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Background: Fruit juice clarification is a challenging aspect of beverage industry which needs to be addressed for economical and hygienic production of fruit juices. Objective: Current study is focused on the complete purification, characterization and thermodynamic analysis of an efficient mannanase enzyme to analyze its applicability in biological clarification fruit juice. Methods: Mannanase production using Aspergillus awamori IIB037 in a 25 L stirred fermenter at pre optimized reaction conditions was carried out. Enzyme purification was carried out via series of steps. Characterization of enzyme along with kinetics and thermodynamic studies was conducted. Purified and characterized enzyme was assessed for its applicability in fruit juice clarification through clarification experiments on fresh apple juice. Results: Purification fold of 3.98 was obtained along with 86.80% purification yield of mannanase with specific activity of 158.16 U/mg. The molecular size of purified enzyme was determined as 66 kDa. The enzyme depicted 56% residual activity at 60°C after 8 hrs. Thermodynamic studies of an enzyme revealed enthalpy of activation (ΔH) and activation energy (Ea) as 30.53KJ/mol, 27.76KJ/mol, respectively. The enzyme activity increased in the presence of ß-mercaptoethanol surprisingly. On the other hand, methyl alcohol, ethanol, Hg2+ and Cu2+ inhibited enzyme activity. The enzyme showed Km and Vmax values of 11.07 mM and 19.08 μM min-1 for Locust Bean Gum (LBG) under optimal conditions. Juice treated with mannanase showed decrease in absorbance and increase in reducing sugar content. Conclusion: The current study demonstrated that mannanase from Aspergillus awamori in its purified form has significant characteristics to be employed industrially for juice clarification.

In this study, complete purification, characterization, and immobilization of polygalacturonase (pectinase) from Penicillium notatum were carried out to achieve an economical and suitable alternative for industrial fruit juice clarification. Biosynthesis of polygalacturonase was carried out under pre-optimized conditions employing solid-state fermentation at a pilot scale using wheat bran. The enzyme was subjected to a series of steps for purification including ion-exchange chromatography. After that, the purified enzyme was characterized and its kinetics and thermodynamic parameters along with the effect of immobilization on its performance were studied. Finally, a purified acidic enzyme was tested for its clarifying abilities on fresh apple juice. Purification fold of 2.98 was attained with increased specific activity of 256U/mg. Purified polygalacturonase showed a molecular weight of 38 kDa, optimum temperature of 50°C, optimum pH of 5, 50% stability at 50°C, and 84% stability at pH 5. The “Vmax” and “Km” of the enzyme were evaluated to be 250U/mg and 0.11mg/mL, respectively for hydrolyzing pectin. From the Arrhenius plot, activation energy (Ea), enthalpy of activation (ΔH), and entropy of activation (ΔS) were found to be 6.35 KJ/mol, 3.67 KJ/mol, and -1.1KJ/mol, respectively. Among metal ions, most of the tested Organic solvents and inhibitors inhibited the activity. Nano emulsion-based pectinase exhibited better stability. The enzyme was found to be an effective agent for the clarification of fresh apple juice.

Phytic acid is not digested by monogastric animals due to deficiency of an enzyme (Phytase) in their digestive tube. Due to this reason, poultry industry needs to add potent phytase in its feed for faster growth rates. Therefore, phytase producing fungal isolates (64) were isolated and subjected to submerged fermentation and maximum production (21.79± 0.05 U/mL/min) was obtained by fungal isolate UJIIB-29, which was identified as Aspergillus oryzae after18S rRNA sequencing. Under optimized conditions, best results were obtained using glucose phosphate broth as a medium after 7 days of incubation, at 30℃, pH 5.5 with 0.5% glucose and 0.3% ammonium sulfate as carbon and nitrogen sources, respectively when it was inoculated with 4% conidial suspension. Purification of phytase by anion exchange chromatography resulted in 89.8% yield, 1.93 folds purification and 53.5 U/mg specific activity. The molecular weight of purified phytase was determined as 53 kDa whereas, Km and Vmax were determined as 3.41 mM and 24.6±0.05 U/mL/min, respectively. Thermodynamic studies of the enzyme revealed activation energy (Ea) and enthalpy of activation (ΔH) as 16.84 KJ/mol and 14.32 KJ/mol, respectively. Phytase showed enzyme activity up to 60℃ but at 30oC and 5.5 pH, the enzyme showed maximum stablitity. The findings of the current study will help in developing an economical process for phytase biosynthesis. In addition, kinetic and thermodynamic studies will help in assessing applicability of phytase in harsh conditions.

Sustainable bio-renewable energy source is a need of an hour to meet the rising global energy demands. Efficient conversion of fermentable sugars by yeast into bioethanol from lingo-cellulosic biomass is one of the most important attribute. In order to get temperature and ethanol tolerant yeast strain, one hundred and seventy one soil samples were collected from adjacent areas of various sugar mills located in Punjab, Pakistan. Fifty six yeast strains were isolated and primarily screened for bioethanol production. Maximum bioethanol producing (3.07 g/L) yeast strain IHZ-26 was identified by sequencing of 18S rRNA ITS region and identified as a new strain of Wickerhamomyces anomalus which was assigned GenBank accession number KT883963. This strain demonstrated substantial tolerance to ethanol (25%) and temperature (50°C) and have the ability to significantly ferment several sugars i.e. glucose, fructose, mannose, maltose, xylose and lactose. To the best of our knowledge, this is first report of multi stress tolerant Wickerhamomyces anomalus from Pakistan. This yeast with these distinct characteristics can be employed in bioethanol industry for more viable and economical process.