IIT Madras is a public technical and research university located in Chennai, Tamil Nadu. Founded in 1959, it is recognised as an Institute of National Importance.

IIT Madras has been ranked as the top engineering institute in India for four years in a row by the National Institutional Ranking Framework of the MHRD

It currently offers undergraduate, postgraduate and research degrees across 16 disciplines in Engineering, Sciences, Humanities and Management. About 596 faculty belonging to science and engineering departments and centres of the Institute are engaged in teaching, research and industrial consultancy.

IIT Madras

Department Of Biotechnology

Department Of Chemical Engineering

Bioprocess Engineering

Behaviour of pectinases under process conditions

Process Biochemistry

Multiresponse analysis of microbiological parameters affecting the production of pectolytic enzymes: a statistical view

The reaction conditions were optimized for enzymatic hydrolysis of pectic substances using pectolytic enzymes produced by Aspergillus niger NCIM 548. Response surface methodology was used to optimize the reaction parameters and the central composite design was employed for this purpose. Pectin was used as the substrate for polymethylgalacturonase and pectinlyase, whereas polygalacturonic acid was the substrate for polygalacturonase. The volume ratio of reactants for maximum hydrolysis was determined under the assay conditions. The optimum amount of substrate and enzyme were found to be 0.2 cm3 and 0.074 cm3 for polymethylgalacturonase, 0.4 cm3 and 0.086 cm3 for polygalacturonase, and 0.63 cm3 and 0.7 cm3 for pectinlyase. The pH and temperature optima were found to be 5.3 and 30°C for polymethylgalacturonase, 6.6 and 26°C for polygalacturonase, and 4.8 and 35°C for pectinlyase. Under optimized conditions the enzyme activities were higher when compared to unoptimized conditions. Copyright (C) 1999 Elsevier Science Inc.The reaction conditions were optimized for enzymatic hydrolysis of pectic substances using pectolytic enzymes produced by Aspergillus niger NCIM 548. Response surface methodology was used to optimize the reaction parameters and the central composite design was employed for this purpose. Pectin was used as the substrate for polymethylgalacturonase and pectinlyase, whereas polygalacturonic acid was the substrate for polygalacturonase. The volume ratio of reactants for maximum hydrolysis was determined under the assay conditions. The optimum amount of substrate and enzyme were found to be 0.2 cm3 and 0.074 cm3 for polymethylgalacturonase, 0.4 cm3 and 0.086 cm3 for polygalacturonase, and 0.63 cm3 and 0.7 cm3 for pectinlyase. The pH and temperature optima were found to be 5.3 and 30°C for polymethylgalacturonase, 6.6 and 26°C for polygalacturonase, and 4.8 and 35°C for pectinlyase. Under optimized conditions the enzyme activities were higher when compared to unoptimized conditions.

GENERAL

Enzyme and Microbial Technology

Performance of pectolytic enzymes during hydrolysis of pectic substances under assay conditions: A statistical approach

Chitin, a homopolymer of N-acetylglucosamine, is obtained from a variety of sources. They form the structural component of fungal cell wall and plants. They are commercially obtained from shrimp and crab shell waste from the fishing industry. Recent advances in understanding the structure and properties of chitin and its derivatives has opened a lot of new avenues for its applications. Improvements in the properties of chitin for a particular application can be easily brought about by chemical modifications. The applicability of chitin in many areas and its easy manipulation has resulted in a considerable amount of research being done on the possible applications of chitinase.

Studies on applications of chitin and its derivatives

Enzymes are deactivated by different ways to an inactive state, which is a major constraint in the development of biotechnological processes. Understanding the complex process of enzyme deactivation will be helpful in enhancing the feasibility of many biological processes. This paper mainly deals with the different ways by which enzymes are inactivated, which includes the role of thermodynamics in deactivation. Different models namely, unified deactivation theory, single exponential model, rapid equilibrium model, isozyme model and bacterial contamination model used to describe the complex deactivation processes are also discussed in this communication. The complete understanding of deactivation process is very essential in commercialization because enzyme activity and stability of the enzyme play a critical role in economics of biotechnological processes. Activity and stability of the enzyme are conflicting properties and trade-off between these factors must be considered in the selection and design of enzymes.

Enhancing the feasibility of many biotechnological processes through enzyme deactivation studies

Response surface methodology was applied for the simultaneous optimization of initial pH and temperature of fermentation for the production of tartaric acid by Gluconobacter suboxydans. A temperature of 32.8 °C and an initial pH of 6.05 was found to be most suitable. Kinetic analysis for the growth of Gluconobacter suboxydans and product formation was done and a logistic model was used to describe the growth of Gluconobacter suboxydans while the Leudeking-Piret model explained the product formation. Parameters of the model were evaluated and tartaric acid formation was found to be non- growth associated.

Optimization of temperature and initial pH and kinetic analysis of tartaric acid production by Gluconobacter suboxydans

The effect of initial glucose concentration on β-1, 3-glucanase production by Trichoderma harzianum NCIM 1185 and cell growth was studied in a batch stirred tank bioreactor. The initial glucose level was varied between 5 g/dm3 and 100 g/dm3. A maximum β-1,3-glucanase production of 0.820 U was obtained using the fully optimized medium which had an initial glucose level of 8.63 g/dm3 and thereafter there was a steady decrease in β-1,3-glucanase production. No β-1,3-glucanase production was observed beyond initial glucose concentrations of 40 g/dm3 which suggests a possible catabolic repression on the enzyme synthesis. The inhibitory effect of increased initial glucose concentrations on cell growth has been studied and the Luong and the Han-Levenspiel models were used to explain the mechanism of inhibition.

Effect of glucose level on the batch production of β-1,3-glucanase by Trichoderma harzianum and cell growth

Hydrolysis of pectic substances by pectinases were carried out under process conditions. Polymethylgalacturonase, polygalacturonase and pectinlyase are three enzymes studies in this regard. Pectin is used as the substrate for polymethylgalacturonase and pectinlyase whereas polygalacturonic acid was used as the substrate for polygalacturonase. The initial concentration was varied between 1.0 and 5.0 kg/m3 for polymethylgalacturonase and polygalacturonase and between 4.0 and 8.0 kg/m3 for pectinylase. Hydrolysis experiments were carried out by varying initial substrate to enzyme ratio and reducing groups formed are measured. The behaviour of pectinases during hydrolysis are studied from the plot of (reducing groups formed)/initial substrate vs. initial substrate to enzyme ratio. This study is helpful in predicting the amount of substrate and enzyme required to produce the required amount of reducing groups within a stipulated time.

Behaviour of pectinases under process conditions

A range of chitinase genes from microorganisms have been cloned and the potential uses of these genetically manipulated organisms are being investigated by various researchers. Fungi and yeast are better producers of chitinase than bacteria. Since fungi grow at a slower rate, there have been efforts to clone the fungal chitinase genes into fast-growing bacteria. This review gives a brief survey of recent progress in the regulation and cloning of microbial chitinase genes. Emphasis is placed on the post-translational modification and localization of the recombinant protein in the host. Various amino acid domains are present in this protein. The mode of catalytic activity of the recombinant protein in comparison to the wild-type protein is discussed in the available literature. The different mechanisms involved in the regulation of chitinase genes from various microorganisms is discussed by the researchers. The scope of future research and conclusions yet to be obtained in this particular area are also outlined in this review.

Applied Microbiology and Biotechnology

Regulation and cloning of microbial chitinase genes

Human placenta is a viable source of many useful products. Some of the products obtained are of use in health care industry. This review article deals with cosmetics, pharmaceuticals, blood products and hormone like substances obtained from human placenta. Various methods of their extraction, purification, characterization and their method of preparation have been discussed in detail.

Useful products from human placenta

A novel optimization procedure has been developed to determine the best combination of variables in large scale fermentation systems. The self- directing optimization or the rotating simplex method of optimization was employed to determine the best suitable combination of parameters, pH (controlled), aeration rate and agitation rate for maximal production of chitinase by Trichoderma harzianum in batch culture. Extracellular enzyme activity increased from an initial value of 0.109 U under unoptimized conditions to a final value of 0.384 U after optimization. A controlled pH of 4.9, an aeration rate of 1.51 l-1 per min, and an agitation rate of 224 rpm was found to be the best combination.

Self-directing optimization of parameters for extracellular chitinase production by Trichoderma harzianum in batch mode

The effect of microbiological parameters, Viz., slant age, inoculum age and amount of inoculum were studied using response surface methodology for the production of pectolytic enzymes, polymethylgalacturonase, polygalacturonase and pectinlyase. The central composite design was used to determine the individual optimum values. Individual optimum values of slant age, inoculum age and amount of inoculum were 49.5 h, 52.3 h and 11.2%, respectively, for polymethylgalacturonase; 62.4 h, 60.5 h and 14.4%, respectively, for polygalacturonase, and 78.3 h, 52.5 h and 1314%, respectively, for pectinlyase. In order to develop a two-stage inoculum possessing the best conditions for the maximum production of all three enzymes, a multiresponse analysis was carried out using a generalized distance approach. The microbiological parameters were optimized simultaneously for maximum production of enzymes. The simultaneous optimal values of slant age, inoculum age and amount of inoculum were 81.4 h, 66.7 h and 15.8%, respectively, for all the three enzymes. After optimization, the fermentation time was reduced from 144 to 120 h.

Multiresponse analysis of microbiological parameters affecting the production of pectolytic enzymes by Aspergillus niger: A statistical view

Production of pectolytic enzymes - a review

The combined effect of pH and temperature on carboxymethyl cellulase from two intergeneric fusants (M 14 and M 62) of Trichoderma reesei QM 9414/Saccharomyces cerevisiae NCIM 3288 was studied using response surface methodology. A central composite design for two variables was employed for the optimization studies. This study was compared with similar studies carried out with Trichoderma reesei QM 9414. The optimal pH and temperature for the enzymes derived from these organisms were: for the fusant M 14 - pH 5.7 and 41.7°C, for the fusant M 62 - pH 5.3 and 43°C, and for Trichoderma reesei QM 9414 - pH 4.31 and 38.3°C.

Journal of Industrial Microbiology and Biotechnology

pH and thermal stability studies of carboxymethyl cellulase from intergeneric fusants of Trichoderma reesei/Saccharomyces cerevisiae

Pectolytic enzymes, viz., polymethylgalacturonase, polygalacturonase and pectinlyase were synthesised by Aspergillus niger NCIM 548 using submerged cultivation. The combined effect of pH and temperature on these enzymes was studied using response surface methodology. A central composite design for two variables was employed to study the combined effect of pH and temperature on stability. The optimum pH and temperature were found to be 2.23 and 23°C respectively for polymethylgalacturonase; 4.8 and 28°C respectively for polygalacturonase and 3.9 and 29°C respectively for pectinlyase. It has also been found that pectinlyase is more thermostable than other two enzymes. © 1998 Elsevier Science S.A. All rights reserved.

Biochemical Engineering Journal

Application of response surface methodology to evaluate some aspects on stability of pectolytic enzymes from Aspergillus niger

Studies on the constitutive β-1,3-glucanase were conducted in submerged as well as in the stationary culture conditions, in the presence and in the absence of lactose and glucose as main carbon sources. In the absence of lactose or glucose, expression of β-1,3-glucanase was observed at 96 h in extracellular, periplasmic, cell wall bound and internal fractions during submerged fermentation. In shake flask culture, enzyme was found in all subcellular fractions using optimal glucose concentration. When Trichoderma harzianum was grown on media containing 55 kg lactose/m3 in submerged culture, activity was found in extracellular, cell wall bound and in the periplasmic fractions. The relative distribution of the enzyme in the cell is independent of the nature of the carbon source and its concentration.

Localization of β-1,3-glucanase in Trichoderma harzianum

The combined effect of sorbitol and yeast extract of the medium on tartaric acid production by Gluconobacter-suboxydans, NCIM 2049, was studied in batch fermentation while keeping the temperature (30 °C) and pH (6.2) constant. Response surface methodology was used to obtain quadratic models for the production of tartaric acid. The multiple coefficients of regression between 0.8945 and 0.9820 was obtained during the process. The optimum medium composition comprising 20 kg/m3 sorbitol and 2 kg/m3 yeast extract was verified experimentally by observing the variation of cell mass and tartaric acid production with time.

Optimization of medium composition by response surface methodology for the production of tartaric acid by Gluconobacter suboxydans

In order to convert cellulosic material to ethanol by single step process a chemofusion method has been followed between protoplasts of Trichoderma reesei, QM 9414, and the spheroplasts of Saccharomyces cerevisiae, NCIM 3288, in the author's laboratory. The fusion was a success and it was observed that endoglucanase was the key enzyme in the success of the fusion. In the present study, characterization of the fusants based on the endoglucanase synthesis, its localization and the distribution in the cells are described and compared with that of Trichoderma reesei, QM 9414, (wild type).

Localization of carboxymethyl cellulase in the intergeneric fusants of trichoderma reesei QM 9414 and Saccharomyces cerevisiae NCIM 3288

Pectolytic enzymes play an important role in food processing industries and alcoholic beverage industries. These enzymes degrade pectin and reduce the viscosity of the solution so that it can be handled easily. These enzymes are mainly synthesized by plants and microorganisms. Aspergillus niger is used for industrial production of pectolytic enzymes. This fungus produces polygalacturonase, polymethylgalacturonase and pectinlyase. This review mainly concerns with the production of pectolytic enzymes using different carbon sources. It also deals with the effect of operating parameters such as temperature, aeration rate, agitation and type of fermentation on the production of these enzymes.

Production of pectolytic enzymes - A review

The requirement of citric acid is increasing every year. Therefore, it is important to have an efficient recovery method following the production of citric acid. At present, most of the manufacturers use the classical method of citric acid recovery which is a precipitation technique using calcium salt followed by filtration and subsequently treated with sulphuric acid. This review focuses on the new developments of recovery of citric acid from the fermentation broth.