The combined effect of pH and temperature on chitinase was investigated using response surface methodology. A central composite design for two variables was employed. The optimal pH and temperature for the least degree of deactivation were found out to be 5.4 and 24°C respectively. The deactivation rate constants and the half life of chitinase were estimated at different pH and temperature combinations. At the optimal pH of 5.4, the rate of the deactivation was found to be the least. Thermodynamic parameters, viz., ΔH*, ΔS*, ΔG* and activation energy of thermal deactivation of chitinase were calculated in the temperature range from 50°C to 60°C.

T Panda

Department Of Chemical Engineering

CHITINASE

article

bioengineering

controlled study

enzyme activity

enzyme analysis

Enzyme Stability

HYPHOMYCETES

nonhuman

priority journal

Temperature

Thermodynamics

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

Bioprocess Engineering

Pectolytic enzymes, viz., polymethylgalacturonase (PMG), polygalacturonase (PG), and pectinlyase (PL) were deactivated at various combinations of pH and temperature. The deactivation process was modelled as first-order kinetics and the deactivation rate constant was found to be minimum at pH 2.2 and 23 °C, respectively for PMG, 4.8 and 28 °C, respectively for PG and 3.9 and 29 °C, respectively for PL. Thermodynamic parameters like ΔG*, ΔH*, ΔS*, and activation energies were evaluated for crude and partially purified enzymes. It was found that entropy values are negative for all the three enzymes. The partially purified enzymes were found to be less stable than the crude enzymes. Hence, the interaction effect among the enzymes and also the effect of protein (BSA) on deactivation were studied. The addition of protein doubled the half-life times of partially purified PMG, PG I and PG II. The interaction effect of pectolytic enzymes on deactivation was found to be significant. © 2003 Elsevier Science B.V. All rights reserved.

Biochemical Engineering Journal

Studies on pH and thermal deactivation of pectolytic enzymes from Aspergillus niger

The critical parameters, such as slant age, mycelium age and contact time of mycelium with lytic enzymes which affects the formation of protoplast from the Penicillium griseofulvum mycelium were studied using a novel technique called response surface methodology. The design used to optimize these parameters was full factorial central composite design. The number of protoplasts, viability of protoplasts and griseofulvin yield are the three responses studied in this communication. The individual optimum values of slant age, inoculum age and contact time obtained are 187.1, 30.81, 21.4 h, respectively, for number of protoplast/ml 68.92, 33.01, 9.88 h, respectively, for percentage viability of protoplasts and 230.2, 73.6, 31.2 h, respectively, for griseofulvin. In order to obtain a single optimum value for all the three responses, a multi-response analysis was carried out using a generalized distance approach. The simultaneous optimum values of slant age, inoculum age and contact time are 133.78, 37.1 and 15.97 h, respectively, for all the three responses. © 2003 Elsevier Science B.V. All rights reserved.

Statistical analysis on some critical parameters affecting the formation of protoplasts from the mycelium of Penicillium griseofulvum

Stability studies and kinetics of β-1,3-glucanase from Trichoderma harzianum NCIM 1185 are reported. The critical parameters, pH and temperature, affecting the stability of β-1,3-glucanase were optimized using response surface methodology. The optimal combination of pH and temperature were 3.6 and 41 °C, respectively. The stability of β-1,3-glucanase was studied at various conditions of pH and temperature above the optimal conditions. Since the final activity was not zero, a non-first order deactivation model was used to explain the experimental data. Enzyme kinetic studies were carried out with and without inhibitors. Cu2+ behaved as a noncompetitive- uncompetitive mixed inhibitor, whereas EDTA and NH4+ behaved as competitive-noncompetitive type mixed inhibitors during the hydrolysis of β-1,3-glucan. © 2002 Elsevier Ltd. All rights reserved.

Process Biochemistry

Stability and kinetics of β-1,3-glucanse from Trichoderma harzianum

The most challenging and emerging field of biotechnology is the tailoring of proteins to attain the desired characteristic properties. In order to increase the stability of proteins and to study the function of proteins, the mechanism by which proteins fold and unfold should be known. It has been debated for a long time how exactly the linear form of a protein is converted into a stable 3-dimensional structure. The literature showed that many theories support the fact that protein folding is a thermodynamically controlled process. It is also possible to predict the mechanism of protein deactivation and stability to an extent from thermodynamic studies. This article reviewed various theories that have been proposed to explain the process of protein folding after its biosynthesis in ribosomes. The theories of the determination of the thermodynamic properties and the interpretation of thermodynamic data of protein stability are also discussed in this article. ©KSBB.

Biotechnology and Bioprocess Engineering

What is the role of thermodynamics on protein stability?

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

A kinetic analysis and optimization of reaction conditions for the enzymatic hydrolysis of chitin using chitinase produced by Trichoderma harzianum NCIM 1185 was carried out. Swollen chitin was used as the substrate for chitinase. The central composite design was followed for this optimization. The required volume ratio of the major reactants for maximum hydrolysis was determined. The pH and temperature optima were found to be 4.75 and 47 °C respectively. K(m) and V(max) for this enzyme were 4.643 kg/m3 and 0.1542 U respectively.

Parameters optimization of chitin hydrolysis by Trichoderma harzianum chitinase under assay conditions

Statistical design was used to determine the optimal levels of medium components, the optimal initial pH of the enzyme production medium, the temperature of fermentation, age of the organism in the slant growth and the age of the inoculum for the production of chitinase in shake flask fermentations. The use of high concentrations of chitin and ammonium sulphate and exclusion of peptone and urea from the medium resulted in the production of higher level of the enzyme. The optimal concentrations of the medium components were 12.5 kg/m3 and 4.2 kg/m3 for the chitin and ammonium sulphate respectively. The effect of the addition of peptone and urea to the optimized medium was studied. The optimal values of initial pH and temperature were 5.6 and 28 °C respectively. The optimal age of the slant and the inoculum were found to be 105 h and 43 h respectively. The highest level of chitinase before optimization of the above variables was 0.054 U which was maximized to the level of 0.197 U.

Optimization of carbon and nitrogen sources in the medium and environmental factors for enhanced production of chitinase by trichoderma harzianum

Efficient synthesis of a large number of viable protoplasts from Trichoderma longibrachiatum (Trichoderma reesei) mycelium has been found to be influenced by a number of factors. In this case, Trichoderma harzianum NCIM 1185 culture filtrate has been used to prepare protoplasts from Trichoderma longibrachiatum QM 9414. A method has been devised to isolate a large number of viable protoplasts from the mycelium. Detailed analysis of different factors affecting the synthesis of protoplasts from T. longibrachiatum has not yet been reported. The most suitable conditions for protoplasting were as follows: age of the organism in slant, 3 days; mycelium age, 20 h; volume of lytic enzymes, 190 ml; mycelial weight (dry equivalent), 1.66 g; time of contact with lytic enzymes, 2 h; temperature of protoplasting, 30°C; phosphate buffer, 25 mm, pH 6.5; KCl as osmotic stabilizer, 0.7 m. Proper spreading of the mycelium in contact with the lytic enzymes was also found to be necessary. © 1992.

Journal	Bioprocess Engineering
ISSN	0178515X
Open Access	No