Purification and some properties of a novel raw starch-digesting amylase fromAspergillus carbonarius

Okolo, B. N.; Ire, Francis Sopuruchukwu; Ezeogu, Lewis I.; Anyanwu, Chukwudi Uzoma; Odibo, F. J. C.

doi:10.1002/1097-0010(200102)81:3<329::aid-jsfa815>3.0.co;2-3

Cited by 36 publications

(8 citation statements)

References 27 publications

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“…Prior to purification, attempts to concentrate the enzyme by ammonium sulphate or acetone precipitation which were reported by Garg & Virupaksha (1970), proved unsuccessful as the enzyme lost activity rapidly even when maintained at low temperature (0-4°C). Sucrose solution was found by experimentation (Okolo et al 2000) to effectively concentrate the protease with minimum loss of activity. The enzyme was purified 7.9-fold to give a 13.4% yield relative to the total activity in the crude extract (Table 1) and a final specific activity of 2128.7 U mg )1 protein.…”

Section: Resultsmentioning

confidence: 99%

Purification and Some Properties of a Metalloprotease from Sorghum Malt Variety KSV8-1

Ogbonna

Okolo

2005

World J Microbiol Biotechnol

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A metalloprotease from sorghum malt variety KSV8-I was purified by a combination of 4-M sucrose fractionation, ion-exchange chromatography on Q-Sepharose (Fast flow), gel-filtration chromatography on Sephadex G-100 and hydrophobic interaction chromatography on phenyl-Sepharose CL-4B. The enzyme was purified 7.9-fold to give a 13.4% yield relative to the total activity in the crude extract and a final specific activity of 2128.7 U mg )1 protein. SDS-PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 35 kDa. The purified enzyme had optimal activity at 60°C and maximal temperature stability between 40 and 60°C but retained over 77% of its initial activity after incubation at 70°C for 30 min. Both pH optimum and maximal stability were at 7.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. Using 0.2 ml of 5 mM solution of each metal ion, the purified protease was slightly (P<0.05) inhibited by Zn 2+ , appreciably (P<0.01) inhibited by Ca 2+ and Co 2+ and highly significantly (P<0.001) inhibited by Ag + , Ba 2+ , Hg 2+ , Mn 2+ and Pb 2+ . The enzyme was equally highly significantly (P<0.001) inhibited by EDTA and hydrolysed casein to give the following kinetic constants: K m = 21.0 mg ml )1 ; V max = 8.2 lmol ml 1 min )1 and K i = 0.390 mM.

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Section: Resultsmentioning

confidence: 99%

Purification and Some Properties of a Metalloprotease from Sorghum Malt Variety KSV8-1

Ogbonna

Okolo

2005

World J Microbiol Biotechnol

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“…Raw potato starch was prepared in our laboratory according to the method outlined by Okolo et al (2001). DNS was purchased from Lancaster, England.…”

Section: Methodsmentioning

confidence: 99%

“…Aspergillus carbonarius capable of producing large amounts of RSDA was earlier isolated (Okolo et al 1996); the amylase was able to degrade different sources of starch including potato starch extensively producing only glucose and maltose (Okolo et al 2001). To further stabilize this novel amylase, the enzyme was immobilized in this study.…”

Section: Introductionmentioning

confidence: 99%

Stabilization of a raw starch digesting amylase from Aspergillus carbonarius via immobilization on activated and non-activated agarose gel

Nwagu

Okolo

Aoyagi

2011

World J Microbiol Biotechnol

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Applications of raw starch digesting amylases (RSDAs) are limited due to instability, product inhibition of enzyme and contamination. RSDA from Aspergillus carbonarius was stabilized through immobilization on agarose gel by adsorption, spontaneous crosslinking and conjugation using glycidol, glutaraldehyde or polyglutaraldehyde. Effects of immobilization on kinetics, catalytic, storage and operational stability of immobilized enzyme were evaluated. Polyglutaraldehyde activated agarose RSDA (PGAg-RSDA) gave the highest immobilization yield (100%) with expressed activity of 86.7% while that of glycidol activated RSDA (GlyAg-RSDA) was 80.4%. A shift in pH from optimum of 5 for the soluble enzyme to 6 for RSDA adsorbed on agarose followed by crosslinking with glutaraldehyde (AgRSDA-CROSS) and simultaneous adsorption and crosslinking (AgRSDA-RET), and pH 7 for PGAg-RSDA was seen. PGAg-RSDA and AgRSDA-CROSS were most pH stable and retained over 82% of their activities between pH 3.5 and 9 compared to 59% for the soluble enzyme. Thermoinactivation studies showed that immobilized RSDAs with the exception of GAg-RSDA retained over 90% of their activities at 60°C for 120 min while soluble enzyme retained only 76% activity under the same condition. AgRSDA-CROSS, PGAg-RSDA, Gly-RSDA and GAg-RSDA retained approximately 100% of their activities after 30 days storage at 4°C. GlyAg-RSDA retained 99.6%, PGAg-RSDA 94%, AgRSDA-CROSS 90%, GAg-RSDA 86.5% and Ag-RSDA-RET 80% activity after 10 batch reactions. Immobilization stabilized RSDA and permits processing at higher temperatures to reduce contamination.

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“…This process is energy-intensive, thus Aspergillus awamori -- [8] Aspergillus awamori var. kawachi -- [9][10][11] Aspergillus awamori KT-11 air α [12] Aspergillus carbonarius rotten cassava β [13] Aspergillus cinnamomeus -GA [14] Aspergillus ficum -α [15] Aspergillus niger -GA [16,17] Aspergillus niger Cassava waste - [18] Aspergillus niger AM07 Soil α, GA [19] Aspergillus niger NIAB 280 -GA [20] Aspergillus niger sl. 1 Soil - [21] Aspergillus oryzae -GA [22] Aspergillus sp.…”

Section: Introductionmentioning

confidence: 99%

Recent Advances in Microbial Raw Starch Degrading Enzymes

Sun

Zhao

Guo

et al. 2009

Appl Biochem Biotechnol

151

117

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Raw starch degrading enzymes (RSDE) refer to enzymes that can directly degrade raw starch granules below the gelatinization temperature of starch. These promising enzymes can significantly reduce energy and simplify the process in starch industry. RSDE are ubiquitous and produced by plants, animals, and microorganisms. However, microbial sources are the most preferred one for large-scale production. During the past few decades, RSDE have been studied extensively. This paper reviews the recent development in the production, purification, properties, and application of microbial RSDE. This is the first review on microbial RSDE to date.

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Purification and some properties of a novel raw starch-digesting amylase fromAspergillus carbonarius

Cited by 36 publications

References 27 publications

Purification and Some Properties of a Metalloprotease from Sorghum Malt Variety KSV8-1

Purification and Some Properties of a Metalloprotease from Sorghum Malt Variety KSV8-1

Stabilization of a raw starch digesting amylase from Aspergillus carbonarius via immobilization on activated and non-activated agarose gel

Recent Advances in Microbial Raw Starch Degrading Enzymes

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