Research Journal of Biotechnology

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In silico exploitation of molecular markers of cancer from wet lab in the mechanism-based screening of potential anticancer compounds

Arif Jamal M.

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Interaction of a Mixed Culture of Two Fungi as a means of improving Laccase Activity

Yan Z.Y., Sun X.W., Li J.1,2, Liu X.F., Yuan Y.X. and Liao Yi Z.

Laccase is an enzyme that is able to catalyze phenolic substances. White rot fungi is known to produce laccase. In this study we set out to find the optimal conditions for the stimulation of laccase activity in 2 strains of white rot fungi and to study the mechanism of action. Laccase activity was improved by co-culturing 2 strains of white rot fungi. Combining strain 55 (Trametes trogii) and strain m-6 (Trametes versicolor) resulted in laccase activity that was many times greater than strain 55 alone and strain m-6 alone. The observation that laccase activity was 7 times higher after adding strain m-6 to strain 55 as compared to the activity after adding strain 55 to strain m-6 suggested that in liquid culture, strain m-6 was found to be more responsible for the increase of laccase activity after combination. The enzyme-producing conditions of combined strain 55 and m-6 were found to be optimal at pH 6.5 and C/N 16:1 using 0.01% of Tween 80. In this study we define the optimal conditions for white rot fungi to stimulate laccase activity. It is thought that this may have applications in areas such as pollution degradation, biopulping, printing and dying.

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Effect of Parameter on Forward Extraction of Amoxicillin by using Mixed Reverse Micelles

Akil Ahmad, Asma Khatoon, Siti Hamidah Mohd-Setapar*, Siti Norazimah Mohamad-Aziz, Muhammad Abbas Ahmad-Zaini and Chuo Sing Chuong

Mixed reverse micellar (MRM) systems of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and non-ionic surfactants (TWEEN 85) in iso-octane were used for the extraction of amoxicillin from the aqueous phase. The process parameters such as concentration of surfactants, addition non-ionic surfactant, feed pH and salt concentration during forward extraction are varied to maximize the solubilisation of amoxicillin in an organic phase. A small addition of TWEEN 85 into AOT solution was found to enhance the degree of amoxicillin extraction and the solubilisation was 120 times higher compared with both single system and the amount of surfactant consumed was also very low in order to enhance the solubilisation of the amoxicillin in mixed reverse micelle which led to the economical factor in term of the surfactant usage.

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Total phenols, antioxidant potential and tyrosinase inhibitory activity of honeybee collected pollen from Turkey

Akin M., Nalbantoglu S. and Saki N.*

The aim of this study was to determine the total phenols content, antioxidant properties and anti tyrosinase activity of honey-bee collected pollen from Bursa-Turkey. The total phenolic contents and antioxidant activity studies in different concentrations were determined by the Folin-Ciocalteu method while the antioxidant properties were evaluated by 2,2-diphenyl-picrylhydrazyl radical scavenging capacity (DPPH) assay. The results were varied between 3.35 and 14.5 mg of gallic acid equivalents/g of extract (mg GAE/g) for the total phenols content and 10.52 and 47.76 % for DPPH scavenging activity. The tyrosinase inhibitory activity of honey-bee collected pollen extract was investigated for L-DOPA oxidation and IC50 value and inhibition constant (Ki) were determined as 0.63 mg/mL and 0.27 respectively. As a part of the enzyme inhibition study, inhibition type was also determined as non-competitive.

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Optimization of pediocin production by batch fermentation of Pediococcus acidilactici NCIM 2292 using goat meat processing waste

Mandal B., Chowdhury R. and Bhattacharjee C.

Goat meat processing waste was enzymatically hydrolyzed using papain to utilize the soluble protein for the growth of Pediococcus acidilactici NCIM 2292 producing pediocin. The suitability of different carbohydrate, namely glucose, lactose, sucrose, maltose and fructose for biomass and bacteriocin production was also investigated. Glucose was observed to be the most preferred carbon source. The experiments based on five-level Central Composite Design (CCD) were conducted to study the effect of hydrolysis time of meat waste, initial pH and initial glucose concentration on the production of biomass and pediocin. The parameters were optimized by Response Surface Methodology (RSM). The optimum values of hydrolysis time, pH and glucose were determined to be 13 h, 6.5 and 20 g/l respectively. The results were analyzed statistically to evaluate analysis of variance (ANOVA). Under optimum condition, the biomass concentration and pediocin activity were measured to be 1.22 g/l and 2285 AU/ml at the end of the exponential growth phase (18 h). Antimicrobial activity of pediocin was assessed by well diffusion method using Staphylococcus aureus NCIM 2127 as indicator organism.

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Competitive Inhibition of Mushroom Tyrosinase by Captopril

Kuo T. C.* and Ho F. M.

In this study, the kinetic parameters and inhibition mechanism of anti-tyrosinase activity of captopril were investigated in order to provide the basis for the development of novel effective tyrosinase inhibitor. The kinetic analysis showed that the inhibition of captopril on tyrosinase activity was competitive. The half-maximal inhibitory concentration (IC50) and inhibition constant (Ki) were estimated to be 590 μg/mL and 294 μM respectively. Furthermore, in vitro studies revealed that tyrosinase did not show any cytotoxicity or nonselective toxicity. These experimental results indicate that captopril may have the potential to be useful in cosmetics or food processing and therefore, it should be investigated further.

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Highly efficient expression of neutral endoglucanases in the cells of Trichoderma reesei to improve the cellulolytic pH profile

Yao Zhu, Fanju Meng, Xihua Zhao, Dandan Lv, Yaran Zhu, Dongzhi Wei1 and Wei Wang*

To improve the cellulolytic pH profile of Trichoderma reesei, neutral endoglucanase genes from Humicolainsolens, Humicolagrisea var. thermoidea and Bacillus subtilis LH were cloned and expressed under the control of the cbh1 promoter. Transformants NE 1 (CMC3), NE 2 (Egl3) and NE3 (EG) were obtained exhibiting a 2.29-, 2.96- and 2.43-fold change in the endoglucanase activity at pH 7.0 respectively as compared to the parent strain, RUT C30. Additionally, the endoglucanase activity of NE2 at pH 5.0 to pH 7.0 was more than 75%, while it was 25% at pH 8.0 to pH 9.0, at which none of the parental strain’s endogenous endoglucanase activity remained. The present results proved that the integration ofneutral endoglucanase was a useful way for improving the cellulase catalytic efficiency and pH optimum of T. reesei and the recombinants developed in this study could be potentially used in biorefinery for achieving high efficiency and also used for neutral endoglucanases production for the food, textile, pulp and paper industries.

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Phylogeography of Red Deer (Cervus Elaphus) in China based on Mtdna Cytochrome B Gene

Liu Yanhua, Zhang Minghai and Ma Jianzhang*

China provides an evolution opportunity for Cervus. The species and subspecies of Cervus are developed and evolved throughout the whole of China. Traditionally, taxonomy and origin of red deer were based on geography, morphology and biological characters which had limitation that caused some incorrect intra-specific taxonomy of species. Through analysis of Cytb gene sequence, the evolution relationship of red deer subspecies in China was studied in this paper. The results showed that Northeast red deer, Tianshan red deer and Alxa red deer had close genetic relationship while Tarim red deer was not close to Alxa red deer, Northeast red deer, Tianshan red deer, Altay red deer, Gansu red deer and Tibert red deer. Meanwhile, we used Cytb gene sequences of red deer form Europe, Mongolia and North America to construct the phylogenetic tree. The phylogenetic tree has two obvious clades: one was composed by Tarim red deer and Europe red deer and another was constituted by the Mongolian red deer, North America red deer, Alxa red deer, North east red deer, Tianshan red deer, Altai red deer, Gansu red deer and Tibet red deer. According to the research results on geology, zoogeography and morphological, with the evidence of molecular biology, it could be estimated that red deer originated in Asia. Part of them migrated eastward and northward and reached North America through the Bering Strait. They and the red deer from Northern Asia (including the Junggar Basin) and North America composed the subspecies of the eastern wild red deer; another part is Tarim wild red deer which migrated westward and eventually settled in Europe and formed the western system of red deer combined with the red deer population which stayed in Central Asia before.

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Fermentative production of thermostable α-amylase from phosphate solubilizing Bacillus thuringiensis using solid substrates: purification and characterization of the enzyme

Bandopadhyay Sandip, Pal Subrata and Gangopadhyay Swati R.

Extracellular α-amylase produced by solid state fermentation from phosphate solubilizing strain of Bacillus thuringiensis was purified by ammonium sulfate precipitation and DE-52 ion exchange column chromatography. Molecular weight from the active fraction of the purified enzyme was estimated to be 52 KDa by SDS-PAGE. The enzyme exhibited highest activity with the pH optima of 6.9 and temperature optima of 60 0C. KM of the enzyme was determined to be 1.01 mg/ml using soluble starch as substrate. Enzyme activity was enhanced in presence of 1mM Ca2+, Mg2+ and Zn2+ ions, whereas enzymatic reaction was inhibited in presence of Cu2+ and Hg2+ ions. Solid State Fermentation (SSF) using different starchy raw materials showed that potato starch was the most cost effective substrate for maximum enzyme production at 72 hours.

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Genetic Transformation of WRKY Gene into Maize by Agrobacterium Tumefaciens

Lian Zeliang, Sun Qingpeng, Yu Yongkun, Han Jun and Pan Jinbao

Based on good corn inbred lines Zheng 58, which is widely used in hybrid breeding, agrobacterium-mediated of LBA4404 with root-carcinoma plasmid is used in transformation to its stem tip. Vacuum suction filter is employed during infection process while acetosyringone is added to improve conversion rates. Co-cultured after 6 days, they are transplanted into earth for growing and further testing. 61 seedlings are obtained through this conversion method. Since the corn roots are kept, new leaves grow fast which greatly reduces the experimental process.

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Comparison of Four Methods to extract Bacterial DNA from Infected Fish Tissue

Cagatay Ifakat Tulay

This study is concerned with single-step bacterial DNA extraction of Renibacterium salmonarium from infected Rainbow Trout (Oncorhynchus mykiss) kidney tissue. We examined and compared four commonly used DNA extraction protocols to determine the reliable and practical method from tissue without isolating bacteria. A total of sixty extracted DNA samples from fish tissues have been tested to show their ability to amplification of R. salmonarium 16S rDNA gene region. Results of our comparison showed that the extraction of bacterial DNA from FTA card technology is more simple, reproducible and inexpensive. We have demonstrated that use of FTA card reduced the time to obtain good quantity of R. salmonarium DNA up to 30 min. Hereby the application of FTA cards for bacterial identification has significantly accelerated the diagnosis of diseases. In conclusion, these cards are convenient to use, easy to transfer and store for long periods of time with providing DNA protection against contamination.

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Pelletization Behavior of Fungal Chlorella Sp. Symbiosis System

Luo Shanshan, Dong Zhijiang, Wu Xiaodan, Liu Yuhuan and Roger Ruan

High cost of microalgae harvesting impeded the development of algae biofuel technology and wastewater treatment to commercial practicality. Filamentous fungi and microalgae could form pellets under certain culture condition which is a novel method to reduce the cost of microalgae harversting. In this study, effects of spore inoculums size, glucose content and initial pH on the pelletization behaviors of fungi-Chlorella sp. are investigated. The results show that with the increase of spore concentration, the final pH value decreased and it is easier to form pellets. Fungi could not grow in BG-11 without glucose, but when the concentration of glucose is over 10g/L, there is no obvious effect on the size of pellets. When the initial pH is higher than 5.5, the pellets stop growing when the pellets reach 0.5mm, but in the case of pH 3.5 and 4.5, pellets could grow continuously until 1-2mm.

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In vitro evaluation of antibacterial and antioxidant activity of Cocus nucifera shell extract

NasimunIslam N., Jemimah Naine S., Mohanasrinivasan V., Panneerselvam A. and Subathra Devi C.

The main objective of the present study was to investigate the antibacterial and antioxidant property of Cocus nucifera crude shell extract. The antibacterial activity of Cocus nucifera aqueous crude shell extract was determined by agar well diffusion assay against 5 pathogenic bacteria at 5mg/mL concentration namely Bacillus cereus MTCC No: 6840 (21mm), Staphylococcus aureus MTCC No: 7405 (15mm), Pseudomonas aeruginosa MTCC No: 4676 (15mm), Escherichia coli MTCC No: 1588 (16mm) and Salmonella typhi MTCC No: 1167 (18mm) Furthermore the capacity to scavenge free radicals was assessed by DPPH assay with 100% inhibition at 1mg/mL concentration. The chemical constitutes of the crude shell extract were further authenticated by GC-MS analysis. These findings demonstrate that the shell extracts of Cocus nucifera present excellent bioactive properties with beneficial virtues.

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Peeling the Drug Target Proteins Network by a Core Decomposition Method

Feng Yanghe and Wang Jiao Teng

Currently as the emergence of high throughput technologies for omics data, such as yeast 2 hybrid protein interactions, researches of protein-protein interaction (PPI) had been explosively conducted on. One of the consensuses is that topographical analysis of the intercellular protein interactions leads to new avenues for drug target prediction. In this paper, a panorama view of drug targets is constructed. We found that the connectivity is often not a sufficient criterion to analyze the function of the drug target. On the other hand, the core of the PPI network poses a systematic way to consider the local and global significance of a protein which indicates the inherent layer structure of the targets interactions. The analysis on core of the interactions shows that most of the drug targets are able to propagate the stimulus to some hub-like proteins and spread the transcription signal to others related proteins indirectly.

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Changes in enzyme activities and soil properties of different altitudes of Changbai Mountain, China in the rhizosphere and non-rhizosphere soils of Rhododendron Chrysanthum Pall.

Xiaojuan Qi, Jianwei Lv, Ensi Wang and Xia Chen

Soil enzyme activities are greatly affected by soil physicochemical properties and often are used as indices of microbial activity and soil fertility. To explore the distribution of soil enzyme activities between rhizosphere and non-rhizosphere soils of Rhododendron Chrysanthum Pall. at four different altitudes of Changbai Mountain, soil parameters were measured and correlations between the soil parameters and soil enzyme activities of rhizosphere soil of Rh. Chrysanthum Pall. along different altitudinal gradients were analyzed. This study suggested that invertase activity was higher in the rhizosphere than in the non-rhizosphere soil at lower altitude (1580m to 2300m), the contrary was found in this study where the activities of phosphatase, catalase and urease were higher in the non-rhizosphere soil. But at the hightest altitude of 2614m, all the 4 enzyme activities were higher in the rhizosphere than in the non-rhizosphere soil. The activities of enzyme were independent on altitude. Moreover, there is a direct correlation between the soil parameters and soil enzyme activities. These results may improve our knowledge to assess the ecosystem function of CMBR and can then be used to gain further information about the survey of medicinal plant ecosystem function.

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Screening of traditional Chinese edible fungi for quorum sensing inhibitors activity

Liaoyuan Zhang*, Zhan Senran, Lizhi Chen, Xiong Guan and Kaihui Hu

The emergence of antibiotic resistance has necessitated new therapeutic approaches for combating persistent bacterial infection. Alternative approaches other than those using antimicrobial agents are required in the fight against infectious diseases. Quorum-Sensing (QS) attracts more attention as a promising antipathogenic drug target, notably preventing the emergence of drug-resistant bacteria because its inhibition could attenuate their pathogenicity instead of growth suppression. In this study, Quorum sensing (QS) inhibitory activities of the methanol-extracts from 13 traditional Chinese edible fungi were evaluated using Chromobacterium violaceum CV026 as the indicator strain. Among the 13 samples tested, Pleurotus eryngii, Flammulina velutipes, Tremella Aurantialba and Lentinula edodes yielded QS inhibitors and that inhibited QS-regulated violacein pigment production in C. violaceum CV026. Flask incubation assay by applying serial dilutions of the four samples showed a significant drop in violacein content which suggested these samples possessed strong anti-QS substances. Meanwhile, the growth of C. violaceum CV026 not inhibited by the extracts revealed that the anti-QS substances were not directly involved in processes essential for the growth. Further, the present investigation also demonstrated obvious inhibitory activity for swarming mobility, another QS-regulated behavior in Serratia marcescens MG1 and Pseudomonas aeruginosa PAO1. These edible fungi may be a rich source of compounds against pathogenic bacteria infection to control the development of antibiotic resistance. Further studies are required to isolate and identify specific substances from the four extracts acting as QS inhibitors.

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Molecular marker-assisted selection for breeding a multiple-allele male sterile line of Brassica campestris ssp. Chinensis

Wang Q. S. and Feng H.*

A multiple-allele male sterile line of Chinese cabbage (Br01) was used as a source of male sterility and an inbred line of Pak-choi, Brassica campestris ssp. chinensis (Br02) was the recurrent parent in a breeding program designed to directionally transfer the multiple-allele male sterility phenotype into Brassica campestris ssp. chinensis. Traditional methods of crossing, backcrossing and selfing were employed to transfer male sterility and the desired botanical and horticultural traits while the genotypes were identified through molecular marker-assisted selection (MAS). The SSR marker locus GSSR1 was found to be closely linked to the male sterility gene Ms and was chosen from a group of 26 SSR markers to use in MAS; this same marker could also be used to screen for the fertility gene ms and the fertility restorer gene Msf. After three generations of backcrossing, we were able to select a new male sterile line of Pak-choi, GMS-4, with 100% male sterile plants that showed 100% male sterility.

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Review Paper: Autosomal STR: From locus information to next generation sequencing technology

Mohammad A. and Imad H.

On legal matters or those that pertain to criminal and civil laws, forensic biology is the scientific approach used to work for the biological evidence. To determine the DNA profile, PCR is used to analyze the specimen which could be blood, semen or saliva. The right detection and screening is required for this purpose. This study deals with some of details of genetic markers. Among them are: microsatellites, common commercial kit used in the United States, short tandem repeats locus information, sample preparation, DNA extraction, quantities of DNA, Amplification using Polymerase Chain Reaction of STRs, Separation and Detection, Interpretation, data analysis of STR by Identity Software, next generation sequencing (NGS) technologies and Linkage between STR marker and genetic diseases. In forensic casework and DNA databases, polymorphism in short tandem repeat as a forensic genetic markers is important. It is very important to increase discrimination power and a new autosomal genetic loci for human forensic testing is essential for this purpose.

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