Francois N. Niyonzima

Isolation, Purification and Characterization of Fungal Extracellular L-Asparaginase from Mucor hiemalis

Isolation, Purification and Characterization of Fungal Extracellular L-Asparaginase from Mucor hiemalis L-asparaginase is an enzyme that deaminates the free L-asparagine to yield aspartic acid and is used as an antileukemic agent. L-asparaginase producing fungus was screened from a local soil sample and identified as Mucor hiemalis based on morphological and microscopic characteristics.

Detergent-Compatible Proteases: Microbial Production, Properties, and Stain Removal Analysis

Proteases are one of the most important commercial enzymes used in various industrial domains such as detergent and leather industries. The alkaline proteases as well as other detergent-compatible enzymes such as lipases and amylases serve now as the key components in detergent formulations. They break down various stains during fabric washing. The search for detergent-compatible proteases with better properties is a continuous exercise. The current trend is to use detergent-compatible proteases that are stable over a wide temperature range. Although the proteases showing stability at elevated pH have the capacity to be used in detergent formulations, their usage can be significant if they are also stable and compatible with detergent and detergent ingredients, and also able to remove protein stains. Despite the existence of some reviews on alkaline proteases, there is no specification for the use of alkaline proteases as detergent additives. The present review describes the detergent-compatible proteases tested as detergent additives. An overview was provided for screening, optimization, purification, and properties of detergent compatible proteases, with an emphasis on the stability and compatibility of the alkaline proteases with the detergent and detergent compounds, as well as stain removal examination methods.

PURIFICATION AND PROPERTIES OF DETERGENT-COMPATIBLE EXTRACELLULAR ALKALINE PROTEASE FROM Scopulariopsis spp.

A fungal alkaline protease of Scopulariopsis spp. was purified to homogeneity with a recovery of 32.2% and 138.1 U/mg specific activity on lectin-agarose column. The apparent molecular mass was 15 ± 1 kD by sodium dodecyl sulfate polyacryalamide gel electrophoresis (SDS-PAGE). It was a homogenous monomeric glycoprotein as shown by a single band and confirmed by native PAGE and gelatin zymography. The enzyme was active and stable over pH range 8.0–12.0 with optimum activity at pH 9.0. The maximum activity was recorded at 50°C and remained unaltered at 50°C for 24 hr. The enzyme was stimulated by Co2+ and Mn2+ at 10 mM but was unaffected by Ba2+, Mg2+, Cu2+, Na+, K+, and Fe2+. Ca2+ and Fe3+ moderately reduced the activity (∼18%); however, a reduction of about 40% was seen for Zn2+ and Hg2+. The enzyme activity was completely inhibited by 5 mM phenylmethylsulfonyl fluoride (PMSF) and partially by N-bromosuccinimide (NBS) and tocylchloride methylketone (TLCK). The serine, tryptophan, and histidine may therefore be at or near the active site of the enzyme. The protease was more active against gelatin compared to casein, fibrinogen, egg albumin, and bovine serum albumin (BSA). With casein as substrate, Km and Vmax were 4.3 mg/mL and 15.9 U/mL, respectively. An activation was observed with sodium dodecyl sulfate (SDS), Tween-80, and Triton X-100 at 2% (v/v); however, H2O2 and NaClO did not affect the protease activity. Storage stability was better for all the temperatures tested (−20, 4, and 28 ± 2°C) with a retention of more than 85% of initial activity after 40 days. The protease retained more than 50% activity after 24 hr of incubation at 28, 60, and 90°C in the presence (0.7%, w/v) of commercial enzymatic and nonenzymatic detergents. The Super Wheel–enzyme solution was able to completely remove blood staining, differing from the detergent solution alone. The stability at alkaline pH and high temperatures, broad substrate specificity, stability in the presence of surfactants and oxidizing and bleaching agents, and excellent compatibility with detergents clearly suggested the use of the enzyme in detergent formulations.

Concomitant production of detergent compatible enzymes by Bacillus flexus XJU-1

A soil screened Bacillus flexus XJU-1 was induced to simultaneously produce alkaline amylase, alkaline lipase and alkaline protease at their optimum levels on a common medium under submerged fermentation. The basal cultivation medium consisted of 0.5% casein, 0.5% starch and 0.5% cottonseedoil as an inducer forprotease, amylase, and lipase, respectively. The casein also served as nitrogen source for all 3 enzymes. The starch was also found to act as carbon source additive for both lipase and protease. Maximum enzyme production occurred on fermentation medium with 1.5% casein, 1.5% soluble starch, 2% cottonseed oil, 2% inoculum size, initial pH of 11.0, incubation temperature of 37 °C and 1% soybean meal as a nitrogen source supplement. The analysis of time course study showed that 24 h was optimum incubation time for amylase whereas 48 h was the best time for both lipase and protease. After optimization, a 3.36-, 18.64-, and 27.33-fold increase in protease, amylase and lipase, respectively was recorded. The lipase was produced in higher amounts (37.72 U/mL) than amylase and protease about 1.27 and 5.85 times, respectively. As the 3 enzymes are used in detergent formulations, the bacterium can be commercially exploited to secrete the alkaline enzymes for use in detergent industry. This is the first report for concomitant production of 3 alkaline enzymes by a bacterium.

Coproduction of detergent compatible bacterial enzymes and stain removal evaluation

Most of the detergents that are presently produced contain the detergent compatible enzymes to improve and accelerate the washing performance by removing tough stains. The process is environment friendly as the use of enzymes in the detergent formulation reduces the utilization of toxic detergent constituents. The current trend is to use the detergent compatible enzymes that are active at low and ambient temperature in order to save energy and maintain fabric quality. As the detergent compatible bacterial enzymes are used together in the detergent formulation, it is important to co‐produce the detergent enzymes in a single fermentation medium as the enzyme stability is assured, and production cost gets reduced enormously. The review reports on the production, purification, characterization and application of detergent compatible amylases, lipases, and proteases are available. However, there is no specific review or minireview on the concomitant production of detergent compatible amylases, lipases, and proteases. In this minireview, the coproduction of detergent compatible enzymes by bacterial species, enzyme stability towards detergents and detergent components, and stain release analysis were discussed.

Detergent-Compatible Bacterial Amylases

Proteases, lipases, amylases, and cellulases are enzymes used in detergent formulation to improve the detergency. The amylases are specifically supplemented to the detergent to digest starchy stains. Most of the solid and liquid detergents that are currently manufactured contain alkaline enzymes. The advantages of using alkaline enzymes in the detergent formulation are that they aid in removing tough stains and the process is environmentally friendly since they reduce the use of toxic detergent ingredients. Amylases active at low temperature are preferred as the energy consumption gets reduced, and the whole process becomes cost-effective. Most microbial alkaline amylases are used as detergent ingredients. Various reviews report on the production, purification, characterization, and application of amylases in different industry sectors, but there is no specific review on bacterial or fungal alkaline amylases or detergent-compatible amylases. In this mini-review, an overview on the production and property studies of the detergent bacterial amylases is given, and the stability and compatibility of the alkaline bacterial amylases in the presence of the detergents and the detergent components are highlighted.

Purification and characterization of phytase from Bacillus lehensis MLB2

Abstract A potent phytase-producing bacterium Bacillus lehensis MLB2 was isolated from bean-grown soil. The optimum conditions recorded after optimization were 24 h incubation time, pH 5.5, 37°C, 2% inoculum level, 0.5% rice bran and 0.5% potassium nitrate. An overall 3.144-fold enhancement in phytase production was achieved after optimization. The use of an inexpensive substrate rice bran and short incubation period make the phytase production cost effective. The purified phytase (152.9 U/mg) had a molecular mass of approximately 98.686 kDa as determined by sodium dodecyl sulphatepolyacryalamide gel electrophoresis and confirmed by liquid chromatography-mass spectrometry, optimum pH of 4.5, and temperature of 37°C. It maintained maximum stability in the acidic region from pH 2.0 to 6.0 and retained 100% at 60◦C or below. It showed an enhanced activity in the presence of 5 mM K+ and Na+. Ca2+, Mg2+, and Ba2+ did not have any effect or slightly activate the phytase. Group-specific reagents indicated the presence of cysteine and tryptophan in or near the active site of the enzyme. Better pH and temperature broad range adaptability, strict sodium phytate specificity and low Km value of 0.1232 mM, and in vitro release of a significant amount of orthophosphate from feedstuffs, and thus reduction of environmental phosphorus pollution, make the B. lehensis MLB2 phytase a good candidate for feed additive applicability.

Biochemical properties of the alkaline lipase of Bacillus flexus XJU-1 and its detergent compatibility

The possibility of using Bacillus flexus XJU-1 lipase in detergent preparations was studied. The enzyme was monomeric protein as confirmed by liquid chromatography-mass spectrometry and its molecular weight was 15.95 kDa. The lipase showed optimum activity at pH 10.0 and was 100% stable for 24 h at pH 10.0 and 11.0. It exhibited maximum activity at 70°C and retained more than 70% of the initial activity at 60, 70 and 80°C for 24 h. The activity was stimulated by Ca2+, Ba2+, Mg2+ and Co2+, whereas 50% of the initial activity was lost with Fe3+ and Hg2+. The activity was inhibited by 10 mM N-bromosuccinimide and tosyl-L-lysylchloromethylketone, while N-ethylmaleimide, phenylmethylsulphonylfluoride and urea did not show any effect. The enzyme significantly hydrolysed olive, cottonseed, sunflower, groundnut, and gingelly oils. With p-nitrophenyl palmitate, Vmax and Km were 62.5 U/mL and 2.25 mM, respectively. The lipase maintained its stability in Tween-80, Triton-100 and H2O2 at 1%, but an activation of 10% and a reduction of 15% in relative activity were observed with NaClO and sodium dodecyl sulphate, respectively. The enzyme retained maximum storage stability for 20 days at −20, 4 and 30°C. In the presence of 0.7% (w/v) Ariel, Henko, Super wheel, Tide plus and Rin, a retention of more than 84.90% initial activity was recorded after 24 h at 60°C. The supplementation of the lipase to the detergents improved the olive oil stain removal. These properties suggested the present enzyme as a potential additive for detergent preparations.

Purification and properties of pendimethalin nitroreductase from Bacillus circulans

A pendimethalin nitroreductase was purified from Bacillus circulans to 52-fold increase with a recovery of 21.1% and had a specific activity of 4.16 U/mg. The molecular weight was 27 kDa. The optimum pH and temperature were 7.5 and 35°C, respectively. Fe2+, Ca2+, Mg2+ and Mn2+ did not cause any effect on the enzyme activity, whereas Ag+, Hg2+ significantly inhibited it. The pendimethalin nitroreductase from B. circulans required NADPH as cofactor. It was not affected by GSH, DTT, L-Cys and β-mercaptoethanol but inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide, and iodoacetamide. EDTA, 1,10-phenanthroline and α,α′-dipyridyl had a moderate effect on the enzyme activity. The nitroreductase reduced all the tested nitroaromatic compounds but was more active towards pendimethalin and trifluralin. The KM and Vmax for the enzyme reaction using pendimethalin as a substrate were 4.35 μM and 620 μmoles mg−1 min−1, respectively. The broad substrate specificity towards dinitroaniline herbicides suggested the enzyme to be used as a potent reducing agent of these toxic compounds. This is the first report for the purification and characterization of pendimethalin nitroreductase from any bacterial or fungal species.