Open Access Original Research Article

Production, Purification and Characterisation of a Purified Low Molecular Weight and Thermo-alkaline Tolerance Xylanase by Aspergillus brasiliensis

Hooi Ling Ho, Li Sha Soh, Soon Hwa Ong

Microbiology Research Journal International, Page 1-25
DOI: 10.9734/BMRJ/2016/20766

Aims: Xylanase is an enzyme which has been used extensively in many different industrial processes. Xylanase also known as endo-1,4-β-xylanase is a glycosidase that catalyses the conversion of xylan to xylose through endohydrolysis of 1,4-β-D-xylosidic linkage which is commonly found in various agro-industrial wastes such as wheat bran. Thus, the objectives of the present study were to produce, purify and characterise cost-effective xylanase from Aspergillus brasiliensis ATCC 16404.

Methodology: Wheat bran as the major sustainable low cost agro-industrial residual was utilised as the sole carbon source for the production of xylanase by A. brasiliensis in submerged fermentation (SmF). Subsequently, a two-step column chromatography was used to purify xylanase followed by step-wise manner of characterisation study on the purified xylanase.

Results: Based on the results, the maximum xylanase production of 7.72 U/mL with spore count of 8.33 × 104 spores/mL at medium pH 6.42 was obtained at 48 h of SmF. Xylanase extracted from A. brasiliensis was then purified with DEAE Sepharose and Sephadex G-75 column chromatography. At the end of purification, xylanase was purified up to 3.6-fold with its recovery yield of 1.68% and specific activity of 116.64 U/mg. Additionally, the purified xylanase was detected to be a low molecular weight protein. Indeed, molecular weight of 36 kDa of the purified xylanase was visualized on SDS-PAGE. The purified xylanase was then subjected to the step-wise manner of characterisation study. Based on the results obtained, xylanase was found to be thermo-tolerance from 40°C to 60°C. In fact, the purified xylanase was detected to be most stable at 50°C whereby 98.33% of its activity was retained even after 3 h of incubation. Furthermore, xylanase from A. brasiliensis was also found to be most active at 50°C where its relative activity increased from 95.24% at 45°C to the maximum activity of 20.51 U/mL at 50°C. Besides that, the pH stability of xylanase was appreciable from pH range of pH 4 to 8. Notably, the purified xylanase showed the highest stability at pH 5 as 94.87% of its activity was retained after 3 h of incubation. Additionally, the activity of the purified xylanase remained relatively higher in pH buffer ranging from pH 3 to 9. Indeed, the purified xylanase reached its maximum activity of 22.18 U/mL at pH 5. In addition, xylanase activity was detected to be the highest, producing 24.46 U/mL when 1% beechwood xylan was used as the optimised substrate for the incubation period of 30 min at 50°C. Besides that, metal ions such as Cu2+, Mn2+ and Zn2+ were identified to enhance xylanase activity, whereas Al3+, Ca2+, K+ and Mg2+ performed otherwise. In particular, Cu2+ was identified as the strongest activator while Al3+ was found to be the toughest inhibitor of xylanase activity. Nonetheless, chelating agent of EDTA inhibited the xylanase activity marginally. Furthermore, the non-ionic detergent of Tween 80 was detected to be a weak enhancer whereas the ionic detergent of SDS was a strong inhibitor of xylanase activity. On the other hand, xylanase activity showed a better tolerance towards glycerol with 51.2% and acetone with 33.9% compared to ethanol with 17.9%.

Conclusion: In a nutshell, some of the characteristics of the purified xylanase by A. brasiliensis in this study revealed its enormous potential as a thermo-alkaline tolerance enzyme in xylan degradation process that is applicable and useful in the manufacturing of animal feeds, fruit juice and paper pulping.

Open Access Original Research Article

Optimization of β-galactosidase Production from Yogurt and Dairy Soil Associated Bacteria Using Different Fermentation Media

Nazish Mazhar Ali, Saiqa Andleeb, Bushra Mazhar, Agha Usman Ali Khan

Microbiology Research Journal International, Page 1-15
DOI: 10.9734/BMRJ/2016/18750

Aims: The aim of current research was the production of β-galactosidase enzyme from Lactobacillus delbruekii subspbulgaricus isolated form yogurt and soil samples.

Study Design: Production of β-galactosidase.

Place and Duration of Study: Microbiology laboratory, Department of Zoology, GC University, Lahore, Pakistan, between 14 Nov, 2013 and 29 Nov, 2014.

Methodology: Bacterial isolates were isolated from yogurt and soil samples. Morphological, biochemical characterization, and 16S rRNA sequencing was used for the identification of the bacterial strains. The optimization and maximum production of β-galactosidase was carried out by using various temperatures, pH, carbon sources (corn flour, wheat flour, rice flour), nitrogen sources (Trypton, Peptone, Yeast extract), and incubation periods.

Results: National Centre for Biotechnology Information (NCBI) Blast analyzed that A11, A13 and A14 strains belongs to Lactobacillus delbruekii subsp. bulgaricus with accession numbers KP256199, KP264120, and KP264121. Maximum enzyme production was obtained after 48 h of incubation at 40-45°C temperature at pH 6.0-7.0. The highest enzyme production was observed in the presence of rice flour and yeast extract. The maximum enzyme activity was shown by A11, A13 and A25 (7.69 U/mL, 7.25 U/mL, and 7.28 U/mL).

Conclusion: Rice flour could be the potential carbon source for the production of β-galactosidase from Lactobacillus delbruekii subsp. bulgaricus whereas nitrogen sources reduced its production. Temperature and pH was important physical parameter for the production of β-galactosidase from yogurt and soil associated bacteria.

Open Access Original Research Article

Cattle Urine as a Fertiliser: Micro-biochemical Changes in Fermenting Cattle Urine and Implications on Plant Nutrient Conservation

Geroge Kilande, John Stephen Tenywa, Mary Christine Rwakaikara-Silver, Alice Amoding-Katushabe

Microbiology Research Journal International, Page 1-10
DOI: 10.9734/BMRJ/2016/18323

Aim: The aim of this study was to evaluate the microbial and biochemical changes in fermenting urine, a practice used by farmers in Sub-Saharan Africa before its application as a soil fertility input.

Methodology: Two 5-litre sterile plastic containers, with a closable ends were each filled with fresh urine to capacity. One container was closed and the other left open. The set-up was replicated three times. Twenty millitres of fresh urine was taken from the bulk collection for microbial and chemical analysis. Urine samples were also taken and analysed at 4-day fermentation intervals till 24 days.

Results: Fresh urine had pH=8.2 and contained Aspergillus spp. and Escherichia coli, with the latter being dominant. After 12 days of fermentation, Penicillium spp. and Pseudomonas spp. emerged and progressively increased, especially under the closed system. Whereas Aspergillus spp. counts increased in both systems, E. coli counts dropped dramatically and eventually disappeared at 16 days. The pH in the open system surged to 9.7, while that of the closed containers remained nearly stable (8.2). Organic N was not significantly (p>0.05) affected by closure of the containers. In the open system, Organic N concentration dropped up to 72%. However, NH4-N concentration increased steadily in the closed system until day 24; but dropped dramatically in the open system. Nitrate concentration increased slightly up to day 8, and thereafter, declined sharply by 97% in the open system. Similarly, in the closed system, this N species dwindled progressively but not to extinction.

Conclusion: There is a shift in microbial communities in urine from Aspergillus spp. and Escherichia coli in fresh urine to Penicillium spp. and Pseudomonas spp. 12 days after the onset of fermentation. Nitrate-N is favoured by the open system, while the ammonium-N increased more in the closed system.

Open Access Original Research Article

Patterns of Initial Drug Resistance of Mycobacterium tuberculosis Isolates from Kashmir Valley, India

Tehmeena Wani, Gulnaz Bashir, Rubina Lone, V. M. Katoch, D. K. Kakru, Azra Shah, Imtiaz Ali Bhat, Mohammad Muzaffar Mirza

Microbiology Research Journal International, Page 1-9
DOI: 10.9734/BMRJ/2016/20234

Aims: We carried out this study to determine the patterns of initial drug resistance in Mycobacterium tuberculosis isolates and prevalence of MDR-TB among category-I pulmonary TB patients in Kashmir Valley. MDR-TB was defined as tuberculosis caused by bacilli showing resistance to at least isoniazid and rifampicin.

Study Design: Prospective study.

Place and Duration of Study: Department of Microbiology, Sher-i-Kashmir Institute of Medical Sciences, Srinagar, Kashmir, J&K, India between May 2007 and April 2010.

Methodology: This study involved 300 category-I pulmonary TB patients attending DOTS clinics in different districts of Kashmir Valley. AFB positive sputum samples were randomly collected in 1% cetylpyridinium chloride from such patients and were subjected to repeat AFB staining and mycobacterial culture in Department of Microbiology, SKIMS. Drug susceptibility testing (DST) to the first line drugs; isoniazid, rifampicin, ethambutol and streptomycin was performed on cultures identified as Mycobacterium tuberculosis using the indirect proportion method on LJ medium.

Results: Out of 300 samples, culture results were available only for 207 samples. Out of 207 samples, 134 (64.73%) were culture positive, 52 sterile (25.12%) and 21(10.14%) showed contamination. Out of the 134 isolates, 123 were identified as MTB and 11 as mycobacteria other than tuberculosis (MOTT). Of the 101 DST results available, 74.25% were sensitive to all four first line drugs, 17.82% showed monoresistance, 7.92% showed polyresistance and 3.96% were MDR.

Conclusion: Resistance to any one drug was 39.60% with a high streptomycin resistance of 20.79%. Since most of these patients harboured organisms susceptible to isoniazid and rifampicin, standard short-course chemotherapy is likely to remain highly effective among the great majority of new TB patients in Kashmir Valley. Prevalence of MDR was relatively low but with a high rifampicin resistance of 6.93% there is a need for restricting use of rifampicin (supervised therapy only for TB and leprosy). It is important to strengthen the capacity of laboratories in Kashmir Valley for TB culture and DST for correct management of TB patients and to prevent emergence of drug resistance. Also, continuous monitoring of resistance in both new and previously treated TB cases needs to be done to know the changing trend of drug resistance in future.

Open Access Original Research Article

Evaluation of Saliva as an Alternative Specimen to Serum for Diagnosis of Hepatitis C Virus Infection

Dalia Saad ElFeky, Alaa Reda Awad, Ahmed Fouad Soliman, Laila Rashed

Microbiology Research Journal International, Page 1-9
DOI: 10.9734/BMRJ/2016/21531

Aim: The study aimed at evaluation of saliva as an alternative specimen to serum for the detection of HCV Abs and HCV RNA.

Study Design: Comparative Study.

Place and Duration of Study: Department of Medical Microbiology and Immunology, and Department of Infectious Diseases and Endemic Hepatic and Gastrointestinal diseases, faculty of medicine, Cairo University, Cairo, Egypt. Between March 2013 and July 2013.

Methodology: The study was conducted on serum and saliva samples collected from 50 HCV-infected patients and 20 healthy controls. All serum and saliva samples were subjected to 3rd generation enzyme linked immune-sorbent assay (ELISA) for detection of HCV antibodies as well as real time RT-PCR assay for detection of HCV RNA. ELISA procedure for saliva samples was done according to a modified protocol, while 3 methods were used to calculate cut-off value (COV) above which saliva samples were considered positive.

Results: HCV antibodies were detected in all serum samples from patients but not in controls. Salivary HCV antibodies results for patients and controls differed according to the three methods used for determining the COV, with sensitivity ranged from 88 to 96% and specificity from 95 to 100%. No correlation existed between positivity of anti-HCV salivary Abs with either serum or salivary viral loads. Salivary real time RT-PCR had sensitivity and specificity of 100% for diagnosis of HCV infection with excellent significant correlation between the HCV viral loads in the saliva and serum.

Conclusions: Saliva can be used as an important substitute to serum for diagnosis of HCV infection either by detection of anti-HCV Abs or HCV RNA.

Open Access Original Research Article

Antibacterial Properties of Snail Mucus on Bacteria Isolated from Patients with Wound Infection

Lawrence B. Etim, Chuku Aleruchi, Godwin Attah Obande

Microbiology Research Journal International, Page 1-9
DOI: 10.9734/BMRJ/2016/21731

Background: Snail mucin has been reported to contain agents with wound healing properties. Mucin obtained from the mucus of snails and epiphgram obtained from species of Achatina fulica and Archachatina marginata have also been reported to show antimicrobial properties. Snail species are abundantly available and widely consumed as a delicacy across Nigeria.

Aim: To assess the antibacterial effects of mucus secretions from different snail types on bacteria isolated from clinically infected wounds.

Place and Duration of Study: The study lasted for a period of four (4) months and was conducted at the Microbiology laboratory of The Cross River State University of Technology in Cross River, Nigeria.

Methodology: The in vitro antibacterial potency of snail mucus secretions obtained from Archachatina marginata saturalis, Archachatina marginata ovum and Achatina fulica on bacterial isolates from wound was investigated. The isolates obtained from twenty eight (28) clinical wound samples were Staphylococcusspp (24:53.3%), Pseudomonas spp (16:33.3%) and Streptococcus spp (6:13.4%). The susceptibility of the isolates to snail mucus secretions was assayed on Muller Hilton Agar by the disc diffusion method, using varied mucus/DMSO concentrations of 100%, 80%, 60%, 40% and 20%. The minimum inhibitory concentration and minimum bactericidal concentration of the mucus secretions were also evaluated.

Results: The viscosity of the mucus secretions were rated as A. marginata saturalis> A. marginata ovum> A. fulica, while their colours were yellow, light brown and dark respectively. Results revealed that Staphylococcus sp was more susceptible to mucus secretion from the A. marginata saturalis (17.4±1.20) than those from A. marginata ovum (15.6±1.44) and A. fulica (15.4±2.04). The minimum inhibitory concentration of mucus secretions from A. marginata saturalis against the test organisms were observed at concentrations of 100% and 20% for Staphylococcus sp, 20% for Pseudomonas sp and 40% for Streptococcus sp respectively. The antibacterial activity of the mucus secretions were observed to be comparable to that of seven (7) different antibiotics used as control.

Conclusion: Snail mucus secretions could be a source for antibacterial agents that can serve as an alternative to the expensive synthetic antibacterial agents used in wound treatment if adequately explored.

Open Access Review Article

Recent Advances in Virus-host Coevolution and Protective Mechanisms against Plant Viruses

Amal Souiri, Mustapha Zemzami, Saaïd Amzazi, Moulay Mustapha Ennaji

Microbiology Research Journal International, Page 1-18
DOI: 10.9734/BMRJ/2016/20439

In plant pathology, the study of the interaction between the plant host and the viral pathogen has been a very active area of research in the last few decades. The infection process of a plant pathogen usually begins with the exchange of molecular signals. With particular emphasis on plant virus evolution, and focusing on quantitative and population genetics, plant virus-host interactions and coevolution allow understanding of the major factors favoring disease emergence. The exploitation of viral interaction phenomena will improve established genetic engineering strategies for viral cross-protection in plants. Also, the study of plant immunity against viral infection, both innate and adaptive (e.g. RNA silencing), has helped in the development of resistant varieties to several plant viruses based on genetic engineering. This review summarizes the recent advances in plant-virus interactions and co-evolution as well as current developments in resistant crop investigations.