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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.169, #5)

Random UV Mutagenesis Approach for Enhanced Biodegradation of Sulfonated Azo Dye, Green HE4B by Swati M. Joshi; Shrirang A. Inamdar; Jyoti P. Jadhav; Sanjay P. Govindwar (pp. 1467-1481).

The objective of the study was to execute mutant bacteria for efficient biodegradation of sulfonated azo dye, Green HE4B (GHE4B). UV irradiation was used to introduce random mutations in Pseudomonas sp. LBC1. Genetic alterations induced by UV irradiation in selected mutant bacteria were confirmed by random amplification of polymorphic DNA technique. The mutant bacteria named as Pseudomonas sp. 1 F reduced the time required for complete degradation of recalcitrant dye GHE4B by 25 % when compared with the wild one. The biodegradation was monitored by UV–Vis spectrophotometric analysis. Activities of enzymes like laccase, lignin peroxidase, veratryl alcohol oxidase, and NADH dichlorophenol indophenol reductase were found to be boosted in mutant bacteria as a consequence of UV-induced mutation. Matrix-assisted laser desorption/ionization-time of flight analysis of differentially expressed proteins of mutant bacteria suggested active role of antioxidant enzymes in the degradation of the dye. The degradation product was analyzed by Fourier transform infrared spectroscopy, high-performance thin-layer chromatography, and gas chromatography–mass spectrometry. Results revealed few variations in the degradation end products of wild-type and mutant bacteria. Phytotoxicity study underlined the safer biodegradation of GHE4B by mutant Pseudomonas sp. 1 F.

Keywords: Degradation; Catalase; Azo dye; Mutation; UV irradiation; RAPD; Decolorization

Characterization of a Novel Glutelin Subunit OsGluBX by the Experimental Approach and Molecular Dynamics Simulations by Ying He; Yubo Zhang; Shuzhen Wang; Hanlai Zeng; Yi Ding (pp. 1482-1496).

Rice glutelin is a multi-subunit storage protein and has high nutritional value. However, many glutelin subunits are still not identified by experiment approach. In this study, a novel subunit (OsGluBX) was discovered by sequence alignment in the UniProtKB database. And then, the OsGluBX of rice from japonica cv. Nipponbare and indica cv. 9311 were cloned and expressed in Escherichia coli system and further identified by Western blotting. The total storage proteins were extracted from the grains of Nipponbare and 9311, and the native OsGluBX were identified. The novel OsGluBX subunit was classified into the subfamily B based on its high homology to the subfamily B members and their immunoblotting identification against the subfamily-specific antibody. Furthermore, two-dimensional electrophoresis analysis showed similarity and difference of the entire glutelin profiles between the two subspecies. Moreover, the atomic coordinate of the OsGluBX was constructed based on homology modeling approach and refined by molecular dynamics simulations. The spatial conformation of the OsGluBX protein was stable during the simulation, and the obvious hydrogen bonds were observed to maintain the integrity and stability of the β-sheets region of the OsGluBX. Research into this novel OsGluBX subunit has improved our understanding of the glutelin family in rice.

Keywords: Japonica and indica rice; Prokaryotic expression; Electrophoresis; OsGluBX; Anti-GluBX antibody; MD simulations; β-Barrel domain

Enhanced Production of Bacterial Cellulose by Using Gluconacetobacter hansenii NCIM 2529 Strain Under Shaking Conditions by Bhavna V. Mohite; Bipinchandra K. Salunke; Satish V. Patil (pp. 1497-1511).

Bacterial cellulose (BC), a biopolymer, due to its unique properties is valuable for production of vital products in food, textile, medicine, and agriculture. In the present study, the optimal fermentation conditions for enhanced BC production by Gluconacetobacter hansenii NCIM 2529 were investigated under shaking conditions. The investigation on media components and culture parameters revealed that 2 % (w/v) sucrose as carbon source, 0.5 % (w/v) potassium nitrate as nitrogen source, 0.4 % (w/v) disodium phosphate as phosphate source, 0.04 % (w/v) magnesium sulfate, and 0.8 % (w/v) calcium chloride as trace elements, pH 5.0, temperature 25 °C, and agitation speed 170 rpm with 6 days of fermentation period are optimal for maximum BC production. Production of BC using optimized media components and culture parameters was 1.66 times higher (5.0 g/l) than initial non optimized media (3.0 g/l). Fourier transform infrared spectroscopy spectrum and comparison with the available literature suggests that the produced component by G. hansenii in the present study is pure bacterial cellulose. The specific action of cellulase out of the investigated hydrolytic enzymes (cellulase, amylase, and protease) further confirmed purity of the produced BC. These findings give insight into conditions necessary for enhanced production of bacterial cellulose, which can be used for a variety of applications.

Keywords: Biopolymer; Media components; Culture parameters; Sucrose; Cellulase

Gene Cloning and Characterization of a Novel Salt-Tolerant and Glucose-Enhanced β-Glucosidase from a Marine Streptomycete by Zhimao Mai; Jian Yang; Xinpeng Tian; Jie Li; Si Zhang (pp. 1512-1522).

The gene BglNH encoding a β-glucosidase was cloned from a marine streptomycete. Sequence analysis revealed that BglNH encoded a 456-aa peptide with a calculated mass of 51 kDa. The deduced amino acid sequence of BglNH showed the highest identities of 61 % with known β-glucosidases and contained a catalytic domain which belonged to the glycoside hydrolase family 1. The gene BglNH was expressed in Escherichia coli and the recombinant enzyme (r-BglNH) was purified. The optimum pH and temperature of r-BglNH were pH 6.0 and 45 °C, respectively. The r-BglNH displayed the typical salt-tolerant and glucose-enhanced characteristics. Its activity was remarkably enhanced in the presence of 0.5 M NaCl (rose more than 1.6-fold) and 0.1 M glucose (rose more than 1.4-fold). Moreover, r-BglNH displayed good pH stability and metal tolerance. It remained stable after incubating with buffers from pH 4.0 to 10.0, and most metal ions had no significant inhibition on its activity. These properties indicate that r-BglNH is an ideal candidate for further research and industrial applications.

Keywords: β-glucosidase; Cellulase; Salt-tolerant; Glucose-enhanced; Streptomyces sp

Flanking Sequence Determination and Event Specific Detection of Transgenic Wheat B72-8-11b Strain by Piqiao Zhang; Junyi Xu; Qiuyue Zheng; Fengxia Luan; Jijuan Cao; Hesheng Hou (pp. 1523-1530).

Exogenous fragment sequence and flanking sequence between exogenous fragment and recombinant chromosome of transgenic wheat B72-8-11b were successfully acquired through PCR amplification with cross-matched primers from exogenous genes. Newly acquired exogenous fragment covered the full-length sequence of transformed genes such as transformed plasmid and corresponding functional genes including marker uidA, promoter ubiquitin, lacZ, 1Dx5, and part of sequence of the wheat genome. A specific PCR detection method for transgenic wheat B72-8-11b strain was established on the basis of primers designed according to flanking sequence. The designed primers revealed specific amplification of 132 bp product of transgenic wheat B72-8-11b strain. This method is characteristics of high specificity, high reproducibility, rapid identification, and excellent accuracy for the identification of transgenic wheat B72-8-11b strain.

Keywords: Transgenic wheat B72-8-11b; Flanking sequence; Chromosome walking; Strain identification; PCR

Ethanol Production Using Whole Plant Biomass of Jerusalem Artichoke by Kluyveromyces marxianus CBS1555 by Seonghun Kim; Jang Min Park; Chul Ho Kim (pp. 1531-1545).

Jerusalem artichoke is a low-requirement sugar crop containing cellulose and hemicellulose in the stalk and a high content of inulin in the tuber. However, the lignocellulosic component in Jerusalem artichoke stalk reduces the fermentability of the whole plant for efficient bioethanol production. In this study, Jerusalem artichoke stalk was pretreated sequentially with dilute acid and alkali, and then hydrolyzed enzymatically. During enzymatic hydrolysis, approximately 88 % of the glucan and xylan were converted to glucose and xylose, respectively. Batch and fed-batch simultaneous saccharification and fermentation of both pretreated stalk and tuber by Kluyveromyces marxianus CBS1555 were effectively performed, yielding 29.1 and 70.2 g/L ethanol, respectively. In fed-batch fermentation, ethanol productivity was 0.255 g ethanol per gram of dry Jerusalem artichoke biomass, or 0.361 g ethanol per gram of glucose, with a 0.924 g/L/h ethanol productivity. These results show that combining the tuber and the stalk hydrolysate is a useful strategy for whole biomass utilization in effective bioethanol fermentation from Jerusalem artichoke.

Keywords: Bioethanol; Jerusalem artichoke; Stalk; Kluyveromyces marxianus ; Simultaneous saccharification and fermentation

hTERT Gene Immortalized Human Adipose-Derived Stem Cells and its Multiple Differentiations: a Preliminary Investigation by L. Wang; K. Song; X. Qu; H. Wang; H. Zhu; X. Xu; M. Zhang; Y. Tang; X. Yang (pp. 1546-1556).

Human adipose-derived adult stem cells (hADSCs) can express human telomerase reverse transcriptase phenotypes under an appropriate culture condition. Because adipose tissue is abundant and easily accessible, hADSCs offer a promising source of stem cells for tissue engineering application and other cell-based therapies. However, the shortage of cells number and the difficulty to proliferate, known as the “Hayflick limit” in vitro, limit their further clinical application. Here, hADSCs were transfected with human telomerase reverse transcriptase (hTERT) gene by the lentiviral vector to prolong the lifespan of stem cells and even immortalize them. Following to this, the cellular properties and functionalities of the transfected cell lines were assayed. The results demonstrated that hADSCs had been successfully transfected with hTERT gene (hTERT-ADSCs). Then, hTERT-ADSCs were initially selected by G418 and subsequently expanded over 20 passages in vitro. Moreover, the qualitative and quantitative differentiation criteria for 20 passages of hTERT-ADSCs also demonstrated that hTERT-ADSCs could differentiate into osteogenesis, chondrogenesis, and adipogenesis phenotypes in lineage-specific differentiation media. These findings confirmed that this transfection could prolong the lifespan of hADSCs.

Keywords: Adipose-derived stem cells; Telomerase reverse transcriptase; Multidifferentiation; Lentiviral vector; Transfection

Isolation of AtNUDT5 Gene Promoter and Characterization of Its Activity in Transgenic Arabidopsis thaliana by Xiu-Chun Zhang; Mei-Ying Li; Meng-Bin Ruan; Yi-Ji Xia; Kun-Xin Wu; Ming Peng (pp. 1557-1565).

AtNUDT5 is a cytosol Nudix that catalyzes the hydrolysis of a variety of substrates. In this report, a 1,387-bp 5′-flanking region of the AtNUDT5 gene was isolated from Arabidopsis thaliana. The tissue-specific activity of the 5′-flanking region was investigated by using the GUS gene as a reporter in transgenic A. thaliana plants. Weak GUS activity appeared in vascular tissues of young plants, strong GUS activity appeared in the axial roots, but no GUS activity was observed in the root cap, lateral roots, rosette leaf, mature silique and reproductive tissues such as stamen, pistil, and petal. Furthermore, by using these transgenic A. thaliana plants, results of the histochemical staining and fluorometric assays of GUS activity showed that the AtNUDT5 promoter can be activated by both avirulent Pst avrRpm1 and virulent Pst strains at 5 h post-infiltration and that the activity of AtNUDT5 promoter increased significantly at 24 h post-infiltration. Taken together, our results demonstrated that the AtNUDT5 promoter is pathogen-responsive. The promoter may be used to develop transgenic plants with an increased tolerance to pathogenic stresses.

Keywords: AtNUDT5 promoter; Pseudomonas syringae pv. Tomato DC3000; Transgenic Arabidopsis thaliana ; Pathogen-responsive

Bovine Serum Albumin Nanospheres Synchronously Encapsulating “Gold Selenium/Gold” Nanoparticles and Photosensitizer for High-Efficiency Cancer Phototherapy by Cong Yu; Fangjie Wo; Yuxiang Shao; Xiangyun Dai; Maoquan Chu (pp. 1566-1578).

Gold nanostructures have generated significant attention in biomedical areas because of their major role in cancer photothermal therapeutics. In order to conveniently combine gold nanostructures and drugs into one nanocomposite, Au₂Se/Au core–shell nanostructures with strong near-infrared-absorbing properties were synthesized using a simple method and embedded inside bovine serum albumin (BSA) nanospheres by using a spray dryer equipped with an ultrasonic atomizer followed by thermal denaturation. The nanospheres with narrow size distribution mainly ranging from 450 to 600 nm were obtained. The Au₂Se/Au-loaded BSA nanospheres (1 mg) adsorbed at least 0.01 mg of water-insoluble zinc phthalocyanine (ZnPc) photosensitizer. After irradiation with a 655-nm laser (20 min), the temperature of the Au₂Se/Au-loaded BSA nanospheres [200 μL, 2 mg/mL, BSA/Au₂Se/Au 10:1 (w/w)] increased by over 20 °C from the initial temperature of 24.82 ± 0.15 °C, and the release of ZnPc was improved compared with a corresponding sample without irradiation. After being incubated with cancer cells (human esophageal carcinoma Eca-109), the nanospheres exhibited photothermal and photodynamic therapy with a synergistic effect upon laser irradiation. This work provides novel Au₂Se/Au-loaded polymer nanospheres prepared by a high-efficiency strategy for incorporating drugs for improving the efficiency in killing cancer cells.

Keywords: Au₂Se/Au core–shell nanoparticles; BSA nanospheres; ZnPc photosensitizer; Light-triggered drug release; Combinational therapy

Advances in RNA Interference Technology and Its Impact on Nutritional Improvement, Disease and Insect Control in Plants by Rajan Katoch; Neelam Thakur (pp. 1579-1605).

This review highlights the advances in the knowledge of RNA interference (RNAi) and discusses recent progress on the functionality of different components RNAi machinery operating in the organisms. The silencing of genes by RNA interference has become the technology of choice for investigation of gene functions in different organisms. The refinement in the knowledge of the endogenous RNAi pathways in plants along with the development of new strategies and applications for the improvement of nutritional value of important agricultural crops through suppression of genes in different plants have opened new vistas for nutritional security. The improvement in the nutritional status of the plants and reduction in the level of toxins or antinutrients was desired for long, but the available technology was not completely successful in achieving the tissue specific regulation of some genes. In the recent years, a number of economically important crop plants have been tested successfully for improving plant nutritional value through metabolic engineering using RNAi. The implications of this technology for crop improvement programs, including nutritional enrichment, reduction of antinutrients, disease, and insect control have been successfully tested in variety of crops with commercial considerations. The enhancement of the nutraceutical traits for the desired health benefits in common crop plants through manipulation of gene expression has been elaborated in this article. The tremendous potential with RNAi technology is expected to revolutionize the modern agriculture for meeting the growing challenges is discussed.

Keywords: RNAi; Gene knockdown; Dicer; miRNA; Crop improvement

Correlations Between FAS Elongation Cycle Genes Expression and Fatty Acid Production for Improvement of Long-Chain Fatty Acids in Escherichia coli by Sunhee Lee; Yeontae Jung; Seunghan Lee; Jinwon Lee (pp. 1606-1619).

Microorganisms have been used for biodiesel (fatty acid methyl ester) production due to their significant environmental and economic benefits. The aim of the present research was to develop new strains of Escherichia coli K-12 MG1655 and to increase the content of long-chain fatty acids by overexpressing essential enzymes that are involved in the fatty acid synthase elongation cycle. In addition, the relationship of β-ketoacyl-acyl carrier protein (ACP) synthase (fabH), β-ketoacyl-ACP reductase (fabG), β-hydroxyacyl-ACP dehydrase (fabZ), and β-enoyl-ACP reductase (fabI) with respect to fatty acid production was investigated. The four enzymes play a unique role in fatty acid biosynthesis and elongation processes. We report the generation of recombinant E. coli strains that produced long-chain fatty acids to amounts twofold over wild type. To verify the results, NAD⁺/NADH ratios and glucose analyses were performed. We also confirmed that FabZ plays an important role in producing unsaturated fatty acids (UFAs) as E. coli SGJS25 (overexpressing the fabZ gene) produced the highest percentage of UFAs (35 % of total long-chain fatty acids), over wild type and other recombinants. Indeed, cis-9-hexadecenoic acid, a major UFA in E. coli SGJS25, was produced at levels 20-fold higher than in wild type after 20 h in culture. The biochemically engineered E. coli presented in this study is expected to be more economical for producing long-chain fatty acids in quality biodiesel production processes.

Keywords: Long-chain fatty acids; FAS elongation cycle genes; FabZ; Fatty acid composition

RNA Preparation of Saccharomyces cerevisiae Using the Digestion Method may Give Misleading Results by Tadahiro Suzuki; Yumiko Iwahashi (pp. 1620-1632).

Zymolyase (lyticase) is used for cell wall digestion in yeast experiments and is needed for incubation processes under moderate experimental conditions. This has been thought to cause unfavorable effects, and many researchers are aware that the enzyme method is unsuitable for RNA preparation following several reports of stress responses to the enzyme process. However, RNA preparation with enzyme digestion continues to be used. This may be because there have been insufficient data directly comparing RNA preparation conditions with previous studies. We investigated the influence of enzyme processes in RNA preparation using a DNA microarray, and compared superoxide dismutase (SOD) activities with a non-treated control and the results of previous research. Gene expressions were commonly changed by enzyme processes, and SOD activities increased only during short-term incubation. Meanwhile, both SOD gene expressions and SOD activity during RNA preparation indicated different results than gained under conditions of long-term incubation. These results suggest that zymolyase treatment surely influences gene expressions and enzyme activity, although the effect assumed by previous studies is not necessarily in agreement with that of RNA preparation.

Keywords: Zymolyase; Yeast; RNA preparation; DNA microarray; SOD activity

Cu/Zn Incorporation During Purification of Soluble Human EC-SOD from E. coli Stabilizes Proper Disulfide Bond Formation by Ji-Young Bae; Bon-Kyung Koo; Han-Bong Ryu; Jung-A Song; Minh Tan Nguyen; Thu Trang Thi Vu; Young-Jin Son; Hyang Kyu Lee; Han Choe (pp. 1633-1647).

Extracellular superoxide dismutase (EC-SOD) is the only enzyme that removes superoxide radical in the extracellular space. The reduction of EC-SOD is linked to many diseases, suggesting that the protein may have therapeutic value. EC-SOD is reported to be insoluble and to make inclusion bodies when overexpressed in the cytoplasm of Escherichia coli. The refolding process has the advantage of high yield, but has the disadvantage of frequent aggregation or misfolding during purification. For the first time, this study shows that fusion with maltose-binding protein (MBP), N-utilization substance protein A, and protein disulfide isomerase enabled the soluble overexpression of EC-SOD in the cytoplasm of E. coli. MBP-tagged human EC-SOD (hEC-SOD) was purified by MBP affinity and anion exchange chromatography, and its identity was confirmed by MALDI-TOF MS analysis. The purified protein showed good enzyme activity in vitro; however, there was a difference in metal binding. When copper and zinc were incorporated into hEC-SOD before MBP tag cleavage, the enzymatic activity was higher than when the metal ions were bound to the purified protein after MBP tag cleavage. Therefore, the enzymatic activity of hEC-SOD is associated with metal incorporation and protein folding via disulfide bond.

Keywords: Human EC-SOD; Soluble overexpression; Purification; E. coli ; Disulfide bond; MALDI-TOF MS; Metal binding

Enhancement of Enzymatic Hydrolysis and Klason Lignin Removal of Corn Stover Using Photocatalyst-Assisted Ammonia Pretreatment by Chang Geun Yoo; Chao Wang; Chenxu Yu; Tae Hyun Kim (pp. 1648-1658).

Photocatalyst-assisted ammonia pretreatment was explored to improve lignin removal of the lignocellulosic biomass for effective sugar conversion. Corn stover was treated with 5.0–12.5 wt.% ammonium hydroxide, two different photocatalysts (TiO₂ and ZnO) in the presence of molecular oxygen in a batch reactor at 60 °C. Various solid-to-liquid ratios (1:20–1:50) were also tested. Ammonia pretreatment assisted by TiO₂-catalyzed photo-degradation removed 70 % of Klason lignin under the optimum condition (12.5 % ammonium hydroxide, 60 °C, 24 h, solid/liquid = 1:20, photocatalyst/biomass = 1:10 with oxygen atmosphere). The enzymatic digestibilities of pretreated corn stover were 85 % for glucan and 75 % for xylan with NH₃-TiO₂-treated solid and 82 % for glucan and 77 % for xylan with NH₃-ZnO-treated solid with 15 filter paper units/g-glucan of cellulase and 30 cellobiase units/g-glucan of β-glucosidase, a 2–13 % improvement over ammonia pretreatment alone.

Keywords: Lignocellulosic biomass; Titanium dioxide (TiO₂); Zinc oxide (ZnO); Photo-degradation; Bioconversion

Disease-Causing Mutation in Extracellular and Intracellular Domain of FGFR1 Protein: Computational Approach by B. Rajith; C. George Priya Doss (pp. 1659-1671).

In-depth computationally based structural analysis of human fibroblast growth factor type 1 (FGFR1) protein carrying disease-causing mutation was performed in this study. Gain or loss of function due to missense mutations in FGFR1 is responsible for a variety of disorders including Kallmann syndrome, Apert syndrome, Pfeiffer syndrome, Crouzon syndrome, etc. The mutant model of the human FGFR1 protein was subjected to various in silico analysis, and most deleterious SNPs were screened out. Furthermore, docking and long molecular dynamics simulations were carried out with an intention of studying the possible impact of these mutations on the protein structure and hence its function. Analysis of various structural properties—especially of those of the functionally important regions: the extracellular immunoglobulin domain and intracellular Tyrosine kinase domain—gave some insights into the possible structural characteristics of the disease mutant and the wild-type forms of the protein. In a nutshell, compared to the wild-type protein, the mutant structures V273M and S685F are associated with significant changes, and the functionally important regions seem to adopt such structures that are not conducive for the wild-type-like functionality.

Keywords: SNPs; FGFR1; Docking; Molecular dynamics

Evaluation of Strategies to Improve the Production of Alkaline Protease PrtA from Aspergillus nidulans by Denise Castro-Ochoa; Carolina Peña-Montes; Amelia Farrés (pp. 1672-1682).

Aspergillus nidulans produces several proteases. The prtA gene encodes a major protease, and two approaches were explored to achieve the overproduction of this enzyme. Molecular cloning of the mature form of this enzyme in Pichia pastoris resulted in the production of an inactive form. In addition, the presence of this enzyme was toxic for the host and resulted in cell lysis. The modification of the culture medium constituents resulted in a 6.4-fold increase in enzyme production. The main effect was achieved through the use of organic nitrogen sources. Although it was previously shown that the PrtA protease shows promiscuous esterase activity, the production of this enzyme was not induced by lipidic sources.

Keywords: Protease; Production; Aspergillus nidulans ; Cloning; Pichia

High-Throughput System for Screening of Cephalosporin C High-Yield Strain by 48-Deep-Well Microtiter Plates by Jun Tan; Ju Chu; Yuyou Hao; Yuanxin Guo; Yingping Zhuang; Siliang Zhang (pp. 1683-1695).

Improvement of microbial strains for the high-production of industrial products has been the hallmark of all commercial fermentation processes. Strain improvement has been conventionally achieved through mutation and selection. However, most of the screenings were performed in shake flasks, which made the screening procedure very complex, time-consuming, and inefficient. Most mutant spore suspension had no chance to be screened due to the low-throughput of shake flasks and had to be sacrificed. In this paper, in order to get a Cephalosporin C (CPC) high-yield stain, traditional mutagenesis was employed to obtain the mutant library and gave them the equal screening chance by a novel mixture culture method combined with high-throughput screening method. The good correlation of fermentation results between differing-scale cultivations confirmed the feasibility of utilizing the 48-deep microtiter plates as a scale-down tool instead of shake flasks for culturing high-aerobic microbes with long cultivation period. The microbioassay based on the antibacterial activity of CPC against Alcaligenes faecalis was used to select mutants. As a result, the high-yield strain W-6 was successfully screened out and the CPC titer was nearly 50 % higher than that of the parental strain in the shake flask. The CPC production of strain W-6 was further validated in 50 l bioreactor, and the CPC production reached 32.0 g/l, twofold higher than that of the wild strain.

Keywords: Cephalosporin C; High-throughput screening; High-throughput bioactivity assay; 48-deep MTP; Mixture culture

Lime Pretreatment and Fermentation of Enzymatically Hydrolyzed Sugarcane Bagasse by Sarita C. Rabelo; Rubens Maciel Filho; Aline C. Costa (pp. 1696-1712).

Sugarcane bagasse was subjected to lime (calcium hydroxide) pretreatment and enzymatic hydrolysis for second-generation ethanol production. A central composite factorial design was performed to determine the best combination of pretreatment time, temperature, and lime loading, as well as to evaluate the influence of enzymatic loadings on hydrolysis conversion. The influence of increasing solids loading in the pretreatment and enzymatic hydrolysis stages was also determined. The hydrolysate was fermented using Saccharomyces cerevisiae in batch and continuous mode. In the continuous fermentation, the hydrolysates were concentrated with molasses. Lime pretreatment significantly increased the enzymatic digestibility of sugarcane bagasse without the need for prior particle size reduction. In the optimal pretreatment conditions (90 h, 90 °C, 0.47 g lime/g bagasse) and industrially realistic conditions of hydrolysis (12.7 FPU/g of cellulase and 7.3 CBU/g of β-glucosidase), 139.6 kg lignin/ton raw bagasse and 126.0 kg hemicellulose in the pretreatment liquor per ton raw bagasse were obtained. The hydrolysate from lime pretreated sugarcane bagasse presented low amounts of inhibitors, leading to ethanol yield of 164.1 kg ethanol/ton raw bagasse.

Keywords: Lignocellulosic; Pretreatment; Lime; Enzymatic hydrolysis; Ethanol

Molecular Cloning, Purification and Functional Implications of Recombinant GST Tagged hGMCSF Cytokine by Nidhi Chaubey; Siddhartha Sankar Ghosh (pp. 1713-1726).

We report the cloning, purification and cell proliferative activity of a novel recombinant GST tagged human granulocyte macrophage colony stimulating factor (GST-hGMCSF). The hGMCSF gene was PCR amplified from the cDNA of ACHN renal carcinoma cells and was cloned into the bacterial expression vector. The GST-hGMCSF was purified to homogeneity using glutathione agarose affinity chromatography and subsequently characterized by Western blot, circular dichroism (CD) and MALDI TOF-TOF analysis. Homology modelling studies revealed the possible binding domains of the recombinant cytokine with cognate receptor. The proliferation of THP-1, Raw 264.7, MCF-7 and U87MG cells upon GST-hGMCSF addition was found to be dose dependent. Hence, this functionally active recombinant cytokine has potential application in cancer therapy for stimulating facile growth recovery of normal cell population.

Keywords: GST-hGMCSF protein; Inclusion bodies; Circular dichroism; MALDI TOF-TOF; Cell proliferation assay; GST-hGMCSF modelling

2.45 GHz Microwave Irradiation-Induced Oxidative Stress Affects Implantation or Pregnancy in Mice, Mus musculus by Saba Shahin; Vineet Prakash Singh; Ritesh K. Shukla; Alok Dhawan; Ravi Kumar Gangwar; Surya Pal Singh; Chandra Mohini Chaturvedi (pp. 1727-1751).

The present experiment was designed to study the 2.45 GHz low-level microwave (MW) irradiation-induced stress response and its effect on implantation or pregnancy in female mice. Twelve-week-old mice were exposed to MW radiation (continuous wave for 2 h/day for 45 days, frequency 2.45 GHz, power density = 0.033549 mW/cm², and specific absorption rate = 0.023023 W/kg). At the end of a total of 45 days of exposure, mice were sacrificed, implantation sites were monitored, blood was processed to study stress parameters (hemoglobin, RBC and WBC count, and neutrophil/lymphocyte (N/L) ratio), the brain was processed for comet assay, and plasma was used for nitric oxide (NO), progesterone and estradiol estimation. Reactive oxygen species (ROS) and the activities of ROS-scavenging enzymes— superoxide dismutase, catalase, and glutathione peroxidase—were determined in the liver, kidney and ovary. We observed that implantation sites were affected significantly in MW-irradiated mice as compared to control. Further, in addition to a significant increase in ROS, hemoglobin (p < 0.001), RBC and WBC counts (p < 0.001), N/L ratio (p < 0.01), DNA damage (p < 0.001) in brain cells, and plasma estradiol concentration (p < 0.05), a significant decrease was observed in NO level (p < 0.05) and antioxidant enzyme activities of MW-exposed mice. Our findings led us to conclude that a low level of MW irradiation-induced oxidative stress not only suppresses implantation, but it may also lead to deformity of the embryo in case pregnancy continues. We also suggest that MW radiation-induced oxidative stress by increasing ROS production in the body may lead to DNA strand breakage in the brain cells and implantation failure/resorption or abnormal pregnancy in mice.

Keywords: Microwave radiation; Reactive oxygen species (ROS); Nitric oxide; Antioxidant enzyme activity; Implantation failure

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.169, #5)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.169, #5)