell sorting. Therefore, this biosensor can be used to isolate novel gene products and optimize pathways involved in amino acid production.
Keywords: DegS; EnvZ; Glutamate; Escherichia coli ; Two-component system

Cytotoxicity of Silica Nanoparticles with Transcaucasian Nose-Horned Viper, Vipera ammodytes transcaucasiana, Venom on U87MG and SHSY5Y Neuronal Cancer Cells by Çiğdem Çelen; Ceren Keçeciler; Mert Karış; Bayram Göçmen; Ozlem Yesil-Celiktas; Ayşe Nalbantsoy (350-357).
Highly bioactive compounds of the snake venom make them particular sources for anticancer agent development. They contain very rich peptide-protein structures. Therefore, they are very susceptible to environmental conditions such as temperature, pH, and light. In this study, Vipera ammodytes transcaucasiana venom was encapsulated in PAMAM-G4 dendrimer by sol-gel method in order to prevent degradation of venom contents from the environmental conditions. For this purpose, nanoparticles were prepared by sol-gel methodology and SEM analyses were performed. U87MG and SHSY5Y neuronal cancer cell lines were treated with different concentrations of venom-containing nanoparticles and cytotoxicity was determined by MTT assay. IC50 values of nanoparticles with snake venom were calculated as 37.24 and 44.64 μg/ml for U87MG and SHSY5Y cells, respectively. The IC50 values of nanoparticles with snake venom were calculated as 10.07 and 7.9 μg/ml for U87MG and SHSY5Y cells, respectively. As a result, nanoparticles with V. a. transcaucasiana venom showed remarkably high cytotoxicity. Encapsulation efficiency of nanoparticles with 1 mg/ml snake venom was determined as %67 via BCA™ protein analysis. In conclusion, this method is found to be convenient and useful for encapsulating snake venom as well as being suitable for drug delivery systems.
Keywords: Snake venom; Encapsulation; Sol-gel; Cytotoxicity; Nerve cells

Establishment of Aromatic Pairs at the Surface of Chondroitinase ABC I: the Effect on Activity and Stability by Mohammad Esmaeil Shahaboddin; Khosro Khajeh; Abolfazl Golestani (358-370).
Removal of chondroitin sulfate glycosaminoglycan (GAG) chains with chondroitinase ABC I (chABC I) in CNS injury models promotes both saxon regeneration and plasticity. It has been suggested that direct interaction between an aromatic pair appears to contribute about − 1.3 kcal/mol to the stability of a folded protein, so introducing an aromatic pair by point mutation might increase the enzyme activity and thermal stability as in the case of mesophilic xylanase, although using this approach destabilized T4 lysozyme. In this study, we used site-directed mutagenesis to investigate the effect of new aromatic pairs on activity and stability of chABC I. We replaced Ile295, Ser581, and Gly730 adjacent to pre-existing aromatic residues with Tyr to obtain new aromatic pairs, i.e., Tyr295/His372, Tyr576/Tyr581, and Tyr623/Tyr730. Results showed that Km values of S581Y and G730Y variants decreased relative to wild-type enzyme while their catalytic efficiency (kcat/Km) increased but I295Y variant was inactive. Also, long-term and thermal stability of the active mutants was decreased. Fluorescence and circular dichroism studies showed that these mutations resulted in a more flexible enzyme structures: a finding which was confirmed by thermal and limited proteolytic studies. In conclusion, the activity of chABC I can be improved by introducing appropriate aromatic pairs at the enzyme surface. This approach did not provide any promising results regarding the enzyme stability.
Keywords: Aromatic pair; Chondroitinase ABC I; Catalytic efficiency; Circular dichroism; Fluorometric assay; Limited trypsinolysis

Diabetes mellitus, which is the result of autoimmune destruction of the insulin-producing β cells, occurs by loss of insulin-secreting capacity. The insufficient source of insulin-producing cells (IPCs) is the major obstacle for using transplantation as diabetes treatment method. The present study suggests a method to form islet-like clusters of IPCs derived from mouse embryonic stem cells (mESCs). This protocol consists of several steps. Before starting this protocol, embryoid bodies (EBs) should be cultured in suspension in conditioned medium of isolated mouse pancreatic islet in combination with activing A to be induced. Then differentiated mESCs were replaced with dishes supplemented with basic fibroblast growth factor (bFGF). Next, bFGF was withdrawn, and cyclopamine and noggin were added. Then the cells were treated with B27, nicotinamide, and islet-conditioned medium for maturation. mESCs, as the control group, were cultured without any treatment. An enhanced expression of pancreatic-specific genes was detected by qRT-PCR and immunofluorescence in the differentiated mESCs. The differentiated mESCsco express other markers of pancreatic islet cells as well as insulin. This method exhibited higher insulin generation and further improvement in IPCs protocol that may result in an unlimited source of ES cells suitable for transplantation. The results indicated that conditioned medium, just as critical components of the stem cell niche associated with other factors, had high potential to differentiate mESCs into IPCs.
Keywords: Co-culture; Diabetes; Differentiation; ES cells; Insulin-secreting cells; Pancreatic development

Chaetominine (CHA), a novel framework tripeptide alkaloid, imparts an attractive cytotoxic against the human leukemia cell line K562, which is produced by Aspergillus fumigatus CY018. However, its pharmacological research is restricted by low yields in submerged culture, which needs to be resolved immediately by biotechnology. In this work, a co-addition strategy was applied to promote CHA production based on related inhibitors’ addition and precursors’ addition, inspired by the biosynthetic pathway analysis of CHA. CHA production reached 53.87 mg/L by addition of 10 mM shikimate, 10 mM anthranilate, 20 mM tryptophan, and 10 mM alanine in shake flask. Compared to the control without addition of precursors, the activity of 3-deoxy-arabino-heptulosonate-7-phospahte (DAHP) synthase was significantly improved and the transcription levels of critical genes in shikimate pathway were up-regulated responded to the co-addition of precursors. The improvement of CHA production by co-addition of precursors was also successfully reproduced in the lab-scale bioreactor (5-L) system, in which CHA production reached 46.10 mg/L. This work demonstrated that precursors’ co-addition was an effective strategy for increasing CHA production, and the information obtained might be useful to the further improvement of CHA on a large scale.
Keywords: Chaetominine; Aspergillus fumigatus ; Shikimate pathway; Co-addition strategy; Submerged fermentation

Valorization of the Crude Glycerol for Propionic Acid Production Using an Anaerobic Fluidized Bed Reactor with Grounded Tires as Support Material by Talita Corrêa Nazareth; Aline Gomes de Oliveira Paranhos; Lucas Rodrigues Ramos; Edson Luiz Silva (400-413).
This study evaluated the propionic acid (HPr) production from crude glycerol (CG) (5000 mg L−1) in an anaerobic fluidized bed reactor (AFBR). Grounded tire particles (2.8–3.35 mm) were used as support material for microbial adhesion. The reactor was operated with hydraulic retention times (HRT) varying from 8 to 0.5 h under mesophilic (30 °C) conditions. The HPr was the main metabolite produced, increasing in composition from 66.5 to 99.6% by decreasing the HRT from 8 to 0.5 h. Other metabolic products were 1,3-propanediol, with a maximum of 29.4% with an HRT of 6 h, ethanol, acetic, and butyric acids. The decrease in HRT from 8 to 0.5 h decreased the HPr yield, with a maximum of 0.48 ± 0.06 g HPr g COD−1 and an HRT of 6 h, and favored HPr productivity, with a maximum of 4.09 ± 1.24 g L−1 h−1 and HRT of 0.5 h. In the biogas, the H2 content increased from 12.5 to 81.2% by decreasing the HRT from 8 to 0.5 h. These results indicate the potential application of the AFBR for HPr production using an immobilized mixed culture.
Keywords: Fermentation; Hydraulic retention time; Organic acids; Propionic acid productivity; Propionic acid yield

Hydrothermal Carbonization of Microalgae (Chlorococcum sp.) for Porous Carbons With High Cr(VI) Adsorption Performance by Yuanyuan Sun; Chang Liu; Yifan Zan; Gai Miao; Hao Wang; Lingzhao Kong (414-424).
Porous carbon adsorbents were prepared from microalgae (Chlorococcum sp.) via directly hydrothermal carbonization coupled with KOH or NH3 activation for Cr(VI) adsorption. KOH-activated porous carbons exhibit high Cr(VI) adsorption capacities than those obtained via NH3 modification (370.37 > 95.70 mg/g). The superior Cr(VI) adsorption capacity is due to high surface areas (1784 m2/g) and pore volumes of porous carbon with mesoporous and macroporous structures. The Cr(VI) adsorption result was well fitted to the Langmuir model, showing that the removal of Cr(VI) was attributed to the monolayer adsorption of activity site on carbon surface.
Keywords: Hydrothermal carbonization; Porous carbons; Microalgae (Chlorococcum sp.); Cr(VI); Adsorption

Enhanced Bioethanol Production from Potato Peel Waste Via Consolidated Bioprocessing with Statistically Optimized Medium by Tahmina Hossain; Abdul Bathen Miah; Siraje Arif Mahmud; Abdullah-Al- Mahin (425-442).
In this study, an extensive screening was undertaken to isolate some amylolytic microorganisms capable of producing bioethanol from starchy biomass through Consolidated Bioprocessing (CBP). A total of 28 amylolytic microorganisms were isolated, from which 5 isolates were selected based on high α-amylase and glucoamylase activities and identified as Candida wangnamkhiaoensis, Hyphopichia pseudoburtonii (2 isolates), Wickerhamia sp., and Streptomyces drozdowiczii based on 26S rDNA and 16S rDNA sequencing. Wickerhamia sp. showed the highest ethanol production (30.4 g/L) with fermentation yield of 0.3 g ethanol/g starch. Then, a low cost starchy waste, potato peel waste (PPW) was used as a carbon source to produce ethanol by Wickerhamia sp. Finally, in order to obtain maximum ethanol production from PPW, a fermentation medium was statistically designed. The effect of various medium ingredients was evaluated initially by Plackett-Burman design (PBD), where malt extracts, tryptone, and KH2PO4 showed significantly positive effect (p value < 0.05). Using Response Surface Modeling (RSM), 40 g/L (dry basis) PPW and 25 g/L malt extract were found optimum and yielded 21.7 g/L ethanol. This study strongly suggests Wickerhamia sp. as a promising candidate for bioethanol production from starchy biomass, in particular, PPW through CBP.
Keywords: Bioethanol; Consolidated bioprocessing; Wickerhamia sp.; Plackett-Burman design; Response surface methodology

Nanofibrous Tubular Membrane for Blood Hemodialysis by Farideh Mohammadi; Afsaneh Valipouri; Dariush Semnani; Fereshteh Alsahebfosoul (443-458).
As the most important components of a hemodialysis device, nanofibrous membranes enjoy high interconnected porosity and specific surface area as well as excellect permeability. In this study, a tubular nanofibrous membrane of polysulfone nanofibers was produced via electrospinning method to remove urea and creatinine from urine and blood serums of dialysis patients. Nanofibrous membranes were electrospun at a concentration of 11.5 wt% of polysulfone (PS) and dimethylformamide (DMF)/tetrahydrofuran (THF) with a ratio of 70/30. The effects of the rotational speed of collectors, electrospinning duration, and inner diameter of the tubular nanofibrous membrane on the urea and creatinine removal efficiency of the tubular membrane were investigated through the hemodialysis simulation experiments. It was found that the tubular membrane with an inner diameter of 3 mm elecrospun at shorter duration with lower collecting speed had the highest urea and creatinine removal efficiency. The hemodialysis simulation experiment showed that the urea and creatinine removal efficiency of the tubular membrane with a diameter of 3 mm were 90.4 and 100%, respectively. Also, three patients’ blood serums were tested with the nanofibrous membrane. The results showed that the creatinine and urea removal rates were 93.2 and 90.3%, respectively.
Keywords: Nanofibrous membrane; Blood hemodialysis; Urea and creatinine removal

Lysozymes are known as ubiquitously distributed immune effectors with hydrolytic activity against peptidoglycan, the major bacterial cell wall polymer, to trigger cell lysis. In the present study, the full-length cDNA sequence of a novel sea urchin Strongylocentrotus purpuratus invertebrate-type lysozyme (sp-iLys) was synthesized according to the codon usage bias of Pichia pastoris and was cloned into a constitutive expression plasmid pPIC9K. The resulting plasmid, pPIC9K-sp-iLys, was integrated into the genome of P. pastoris strain GS115. The bioactive recombinant sp-iLys was successfully secreted into the culture broth by positive transformants. The highest lytic activity of 960 U/mL of culture supernatant was reached in fed-batch fermentation. Using chitin affinity chromatography and gel-filtration chromatography, recombinant sp-iLys was produced with a yield of 94.5 mg/L and purity of > 99%. Recombinant sp-iLys reached its peak lytic activity of 8560 U/mg at pH 6.0 and 30 °C and showed antimicrobial activities against Gram-negative bacteria (Vibrio vulnificus, Vibrio parahemolyticus, and Aeromonas hydrophila) and Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis). In addition, recombinant sp-iLys displayed isopeptidase activity which reached the peak at pH 7.5 and 37 °C with the presence of 0.05 M Na+. In conclusion, this report describes the heterologous expression of recombinant sp-iLys in P. pastoris on a preparative-scale, which possesses lytic activity and isopeptidase activity. This suggests that sp-iLys might play an important role in the innate immunity of S. purpuratus.
Keywords: Invertebrate-type lysozyme; Strongylocentrotus purpuratus ; Pichia pastoris ; Antimicrobial activity; Isopeptidase activity

Resveratrol is extensively being used as a therapeutic moiety, as well as a pharmacophore for development of new drugs due to its multifarious beneficial effects. The objective of the present study was to isolate and screen the resveratrol-producing endophytic fungi from different varieties of Vitis vinifera. A total of 53 endophytic fungi belonging to different fungal genera were isolated from the stem and leaf tissues of Vitis vinifera (merlot, wild, pinot noir, Shiraz, muscat) from different grape-producing locations of India. Only 29 endophytic fungal isolates exhibited a positive test for phenolics by phytochemical methods. The resveratrol obtained after ethyl acetate extraction was confirmed using standard molecule on thin layer chromatography (TLC) with a retention factor (R f) of 0.69. The purified and standard resveratrol were visualized under UV light as a violet-colored spot. In HPLC analysis of the ethyl acetate extract of culture broth of 11 endophytic isolates, the highest resveratrol content was found in #12VVLPM (89.1 μg/ml) followed by #18VVLPM (37.3 μg/ml) and 193VVSTPM (25.2 μg/ml) exhibiting a retention time of 3.36 min which corresponded to the standard resveratrol. The resveratrol-producing isolates belong to seven genera viz. Aspergillus, Botryosphaeria, Penicillium, Fusarium, Alternaria, Arcopilus, and Lasiodiplodia, and using morphological and molecular methods, #12VVLPM was identified as Arcopilus aureus.
Keywords: Endophyte; Fungi; Grapes; Arcopilus sp.; Resveratrol

Efficient Biotransformation of Phytosterols to Dehydroepiandrosterone by Mycobacterium sp. by Pei Zhou; Ya-kun Fang; Hao-ke Yao; Hua Li; Gang Wang; Yu-peng Liu (496-506).
In this study, a method for the efficient production of dehydroepiandrosterone (DHEA) from phytosterols in a vegetable oil/aqueous two-phase system by Mycobacterium sp. was developed. After the 3-hydroxyl group of phytosterols was protected, they could be converted into DHEA with high yield and productivity by Mycobacterium sp. NRRL B-3683. In a shake flask biotransformation, 15.05 g l−1 of DHEA and a DHEA yield of 85.39% (mol mol−1) were attained after 7 days with an initial substrate concentration of 25 g l−1. When biotransformation was carried out in a 30-l stirred bioreactor with 25 g l−1 substrate, the DHEA concentration and yield was 16.33 g l−1 and 92.65% (mol mol−1) after 7 days, respectively. The results of this study suggest that inexpensive phytosterols could be utilized for the efficient production of DHEA.
Keywords: Phytosterols; Dehydroepiandrosterone; Oil/aqueous two-phase system biotransformation; Mycobacterium