Showing papers on "Enzyme assay published in 2022"

Selective Inhibition toward Dual Enzyme-like Activities of Iridium Nanozymes for a Specific Colorimetric Assay of Malathion without Enzymes.

Abstract: A colorimetric assay based on an enzyme-inhibition strategy is promising for the on-site detection of pesticide residues. Due to the high cost and low stability of enzymes, nanozymes (nanomaterials with enzyme-like activities) are widely developed as substitutes of enzymes. However, the inhibition of pesticides toward enzymes and nanozymes generally lacks selectivity. It is of great significance and challenge to design a specific pesticide assay based on an activity-inhibition strategy. Here, we discovered that iridium nanoparticles possess both peroxidase-like and oxidase-like activities under the same conditions, and their catalytic mechanisms are different. The synergistic effect of dual enzyme-like activities enhanced the colorimetric signal. Interestingly, the dual enzyme-mimicking activities could be simultaneously inhibited, and the inhibition effect exhibited high selectivity toward malathion. Considering the popularity and the hazards of malathion, a malathion assay method based on activity inhibition was established without enzymes and a redundant process. The synergistic effect of the selective inhibition of dual enzyme-like activities enhanced the selectivity and sensitivity. The proposed assay strategy opens up an avenue for specific assay of various pesticides.

Soil Enzyme Activity Response under the Amendment of Different Types of Biochar

Abstract: Biochar (BC) is a material that finds many applications in agriculture and environmental activities. The aim of the study was to define the influence of biochar produced from various organic materials: mellow compost (MC), stabilized municipal sewage sludge (MSS), pine sawdust (PS), sycamore sawdust (SS) and oak leaves (OL) on soil enzyme activity, as well as its relations with carbon and nitrogen content. After a 60-day incubation of soil and BC, the activity of dehydrogenases (DEH), catalase (CAT), alkaline (AlP) and acid (AcP) phosphatases was investigated. The basic parameters of soil were also determined: TOC, TN, DOM, pH in H2O, available phosphorus (AP). The highest AP content was obtained in the S + MSS, S + OL and S + MC variants. Enzyme activity was highest in soil with MSS BC, regardless of incubation time. After 60 days, the activity of soil enzymes was inhibited. The obtained results indicate that the response of enzymatic activity to biochar depends on the feedstock material and the incubation time. When using BC as an exogenous matter, it is necessary to determine the TOC/TN ratio. For the very wide range of this parameter, supplemental nitrogen fertilization or mixtures of different biochars should be applied.

Insights into Asparaginase from Endophytic Fungus Lasiodiplodia theobromae: Purification, Characterization and Antileukemic Activity

Abstract: Endobiotic fungi are considered as a reservoir of numerous active metabolites. Asparaginase is used as an antileukemic drug specially to treat acute lymphoblastic leukaemia. The presented study aims to optimize the media conditions, purify, characterize, and test the antileukemic activity of the asparaginase induced from Lasiodiplodia theobromae. The culture medium was optimized using an experiment designed by The Taguchi model with an activity ranging from 10 to 175 IU/mL. Asparaginase was induced with an activity of 315 IU/mL. Asparaginase was purified with a specific activity of 468.03 U/mg and total activity of 84.4 IU/mL. The purified asparaginase showed an approximate size of 70 kDa. The purified asparaginase showed an optimum temperature of 37 °C and an optimum pH of 6. SDS reduced the activity of asparaginase to 0.65 U/mL while the used ionic surfactants enhanced the enzyme activity up to 151.92 IU/mL. The purified asparaginase showed a Km of 9.37 µM and Vmax of 127.00 µM/mL/min. The purified asparaginase showed an IC50 of 35.2 ± 0.7 IU/mL with leukemic M-NFS-60 cell lines and CC50 of 79.4 ± 1.9 IU/mL with the normal WI-38 cell line. The presented study suggests the use of endophytic fungi as a sustainable source for metabolites such as asparaginase, provides an opportunity to develop a facile, eco-friendly, cost-effective, and rapid synthesis of antileukemic drugs, which have the potential to be used as alternative and reliable sources for potent anticancer agents.

Effects of Nitrogen and Phosphorus Addition on Soil Extracellular Enzyme Activity and Stoichiometry in Chinese Fir (Cunninghamia lanceolata) Forests

Abstract: Soil extracellular enzymes play an important role in microbial functions and soil nutrient cycling in the context of increasing N deposition globally. This is particularly important for Chinese fir (Cunninghamia lanceolata) forests because of the decline in soil fertility induced by successive rotation. In this study, we aimed to determine the effects of simulated N deposition (N30: 30 kg ha−2 year−1; N60: 60 kg ha−2 year−1) and phosphorus addition (P20: 20 mg kg−1; P40: 40 mg kg−1) on the activity and stoichiometry of soil extracellular enzymes related to soil C, N, and P cycling in Chinese fir. The results showed that N addition alone increased the activity of soil β-1,4 glucosidase (BG) but decreased the activity of N-acetyl-β-d-glucosidase (NAG) and leucine aminopeptidase (LAP). N addition increased the ratios of soil enzymes, C:N and C:P, alleviated microbial N-limitation, and aggravated microbial C-limitation. P addition alone increased enzyme activity, and P40 addition increased the ratio of BG to soil microbial biomass carbon (MBC), and (NAG + LAP):MBC activity ratio, thereby aggravating C restriction. N and P co-addition significantly affected soil extracellular enzyme activity and stoichiometry. For instance, BG activity and BG:MBC activity ratio increased significantly under the N30 + P40 treatment, which intensified C-limitation. Soil pH was the main factor influencing enzyme activity, and these variables were positively correlated. The stoichiometric relationships of enzyme reactions were coupled with soil pH, total nitrogen (TN), and available phosphorus (AP). Our results indicate that changes in soil characteristics induced by N and P inputs influence the activities of soil microorganisms and result in changes in microbial resource acquisition strategies. This study provides useful insights into the development of management strategies to improve the productivity of Chinese fir forests under scenarios of increasing N deposition.

The Stability Improvement of α-Amylase Enzyme from Aspergillus fumigatus by Immobilization on a Bentonite Matrix

Abstract: The stability of the α-amylase enzyme has been improved from Aspergillus fumigatus using the immobilization method on a bentonite matrix. Therefore, this study aims to obtain the higher stability of α-amylase enzyme from A. fumigatus; hence, it is used repeatedly to reduce industrial costs. The procedures involved enzyme production, isolation, partial purification, immobilization, and characterization. Furthermore, the soluble enzyme was immobilized using 0.1 M phosphate buffer of pH 7.5 on a bentonite matrix, after which it was characterized with the following parameters such as optimum temperature, Michaelis constant (KM), maximum velocity (Vmax), thermal inactivation rate constant (ki), half-life (t1/2), and the change of energy due to denaturation (ΔGi). The results showed that the soluble enzyme has an optimum temperature of 55°C, KM of 3.04 mg mL−1 substrate, Vmax of 10.90 μmole mL−1 min−1, ki of 0.0171 min−1, t1/2 of 40.53 min, and ΔGi of 104.47 kJ mole−1, while the immobilized enzyme has an optimum temperature of 70°C, KM of 8.31 mg mL−1 substrate, Vmax of 1.44 μmole mL−1 min−1, ki of 0.0060 min−1, t1/2 of 115.50 min, and ΔGi of 107.37 kJ mole−1. Considering the results, the immobilized enzyme retained 42% of its residual activity after six reuse cycles. Additionally, the stability improvement of the α-amylase enzyme by immobilization on a bentonite matrix, based on the increase in half-life, was three times greater than the soluble enzyme.

The Stimulation of Superoxide Dismutase Enzyme Activity and Its Relation with the Pyrenophora teres f. teres Infection in Different Barley Genotypes

Abstract: Changes in superoxide dismutase (SOD) enzyme activity were examined in infected barley seedlings of five cultivars with the goal to study the role of SOD in the defense mechanism induced by Pyrenophora teres f. teres (PTT) infection. Our results showed that although there were differences in the responses of the cultivars, all three PTT isolates (H-618, H-774, H-949) had significantly increased SOD activity in all examined barley varieties at the early stages of the infection. The lowest SOD activity was observed in the case of the most resistant cultivar. Our results did not show a clear connection between seedling resistance of genotypes and SOD enzyme activity; however, we were able to find strong significant correlations between the PTT infection scores on the Tekauz scale and the SOD activity. The measurement of the SOD activity could offer a novel perspective to detect the early stress responses induced by PTT. Our results suggest that the resistance of varieties cannot be estimated based on SOD enzyme activity alone, because many antioxidant enzymes play a role in fine-tuning the defense response, but SOD is an important member of this system.

Chemical Modification for the "off-/on" Regulation of Enzyme Activity.

Abstract: Enzymes with excellent catalytic performance play important roles in living organisms. Advances in strategies for enzyme chemical modification have enabled powerful strategies for exploring and manipulating enzyme functions and activities. Based on the development of chemical enzyme modifications, incorporating external stimuli-responsive features-for example, responsivity to light, voltage, magnetic force, pH, temperature, redox activity, and small molecules-into a target enzyme to turn "on" and "off" its activity has attracted much attention. The ability to precisely control enzyme activity using different approaches would greatly expand the chemical biology toolbox for clarification and detection of signal transduction and in vivo enzyme function and significantly promote enzyme-based disease therapy. This review summarizes the methods available for chemical enzyme modification mainly for the off-/on control of enzyme activity and particularly highlights the recent progress regarding the applications of this strategy. This article is protected by copyright. All rights reserved.

No thermal adaptation in soil extracellular enzymes across a temperate grassland region

Abstract: Soil organic matter decomposition rates are thermally adapted, in relation to the ambient mean annual temperature of the soil environment. This could be because of temperature sensitivity of soil extracellular enzymes involved in decomposition. The objective of this study was to determine if soil extracellular enzyme activity and its temperature sensitivity (measured as Q10) were related to the mean annual temperature across a grassland region. We collected surface soil (0-10 cm) from 57 temperate grassland sites along a latitudinal gradient in northern China. The Q10 of soil extracellular enzymes (β-1,4-glucosidase, β-1,4-N acetylglucosaminidase, leucine aminopeptidase and acid phosphatase) was measured at four assay temperatures (5 °C, 15 °C, 25 °C, and 35 °C). There was no relationship between the mean annual temperature and standardized extracellular enzyme activity when other influential factors (soil pH, substrates, texture) were controlled, implying no thermal adaption of extracellular enzyme activity. Soil edaphic factors like soil pH, substrates and texture explained more of the spatial variation in the standardized extracellular enzyme activity than the mean annual temperature. Although the Q10 of all four enzymes increased significantly with mean annual temperature, the data converged at the maximum activity, and the average Q10 values were 1.17 for β-1,4-glucosidase, 1.21 for β-1,4-N acetylglucosaminidase, 1.11 for leucine aminopeptidase and 1.08 for acid phosphatase. The positive correlation between the Q10 of extracellular enzyme activity and mean annual temperature is not consistent with the thermodynamic theory, which predicts higher Q10 at sites with lower mean annual temperature. This may be due to the fact that extracellular enzymes operate outside of biological cells and thus are not directly related to physiological process rates of the microbial biomass. This study contributes to our understanding of the thermal adaptation of soil extracellular enzymes, and show that grassland sites with lower mean annual temperature do not necessarily induce higher temperature sensitivity of extracellular enzymes.

Enzymatic hydrolysis of lignocellulosic biomass using a novel, thermotolerant recombinant xylosidase enzyme from Clostridium clariflavum: a potential addition for biofuel industry

Abstract: The present study describes the cloning, expression, purification and characterization of the xylosidase gene (1650 bp) from a thermophilic bacterium Clostridium clariflavum into E. coli BL21 (DE3) using the expression vector pET-21a(+) for utilization in biofuel production. The recombinant xylosidase enzyme was purified to homogeneity by heat treatment and immobilized metal ion affinity chromatography. SDS-PAGE determined that the molecular weight of purified xylosidase was 60 kDa. This purified recombinant xylosidase showed its maximum activity at a temperature of 37 °C and pH 6.0. The purified recombinant xylosidase enzyme remains stable up to 90 °C for 4 h and retained 54.6% relative activity as compared to the control. The presence of metal ions such as Ca2+ and Mg2+ showed a positive impact on xylosidase enzyme activity whereas Cu2+ and Hg2+ inhibit its activity. Organic solvents did not considerably affect the stability of the purified xylosidase enzyme while DMSO and SDS cause the inhibition of enzyme activity. Pretreatment experiments were run in triplicate for 72 h at 30 °C using 10% NaOH. Saccharification experiment was performed by using 1% substrate (pretreated plant biomass) in citrate phosphate buffer of pH 6.5 loaded with 150 U mL−1 of purified recombinant xylosidase enzyme along with ampicillin (10 μg mL−1). Subsequent incubation was carried out at 50 °C and 100 rpm in a shaking incubator for 24 h. Saccharification potential of the recombinant xylosidase enzyme was calculated against both pretreated and untreated sugarcane bagasse and wheat straw as 9.63% and 8.91% respectively. All these characteristics of the recombinant thermotolerant xylosidase enzyme recommended it as a potential candidate for biofuel industry.

Localization, purification, and characterization of a novel β-glucosidase from Hanseniaspora uvarum Yun268.

Abstract: β-Glucosidase is a key enzyme that hydrolyzes nonvolatile glycosylated precursors of aroma compounds and enhances the organoleptic quality of wines. In this study, a novel β-glucosidase from Hanseniaspora uvarum Yun268 was localized, purified, and characterized. Results indicated that β-glucosidase activity was mainly distributed within the cells. After purification via ammonium sulfate precipitation combined with chromatography, β-glucosidase specific activity increased 8.36 times, and the activity recovery was 56.90%. The enzyme had a molecular mass of 74.22 kDa. It has a Michaelis constant (Km ) of 0.65 mmol/L, and a maximum velocity (Vmax ) of 5.1 nmol/min under optimum conditions; and Km of 0.94 mmol/L, and Vmax of 2.8 nmol/min under typical winemaking conditions. It exhibited the highest activity at 50°C and pH 5.0 and was stable at a temperature range of 20-80°C and pH range of 3.0-8.0. The enzyme has good tolerance to Fe3+ , especially maintaining 93.68% of its activity with 10 mmol/L of Fe3+ . Ethanol (<20%) and glucose (<150 g/L) inhibited its activity only slightly. Therefore, β-glucosidase from H. uvarum Yun268 has excellent biochemical properties and a good application potential in winemaking. PRACTICAL APPLICATION: Winemaking is a biotechnological process in which exogenous β-glucosidase is used to overcome the deficiency of endogenous β-glucosidase activity in grapes. By localizing, purifying, and characterizing of β-glucosidase from Hanseniaspora uvarum Yun268, it is expected to reveal its physical and chemical characteristics to evaluate its oenological properties in winemaking. The results may provide the basis for promoting the release of varietal aroma and improving wine sensory quality in the wine industry.

Genetic Variants of Glucose-6-Phosphate Dehydrogenase and Their Associated Enzyme Activity: A Systematic Review and Meta-Analysis

Abstract: Low glucose-6-phosphate dehydrogenase enzyme (G6PD) activity is a key determinant of drug-induced haemolysis. More than 230 clinically relevant genetic variants have been described. We investigated the variation in G6PD activity within and between different genetic variants. In this systematic review, individual patient data from studies reporting G6PD activity measured by spectrophotometry and corresponding the G6PD genotype were pooled (PROSPERO: CRD42020207448). G6PD activity was converted into percent normal activity applying study-specific definitions of 100%. In total, 4320 individuals from 17 studies across 10 countries were included, where 1738 (40.2%) had one of the 24 confirmed G6PD mutations, and 61 observations (3.5%) were identified as outliers. The median activity of the hemi-/homozygotes with A-(c.202G>A/c.376A>G) was 29.0% (range: 1.7% to 76.6%), 10.2% (range: 0.0% to 32.5%) for Mahidol, 16.9% (range 3.3% to 21.3%) for Mediterranean, 9.0% (range: 2.9% to 23.2%) for Vanua Lava, and 7.5% (range: 0.0% to 18.3%) for Viangchan. The median activity in heterozygotes was 72.1% (range: 16.4% to 127.1%) for A-(c.202G>A/c.376A>G), 54.5% (range: 0.0% to 112.8%) for Mahidol, 37.9% (range: 20.7% to 80.5%) for Mediterranean, 53.8% (range: 10.9% to 82.5%) for Vanua Lava, and 52.3% (range: 4.8% to 78.6%) for Viangchan. A total of 99.5% of hemi/homozygotes with the Mahidol mutation and 100% of those with the Mediterranean, Vanua Lava, and Viangchan mutations had <30% activity. For A-(c.202G>A/c.376A>G), 55% of hemi/homozygotes had <30% activity. The G6PD activity for each variant spanned the current classification thresholds used to define clinically relevant categories of enzymatic deficiency.

Immobilization of an Industrial β-Glucosidase from Aspergillus fumigatus and Its Use for Cellobiose Hydrolysis

Abstract: In this study, several covalent methods of immobilization based on acrylic supports, Schiff bases and epoxides have been applied to a commercial cocktail with a high β-glucosidase activity secreted by Aspergillus fumigatus. This cocktail was preliminary compared to a commercial secretome of Aspergillus niger, which was also subjected to the aforementioned immobilization methods. Due to its higher activity, the cocktail from A. fumigatus immobilized on ReliZyme™ HA403 activated with glutaraldehyde was employed for pNPG and cellobiose hydrolysis in diverse operational conditions and at diverse enzyme loadings, showing a very high activity at high enzyme load. A kinetic model based on the Michaelis–Menten hypothesis, in which double inhibition occurs due to glucose, has been selected upon fitting it to all experimentally retrieved data with the lowest-activity immobilized enzyme. This model was compared to the one previously established for the free form of the enzyme, observing that cellobiose acompetitive inhibition does not exist with the immobilized enzyme acting as the biocatalyst. In addition, stability studies indicated that the immobilized enzyme intrinsically behaves as the free enzyme, as expected for a one-bond low-interaction protein-support immobilization.

The Important Role of Enzyme Adsorbing Capacity of Soil Minerals in Regulating β‐Glucosidase Activity

Abstract: Mineral surfaces are known for their ability to adsorb enzymes, but their effects on enzyme activity are not well studied. This study explores the extent to which interaction with primary silicates and secondary minerals affects the activity and longevity of a model soil enzyme β-glucosidase (BG). The BG activity was correlated with the capacity-related properties of minerals (controlling space for adsorption), including specific surface area (SSA) and the SSA-normalized amount of BG adsorption, but not with the functional properties (controlling the mechanisms of adsorption) such as surface charge. BG conformational change is a function of SSA-normalized amount of adsorption, suggesting that spatially more distributed enzyme molecules would have a higher probability of encounter with the substrate than the congested molecules. The highest decay rate of BG activity was observed in the presence of Mn-oxide. Our results shed new light on the effect of mineral surface on enzyme activity.

The Neutral Protease Immobilization: Physical Characterization of Sodium Alginate-Chitosan Gel Beads

Abstract: Sodium alginate and chitosan were cross-linked to form composite gel spheres, which were entrapped to immobilize the free neutral protease. The matrix of the immobilized neutral protease was detected and characterized by using Fourier transform infrared spectroscopy and energy-dispersive X-ray. The optimum immobilization conditions were determined by orthogonal test, in which the concentration of sodium alginate was 3.5%, CaCl2 2.5%, chitosan 2.5%, and immobilizing time 1.5 h. Meanwhile, the activities of immobilized neutral protease and free enzyme were compared. The results showed that the pH value of immobilized enzyme was 5-8, the relative activity was above 90%, the free enzyme was above 80%, the relative activity of immobilized enzyme was above 80% in 30-80 °C, and the free enzyme was above 64% in 40-80 °C. The immobilized enzyme is better than the free enzyme in the constant of pH and temperature. The relative activity of immobilized enzyme was 50% after six hydrolysis cycles, and 80% after 11 days of storage.

The effects of mepiquat chloride (DPC) on the soluble protein content and the activities of protective enzymes in cotton in response to aphid feeding and on the activities of detoxifying enzymes in aphids

Abstract: Mepiquat chloride (DPC) enhances the resistance of cotton plants, and it is widely used as a growth regulator. DPC can stimulate photosynthesis, stabilize the structure of cotton leaves, and affect population reproduction and energy substances in Aphis gossypii Glover (cotton aphids), but interactions between DPC and cotton aphids remain unclear. In this study, we analyzed the physiological responses of cotton to DPC, and the toxicity of DPC toward cotton aphids, before and after feeding, to explore the DPC-induced defense mechanism against cotton aphids.Measurements of protective enzyme activity in cotton showed that the soluble protein contents, peroxidase (POD) activity, and catalase (CAT) activity in cotton treated with different concentrations of DPC were higher than in the control. Superoxide dismutase (SOD) activity was higher than that of the control when the concentration of DPC was < 0.1 g/L. Under aphid feeding stress, POD activity in cotton treated with a low insect population density was significantly lower than in the controls, but the reverse was true for cotton treated with a high insect population density, and SOD activity was positively correlated with population density. The activities of detoxification enzymes in field and laboratory experiments showed that DPC promoted the specific activity of glutathione S-transferase (GST) in cotton aphids, while the specific activities of carboxylesterase (CarE) and acetylcholinesterase (AchE) were decreased.DPC enhanced the aphid resistance in cotton by increasing the soluble protein content and the activity of protective enzymes. It also had a toxic effect on cotton aphids by increasing GST activity (the main DPC target). DPC increased the soluble protein content and protective enzymes activity in cotton under aphid stress, and thereby enhanced tolerance to cotton aphids. It conclude that DPC interferes with cotton aphids through indirect (DPC induced cotton defense responses) and direct (DPC toxicity to cotton aphids) ways, which plays a positive role in interfering with cotton aphids.

The effects of mepiquat chloride (DPC) on the soluble protein content and the activities of protective enzymes in cotton in response to aphid feeding and on the activities of detoxifying enzymes in aphids

Abstract: Mepiquat chloride (DPC) enhances the resistance of cotton plants, and it is widely used as a growth regulator. DPC can stimulate photosynthesis, stabilize the structure of cotton leaves, and affect population reproduction and energy substances in Aphis gossypii Glover (cotton aphids), but interactions between DPC and cotton aphids remain unclear. In this study, we analyzed the physiological responses of cotton to DPC, and the toxicity of DPC toward cotton aphids, before and after feeding, to explore the DPC-induced defense mechanism against cotton aphids.Measurements of protective enzyme activity in cotton showed that the soluble protein contents, peroxidase (POD) activity, and catalase (CAT) activity in cotton treated with different concentrations of DPC were higher than in the control. Superoxide dismutase (SOD) activity was higher than that of the control when the concentration of DPC was < 0.1 g/L. Under aphid feeding stress, POD activity in cotton treated with a low insect population density was significantly lower than in the controls, but the reverse was true for cotton treated with a high insect population density, and SOD activity was positively correlated with population density. The activities of detoxification enzymes in field and laboratory experiments showed that DPC promoted the specific activity of glutathione S-transferase (GST) in cotton aphids, while the specific activities of carboxylesterase (CarE) and acetylcholinesterase (AchE) were decreased.DPC enhanced the aphid resistance in cotton by increasing the soluble protein content and the activity of protective enzymes. It also had a toxic effect on cotton aphids by increasing GST activity (the main DPC target). DPC increased the soluble protein content and protective enzymes activity in cotton under aphid stress, and thereby enhanced tolerance to cotton aphids. It conclude that DPC interferes with cotton aphids through indirect (DPC induced cotton defense responses) and direct (DPC toxicity to cotton aphids) ways, which plays a positive role in interfering with cotton aphids.

Production, statistical optimization, and functional characterization of alkali stable pectate lyase of Paenibacillus lactis PKC5 for use in juice clarification

Abstract: Abstract Pectate lyase is a hydrolytic enzyme used by diverse industries to clarify food. The enzyme occupies a 25% share of the total enzyme used in food industries, and their demand is increasing gradually. Most of the enzymes in the market belong to the fungal origin and take more time to produce with high viscosity in the fermentation medium, limiting its use. The bacteria belonging to the genus Bacillus have vast potential to produce diverse metabolites of industrial importance. The present experiment aimed to isolate pectate lyase-producing bacteria that can tolerate an alkaline environment at moderate temperatures. Bacillus subtilis PKC2, Bacillus licheniformis PKC4, Paenibacillus lactis PKC5, and Bacillus sonorensis ADCN produced pectate lyase. The Paenibacillus lactis PKC5 gave the highest protein at 48 h of incubation that was partially purified using 80% acetone and ammonium sulphate. Purification with 80% acetone resulted in a good enzyme yield with higher activity. SDS-PAGE revealed the presence of 44 kDa molecular weight of purified enzyme. The purified enzyme exhibits stability at diverse temperature and pH ranges, the maximum at 50 °C and 8.0 pH. The metal ions such as Mg 2+ , Zn 2+ , Fe 2+ , and Co 2+ significantly positively affect enzyme activity, while increasing the metal ion concentration to 5 mM showed detrimental effects on the enzyme activity. The organic solvents such as methanol and chloroform at 25% final concentration improved the enzyme activity. On the other hand, detergent showed inhibitory effects at 0.05% and 1% concentration. Pectate lyase from Paenibacillus lactis PKC5 had K m and V max values as 8.90 mg/ml and 4.578 μmol/ml/min. The Plackett–Burman and CCD designs were used to identify the significant process parameters, and optimum concentrations were found to be pectin (5 gm%) and ammonium sulphate (0.3 gm%). During incubation with pectate lyase, the clarity percentage of the grape juice, apple juice, and orange juice was 60.37%, 59.36%, and 49.91%, respectively.