Sayyed, R. Z. | bioRxiv Science

bioRxiv2020

Tree bark scrape fungus: A potential source of laccase for application in bioremediation of non-textile dyes

Although laccase has been recognized as a wonder molecule, and green enzyme, the use of low yielding fungal strains, poor production, purification, and low enzyme kinetics have hampered its larger-scale applications. Hence the present research was aimed to select high yielding fungal strains and to optimize the production, purification, and kinetics of laccase of Aspergillus sp. HB_RZ4. Aspergillus sp. HB_RZ4 produced a copious amount of laccase on under meso-acidophillic shaking conditions in a medium containing glucose and yeast extract. A 25 {micro}M of CuSO4 enhanced the enzyme yield. The enzyme was best purified on Sephadex G-100 column. Purified enzyme resembled with the laccase of A. flavus. Kinetics of purified enzyme revealed the high substrate specificity and good velocity of reaction with ABTS as substrate. The enzyme was stable over a wide range of pH and temperature. The peptide structure of the purified enzyme resembled with the laccase of A. kawachii IFO 4308. The fungus decolorized various dyes independent of the requirement of a laccase mediator system (LMS). Aspergillus sp. HB_RZ4 came out as a potent natural producer of laccase, it decolorized the dyes even in absence of LMS and thus can be used for bioremediation.

biochemistry↗

bioRxiv2019

Production, purification and evaluation of biodegrading potential of PHB depolymerase of Stenotrophomonas sp. RZS 7

There are numerous reports on PHB depolymerases produced by a wide variety of microorganisms isolated from various habitats, however, reports on PHB depolymerase isolated from plastic contaminated sites are scares. Thermophilic PHB polymerase produced by isolates obtained from plastic contaminated sites is expected to have better relevance for its application in plastic/bioplastic degradation. Although PHB has attracted commercial significance, the inefficient production and recovery methods, inefficient purification of PHB depolymerase and lack of ample knowledge on PHB degradation by PHB depolymerase have hampered its large scale commercialization. Therefore, to ensure the biodegradability of biopolymers, it becomes imperative to study the purification of the biodegrading enzyme system. We report the production, purification, and characterization of extracellular PHB depolymerase from Stenotrophomonas sp. RZS 7 isolated from a plastic contaminated site. The isolate produced extracellular poly-{beta}-hydroxybutyrate (PHB) depolymerase in the mineral salt medium at 30oC during 4 days of incubation under shake flask condition. Purification of the enzyme was carried out by three different methods using PHB as a substrate. Purification of PHB depolymerase by ammonium salt precipitation, column chromatography, and solvent purification method was successfully carried out. Among the purification method tested, the enzyme was best purified by column chromatography on Octyl-Sepharose CL-4B column with maximum (0.7993 U mg-1 ml-1) purification yield. The molecular weight of purified PHB depolymerase (40 kDa) closely resembled with PHB depolymerase of Aureobacterium saperdae. Experiments on assessment of biodegradation of PHB in liquid culture medium and under natural soil conditions confirmed PHB biodegradation potential of Stenotrophomonas sp. RZS 7. The results obtained in FTIR analysis, HPLC study and GC-MS analysis confirmed the biodegradation attempt in liquid medium by Stenotrophomonas sp. RZS 7. Changes in surface morphology of PHB film in soil burial as observed in FE SEM analysis confirmed the biodegradation of PHB. The isolate was capable of degrading PHB and resulted in 87.74% degradation. Higher rate of degradation under natural soil condition is the result of activity of soil microbes that complemented the degradation by Stenotrophomonas sp. RZS 7.

microbiology↗