By far the largest proportion of the Earth's biosphere is comprised of organisms that thrive in cold environments (psychrophiles). Their ability to proliferate in the cold is predicated on a capacity to synthesize cold-adapted enzymes. These enzymes have evolved a range of structural features that confer a high level of flexibility compared to thermostable homologs. High flexibility, particularly around the active site, is translated into low-activation enthalpy, low-substrate affinity, and high specific activity at low temperatures. High flexibility is also accompanied by a trade-off in stability, resulting in heat lability and, in the few cases studied, cold lability. This review addresses the structure, function, and stability of cold-adapted enzymes, highlighting the challenges for immediate and future consideration. Because of the unique properties of cold-adapted enzymes, they are not only an important focus in extremophile biology, but also represent a valuable model for fundamental researc...

Cold-adapted enzymes.

The main purpose of this study is to reduce the production cost of cellulase by optimizing the production medium and using an alternative carbon source such as municipal solid waste residue. In the present investigation, we aim to isolate the two novel cellulase producing fungi (Aspergillus niger and Trichoderma sp.) from municipal solid waste. Municipal solid waste residue (4-5% (w/v)) and peptone and yeast extract (1.0% (w/v)) were found to be the best combination of carbon and nitrogen sources for the production of cellulase by A. niger and Trichoderma sp. Optimum temperature and pH of the medium for the cellulase production by A. niger were 40°C and 6-7, whereas those for the production of cellulase by Trichoderma sp. were 45°C and 6.5. Cellulase production from A. niger and Trichoderma sp. can be an advantage as the enzyme production rate is normally higher as compared to other fungi.

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Optimization for the production of cellulase enzyme from municipal solid waste residue by two novel cellulolytic fungi.

3,5-Dinitrosalicylic acid (DNS) reagent is widely used in the estimation of reducing sugars. The reagent shows a differential behaviour towards mono- and di-saccharides. This phenomenon has been misinterpreted in the literature. Contrary to the facts, it has been reported that the DNS test is less sensitive for the estimation of cellobiose than it is for the estimation of glucose. This communication clarifies the concept. In addition, the study also compares the reaction of different mono- and di-saccharides and discusses the difference in their reactivity.

Differential behaviour of the dinitrosalicylic acid (DNS) reagent towards mono- and di-saccharide sugars

Glucoamylase is one of the oldest and widely used biocatalysts in food industry. The major application of glucoamylase is the saccharification of partially processed starch/dextrin to glucose, which is an essential substrate for numerous fermentation processes and a range of food and beverage industries. Glucoamylase for commercial purposes has traditionally been produced employing filamentous fungi, although a diverse group of microorganisms is reported to produce glucoamylase, since they secrete large quantities of the enzyme extracellularly. The commercially used fungal glucoamylases have certain limitations such as moderate thermostability, acidic pH requirement, and slow catalytic activity that increase the process cost. Consequently, the search for newer glucoamylases and protein engineering to improve pH and temperature optima leading to amelioration in catalytic efficiency of existing enzymes have been the major areas of research over the years. The present review focuses attention on the recent advances in molecular biology and protein engineering of glucoamylase to improve its production and functional properties including the so far success achieved in isolating mutants with enhanced thermostability and selectivity, higher pH optimum and improved catalytic activity. A comprehensive account is included on the diversity, regulation of production, classification, purification and properties, and potential applications of microbial glucoamylases to provide an overview on all the important aspects of the enzyme.

Microbial glucoamylases: characteristics and applications

Municipal solid waste contains high amounts of cellulose, which is an ideal organic waste for the growth of most of microorganism as well as composting by potential microbes. In the present study, Congo red test was performed for screening of microorganism, and, after selecting a potential strains, it was further used for biodegradation of organic municipal solid waste. Forty nine out of the 250 different microbes tested (165 belong to fungi and 85 to bacteria) produced cellulase enzyme and among these Trichoderma viride was found to be a potential strain in the secondary screening. During the biodegradation of organic waste, after 60 days, the average weight losses were 20.10% in the plates and 33.35% in the piles. There was an increase in pH until 20 days. pH however, stabilized after 30 days in the piles. Temperature also stabilized as the composting process progressed in the piles. The high temperature continued until 30 days of decomposition, after which the temperature dropped to C and below during the maturation. Good quality compost was obtained in 60 days.

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Diversity of cellulolytic microbes and the biodegradation of municipal solid waste by a potential strain.

Carboxyl group modification significantly altered the kinetic properties of purified carboxymethylcellulase from Aspergillus niger.

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Optimization of Media for Enhanced Glucoamylase Production in Solid-State Fermentation by Fusarium solani

Purification and characterization of a novel glucoamylase from Fusarium solani

The carboxyl groups of purified carboxymethylcellulase (CMCase) from Aspergillus niger NIAB280 were modified by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) in the presence of glycinamide for 15 min (GAM15) and glycinamide plus cellobiose for 75 min (GAM75). The half-lives of GAM15 at different temperatures were significantly enhanced whereas those of GAM75 were reduced as compared with the native CMCase. The activation energies of denaturation of native, GAM15 and GAM75 were 40, 35 and 59kJ mol respectively. Native CMCase and GAM15 showed no compensation effect, whereas native and GAM75 gave temperature of compensation of 44iC. Gibb's free energy of activation for denaturation (DG*) of GAM15 was increased as compared with native CMCase. Surprisingly the entropies (DS*) of activation for denaturation were negative for native and GAM75 and decreased further for GAM15 between the temperature range of 45 to 65iC. A possible explanation for the thermal inactivation of native and increased thermal stability of GAM15 is also discussed.

Thermostabilization of carboxymethylcellulase from Aspergillus niger by carboxyl group modification

A simple, sensitive, accurate and more informative assay for determining the number of modified groups during the course of carboxyl group modification is described. Monomeric carboxymethylcellulase (CMCase) from Aspergillus niger was modified by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of glycinamide. The different time-course aliquots were subjected to non-denaturing PAGE and the gel stained for CMCase activity. The number of carboxyl groups modified are directly read from the ladder of the enzyme bands developed at given time. This method showed that after 75 min of modification reaction there were five major species of modified CMCases in which 6 to 10 carboxyls were modified.

Mohammad Hamid Rashid

Papers

Carboxyl group modification significantly altered the kinetic properties of purified carboxymethylcellulase from Aspergillus niger.

Optimization of Media for Enhanced Glucoamylase Production in Solid-State Fermentation by Fusarium solani

Purification and characterization of a novel glucoamylase from Fusarium solani

Thermostabilization of carboxymethylcellulase from Aspergillus niger by carboxyl group modification

Native Enzyme Mobility Shift Assay (NEMSA): a new method for monitoring carboxyl group modification of carboxymethylcellulase from Aspergillus niger