RESUMO
In this work, the polygalacturonase (TL-PG1) from the thermophilic fungus Thermomyces lanuginosus was heterologously produced for the first time in the yeast Komagataella phaffii. The TL-PG1 was successfully expressed under the control of the AOX1 promoter and sequentially purified by His-tag affinity. The purified recombinant pectinase exhibited an activity of 462.6â¯U/mL toward polygalacturonic acid under optimal conditions (pH 6 and 55 ËC) with a 2.83â¯mg/mL and 0.063 µmol/minute for Km and Vmax, respectively. When used as supplementation for biomass hydrolysis, TL-PG1 demonstrated synergy with the enzymatic cocktail Ctec3 to depolymerize orange citrus pulp, releasing 1.43â¯mg/mL of reducing sugar. In addition, TL-PG1 exhibited efficiency in fabric bioscouring, showing potential usage in the textile industry. Applying a protein dosage of 7â¯mg/mL, the time for the fabric to absorb water was 19.77â¯seconds (ten times faster than the control). Adding the surfactant Triton to the treatment allowed the reduction of the enzyme dosage by 50% and the water absorption time to 6.38â¯seconds. Altogether, this work describes a new versatile polygalacturonase from T. lanuginosus with the potential to be employed in the hydrolysis of lignocellulosic biomass and bioscouring.
Assuntos
Proteínas Fúngicas , Poligalacturonase , Saccharomycetales , Biomassa , Eurotiales/enzimologia , Eurotiales/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hidrólise , Cinética , Poligalacturonase/metabolismo , Poligalacturonase/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Saccharomycetales/genética , Saccharomycetales/enzimologia , Saccharomycetales/metabolismo , Indústria Têxtil , TêxteisRESUMO
Abstract Alkaline pectinase is the utmost significant industrial enzyme of the bioscouring process. By considering bio scouring of cotton, 30 microbial isolates from fruit and vegetable waste rich dump soil of Solang Valley and Vasishta (Manali, Himachal Pradesh, India) were isolated and screened for the alkaline pectinase production in the current research work. Only four isolates P3, P16, P21, and P27 were capable to produce extracellular alkaline pectinase at pH 9. Further by applying submerged fermentation, the alkaline pectinase production was quantitatively screened. The most efficient isolate was P3 identified as Bacillus tropicus, based on morphological, biochemical, and molecular characterization. Molecular characteristics confirmed by 16S rDNA sequence analysis. The nucleotide sequence of the isolate was novel with a 97% similarity index and submitted to the GenBank with accession number MK332379. The Bacillus strain selected was active at broad pH range from 8-10.5 and a temperature range from 25-50 oC. Optimum pH and temperature observed were 9 and 37 oC respectively and can be suitably used for the bio scouring process for the pretreatment of the fabrics.