Production, statistical optimization, and functional characterization of alkali stable pectate lyase of Paenibacillus lactis PKC5 for use in juice clarification

Abstract

Pectate lyase is a hydrolytic enzyme used by diverse industries to clarify food. The enzyme occupies a 25% share of the total enzyme used in food industries, and their demand is increasing gradually. Most of the enzymes in the market belong to the fungal origin and take more time to produce with high viscosity in the fermentation medium, limiting its use. The bacteria belonging to the genus Bacillus have vast potential to produce diverse metabolites of industrial importance. The present experiment aimed to isolate pectate lyase-producing bacteria that can tolerate an alkaline environment at moderate temperatures. Bacillus subtilis PKC2, Bacillus licheniformis PKC4, Paenibacillus lactis PKC5, and Bacillus sonorensis ADCN produced pectate lyase. The Paenibacillus lactis PKC5 gave the highest protein at 48 h of incubation that was partially purified using 80% acetone and ammonium sulphate. Purification with 80% acetone resulted in a good enzyme yield with higher activity. SDS-PAGE revealed the presence of 44 kDa molecular weight of purified enzyme. The purified enzyme exhibits stability at diverse temperature and pH ranges, the maximum at 50 °C and 8.0 pH. The metal ions such as Mg2+, Zn2+, Fe2+, and Co2+ significantly positively affect enzyme activity, while increasing the metal ion concentration to 5 mM showed detrimental effects on the enzyme activity. The organic solvents such as methanol and chloroform at 25% final concentration improved the enzyme activity. On the other hand, detergent showed inhibitory effects at 0.05% and 1% concentration. Pectate lyase from Paenibacillus lactis PKC5 had Km and Vmax values as 8.90 mg/ml and 4.578 μmol/ml/min. The Plackett–Burman and CCD designs were used to identify the significant process parameters, and optimum concentrations were found to be pectin (5 gm%) and ammonium sulphate (0.3 gm%). During incubation with pectate lyase, the clarity percentage of the grape juice, apple juice, and orange juice was 60.37%, 59.36%, and 49.91%, respectively.

An Extracellular Pectinase from ISH16 Bacteria Isolated Induced by Coffee Pulp Waste Substrate

“…This indicates that metal ions such as Mg 2+ play an important role in maintaining the active conformation of pectinase to stimulate enzyme activity (Oumer and Abate, 2017). The results are the same as the increase in pectinase activity by Mg 2+ ions, namely Bacillus subtilis isolated from kimchi (traditional Korean food) (Rahman et al, 2020), Bacillus subtilis strain Btk 27 (Oumer and Abate, 2017), and Paenibacillus lactis isolated from the coast (Sheladiya et al, 2022). In contrast, different results were reported that the pectinase activity of Bacillus subtilis isolated from the sea was inhibited by 10 mM Mg 2+ ions, and the pectinase was increased when Fe 2+ ions were added (Joshi et al 2015).…”

“…These results indicate that enzymes from different sources require various metal ions at certain concentrations to carry out the activity. In addition, the evolution of enzymes also determines the need for metal ions at the active site because microorganisms evolve according to the surrounding environment to survive and grow well so they tend to modify their metabolic pathways or enzymes (Sheladiya et al, 2022).…”

Pseudomonas aeruginosa inhibits quorum-sensing mechanisms of soft rot pathogen Lelliottia amnigena RCE to regulate its virulence factors and biofilm formation

“…The DNA of bacteria was isolated by the LSP buffer method (Sheladiya et al, 2022 ). The 16S rRNA gene-specific primer; 27F 5‘AGAGTTTGATCCTGGCTCAG-3' and 1492R 5‘GGTTACCTTGTTACGACTT 3' were used to amplify the target sequence of 1,500 bp.…”

Utilization of wild Cressa cretica biomass for pectinase production from a halo‐thermotolerant bacterium

“…Post-incubation, CFCS aliquots were drawn at 0 and 48 h to assess pectinase activity, amount of reducing sugar, [26] and the change in pH.…”