INTRODUCTION:

Pectin is an important component of the middle lamella and primary cell wall of higher plants. Pectins are high molecular weight acidic heteropolysaccharide primarily made up of α (1-4) linked D-galacturonic acid residues.¹ Three major pectic polysaccharides groups are recognized, all containing d-galacturonic acid to a greater or a lesser extent. They are homogalacturonan (HG), rhamnogalacturonan I (RGI), and rhamnogalacturonan II (RGII).²

These enzymes classified according to the criteria whether Pectinase act by tranelimination or hydrolysis or whether cleavage is random. They are pectin esterase (PE), depolymerizing enzymes and propectinase.³ More than 30 different genera of bacteria, yeasts and moulds have been used for the production of pectinase. However, Erwinia, Bacillus, Saccharomyces, Kluyveromyces, Aspergillus, Penicillium, Fusarium and Rhizopus have been the genera most frequently studied in the last 15 years.⁴

Selection of the microbial source for pectinase production depends upon the type of microorganisms, fermentation condition such as pH, incubation time or cultivation time, carbon and nitrogen source, types and concentration of substrate, temperature, agitation, and use of different enzyme preparations.⁵ The current study was aimed for development and validation of fermentation conditions for pectinase production in submerged fermentation conditions.

MATERIALS AND METHODS:

Soil Sampling:

A total of 12 soil samples comprising of fruit and vegetable wastes, decaying coconut husks and agricultural waste were collected from six different places for the isolation of pectinase producing microbial strains. The soil samples were collected via sieving through a 2mm sieve from 1-15 inches depth below the soil where there was moisture which supports ideal environment for the growth of microbes. The samples were dispensed into bags and were brought to the laboratory.

Enrichment of the soil sample:

One hundred grams of the soil sample was taken in a sterile beaker and to this soil sample, 1g of the pectin and a small quantity of water (equivalent to 60% of the water holding capacity) was added and incubated for 30 days for enrichment.

Screening:

A serial dilution of the 12 soil samples was done with saline. Screening media was sterile nutrient agar for 24h at 37°C for the growth of microorganisms. After 24 hours incubation, the colony characteristics were studied. We identified 51 microbes showing clear zone which is the zone of hydrolysis. We proceeded for our further studies with bacterial isolates.

Enrichment Broth for Isolation of Pectinolytic Bacteria:

One gram of the soil sample of different samples that were enriched with 1% of pectin was taken in a conical flask containing 100 ml of sterilized enrichment broth which had the following composition (gms/L).

Table 1: Composition of the Enrichment Broth for pectinase

Pectin (1%)	1gm
Yeast extract	0.1gm
Peptone	0.5gm
CaCO₃	0.2gm
NaCl	0.2gm
Distilled Water	100ml

The pectin was supplemented as the carbon source and the peptone as the source of nitrogen. The enrichment broth was inoculated with the enriched soil sample and placed on the rotary shaker at 200 rpm for 10 days.

Serial dilution technique:

The microbes were isolated from the collected soil samples by subjecting the soil for enrichment with Pectin. 10 fold serial dilutions (10^-1 to 10^-8) of the enriched soil sample were prepared. 1ml portion of the dilution (10^-4 to 10^-6) was inoculated into 100 ml of the selective broth (Vincent’s mineral salts broth) at pH 7.

Selective Media:

A selective medium is the one that allows the growth of microorganisms of interest. Selective media are very important in primary isolation of a specific type of microorganism from soil sample that harbor millions of microbes of different types. They hasten isolation by suppressing the unwanted background organisms and favour the growth of desired ones. Thus, a selective medium i.e. Vincent's mineral salts broth (Vincent, 1970) was employed here which allows only the desired microbes to utilize pectin as the sole carbon source and inhibits the growth of the bacteria that cannot use pectin as the carbon source. The bacterial isolates were inoculated and grown in Vincent’s mineral salts broth at pH 7 (Culture broth) and incubated.

Table 2:The composition of the medium (g/L) (Vincent's mineral salts broth at pH 7)

Ingredients	Quantity
Sucrose	1gm
KNO₃	0.6gm
KH₂PO₄	1gm
MgSO₄ 7H₂O	0.25gm
CaCl₂	0.1gm
NaNO₃	2gm
K₂HPO₄	0.5gm
KCl	0.5gm
Yeast extract	1gm
Pectin (1%)	10gm
Distilled water	1000 ml

All the above ingredients were weighed to the desired volume and made up to 250 ml in Erlenmeyer flask and sterilized at 121°C for 15 minutes according to the standard procedure.

Vincent’s Agar Medium:

Vincent’s Agar Medium is a selective medium in which the pectin has been supplemented as a carbon source. All the collected soil samples were enriched and diluted. Aliquots of 0.5 ml diluted (10^-⁴, 10^-5) culture broth was inoculated under aseptic conditions into Petri plates containing the Vincent’s agar medium and the plates were incubated at 37°C for 48 hrs in inverted position. These plates were maintained in replicates. The secretion of pectinase was identified by the clearing zones of the medium after the addition of Logule’s iodine solution. The ratio of the clear zone diameter to colony diameter was measured in order to select for the highest pectinase activity producer. The largest ratio was assumed to contain the highest activity. The composition of the Vincent’s agar medium is as follows:

Ingredients	Quantity
Sucrose	1gm
KNO₃	0.6gm
KH₂PO₄	1gm
MgSO₄ · 7H₂O	0.25gm
CaCl₂	0.1gm
NaNO₃	2gm
K₂HPO₄	0.5gm
KCl	0.5gm
Yeast extract	1gm
Pectin (1%)	10gm
Distilled water	1000 ml
Agar	20gm

Validation of Partially Purified Pectinase Enzyme

Effect of Temperature on Enzyme Activity:

For temperature assessment, the production process was carried out by shake flask method by placing the flasks on a rotary shaker. The production process was carried out at various temperatures such as 28°C, 37°C, 40°C, 45°C, 50°C, 55°C and 60°C to study the effect of temperature on enzyme production. All the experiments were carried out in triplicates and the average values were recorded. The optimum range of temperature achieved by this step was followed for all subsequent experiments. The enzyme activity was monitored by the DNS assay method.

Effect of different substrate on Enzyme Activity:

Since all the plant waste material contains pectin as the cell biomass which can be used as substrates for the production of the pectinase, apart from cassava waste, Tea waste, Molassess, wheat bran and agricultural waste, different natural substrates such as orange peel powder were also used in the present study. The production medium was supplemented with the different substrates as the source of the carbon and inoculum of Bacillus subtilis were added into 250 ml production medium for pectinase production. Orange peel is the good source of pectin, therefore used as a substrate for pectinase enzyme production. The study was carried out in triplicates by maintaining temperature, pH at their optimum level and the average values of pectinase production were recorded. The best substrate which gave highest enzyme activity was identified and that substrate was used for all other parameters. Determination of the pectinase activity was done by the DNS assay method.

Effect of pH on Enzyme Activity:

Optimization of the pH of the production media yeast extract pectin (YEP) for Pectinase enzyme production was adjusted to 4, 5, 6, 7, 8 and 9. The effect of pH on enzyme activity was tested using sodium acetate buffer (0.1 M; pH range, 3.2–5.8), sodium phosphate buffer (0.1 M; pH range, 5.9–7.1) and Tris-HCl buffer (0.1 M; pH range, 7.2–9.0). Tubes containing 0.5 mL respective buffers were mixed with 0.5 mL of the enzyme. Then, 1 mL of 0.3% (w/v) pectin solution was added and all the tubes were incubated at 30C for 10 min. Afterward, 2 mL of the DNS reagent was added and incubated in boiling water for 15 min. The enzyme activity was measured by DNS assay method.

Pectinase Assay by DNS method (3,5-dinitrosalicyIic acid):

The pectinase activity was assayed by DNS method. The DNS method is the most sensitive method for assay of the all the enzymes including pectinase. Pectinase activity was measured by the DNS method (3,5-dinitrosalicylic acid), through the determination of the amount of reducing sugars liberated from carboxymethylcellulose (CMC) solubilized in 50 mM Tris-HCI buffer, pH 7.0. This mixture was incubated for 20 min at 70°C. For crystalline cellulose substrates, incubation times were extended to 2 h and the reaction was stopped by the addition of DNS solution. The treated samples were boiled for 10 min, cooled in water for color stabilization, and the optical density was measured at 550 nm. The cellulose and pectinase activity was determined by using a calibration curve for glucose (Sigma-Aldrich, UK). One unit of enzyme activity was defined as the amount of enzyme that was released 1 pmol of glucose/ml/min. The substrate specificity of the crude enzyme was determined by performing the assay with different substrates such as sugarcane bagasse and orange peel waste powder as the substrates. The activity of the enzyme was expressed in pmol/ml/min, by the following formula:

Enzyme activity = µ moles of the product liberated per mole of enzyme per ml per minute

Enzyme activity = Concentration of Glucose liberated / Incubation time = µ grams

Conversion of µ grams to µ moles

µ moles = µ grams / molecular weight of glucose = µ moles / ml / min

RESULTS:

Isolation, Screening, and Identification of the bacterial Isolates:

A total of 51 microbes were isolated and screened primarily. Based on the zone of hydrolysis, only 12 strains were subjected to secondary screening. During secondary screening, one strain was morphologically and culturally identified as Bacillus subtilis. Based on primary and secondary screening, Bacillus subtilis was found to be a potent strain and hence was selected for further study.

Optimization of the Fermentation Conditions for Maximum Enzyme Production:

Bacillus subtilis was used to study the optimum conditions for maximum enzyme production.

DISCUSSION:

Temperature:

At 37°C, activity of Bacillus subtilis was reported to be the highest and produces maximum pectinase. At a higher temperature, enzyme production declined. Enzymes are usually denatured at higher temperature resulting decreased activity.⁶ Previous studies have reported that the maximum yield of pectinase enzyme from the members of genus Bacillus subtilis occur at the temperature range of 30–40°C.^7-9

Different Substrates:

With appropriate substrate concentration, Bacillus subtilis produces maximum pectinase. The enzyme production was found to be decreased when other substrates were used. A higher substrate concentration increases the viscosity of culture media and creates nutrient rich environment. Higher nutrients and substrate in the fermentation media inhibit microbial growth lowering enzyme production.¹⁰ Previous researches have also stated that lower substrate concentration is effective for enzyme production.¹¹ The requirement of low substrate concentration might be cost effective for large scale production of pectinase enzyme.

pH specificity:

Pectinase enzyme showed higher activity in the slightly acidic range. The enzyme activity was different with various pH. Pectinolytic enzymes have shown to exhibit higher enzymatic activity at pH range of 4.0–7.0].¹² Although the enzyme showed highest activity at pH 5.8, a peak at pH 8.0 was observed. This may be due to enzyme’s activity and stability. Bacterial pectinases are found to be stable from acidic to alkaline pH ranges (4.0–8.0) but their activity and stability may have different peaks at different pH ranges. In a similar study, they observed the stability and optimum activity of bacterial pectinases at different ranges, i.e., stability pH 4.0, whereas the optimum activity was found at pH 10.5–11.¹³

From these optimization data, this study found that 48 h of incubation period, appropriate substrate, ph 5.8 and 37°C temperature could be the optimum cultural conditions for Bacillus subtilis to produce maximum pectinase enzyme.

This bacterium showed the highest pectinase production at 48 h of incubation period. After 48 h, the enzyme production was found to be decreased. Various other studies have reported the highest enzyme activity of Bacillus subtilis at 48–72 h, after which the enzyme production was found to be declined. The cause of decrease in enzyme production after certain time interval during incubation might be due to the exhaustion of essential supplements and/or accumulation of toxic metabolites in the culture medium. The shorter fermentation period of 48h could be advantageous for the production of pectinase at industrial scale.

In conclusion, Bacillus subtilis was identified as the promising strain for the production of pectinase enzyme via submerged fermentation. The maximum pectinase production was obtained under optimal cultivation conditions at 48 h of incubation period, appropriate substrate, and 37°C temperature. The pectinase enzyme produced from strain Bacillus subtilis reported maximum enzymatic activity at 37°C temperature and pH 5.8. However, further studies must be performed to identify the strain in genetic levels.

ACKNOWLEDGMENTS:

We acknowledge for all the helping hands who helped to conduct this study.

CONFLICTS OF INTEREST:

The authors declare that they have no conflict of interest.

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Received on 28.10.2020 Modified on 30.12.2020

Research J. Pharm.and Tech 2021; 14(12):6665-6668.

DOI: 10.52711/0974-360X.2021.01151