Agro-industrial residues are primarily composed of complex polysaccharides that strengthen microbial growth for the production of industrially important enzymes. Pectinases are one of the most widely disseminated enzymes in bacteria, fungi and plants. Czapeck media supplemented with orange waste peel as carbon source under submerged fermentation process Aspergillus niger presenting the preeminent enzymatic production. On partial optimization culture showed the maximum enzyme yield (117.1 ± 3.4 μM/mL/min) at 30 °C in an orange waste peel medium having pH 5.5 and substrate concentration (4%) after 5th day of fermentation. The produced enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography. A purification fold of 5.59 with specific activity and % recovery of 97.2 U/mg and 12.96% was achieved respectively after gel filtration chromatographic technique. The molecular weight of purified pectinase from A . niger was 30 kDa evidenced by SDS-PAGE. Pectinase activity profile showed purified enzyme was optimally active at pH = 7 and 55 °C. The maximum production of pectinase in the presence of cheaper substrate at low concentration makes the enzyme useful in industrial sectors especially for textile and juice industry.

Pectin comprises of d -galacturonic acid occurred in α (1–4)-chain, naturally esterifies with methoxy groups and natural sugars occupy the side chains. Occasionally, rhamnose group present in chain unsettles the chain helix formation ( Shembekar & Dhotre, 2009 ). In the last two decades of twentieth century, an industrial revolution happened in developing countries in citrus producing and processing area (Juices, Oils and other by-products). Pakistan being an agricultural country produces millions of tons of fruits and vegetables annually. Mango, citrus, apple, banana and dates are the highest yielding fruits in Pakistan. The cell wall and middle lamella are enriched of pectin ingredients ( Alkorta, Garbisu, Llama, & Serra, 1998 ). Carbohydrates and proteins are the major but the fat contents are in low amount in dried citrus peel. Significant quantity of pectin in citrus peel urges us to use them as a substrate for microorganism to produce pectinolytic enzymes. Microbial systems enhance pectinolytic production using pectin as inducer ( Aguilar & Huitron, 1993 ).

During the raw agriculture material processing for food, a bulk quantity of agro wastes generated. Thus, agro residues of coffee, orange, rice and sugarcane provide a suitable platform for bio-production of different enzymes using the fermentation process, thereby making valuable of these waste products ( Giese, Dekker, & Barbosa, 2008 ). Orange bagasse contains large amounts of soluble carbohydrates, particularly fructose, glucose, sucrose, and pectin, as well as insoluble cellulose, and has been used as a fermentation products including enzymes ( Rosales, Couto, & Sanroman, 2002 ). Microorganisms have noteworthy collaboration in food (dairy and meat) and alcoholic beverages industries ( Buyukkileci, Tari, & Fernandez-Lahore, 2011 ). The usage of bio-produced food and additives makes them more appropriate rather than the synthetically produced ones ( Susana and Sanroman, 2006 ). Citrus peel is known as the major source of pectinases in several industries instead of apple pomace. The potential applications of pectinases in different industries craving us to conduct the present study.

The pure culture of Aspergillus niger was available in our lab as previously reported by Ahmed, Zia, Iftikhar, and Iqbal (2011) . Fungal spore suspension was prepared by using the method of Dhillon, Gill, Gill, and Singh (2004) . In brief; Spores of A . niger were cultured in 250 mL flask holding 30 mL of Potato Dextrose broth at 30 ± 1 °C for 4 days. To achieve the maximum concentration of spores in the solution, we used saline solution (0.9% w/v) on the fungal spores and shaken flask to accelerate the release of spores into the surrounding saline solution. Orange peel waste was obtained from local fruit market Faisalabad, Pakistan. The substrate was dried (60 °C), ground and kept in air tight plastic jars to lessen the moisture contents.

Substrate screening was done by taking 4 g orange peel waste into 250 mL flasks of three replicates and inoculated with spore suspension of A . niger . All the experimental flasks were incubated at 30 ± 1 °C in a shaking incubator at 180 rpm for 5 days of fermentation period. The ingredients of the flasks were filtered and clear supernatant having enzyme solution was subjected to further analytical studies.

2.3.1. Determination of % moisture content

The moisture contents of sour orange peel was determined by taking 2 g sample in a sterilized bottle (W1) and dried at 100 °C for 2 h. The sample was cooled and weighed in desiccators, after that sample again dried until constant weight (W2). The moisture percentage was calculated by using the following formula: equation ( E1 ) ( % ) Moisture = ( W 1 ) − ( W 2 ) ( W 1 ) × 100

2.3.2. Determination of % ash content

Pre-weight crucible containing 5 mL HNO₃ and 2 g of substrate (W1) was used to heat the material until char forming. After that sample put in muffle furnace for 6 h at 555 °C and weighed (W2) to determine the percentage ash contents of sample. equation ( E2 ) ( % ) Ash = ( W 1 ) − ( W 2 ) Weight of sample × 100