Periasamy Anbu

Purification, characterization and crystallization of an extracellular alkaline protease from Aspergillus nidulans HA-10

Aspergillus nidulans is a highly potent fungus used in the production of alkaline protease. Extracellular alkaline protease was purified from A. nidulans in a two‐step procedure involving ammonium sulphate precipitation and Sephadex G‐100 column chromatography. The molecular mass of the enzyme was determined to be 42 kDa by SDS‐PAGE. The enzyme activity was also analyzed by zymogram with gelatin. The enzyme was more stable over a wide range of pH (6–10) and the temperatures up to 50 °C. It showed optimum enzyme activity at pH 8.0 and a temperature of 35 °C. The protease enzyme was completely inhibited by the serine protease inhibitor of phenylmethylsulfonyl fluoride (PMSF). The crystallization of the purified enzyme was performed by hanging drop vapour diffusion method using PEG 6000 as the precipitant. The micro crystals occurred in 40% of PEG 6000. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Formations of calcium carbonate minerals by bacteria and its multiple applications

Abstract Biomineralization is a naturally occurring process in living organisms. In this review, we discuss microbially induced calcium carbonate precipitation (MICP) in detail. In the MICP process, urease plays a major role in urea hydrolysis by a wide variety of microorganisms capable of producing high levels of urease. We also elaborate on the different polymorphs and the role of calcium in the formation of calcite crystal structures using various calcium sources. Additionally, the environmental factors affecting the production of urease and carbonate precipitation are discussed. This MICP is a promising, eco-friendly alternative approach to conventional and current remediation technologies to solve environmental problems in multidisciplinary fields. Multiple applications of MICP such as removal of heavy metals and radionuclides, improve the quality of construction materials and sequestration of atmospheric CO2 are discussed. In addition, we discuss other applications such as removal of calcium ions, PCBs and use of filler in rubber and plastics and fluorescent particles in stationary ink and stationary markers. MICP technology has become an efficient aspect of multidisciplinary fields. This report not only highlights the major strengths of MICP, but also discusses the limitations to application of this technology on a commercial scale.

Strategies to characterize fungal lipases for applications in medicine and dairy industry.

Lipases are water-soluble enzymes that act on insoluble substrates and catalyze the hydrolysis of long-chain triglycerides. Lipases play a vital role in the food, detergent, chemical, and pharmaceutical industries. In the past, fungal lipases gained significant attention in the industries due to their substrate specificity and stability under varied chemical and physical conditions. Fungal enzymes are extracellular in nature, and they can be extracted easily, which significantly reduces the cost and makes this source preferable over bacteria. Soil contaminated with spillage from the products of oil and dairy harbors fungal species, which have the potential to secrete lipases to degrade fats and oils. Herein, the strategies involved in the characterization of fungal lipases, capable of degrading fatty substances, are narrated with a focus on further applications.

Microbial Enzymes and Their Applications in Industries and Medicine 2014.

Enzymes are considered as a potential biocatalyst for a large number of reactions. Particularly, the microbial enzymes have widespread uses in industries and medicine. The microbial enzymes are also more active and stable than plant and animal enzymes. In addition, the microorganisms represent an alternative source of enzymes because they can be cultured in large quantities in a short time by fermentation and owing to their biochemical diversity and susceptibility to gene manipulation. Industries are looking for new microbial strains in order to produce different enzymes to fulfil the current enzyme requirements. This special issue covers ten articles including three review articles, mainly highlighting the importance and applications of biotechnologically and industrially valuable microbial enzymes. M. Dinarvand et al. in their paper optimized the conditions for overproduction of intraextracellular inulinase and invertase from the fungus Aspergillus niger ATCC 20611. Optimization is one of the most important criteria in developing any new microbial process. Response surface analysis is one of the vital tools to determine the optimal process conditions. This kind of design of a limited set of variables is advantageous compared to the conventional method. The response surface methodology was used for this optimization and achieved the increment until 16 times. This study would be highly useful for the potential application in fermentation industries. In this review, N. Gurung et al. have made an attempt to highlight the importance of different enzymes with a special focus on amylase and lipase. Enzymes generally increase the reaction rates by several million times than normal chemical reactions. Lipases play an important role in the food, detergent, chemical, and pharmaceutical industries. In the past, microbial lipases gained significant attention in the industries due to their substrate specificity and stability under varied conditions. Amylase is an enzyme that catalyses the breakdown of starch into sugars, abundant in the process of animal and human digestion. The major advantage of microbial amylases is being economical and easy to manipulate. Currently, much attention is paid to rapid development of microbial enzyme technology, and these enzymes are relatively more stable than the enzymes derived from plants and animals. P. Mukherjee and P. Roy in their paper have purified and characterized the enzyme hydrocarbon dioxygenase from Stenotrophomonas maltophilia PM102, which has a broad substrate specificity. They found that the presence of copper induces the enzyme activity to be 10.3-fold higher, and NADH induces the increment to be 14.96-fold. Proposed copper enhanced monooxygenase activity and Fourier transform-infrared (FT-IR) characterization of biotransformation products from trichloroethylene satisfy the production of industrially and medically important chemicals and make bioremediation more attractive by improving the development of this technology. C. Huynen et al. in their review paper discuss the importance of protein scaffold to develop hybrid enzymes. The paper discusses the use of class A betalactamase as versatile scaffolds to design hybrid enzymes mentioned as betalactamase hybrid proteins (BHPs), in which an external polypeptide, peptide, protein, or their fragment is inserted at various suitable positions. The paper highlights further how BHPs can be specifically designed to develop as bifunctional proteins to produce and characterize the proteins otherwise difficult to express, to determine the epitope of specific antibodies, to generate antibodies against nonimmunogenic epitopes, and to understand the structure/function relationship of proteins. The hybrid proteins can be applied to produce difficult-to-express peptides/proteins/protein fragments, to map epitopes, to display antigens, and to study protein structure/function relationships. Among other applications, BHPs could be an important player in biosensors and in affinity chromatography, drug screening, and drug targeting. P. Manivasagan et al. in their paper focus on purification and characterization of the protease from Streptomyces sp. MAB18. The authors have optimized the conditions for overproduction of protease using response surface methodology. They have also determined the molecular mass of purified enzyme and great activity and stability of enzyme in different pH and temperatures. Furthermore, the authors confirmed that the protease has an antioxidant ability. In industries, the poultry waste derived protease will be useful as a protein or as an antioxidant. The paper titled “β-Glucosidases from the fungus Trichoderma: an effeicient cellulose machinery in biotechnological applications” is a detailed review on β-glucosidases which are members of the cellulose enzyme complex described by P. Tiwari et al. The authors especially focus on β-glucosidases from the fungus Trichoderma, mostly used for the saccharification of cellulosic biomass for biofuel production. They describe the enzyme family, their classification, structural parameters, properties, and studies at the genomics and proteomics levels. In addition, by bypassing the low enzyme production with hypersecretory strains, they give an insight on using these strains for renewable energy sources like bioethanol production. They imply the importance of fungal β-glucosidases which might be successful for biofuel production in order to meet the need in energy crisis. A. Khoramnia et al. in their paper discuss yeast enzyme application for medium chain fatty acids (MCFAs) modification for industrial purpose and antibacterial applications. The paper focuses on the conceptualization, design, and assay of the enzyme produced from a Malaysian strain of Geotrichum candidum. With the modification on fatty acid processing using a naturally derived enzyme, a free lauric acid rich MCFAs can be obtained which can become a source of antibacterial use for both Gram-positive (Staphylococcus aureus) and Gram-negative (E. coli) bacteria which are difficult microbes due to some of their strains becoming drug resistant. They also describe that the higher lipolysis by the strain specific enzyme is associated with the increased moisture content in the reaction environment on coconut oil hydrolysis. M. A. Hassan et al. in their paper discuss isolation of Bacillus amyloliquefaciencs and B. subtilis from soil and production and characterization of keratinolytic protease. These bacteria were able to degrade the wool completely within 5 days and also produced the highest enzyme activity. The characterization studies confirmed that the enzyme is stable in a broad range of pH and temperatures. Furthermore, they confirmed that the keratinolytic proteases from isolated bacteria are stable in various organic solvents. In this review article, S. C. B. Gopinath et al. put different strategies to characterize fungal lipases for their role in industry and medicine. The advantage of fungal lipases is bestowed with their extracellular nature of production thus reducing the complexities and high operation cost comparing to other bacterial enzymes. The authors provide several illustrations to show how lipolysis can be utilized and put strategies for the characterization of fungal lipases that are capable of degrading fatty substances from different sources, with an effort to highlight further applications. This review would contribute to the isolation and characterization of lipase from various fungal sources and application of lipase for medical and dairy industry and degradation of fatty substance from oil spillages. A. Knob et al. in their paper focus on xylanses and discuss the purification and characterization of a xylanase produced by Penicillium glabrum using brewers spent grain as a substrate in their paper. This study is the first report as the characterization of xylanase was carried out by using such an agroindustrial waste. Furthermore, the researchers also determined the molecular mass of the purified xylanase, the enzyme activity and stability on various pH and temperature ranges, the optimal enzyme production conditions, and the effect of some metal ions and inhibitors on xylanase activity. The authors concluded that the use of substrate brewers spent grain for xylanase production not only decreased the amount of this waste but also reduced the xylanase production cost as desired in biotechnological processes. Periasamy Anbu Subash C. B. Gopinath Arzu Coleri Cihan Bidur Prasad Chaulagain

Biotechnological Aspects and Perspective of Microbial Keratinase Production

Keratinases are proteolytic enzymes predominantly active when keratin substrates are available that attack disulfide bridges in the keratin to convert them from complex to simplified forms. Keratinases are essential in preparation of animal nutrients, protein supplements, leather manufacture, textile processing, detergent formulation, feather meal processing for feed and fertilizer, the pharmaceutical and biomedical industries, and waste management. Accordingly, it is necessary to develop a method for continuous production of keratinase from reliable sources that can be easily managed. Microbial keratinase is less expensive than conventionally produced keratinase and can be obtained from fungi, bacteria, and actinomycetes. In this overview, the expansion of information about microbial keratinases and important considerations in keratinase production are discussed.

Biopolymers Regulate Silver Nanoparticle under Microwave Irradiation for Effective Antibacterial and Antibiofilm Activities.

In the current study, facile synthesis of carboxymethyl cellulose (CMC) and sodium alginate capped silver nanoparticles (AgNPs) was examined using microwave radiation and aniline as a reducing agent. The biopolymer matrix embedded nanoparticles were synthesized under various experimental conditions using different concentrations of biopolymer (0.5, 1, 1.5, 2%), volumes of reducing agent (50, 100, 150 μL), and duration of heat treatment (30 s to 240 s). The synthesized nanoparticles were analyzed by scanning electron microscopy, UV-Vis spectroscopy, X-ray diffraction, and Fourier transform infrared spectroscopy for identification of AgNPs synthesis, crystal nature, shape, size, and type of capping action. In addition, the significant antibacterial efficacy and antibiofilm activity of biopolymer capped AgNPs were demonstrated against different bacterial strains, Staphylococcus aureus MTCC 740 and Escherichia coli MTCC 9492. These results confirmed the potential for production of biopolymer capped AgNPs grown under microwave irradiation, which can be used for industrial and biomedical applications.

Characterization of solvent stable extracellular protease from Bacillus koreensis (BK-P21A)

A total of 18 protease producing bacterial strains were isolated from detergent effluent in South Korea using skim milk agar medium. A strain (BK-P21A) was selected and identified as Bacillus koreensis based on morphological, biochemical and molecular characterizations (16S rRNA gene sequence analysis). Optimized culture conditions for the production of protease were pH 8.5, 30 °C, sucrose (2%) and yeast extract (0.2%) during 36 h of incubation. Furthermore, the protease was partially purified by ammonium sulphate precipitation (80%) and again by Superdex 200 10/300 GL and Superdex 75 10/300 GL column chromatography, which resulted in 5.0 fold purification and a yield of 23%. The molecular mass of the protease was estimated to be 48 kDa by SDS-PAGE. The purified enzyme was further characterized and found to be most active at pH 9.0 and 60 °C. The activity of the purified protease was enhanced by CaCl₂ and CoCl₂, but inhibited by PMSF, which indicated it was a serine type protease. Moreover, the protease was moderately stable in surfactants and 81% stable in H₂O₂. Finally, the enzyme was more active and stable (94-126.5%) in various hydrophilic organic solvents. Considering the stability of protease towards the alkaline pH, high temperature and organic solvents (50%), the enzyme from B. koreensis can be used as an alternative biocatalyst for several industrial applications mainly for peptide synthesis in nonaqueous solvents.

The effects of culture condition on the growth property and docosahexaenoic acid production from Thraustochytrium aureum ATCC 34304

The effect of the composition of artificial sea water (ASW) medium on the growth properties and docosahexaenoic acid (DHA) production from Thraustochytrium aureum ATCC 34304 was investigated. A maximum dry cell weight (DCW) of 3.71 g/l was obtained when the NaCl and MgSO4 concentration in the ASW media were 15 and 0 g/l, respectively. The maximum initial specific growth (ISG) rate of 0.16 was attained at 7.5 g/l NaCl and 18 g/l MgSO4, while the minimum ISG rate (0.02) was obtained at 5.0 g/l NaCl and 4.5 g/l MgSO4. The least doubling time required for biomass production was 4.3 h at 7.5 g/l NaCl and different MgSO4 concentrations. A maximum of 7.9 g/l DCW was obtained on the fourth day of cultivation at 30 g/l glucose and 2.5 g/l (each) yeast extract (YE) and peptone. The DHA content in the lipids was significantly affected by the concentration of glucose and nitrogen sources (YE and peptone) in the ASW medium. At the lowest glucose (10 g/l) and YE/peptone (0.5 g/l) concentration and highest glucose (30 g/l) and YE/peptone (2.5 g/l) concentration, the DHA content was 34.725 and 40.33%, respectively, relative to total lipid content. However, the DHA content in the lipid was not affected by the NaCl and MgSO4 concentration. At the lowest NaCl (2.5 g/l) and MgSO4 (4.5 g/l) concentration and highest NaCl (60 g/l) and MgSO4 (18 g/l) concentration the DHA content was 39.62 and 38.48%, respectively. The maximum DHA content in the lipid was 49.01% after four days of cultivation when 7.5 g/l NaCl and 4.5 g/l MgSO4 were in the ASW medium. The growth properties of T. aureum ATCC 34304 for biomass production and DHA yield in the lipid content were found to be affected by NaCl and glucose concentration.

Investigation of the physiological properties and synthesis of PUFAs from Thraustochytrids and its electrophoretic karyotypes

Physiological properties of organism, such as the number of chromosomes, genome size, fatty acid profile and the activities of desaturases and elongases were investigated for four differentThraustochytrium species. The investigation revealed thatThraustochytrium aureum could synthesize a significant level of polyunsaturated fatty acids (PUFAs), particularly docosa-hexaenoic acid (DHA), when compared to the other threeThraustochytrium species tested. A higher level of saturated fatty acids was observed byT. striatum followed byThraustochytrium sp. ATCC 26185. The PUFA accumulation rate was higher in the n-3 than in the n-6 pathway. A comparison of the activities for these desaturases and elongases of the four different species were also studied. Further, the electrophoretic karyotypes of Thraustochytrids were separated by pulsed-field gel electrophoresis (PFGE). The separation condition of PFGE was developed to obtain the different chromosomes from the variousThraustochytrium species. The number of chromosomes inT. aureum, T. striatum, Thraustochytrium sp. ATCC 20891 andThraustochytrium sp. ATCC 26185 were 13, 17, 10. 8, and the whole genome size of those species were estimated to be 12.9, 11.7, 11.3 and 9.93 Mbp, respectively.

Production of alkaline protease from a newly isolated Exiguobacterium profundum BK-P23 evaluated using the response surface methodology

Protease producing bacteria were isolated from soil in South Korea. These bacteria were screened in skim milk agar medium using skim milk as the substrate. The highest clear zone producing bacterial strain (BK-P23) was selected for further optimization studies. The strain was identified as Exiguobacterium profundum BK-P23 based on morphological, biochemical and molecular characterizations. The results of the 16S rRNA analysis showed that this strain was highly similar to E. profundum. The strain was able to grow under alkaline conditions at pH 8.5 and a temperature of 30°C. In the preliminary optimization experiments, five different parameters, i.e. carbon source (lactose), nitrogen source (corn steep solid), pH, temperature and incubation period were varied with the goal of optimizing enzyme production in low cost medium using the Box-Behnken design combined with response surface methodology. The optimal conditions were determined to be pH 9.0, a temperature of 30°C, lactose (1.0%) as the carbon source and corn steep solid (1.0%) as the cheap additional nitrogen source. In addition, 24 h of incubation was shown to produce the highest protease yield. Overall, the amount of enzyme produced was significantly higher in the optimized medium when compared with the original medium.