Improved Production of Mannanase byStreptomyces lividans (original) (raw)
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Different actinomycete isolates were screened for the production of mannanase under submerged state fermentation. Streptomyces sp. AZA12 was selected as the most potent isolate with highest enzyme activity and was therefore selected for further studies. The nutritional requirements of Streptomyces sp. AZA12 for the production of mannanase in submerged fermentation were investigated. Different agricultural wastes were screened as substitutes to commercial substrate for mannanase production. Soyastraw was found to be the most effective carbon source with highest mannanase activity of 48.33 U/ml. Aspargine was obtained to be the best nitrogen source with highest activity of 93.38 U/ml out of all the nitrogen sources screened in this study.Process parameters like pH, temperature, time of incubation, affecting enzyme production were optimized using one-factor-at-a-time approach. Mannanase production was conducted in mineral salt medium and enzyme activity was determined by dinitrosalicylic acid method.The optimal culture conditions obtained from this study helped to standardize the requirements for optimum production of mannanase using agrowastes.
Optimization of mannanse production from Streptomyces sp. PG-08-03 in submerged fermentation
Bioresources, 2009
Streptomyces sp. PG-08-3 was isolated from the desert of Rajasthan (India). The organism produced mannanase (15 Umg-1 protein) in the presence of 0.5% guar gum as a sole carbon source in minimal media by submerged fermentation (SmF). Enzyme production was enhanced by 7.3-fold when 0.5% soyabean meal and 0.25% of leucine were added to the minimal media. Increasing the guar gum concentration in the media by 0.1-1.0% resulted in linearly enhanced the production of mannanase.
Chemical Mutagenesis of Bacillus subtilis for Improved Mannanase Biosynthesis
Journal of Advances in Microbiology, 2017
The aim of the present study was to isolate bacterial associated with abattoir waste water, compost saw dust, soil and water samples from Ilaje Lake, Ondo State, Nigeria. The microbial isolates were identified using standard microbiological method. The bacterial isolates were screened for mannanase production. Mannanase activity was determined by dinitrosalicylic acid (DNSA) method while protein concentration in the fermentation broth was quantified by Lowry's method. Isolate designated 2k tentatively identified as Bacillus subtilis had the highest mannanase activity. The isolate with highest mannolytic activity was then subjected to different mutagens. The mutants of Bacillus subtilis generated were screened for mannanase production in comparison with the wild type in submerged state fermentation. All the mutant strains generated from B. subtilis had their mannolytic activities repressed in comparison with the wild strain. Out of mutants screened, mutant designated CH017 have the highest mannolytic activity 1.20 U/mg. The mannanase activity produced by CH017 was approximately 44% lower than the wild strain. The Original Research Article Adeleke et al.; JAMB, 3(2): 1-6, 2017; Article no.JAMB.30911 2 pretreatment of B. subtilis with nitrous acid caused enzyme repression. Therefore, another chemical mutagen should be worked upon whether it would result in appreciable yield of mannanase.
Optimization of Cultural Conditions for β-mannanase Production by a Local Aspergillus niger Isolate
fspublishers.org
A survey of eight locally isolated fungal strains was carried out to select the most potent fungus producing an extracellular βmannanase activity. In both static and shaking cultures, Aspergillus niger recorded the highest β-mannanase activity (3.00 & 2.69 Um L-1 , respectively) among the eight fungal isolates. Optimization of the fermentation medium for maximization of βmannanase activity in A. niger batch culture was performed. The highest β-mannanase activity (3.00 U mL-1) was achieved after 6 days of static incubation. Coconut at a concentration of 5 g 50 mL-1 proved an optimum natural substrate for reaching the highest β-mannanase activity (8.56 U mL-1), and protein content (1.689 mg mL-1). Ammonium chloride (as the best nitrogen source) and di-potassium hydrogen phosphate at concentrations of 2.5 and 1 g L-1 culture medium, respectively yielded the highest β-mannanase activity (20.91 U mL-1). On the other hand, the use of magnesium sulphate at 1.25 g L-1 resulted in maximum enzyme activity (25.64 U mL-1) as well as maximum protein content and growth. Maximum enzyme activity (26.86 U mL-1) protein content and growth were all obtained when using potassium chloride at concentration of 0.75 g L-1. β-mannanase activity increased by 9.6-fold yielding 28.98 U mL-1 when the optimized culture medium was initially adjusted to pH 6.0, inoculated with 2 x 10 6 spore mL-1 and incubated at 30°C.
Cloning and characterization of a new β-mannosidase from Streptomyces sp. S27
Enzyme and Microbial Technology, 2011
A new -mannosidase gene, designated as man2S27, was cloned from Streptomyces sp. S27 using the colony PCR method and expressed in Escherichia coli BL21 (DE3). The full-length gene consists of 2499 bp and encodes 832 amino acids with a calculated molecular mass of 92.6 kDa. The amino acid sequence shares highest identity of 62.6% with the mannosidase Man2A from Cellulomonas fimi which belongs to the glycoside hydrolase family 2. Purified recombinant Man2S27 showed optimal activity at pH 7.0 and 50 • C. The specific activity, K m , and k cat values for p-nitrophenyl--d-mannopyranoside (p-NP--MP) were 35.3 U mg -1 , 0.23 mM, and 305 s -1 , respectively. Low transglycosylation activity was observed when Man2S27 was incubated with p-NP--MP (glycosyl donor) and methyl-␣-d-mannopyranoside (p-NP-␣-MP) (acceptor) at 50 • C and pH 7.0, and a small amount of methylmannobioside was synthesized. Using locust bean gum as the substrate, more reducing sugars were liberated by the synergistic action of Man2S27 and -mannanase (Man5S27), and the synergy degree in sequential reactions with Man5S27 firstly and Man2S27 secondly was higher than that in the simultaneous reactions.
Molekuliarnaia genetika, mikrobiologiia i virusologiia, 2012
Mannanases can be useful in the food, feed, pulp and paper industries. In this research a Bacillus subtilis strain (named Bs5) which produced high-level beta-mannanase was isolated. Maximum level of beta-mannanase (1231.41 U/ml) was reached when Bacillus subtilis Bs5 was grown on konjac powder as the carbon source for nine hours at 32 degrees C. The beta-mannanase was a typical cold-active enzyme and its optimal temperature of 35 degrees C was the lowest among those of the known mannanases from bacteria. In addition, the optimal pH was 5.0 and much wide pH range from 3.0-8.0 was also observed in the beta-mannanase. These properties make the beta-mannanase more attractive for biotechnological applications. The DNA sequence coding the beta-mannanase was cloned and the open reading frame consisted of 1089 bp encoding 362 amino acids. A phylogenetic tree of the beta-mannanase based on the similarity of amino acid sequences revealed that the beta-mannanase formed a cluster with the beta-...
SpringerPlus, 2014
The mannanase gene of B. circulans NT 6.7 was cloned and expressed in an Escherichia coli expression system. The B. circulans NT 6.7 mannanase gene consists of 1,083 nucleotides encoding a 360-amino acid residue long polypeptide, belonging to glycoside hydrolase family 26. The full-length mannanase gene including its native signal sequence was cloned into the vector pET21d and expressed in E. coli BL21 (DE3). β-Mannanase activities in the culture supernatant and crude cell extract were 37.10 and 515 U per ml, respectively, with most of the activity in the cell extract attributed to the periplasmic fraction. In contrast, expression of mannanase was much lower when using the B. circulans NT 6.7 mannanase gene without its signal sequence. The optimum temperature of recombinant β-mannanase activity was 50°C and the optimum pH was 6.0. The enzyme was very specific for β-mannan substrates with a preference for galactomannan. Hydrolysis products of locust bean gum were various mannooligosaccharides including mannohexaose, mannopentaose, mannotetraose, mannotriose and mannobiose, while mannose could not be detected. In conclusion, this expression system is efficient for the secretory production of recombinant β-mannanase from B. circulans NT 6.7, which shows good characteristics for various applications.
Isolation of mannan-utilizing bacteria and the culture conditions for mannanase production
World Journal of Microbiology & Biotechnology, 1994
A locally isolated strain, Bacillus subtilis NM-39, was selected as an active mannan-utilizing bacterium based on high saccharifying activities on coconut residue and locust bean gum galactomannan. The optimal pH and temperature ranges for activity of the crude enzyme were 5.0 to 6.0 and 50 to 60°C, respectively. The organism gave maximum mannanase activity when grown in liquid mineral salts medium containing 1% (w/v) each of coconut residue and soybean flour, as carbon and nitrogen sources, respectively, at pH 7.0 and in aerobic growth for 28 h at 37°C. High saccharifying activity on coconut mannan was also observed.
Journal of Biotechnology, 1999
A number of wild-type isolates of Sclerotium (Athelia) rolfsii and S. coffeicola were studied for their ability to produce endo-i-1,4-mannanase, endo-i-1,4-xylanase, and endo-i-1,4-glucanase activity when grown on cellulose-or glucose-based media. Whereas the presence of the inducer cellulose was strictly necessary for increased xylanase and endoglucanase production by both S. rolfsii (208 and 599 U ml − 1 , respectively) and S. coffeicola (102 and 330 U ml − 1 , respectively), elevated activities of mannanase (up to 96.6 U ml − 1 ) were formed even when employing glucose as the only carbohydrate substrate. Significant production of mannanases as well as of auxiliary mannan-degrading enzymes (i-mannosidase, i-glucosidase, h-galactosidase, acetyl esterase) was only observed, however, under derepressed conditions, i.e. after glucose had been consumed from the medium. By applying a fed-batch strategy, in which a glucose solution was continuously fed to a cultivation of S. rolfsii CBS 191.62 so that the glucose concentration in the medium never exceeded a certain low, critical value, production of mannanase could be almost doubled as compared to a batch cultivation on glucose (462 versus 240 U ml − 1 ). Mannanase preparations produced by several S. rolfsii and S. coffeicola strains under inductive and noninductive conditions (i.e. using cellulose or glucose as the substrates, respectively) were further analyzed with respect to the patterns of isoformic mannanases formed under these different growth conditions. Multiple mannanases were secreted by all isolates investigated. Certain mannanase isoenzymes were only formed by S. rolfsii in the presence of the inducer cellulose, indicating a complex and separated regulation of the synthesis of mannanase isoenzymes in this strain.
Biotechnology Progress, 2009
To be utilized in biomass conversion, including ethanol production and galactosylated oligosaccharide synthesis, namely prebiotics, the gene of extracellular endo-β-1,4-mannanase (EC 3.2.1.78) of Aspergillus fumigatus IMI 385708 (formerly known as Thermomyces lanuginosus IMI 158749) was expressed first in Aspergillus sojae and then in Pichia pastoris under the control of the glyceraldehyde triphosphate dehydrogenase (gpdA) and the alcohol oxidase (AOX1) promoters, respectively. The highest production of mannanase (352 U mL−1) in A. sojae was observed after 6 days of cultivation. In P. pastoris, the highest mannanase production was observed 10 h after induction with methanol (61 U mL−1). The fold increase in mannanase production was estimated as ∼12-fold and ∼2-fold in A. sojae and P. pastoris, respectively, when compared with A. fumigatus. Both recombinant enzymes showed molecular mass of about 60 kDa and similar specific activities (∼350 U mg−1 protein). Temperature optima were at 60°C and 45°C, and maximum activity was at pH 4.5 and 5.2 for A. sojae and P. pastoris, respectively. The enzyme from P. pastoris was more stable retaining most of the activity up to 50°C, whereas the enzyme from A. sojae rapidly lost activity above 40°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009