Immobilization of Bacillus amyloliquefaciens SP1 and its alkaline protease in various matrices for effective hydrolysis of casein

Abstract

An extracellular alkaline protease producing B. amyloliquefaciens SP1 was isolated from apple rhizosphere having multifarious plant growth-promoting activities. B. amyloliquefaciens SP1 protease was immobilized using various concentrations of calcium alginate, agar and polyacrylamide to determine the optimum concentration for formation of the beads. Enzyme activity before immobilization (at 60 °C, pH 8.0 for 5 min) was 3580 µg/ml/min. The results of immobilization with various matrices revealed that 3 % calcium alginate (2829.92 µg/ml/min), 2 % agar (2600 µg/ml/min) and 10 % polyacrylamide (5698.99 µg/ml/min) were optimum concentrations for stable bead formation. Immobilized enzyme reusability results indicated that calcium alginate, agar and polyacrylamide beads retained 25.63, 22.05 and 34.04 % activity in their fifth repeated cycle, respectively. In cell immobilization technique, the free movement of microorganisms is restricted in the process, and a semi-continuous system of fermentation can be used. In the present work, this technique has been used for alkaline protease production using different matrices. Polyacrylamide (10 %) was found with the highest total alkaline protease titer, i.e., 24,847 µg/ml/min semi-continuously for 18 days as compared to agar (total enzyme titer: 5800 in 10 days) and calcium alginate (total enzyme titer: 13,010 in 15 days). This present study reported that polyacrylamide (10 %) among different matrices has maximum potential of immobilization of B. amyloliquefaciens SP1 and its detergent stable alkaline protease with effective application in bloodstain removal.

Microbial proteases: ubiquitous enzymes with innumerable uses

“…So, even now more and more reports about alkaline protease from novel strains of Bacillus sp. as well as novel proteases from already discovered strains are being published from around the globe (Guleria et al 2016a ; Hakim et al 2018 ; Dorra et al 2018 ; Jadhav et al 2020 ; Qamar et al 2020 ; Far et al 2020 ). Mothe and Sultanpuram ( 2016 ) carried out the purification and characterization of serine alkaline protease from a novel B. caseinilyticus .…”

“…Many of these enzymes also display higher thermostability and activity at elevated temperatures (Naveed et al 2021 ). Being capable of showing activity in the alkaline pH range, the alkaline proteases have been very much in use as an additive to the detergents, for enhancement of the stain removal properties (Guleria et al 2016a ; Asha and Palaniswamy 2018 ). Similarly, leather processing is carried out at high pH, hence the alkaline proteases can act as suitable additives for the tanning industries (Hussain et al 2017 ; Hakim et al 2018 ).…”

“…As per the Enzyme Commission classification, proteases are placed in the class 3(hydrolases), sub-class 4 with each proteolytic enzyme assigned unique number as EC 3.4.x.x (Contesini et al 2017 ). These enzymes have been categorized on the basis of various parameters like the site of action, the type of substrate, active pH range, mechanism of action involving particular amino acid present in the active site (Guleria et al 2016a , b ). Depending on the site of action, broadly these enzymes can be classified as endopeptidase and exopeptidase.…”

Optimization of immobilization process and survival study of microbial sensing strains used for aromatic hydrocarbon detection in industrial wastewater

“…Then the solution containing beads was incubated at 37°C for 30 min with shaking followed by vortexing to ensure the complete dissolution of beads. CFU (colony‐forming unit) count was performed using the serial dilution method from this prepared solution (Taha et al ., 2013; Guleria et al ., 2016). All the experiments were performed in triplicates and the results reported are mean of the three observations unless otherwise mentioned.…”

“…CFU count was performed by the preparation of the dilution series and plating for the determination of viable cells present in the cubes. A similar process was also used to immobilize these strains in the agar matrix (Pernetti et al ., 2003; Poddar et al ., 2011; Guleria et al ., 2016).…”