Cloning and expression of the gene encoding industrial alfa-amylase from Bacillus subtilis ORBA97 in Escherichia coli and Bacillus subtilis YB886

The a-amylase encoding gene from Bacillus subtilis ORBA97 strain was cloned and expressed in Escherichia coli with the plasmid vector called pUC18 as well as in the a-amylase negative Bacillus subtilis YB886 strain by using the pUB110 vector. The specific activity of the enzyme was determined to be 5.052 mmol (mg protein min)-1. Zymogram analysis revealed no sign of protease degradation in the YB886 strain. The optimum temperature and pH of the enzyme were found to be 50°C and 5 respectively. Relative enzyme activity decreased to 36% of the original activity when it was subjected to a temperature of 90°C for 15 minutes. Calcium chloride stimulated the enzyme activity and resulted in an approximately 23% increase in the activity. The pH tolerance of the enzyme seems to be suitable for the poultry digestive system. However, the thermostability of the enzyme may restrict its application in pelleted feeds.The a-amylase encoding gene from Bacillus subtilis ORBA97 strain was cloned and expressed in Escherichia coli with the plasmid vector called pUC18 as well as in the a-amylase negative Bacillus subtilis YB886 strain by using the pUB110 vector. The specific activity of the enzyme was determined to be 5.052 mmol (mg protein min)-1. Zymogram analysis revealed no sign of protease degradation in the YB886 strain. The optimum temperature and pH of the enzyme were found to be 50°C and 5 respectively. Relative enzyme activity decreased to 36% of the original activity when it was subjected to a temperature of 90°C for 15 minutes. Calcium chloride stimulated the enzyme activity and resulted in an approximately 23% increase in the activity. The pH tolerance of the enzyme seems to be suitable for the poultry digestive system. However, the thermostability of the enzyme may restrict its application in pelleted feeds

Maya Saccharomyces cerevisiae'yede farklı genlerin exspresyonu

Both an $/alpha$-amylase gene of Bacillus subtilis RSKK246 and a gene encoding $/beta$-glucanase from B.subtilis RSKK243 were cloned and expressed in both E.coli XL1-Blue MRF and B.subtilis YB886 by using the vectors pUC18 and pUB110 respectively. These genes were also cloned into the E.coli-yeast shuttle vectors pRS406 and pRS416 for transfer into the yeast Saccharomyces cerevisiae.These constructs carrying $/alpha$ -amylase and $/alpha$ -glucanase,genes which were cloned into the pRS406 vector,were expressed by integration into the yeast chromosome.These genes were also expressed in the yeast by autonomous replication of the ARS/CEN plasmid pRS416.This work demonstrates that genes belonging to B.subtilis can replicate in both E.coli and in the yeast S. cerevisiae.Both an $/alpha$-amylase gene of Bacillus subtilis RSKK246 and a gene encoding $/beta$-glucanase from B.subtilis RSKK243 were cloned and expressed in both E.coli XL1-Blue MRF and B.subtilis YB886 by using the vectors pUC18 and pUB110 respectively. These genes were also cloned into the E.coli-yeast shuttle vectors pRS406 and pRS416 for transfer into the yeast Saccharomyces cerevisiae.These constructs carrying $/alpha$ -amylase and $/alpha$ -glucanase,genes which were cloned into the pRS406 vector,were expressed by integration into the yeast chromosome.These genes were also expressed in the yeast by autonomous replication of the ARS/CEN plasmid pRS416.This work demonstrates that genes belonging to B.subtilis can replicate in both E.coli and in the yeast S. cerevisiae

Cloning of bacillus subtilis RSKK243 bifunctional xylanase gene in E. coli and B. subtilis and enzyme characterization

A bifunctional gene xylanase of B. subtilis RSKK243 encoding detectable beta(1,4) glucanase (CMCase) activity in addition to a high level of main xylanase activity was cloned and expressed in Escherichia coli with pUC18 plasmid and in various Bacillus subtilis strains with pUB110 vector. For enzymatic analysis and plasmid isolation, the gene was also expressed in xylanase and beta glucanase negative B.subtilis YB886 strain with pUB110 vector. The pUB110 with bifunctional xylanase gene was transferred into B. subtilis RSKK243 for maximizing the enzyme yield, as well as into other RSKK strains to create novel strains with multiple enzyme capacities. Recombinant strains were compared with their original counterparts with respect to their enzyme yield and multiple enzyme activities. In addition, molecular size determination and zymogram analysis were performed on SDS-PAGE and SDS-CMC-PAGE and SDS-Xylan-PAGE gels. The temperature and optimum pH of the enzyme were determined to be 50şC and 5 respectively. The enzyme was totally inactivated when it was exposed to a temperature of 90şC for 15 minutes.A bifunctional gene xylanase of B. subtilis RSKK243 encoding detectable beta(1,4) glucanase (CMCase) activity in addition to a high level of main xylanase activity was cloned and expressed in Escherichia coli with pUC18 plasmid and in various Bacillus subtilis strains with pUB110 vector. For enzymatic analysis and plasmid isolation, the gene was also expressed in xylanase and beta glucanase negative B.subtilis YB886 strain with pUB110 vector. The pUB110 with bifunctional xylanase gene was transferred into B. subtilis RSKK243 for maximizing the enzyme yield, as well as into other RSKK strains to create novel strains with multiple enzyme capacities. Recombinant strains were compared with their original counterparts with respect to their enzyme yield and multiple enzyme activities. In addition, molecular size determination and zymogram analysis were performed on SDS-PAGE and SDS-CMC-PAGE and SDS-Xylan-PAGE gels. The temperature and optimum pH of the enzyme were determined to be 50şC and 5 respectively. The enzyme was totally inactivated when it was exposed to a temperature of 90şC for 15 minutes

Bacillus subtilis RSKK246 şuşundan bir alfa amilaz geninin moleküler klonlanması ve Escherichia coli ve Bacillus subtilis şuşlarında ekspresyonu

Bacillus subtilis RSKK246 was found to produce approximately a 65-kDa $\alpha$-amylase enzyme. A gene was isolated encoding $\alpha$-amylase activity that corresponded to this size and was inserted into pUC18 plasmid which was transferred to Escherichia coli. An approximately 1.7kbp fragment, which contains a whole a-amylase gene, was excised and inserted into pUB110 and then transferred into the different B. subtilis strains including RSKK246, RSKK243, RSKK244, YB886 and ORBAM. The a-amylase gene was cloned into the plasmids and expressed with its own promoter, and this promoter sequence seemed to function in the E. coli and in all B. subtilis strains. Specific activity of the cloned enzyme was found to be higher than the native enzyme and molecular weight of the gene product remained the same in all other strains suggesting that it is resistant to the proteolytic attacks of these organisms.Bacillus subtilis RSKK246 was found to produce approximately a 65-kDa $\alpha$-amylase enzyme. A gene was isolated encoding $\alpha$-amylase activity that corresponded to this size and was inserted into pUC18 plasmid which was transferred to Escherichia coli. An approximately 1.7kbp fragment, which contains a whole a-amylase gene, was excised and inserted into pUB110 and then transferred into the different B. subtilis strains including RSKK246, RSKK243, RSKK244, YB886 and ORBAM. The a-amylase gene was cloned into the plasmids and expressed with its own promoter, and this promoter sequence seemed to function in the E. coli and in all B. subtilis strains. Specific activity of the cloned enzyme was found to be higher than the native enzyme and molecular weight of the gene product remained the same in all other strains suggesting that it is resistant to the proteolytic attacks of these organisms

Partial Characterization of CMCase, Xylanase and Lichenase Enzymes ProducedPartial Characterization of CMCase, Xylanase and Lichenase Enzymes Produced by Newly Isolated Bacillus sp. CXL by Newly Isolated Bacillus sp. CXL

In the study, cellulase, xylanase and lichenase producing bacterium Bacillus sp. CXL was isolated from the soil samples collected from agricultural greenhouse. The optimum enzyme activities were observed at 50 °C for CMCase and lichenase, whereas at 40 °C for xylanase. On the other hand, optimum pH values for CMCase, xylanase and lichenase were 8.0, 6.0 and 7.0, respectively. CMCase, xylanase and lichenase were showed 71, 63, and 78% residual activity after pre-incubation at 80 °C for 15 min, respectively. The relative residual activities between 40-80 °C were occurred as 84.4, 86.6, and 92.6%, in the same order. Maximum CMCase, xylanase and lichenase productions of isolate were observed after 36, 24, and 12 hours later from inoculation time. All three enzyme activities were stimulated by CaCl2 , MnCl2 , CoCl2 , and MnCl2 , whereas inhibited by HgCl2 , and FeCl3 . While ZnCl2 and EDTA stimulated the CMCase and xylanase activities, CuCl2 stimulated CMCase and lichenase. If xylanase activity is accepted as 100%, the activities of CMCase and lichenase in comparison to the xylanase remain 34 and 56%, respectively

Isolation of CMCase, Xylanase and Lichenase Producer Bacillus Strain and Partial Characterization of the Enzymes

The importance of the study was to gain new feed additive enzymes produced by newly isolated microorganisms for animal nutrition. Isolation of carboxymethyl cellulase (CMCase), xylanase and lichenase producer Bacillus strain from soil sample, partial characterization and investigation of the potential availability of the enzymes for animal nutrition were main scopes of the study. The aim of the study was isolation of the Bacillus strain producing CMCase, xylanase and lichenase enzymes which can be used in animal nutrition. The soil sample was pasteurised at 80 °C for 10 min for isolation of spore forming and aerobic bacteria. CMCase, xylanase and lichenase producer strains were determined on LB-agar plates including certain substrates by Congo-red staining. Dinitrosalicylic acid method was used for partial characterization of the enzymes. In the study, Bacillus sp. strain was isolated from the soil sample collected from agricultural area in Turkey. The isolate which exhibiting CMCase, xylanase and lichenase, was entitled as Bacillus sp. BMN-2. The optimum pH values of CMCase was 7.0, whereas 6.0 for xylanase and lichenase. However, the optimum temperature value of CMCase was 40 °C, whereas 50 °C for xylanase and lichenase. The enzymes retained most of their activities after pre-incubation at 70 °C for 15 min. Bacillus sp. BMN-2 reached highest enzyme production levels for all enzymes within 12-36 hours later from bacterial inoculation. The common activators for all enzymes were MnCl2, CaCl2 CoCl2, and EDTA. All enzymes were strongly inhibited by HgCl2. These properties of the enzymes indicated that they could be used as enzyme supplement for animal fee

Characterization of thermostable α-amylase from thermophilic andAlkaliphilic bacillus sp. isolate DM-15

Bacillus sp. DM-15 was isolated from Çiftehan Thermal Spring, Turkey produced thermostable α-amylase at 55°C at pH 9. Analysis of the enzyme for molecular mass and amylolytic activity carried out by SDS-Starch-PAGE electrophoresis revealed a single band with high molecular mass 126 kDa. After extraction, the enzyme remained stable in a range of temperature and pH between 40-100°C and 4.5-10, respectively. The optimum enzyme activity was displayed at 60°C and pH 5.5-6.0. α-amylase production by thermophilic Bacillus sp. strain cultivated in liquid media reached a maximum at 12 h with levels of 27.5 μmol mg-1 protein min-1. The enzyme was stable for 15 min at 60°C while 24% of the original activity was lost at 100°C. Enzyme activity was increased in the presence of 5 mM CaCl2, Na2SO3 and KCl (105%) and inhibited in the presence of 5 mM EDTA, CuSO4, FeSO4, SDS (1%), Urea (8 M), Co up to 98, 79, 57, 36, 33 and 10%, respectively. The DM-15 α-amylase may be suitable for in liquefaction of starch in detergent and textile industries and in other industrial applications

Comparison of enzyme production by halophilic bacteria in sterilized and non-sterilized conditions

Asan Ozusağlam, Meltem ( Aksaray, Yazar )In present study, we aimed to determine the enzymes of moderately halophilic bacteria isolated from Serefl ikochisar Lake the largest salt lake in Turkey and compared their enzymes in sterilized and non-sterilized conditions to obtain any contamination from non-sterilized growth condition. The enzymes of moderately halophilic bacteria isolated from Şerefl ikoçhisar Lake were determined by SDS-PAGE. In addition of zymogram analysis, the enzymes from the moderately halophilic bacteria cultured in sterilized and non-sterilized growth condition were compared on SDS-PAGE to determine any contamination in non-sterilized growth condition. The enzyme bands in both conditions were similar and therefore the investigation suggests that the moderately halophilic isolates can be easily cultured in non-sterilized growth conditions without sterilization of growth medium and thereby energy cost can be reduce to produce such enzymes for industrial applications

Comparative analysis of multi-criteria decision making methodologies and implementation of a warehouse location selection problem

For the solution of decision making problems with multi criteria, the literature presents many methodologies under the title of decision theory. In this context, AHP, TOPSIS, ELECTRE and Grey Theory are well-known and the most acceptable methodologies. Firstly, in this study; these methodologies are compared in terms of main characteristic of decision theory and thus advantages and disadvantages of these methodologies are offered. Later, the application of these methodologies on the warehouse selection problem, which is one of the main topics of logistics management that has a wide range of applications with multi-criteria decision making methodologies, is presented as a case study which is characterized in retail sector, that maintains high uncertainity and product variety and then how to choose the best warehouse location among many alternatives has been shown. (C) 2011 Elsevier Ltd. All rights reserved