Citation: Shet, A.R.; Muhsinah, A.B.; Alhazmi, A.Y.; Achappa, S.; Desai, S.V.; Mahnashi, M.H.; Muddapur, U.M.; Khan, A.A.; Shaikh, I.A. Bioprocessing of Agro-Industrial Waste for Maximization of Pectinase Production by a Novel Native Strain Aspergillus cervinus ARS2 Using Statistical Approach. Separations 2022, 9, 438. https://doi.org/10.3390/ separations9120438 Academic Editor: Victoria Samanidou Received: 10 November 2022 Accepted: 8 December 2022 Published: 13 December 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). separations Article Bioprocessing of Agro-Industrial Waste for Maximization of Pectinase Production by a Novel Native Strain Aspergillus cervinus ARS2 Using Statistical Approach Anil R. Shet 1 , Abdullatif Bin Muhsinah 2 , Abdulfattah Y. Alhazmi 3 , Sharanappa Achappa 1 , Shivalingsarj V. Desai 1, * , Mater H. Mahnashi 4 , Uday M. Muddapur 1 , Aejaz Abdullatif Khan 5 and Ibrahim Ahmed Shaikh 6, * 1 Department of Biotechnology, KLE Technological University, Hubballi 580031, India 2 Department of Pharmacognosy, College of Pharmacy, King Khalid University, Abha 61441, Saudi Arabia 3 Clinical Pharmacy Department, Umm Al-Qura University, Makkah 24382, Saudi Arabia 4 Department of Pharmaceutical Chemistry, College of Pharmacy, Najran University, Najran 66462, Saudi Arabia 5 Department of General Science, Ibn Sina National College for Medical Studies, Jeddah 21418, Saudi Arabia 6 Department of Pharmacology, College of Pharmacy, Najran University, Najran 66462, Saudi Arabia * Correspondence: desaisv@kletech.ac.in (S.V.D.); i.ibrahimshaikh09@gmail.com (I.A.S.) Abstract: The demand for microbial pectinase has increased due to its vast applications in different industries. The current study dealt with the synthesis of pectinase by a novel native strain Aspergillus cervinus ARS2 using agro-industrial waste. Comparative studies conducted on pectinase produc- tion by submerged fermentation (SmF) and solid-state fermentation (SSF) showed that pectinase activity was more increased in SSF (44.51 ± 1.33 IU/mL) than in SmF (40.60 ± 1.15 IU/mL) when using orange peel as a substrate. Using SSF, one-factor-at-a-time (OFAT) studies were conducted, considering different process variables such as inoculum size, initial pH, incubation time, moisture content, incubation temperature, and substrate particle size, all of which affected the pectinase activity. OFAT results showed the highest pectinase activity at an inoculum size of 10 6 spores/mL (43.11 ± 1.06 U/mL), an incubation time of 6 days (43.81 ± 1.21 U/mL), a moisture content of 100% (44.30 ± 1.69 U/mL), a substrate particle size of 1.7 mm (42.06 ± 1.20 U/mL), an incubation tem- perature of 37 °C (45.90 ± 1.33 U/mL), and an initial pH of 4 (43.31 ± 0.89 U/mL). The identified significant process variables were then optimized by response surface methodology (RSM)-central composite design (CCD). The results showed optimum pectinase activity of 107.14 ± 0.71 IU/mL for a substrate particle size of 2 mm, an incubation temperature of 31.5 ◦ C, an initial pH of 4.9, and a moisture content of 107%, which was obtained from the Minitab optimizer. By using statistical optimization, the pectinase production from the isolated novel fungal strain A. cervinus ARS2 was increased 2.38-fold. Therefore, the A. cervinus ARS2 strain can be further explored for large-scale pectinase production which could meet the growing industrial demands. Keywords: Aspergillus cervinus; agro-industrial waste; optimization; pectinase; process variables; solid-state fermentation; response surface methodology 1. Introduction Enzymes are substances which speed up chemical reactions without undergoing any change themselves during the reaction. Enzymes have many advantages over chemical catalysts, such as adjustable activity, high specificity, and high catalytic efficiency, which encourages their use in pharmaceutical, chemical, and food industries [1]. Due to their vast applications, the demand for industrial enzymes has increased, which has led to continuous research and development in optimizing the production of enzymes and in reducing this production cost [2]. Amongst industrially vital enzymes, pectinases have gained importance Separations 2022, 9, 438. https://doi.org/10.3390/separations9120438 https://www.mdpi.com/journal/separations