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.

T Panda

Department Of Chemical Engineering

G. Sathya Narayana Naidu

Chemical activation

entropy

pH effects

Purification

Rate constants

Reaction kinetics

PECTOLYTIC ENZYMES

Enzymes

fungal enzyme

PECTIN LYASE

POLYGALACTURONASE

POLYMETHYLGALACTURONASE

unclassified drug

article

Aspergillus niger

enzyme inactivation

Enzyme kinetics

Enzyme Stability

half life time

heating

nonhuman

priority journal

temperature dependence

Thermodynamics

Aspergillus

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

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

Biochemical Engineering Journal

Xylose reductase (XR) is a biocatalyst that converts xylose to xylitol and it has a potential application in the enzyme based production of xylitol from lignocellulosic hydrolysates. However, the development of a successful XR based bioprocess for xylitol production is challenging, due to the presence of inhibitory lignocellulose derived by-products (LDBs) in hydrolysates. Though various methods have been developed to mitigate the toxic effects of LDBs, none of them were promising. One of the reasons for this, could be the lack of knowledge on the enzyme inhibition mechanisms. Therefore, for the first time, here we investigated mechanisms of XR inhibition by major LDBs using XR from Debaryomyces nepalensis. We found that phenol showed competitive inhibition of XR whereas gallic acid, vanillin, furfural, 5-hydroxymethylfurfural (HMF) and acetate exhibited mixed inhibition. The inhibitory constants (KI) of vanillin, phenol, gallic acid, furfural, HMF and acetate were found to be 0.1, 11, 32, 54, 45 and 100 mM respectively. Moreover, the enzyme stability was drastically affected in the presence of phenols. In addition, molecular docking simulations performed using AutoDock 4.2.6 program revealed the putative binding sites of LDBs on XR and corroborated the experimental data. © 2017 Elsevier B.V.

Inhibition of Debaryomyces nepalensis xylose reductase by lignocellulose derived by-products

Aldose reductase (AR) catalyzes the conversion of aldoses to their corresponding polyols in yeasts and filamentous fungi. ARs have the potential to be exploited for the enzymatic production of xylitol, thus the identification and characterization of ARs from novel strains have gained interest. In this study, we chose the novel yeast Debaryomyces nepalensis as a source for an AR gene. For the first time, here we isolated the AR gene from D. nepalensis (DnAR) that encodes a protein of 320 amino acids with a predicted molecular weight of 36.7 kDa using the RACE technique. It was heterologously expressed in Escherichia coli as a His-tagged fusion protein and purified. The enzyme showed strict NADPH dependence and broad substrate specificity with high catalytic efficiency for arabinose, xylose and 3-nitro benzaldehyde. Remarkably, it was active and stable in the presence of high concentrations of salts (KCl/NaCl), thus exhibiting halotolerance. It showed 75% and 45% activity at 0.5 and 1 M concentration of salts respectively. Enzyme half-lifetime at 1 M KCl and 1 M NaCl was found to be 30 h and 16.5 h respectively. Furthermore, to explore the structural basis of its halotolerance, we built a homology model of DnAR. Surprisingly, we found that the existence of a uniform negative electrostatic potential over the protein surface, which is one of the known mechanisms governing protein halotolerance. Therefore, DnAR could be exploited as a biocatalyst to develop an enzyme based bioprocess for xylitol production from lignocelluloses. Moreover, this is the first report providing the genetic sequence and biochemical characteristics of a halotolerant aldose reductase. © The Royal Society of Chemistry.

Fulltext

RSC Advances

A halotolerant aldose reductase from Debaryomyces nepalensis: gene isolation, overexpression and biochemical characterization

Studies on laccase production by Daedalea flavida were carried out in static and low-speed shake cultures. The enzyme production was reduced drastically at a high speed of shaking. Optimal production conditions are necessary to assess the quality of laccase suitable for a specific application. Thus, the production of laccase was optimized by the application of response surface methodology. Laccase production was 8-fold and 7.5-fold more in static and low-speed shake conditions, respectively, in an optimal medium composition than in an unoptimized medium. Laccase obtained using the optimal culture medium of D. flavida was tested for its stability at different temperatures and pH conditions. The partially purified enzyme was most stable at 30°C and pH 5. The half-life of laccase is 87 min at 60°C and at pH 6. The kinetic and thermodynamic parameters were evaluated for the inactivation of the partially purified laccase. The entropy change of inactivation of the enzyme is least at pH 4. © 2015 Taylor and Francis Group, LLC.

Preparative Biochemistry and Biotechnology

Optimization of Laccase Fermentation and Evaluation of Kinetic and Thermodynamic Parameters of a Partially Purified Laccase Produced by Daedalea flavida

A Xylose reductase (XR) from the halotolerant yeast, Debaryomyces nepalensis NCYC 3413 was purified to apparent homogeneity. The enzyme has a molecular mass of 74. kDa with monomeric subunit of 36.4. kDa (MALDI-TOF/MS) and pI of 6.0. The enzyme exhibited its maximum activity at pH 7.0 and 45°C (21.2. U/mg). In situ gel digestion and peptide mass fingerprinting analysis showed 12-22% sequence homology with XR from other yeasts. Inhibition of the enzyme by DEPC (diethylpyrocarbonate) confirmed the presence of histidine residue in its active site. The enzyme exhibited high preference for pentoses over hexoses with greater catalytic efficiency for arabinose than xylose. The enzyme also showed absolute specificity with NADPH over NADH. The enzyme retained 90% activity with 100. mM of NaCl or KCl and 40% activity with 1. M KCl which suggest that the enzyme is moderately halotolerant and can be utilized for commercial production of xylitol under conditions where salts are present. © 2011 Elsevier Ltd.

Bioresource Technology

Purification and biochemical characterization of a moderately halotolerant NADPH dependent xylose reductase from Debaryomyces nepalensis NCYC 3413

Pectin and other pectic substances are complex polysaccharides, which contribute firmness and structure to plant tissues as a part of the middle lamella. The basic unit in pectic substances is galacturonan (α-D-galacturonic acid). Pectic substances are classified into two types; homogalacturonan and heterogalacturonan (rhamnogalacturonan). In homogalacturonan, the main polymer chain consists of α-D-galacturonate units linked by (1 → 4) glycosidic bonds, whereas in rhamnogalacturonan, the primary chain consist of (1→4) linked α-D-glacturonates and with about 24% L-rhamnose units that are β(1→2) and β(1→4) linked to D-galacturonate units (Whitaker, 1991). The side chains of rhamnogalacturonans usually consist of L-arabinose or D-galacturonic acid units. In plant tissues, about 6070% of the galacturonate units are esterified with methanol and occasionally with ethanol. Based on the degree of esterification, pectic substances are classified into protopectin, pectinic acid, pectin and polygalacturonic acid (Table 1). Molecular size, degree of esterification and weight distribution of polygalacturonic acid residues are important factors that contribute to heterogeneity in pectic substances. Relative molecular masses of pectic substances isolated from various sources such as citrus fruits, apple and plums, range from 25 to 350 kDa. Pectinases are a complex and diverse group of enzymes involved in the degradation of pectic substances. The diversity of forms of pectic substances in plant cells probably accounts for the existence of various forms of these enzymes. Pectinases are classified depending on their substrate and mode of enzymatic reaction (Fig. 1). Pectinases act as carbon recycling agents in nature by degrading pectic substances to saturated and unsaturated galacturonans, which are further catabolized (Table represented) to 5-keto-4-deoxy-uronate and finally to pyruvate and 3-phosphoglyceraldehyde (Vincent-Sealy et al., 1999). Pectinases from phytopathogenic fungi such as Aspergillus flavus, Fusarium oxysporum and Botrytis cinerea are also known to play a vital role in plant pathogenicity or virulence by degrading pectic compounds present in cell wall (Lang and Drenberg, 2000; DiPietro and Roncero, 1996; Ten Have et al., 1998). Pectinases, especially polygalacturonase, is known to play a major role in pectin breakdown during the final stages of fruit ripening (Sozzi- Quiroga and Fraschina, 1997; Chin et al., 1999). Polymethylgalacturonase (PMG), polygalacturonase (PG), pectin lyase (PL), polygalacturonate lyase (PGL) and pectinesterase (PME) are industrially important pectinases discussed in this chapter. © 2007 Springer.

Industrial Enzymes: Structure, Function and Applications

Structural and biochemical properties of pectinases

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.

Enzyme and Microbial Technology

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

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.

Bioprocess Engineering

Production of pectolytic enzymes - A review

The synthesis of pectinase using Aspergillus niger, NCIM 548, has been enhanced by optimizing the carbon and nitrogen sources present in the medium. Enzyme synthesis was about 40% more using the optimized medium than using the unoptimized medium. The study provided information on the appropriate carbon source as well as the optimum ratio of carbon to nitrogen, yielding the highest enzyme levels.

Statistical optimization of medium components for improved synthesis of pectinase by Aspergillus niger

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.

pH and thermal stability studies of chitinase from Trichoderma harzianum: A thermodynamic consideration

Applied Microbiology and Biotechnology

Microbial alkaline pectinases and their industrial applications: a review

Journal	Data powered by TypesetBiochemical Engineering Journal
Publisher	Data powered by TypesetElsevier
ISSN	1369703X
Open Access	No