Isolation, Screening and Molecular Cloning Studies of Xylanase Producing Bacillus Pumilus Strains

Cellulosic plant materials are an excellent source of hemicellulolytic microorganisms. Five strains of Bacillus pumilus PJ19, P2, K52A, K51 and K5B, two strains of B. subtilis B2 and PJ18 and one Cellulomonas sp. which produced endoxylanase (1,4-B-D-xylan xylanohydrolase, EC 3.2.1.8) have been isolated locally from plant materials. B. pumilus PJ19 produced the highest xylanase activity when grown in shake flask in yeast tryptone broth (YIB) at 200 rpm, 37°C which yielded activity of 265 U/ml. The enzyme was induced in Dubois media by the addition of xylan as carbon source and was repressed by xylose, glucose, fructose, maltose and sucrose. B. pumilus strain PJ19 and K5B showed maximum enzyme activity when grown in YIB (PH 7.2), 37°C after 36 hours, P2 (28 hours), while K51 and K52A after 32 hours incubation. The xylanase from B. pumilus PJ19 was purified to homogeneity by ammonium sulphate precipitation and gel filtration of CM-Sepharose and Sephacryl S-200. The molecular weight of the purified xylanase was estimated to be 23,000 D by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and 22,SIS D by mass spectrometry. The isoelectric point of the enzyme was 9.3. The optimum pH and temperature for hydrolysis of oat spelt xylan were 6.5 and 60°C, respectively. The enzyme was stable at a pH range of 7.5 to 8.5 and at a temperature of up to 45˚C but lost 50% of its activity at 58°C after 10 minutes of incubation. The purified enzyme had a Km of 1.42 mg/ml and V max of 107 µmol/ minlmg for oat spelt xylan and Km of 2.15 mg/ml and V max of 29.22 µmol/min/mg for birchwood xylan. The major end products of oat spelt xylan hydrolysis were xylobiose, xylotriose and higher oligosaccharides while for birchwood xylan were xylotriose with some xylobiose determined by thin layer chromatography and high performance liquid chromatography. Xylose was not produced as a product of hydrolysis and trans xylosidation was detected. The activity of the enzyme was enhanced in the presence of Mg2+, Ca2+ and K+ but was inhibited by EDTA, Cu2+, Ag+, Zn2+, Fe2+ and Hg2+. The enzyme was competitively inhibited in the presence of xylose with Kj of 1 98 mM. A complete DNA sequence of the xylanase gene was amplified by polymerase chain reaction and cloned into E. coli INVaF' using pCRII cloning vector. The complete DNA sequence was also determined. The structural xylanase gene which started from an ATG initiation codon, consists of an open reading frame of 684 bp, which encoded 202 amino acid residues. The molecular weight of the xylanase was estimated from the amino acid composition to be 22,474 D and is in agreement with the results obtained from SDS-PAGE of the purified xylanase. The xylanase was expressed constitutively by the cloned gene in the absence of xylan. The enzyme was located primarily in the cytoplasm probably because of the incompatibility of the Gram-positive signal peptide in E. coli to direct the enzyme extracellularly as in the donor strain

Effects of Paraquat and Alachlor on Soil Microorganisms in Peat Soil

A study was carried out to investigate the effects of alachlor and paraquat on microbial activities in peat soil. Effects of the herbicides on CO2 evolution and phosphatase activity were monitored for 12 weeks in ambient conditions. The results showed that paraquat and alachlor caused an initial increase in CO2 released and subsequently decreased after 53 days of incubation. Comparatively, more CO2 was released from the soil treated with alachlor than that treated with paraquat. An initial increase in phosphatase activity was observed for both herbicides but the level of activity was substantially reduced after 12 days of incubation. Fungal and bacterial populations in the soil were also affected by both herbicides. At 250 ppm, alachlor and paraquat caused a reduction in bacterial population of 78%and 95% respectively. Alachlor was shown to be more toxic to fungal populations in the soil than paraquat

Biodegradation of Tapis crude oil using consortium of bacteria and fungi: optimization of crude oil concentration and duration of incubation by Response Surface Methodology

Response surface analysis was conducted to optimize the concentrations of Tapis crude oil and duration of incubation in order to achieve optimal microbial growth and crude oil biodegradation. Central Composite Rotatable Design (CCRD) was employed, where the design contained 13 experimental runs with different combinations of incubation time and crude oil concentration. The cultures containing mineral salt medium (MSM) with varying crude oil concentrations were incubated at 30°C, pH6.5 with 150 rpm agitation for 120-336 h. The inoculum contained a consortium of previously identified as oil degrading bacteria and fungi, namely Pseudomonas aeruginosa UKMP-14T, Acinetobacter baumannii UKMP-12T and Trichoderma sp. UKMP-1M and UKMP-2M. The analysis showed the duration of incubation plays a significant role (p<0.05) in affecting the bacterial growth and percentage of total petroleum hydrocarbon (TPH) biodegradation, meanwhile concentrations of Tapis crude oil has insignificant effect on the responses. Interaction of the two variables was found to be significant in affecting all the three responses, namely bacterial population, fungal biomass and percentage of TPH biodegradation. It was predicted through the CCRD that the percentage of biodegradation can be optimized to reach 86% on the 270th h when 5% (v/v) crude oil was used. This predicted value was verified to be achievable and reproducible through validation experiments

Kecekapan filem kanji/minyak kayu manis sebagai pembungkus makanan dengan sifat antimikrob

Dalam kajian ini, filem kanji/minyak kayu manis telah dihasilkan menggunakan kaedah pengacauan larutan dengan kandungan minyak kayu manis (MKM) berbeza untuk mengkaji kesan MKM terhadap sifat antimikrob filem kanji. Sifat kelutsinaran filem didapati berkurang apabila peratus kandungan MKM meningkat yang telah menghalang penembusan cahaya. Sementara itu, analisis ATR-FTIR mendapati bahawa tiada penyesaran puncak atau pembentukan puncak baharu yang menunjukkan bahawa MKM dan filem kanji tidak terikat secara kimia. Kajian aktiviti antimikrob filem ini terhadap Bacillus cereus dan Aspergillus niger telah mendapati berlakunya peningkatan kecekapan aktiviti antimikrob bagi penambahan minyak kayu manis (MKM) dengan peningkatan diameter zon perencatan. Didapati berlaku perencatan pada kepekatan minimum MKM ditingkatkan bermula 16% dalam filem kanji mencatatkan corak pertumbuhan dalam zon rencatan dengan diameter 24-44.2 mm dengan peningkatan 28.23-37.88% dan 34.12-52%, masing-masing bagi zon perencatan terhadap B. cereus dan A. niger berbanding tiada sebarang zon perencatan untuk filem kanji tanpa penambahan MKM. Pemerhatian morfologi menunjukkan pembentukan liang dan pemisahan fasa yang heterogen antara MKM dan kanji. Tegangan maksimum filem pula menunjukkan pengurangan kekuatan tegangan apabila kepekatan MKM bertambah walaupun tidak ketara pada peningkatan MKM sehingga 24%. Sebaliknya, terikan pada takat putus filem didapati meningkat sebanyak 102-252% selari dengan penambahan MKM yang bertindak sebagai agen pemplastikan. Keseluruhannya, filem kanji/MKM telah berjaya disediakan dan pencirian sifat fizikal dan aktiviti antimikrob bagi filem ini menunjukkan potensinya dalam penggunaan pembungkusan makanan

Starch film incorporated with cinnamon oils optimally prepared by using response surface methodology

Recent progress in the food industry emphasizes on active packaging that integrates antimicrobial feature into food packaging made from biodegradable films. In this study, antimicrobial films were prepared from starch biopolymer incorporated with cinnamon oil as antimicrobial agent. Many studies have reported on the increasing antimicrobial properties of biofilms when the concentration of antimicrobial agent is increased, yet their tensile strength would be lowered. Therefore, the preparation of these films requires comprehensive optimization to ensure optimum properties of the resulting films. The aim of this study was to optimize the mechanical and antimicrobial property of film after adding cinnamon oil using response surface methodology (RSM). The optimization of variables to prepare the films was successfully accomplished by manipulating cinnamon oil loadings and mixing temperature as proposed by RSM. Several models were generated to associate those parameters with the responding variables such as tensile strength and antimicrobial activity against Bacillus cereus, Bacillus subtilis, Aspergillus niger, and Aspergillus brasiliensis with R2 values of 0.9835, 0.9816, 0.9709, 0.9930, and 0.9950, respectively. Subsequently, the optimum conditions for the preparation of starch/cinnamon oil films were found to be at 24.02% of cinnamon oil and 49.35 °C of mixing temperature. Our study has demonstrated a novel, statistical experimental design and elaborate discussion on the effects of processing parameters in preparing films composing of starch and cinnamon oil. In summary, the active films prepared from this study have displayed promising qualities as potential food packaging against most common food borne microorganism during the food storage

Substrate specificity of lipases from four species of Aspergillus towards hydrolysis of homoacid triacylglycerols and vegetable oils in non-aqueous system

Among the four species of Aspergillus, Aspergillus oryzea lipase demonstrates high preference towards medium chain triacylglycerols (C10) and discriminates against triunsaturated triacylglycerol (TAG) e.g. triolein. The great discriminating power of its lipase against triolein was shown in comparison with its ability to catalyse the hydrolysis of medium chain (C10) TAG e.g. tricaprin and less shown when hydrolysing saturated long chain TAG i.e. tripalmitin. The discriminating power of these lipases are in this order: triolein > tripalmitin > trilaurin > tricaprin. Similar phenomenon was noted when mycelium-bound lipases of Aspergillus sp. were used to catalyse the hydrolysis of coconut oil, palm olein, olive oil and flaxseed oil. In most cases, relative percentage of monounsaturated fatty acid (C18:1) in non-hydrolysed fractions of oils increased after 12 days of hydrolysis. Hydrolysis of flaxseed and olive oil showed that Aspergillus murarum, Aspergillus oryzea and Aspergillus flavus lipases have high preference towards polyunsaturated fatty acids i.e. linoleic acid (C18:2) in olive oil and linolenic acid (C18:3) in flaxseed oil and no preference for monounsaturated and saturated long chain fatty acids

Screening, isolation and identification of microbial lipase producers from palm oil mills

Screening, isolation and identification of microbial lipase producers from 16 locations of palm oil mills in Malaysia were successfully conducted. A total of 52 different strains were found to be lipase producers. These isolates were identified and divided into three different groups i.e. bacteria, yeast and mould. Twenty-five isolates were bacteria, 10 were yeast and the remainder were mould. Out of the 52 isolates, four were found to be new lipase producers and identified as Klebsiella pneumoniae rhinoschlero, CDC gr. IV C-2, Geotrichum penicillatum and Rahnella aquaticus. Lipases from Bacillus lentus, Bacillus sphaericus, Tatumella ptyseos, Geotrichum capitatum and Thielaviopsis sp. have been less reported and studied as lipase producers. Both Bacillus lentus and Bacillus sphaericus were found to be thermophilic bacteria and produce alkaline tolerantlipase

Penilaian kualiti air bawah tanah di Kelantan dan Pahang, Malaysia

Air bawah tanah yang menjadi sumber kegunaan domestik kepada pengguna memerlukan penilaian air bawah tanah bagi mengelakkan berlakunya isu kesihatan. Kajian ini dijalankan untuk menilai kualiti air telaga tiub dalam aspek kandungan logam berat, bahan radioaktif semula jadi (NORM) dan bakteria patogen di Kelantan dan Pahang, Malaysia. Hasil kajian ini menunjukkan kandungan Fe (90-42100 µg/L) dan Mn (3-478 µg/L) yang diperoleh adalah tinggi dan boleh menjejaskan kualiti air secara keseluruhan. Kandungan NORM yang terkandung dalam air telaga adalah 0.02-0.08 Bq/L bagi jumlah pengaktifan Alpha dan 0.02-0.15 Bq/L bagi jumlah pengaktifan beta, yang berada di bawah paras selamat yang telah ditetapkan oleh WHO. Kandungan NORM dalam sampel tanah adalah dalam julat 401-1348 Bq/kg untuk 40K, 19-175 Bq/kg untuk 232Th dan 28-318 Bq/kg untuk 238U yang boleh diterima di Malaysia. Anggaran dos sinaran tahunan adalah antara 52 µSv/tahun hingga 379 µSv/tahun dan berada dalam julat selamat untuk orang ramai. Kesemua sampel air dari Kelantan dan dua sampel air dari Pahang mengandungi bakteria enteropatogen terutamanya Escherichia coli dan Salmonella sp. yang melebihi piawaian Kualiti Air Kebangsaan. Boleh disimpulkan bahawa kualiti air bawah tanah selamat untuk digunakan semasa kecemasan, tetapi ia perlu dirawat sebelum diminum

Screening and optimization of biosurfactant production by the hydrocarbon-degrading bacteria

Biosurfactants are amphiphilic compounds produced by microorganisms as secondary metabolite. The unique properties of biosurfactants make them possible to replace or to be added to synthetic surfactants which are mainly used in food, cosmetics and pharmaceutical industries and in environmental applications. In this study twenty hydrocarbon-degrading bacteria were screened for biosurfactant production. All of the bacterial isolates were grown in mineral salt medium (MSM) with addition of 1% (v/v) Tapis crude oil as carbon source. The presence of biosurfactant was determined by the drop-collapse test, microplate analysis, oil spreading technique, emulsification index (%EI24) and surface tension measurement. Only one isolate, Pseudomonas aeruginosa UKMP14T, was found to be positive for all the qualitative tests and reducing the surface tension of the medium to 49.5 dynes/cm with emulsification index of 25.29%. This isolate produced biosurfactant optimally at pH9.0 and incubation temperature of 37°C. Furthermore, P. aeruginosa UKMP14T when grown in MSM with addition of 1% (v/v) glycerol and 1.3 g/L ammonium sulphate with C/N ratio 14:1 produced biosurfactant with percentage of surface tension reduction at 55% or 30.6 dynes/cm with %EI24 of 43%. This percentage of surface tension reduction represents an increasing reduction in surface tension of medium by 39% over the value before optimization. This study showed that P. aeruginosa UKMP14T has the ability to biodegrade hydrocarbon and concurrently produce biosurfactant

Detection of toluene degradation in bacteria isolated from oil contaminated soils

Toluene (C7H8) a hydrocarbon in crude oil, is a common contaminant in soil and groundwater. In this study, the ability to degrade toluene was investigated from twelve bacteria isolates which were isolated from soil contaminated with oil. Out of 12 bacterial isolates tested, most of Pseudomonas sp. showed the capability to grow in 1 mM of toluene compared with other isolates on the third day of incubation. Based on enzyme assays towards toluene monooxygenase, Pseudomonas aeruginosa UKMP-14T and Bacillus cereus UKMP-6G were shown to have the highest ability to degrade toluene. The toluene monoxygenase activity was analysed by using two calorimetric methods, Horseradish peroxidase (HRP) and indole-indigo. Both of the methods measured the production of catechol by the enzymatic reaction of toluene monooxygenase. In the HRP assay, the highest enzyme activity was 0.274 U/mL, exhibited by Pseudomonas aeruginosa UKMP-14T. However, for indole-indigo assay, Bacillus cereus UKMP-6G produced the highest enzyme activity of 0.291 U/ml. Results from both experiments showed that Pseudomonas aeruginosa UKMP-14T and Bacillus cereus UKMP-6G were able to degrade toluene