Das Arijit, Bhattacharya Sourav, Panchanan Gangotri, Navya B S, Nambiar Parvathi
Department of Microbiology, Center for Post Graduate Studies, Jain University, Bangalore 560011, Karnataka, India.
J Genet Eng Biotechnol. 2016 Dec;14(2):281-288. doi: 10.1016/j.jgeb.2016.09.007. Epub 2016 Oct 26.
A laccase produced by MTCC 142 under solid-state fermentation using co-substrates of paddy straw and corn husk (1.5:1.5, g w/w) showed an activity of 2.54 U gds. Laccase activity was determined spectrophotometrically using 0.5 mM 2,2'- azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). Supplementation with fructose and potassium nitrate resulted in maximum enzyme production at initial pH 5.8 ± 0.2 and initial moisture content of 70%. A carbon: nitrogen ratio of 0.5:0.1 yielded highest laccase activity in the presence of surfactant Tween 20 (0.05%, w/v). Incorporation of vanillin (5 mM) and copper sulphate (10 mM) facilitated enhanced synthesis of laccase. A 4.8-fold increase in enzyme activity was recorded after optimization of nutritional parameters. The apparent molecular mass of this enzyme was revealed as 43 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The laccase showed optimal activity at pH 3 and 35 °C with 82.8% residual activity after 1 h of incubation. The and values on ABTS were found to be 0.52 mM and 9.33 U gds, respectively. The enzyme activity was enhanced by Cu and remained unaffected with Ba, Mn, Pb, Mg, Ca and Fe. However, pre-incubation of the enzyme with reagents like sodium azide, sodium lauryl sulphate and 2-mercaptoethanol demonstrated an inhibition of its activity. Addition of crude laccase to Congo red dye solution resulted in 36.84% decolourization after 20 h of incubation at 35 ± 2 °C. This study discusses the production and characterization of a laccase from strain with potential for azo dye decolourization.
MTCC 142利用稻草和玉米皮(1.5:1.5,克/克)作为共底物通过固态发酵产生的漆酶,其活性为2.54 U/g干物质。漆酶活性采用分光光度法,以0.5 mM 2,2'-联氮-双(3-乙基苯并噻唑啉-6-磺酸)(ABTS)进行测定。添加果糖和硝酸钾后,在初始pH 5.8±0.2和初始水分含量70%的条件下酶产量最高。在表面活性剂吐温20(0.05%,w/v)存在的情况下,碳氮比为0.5:0.1时漆酶活性最高。加入香兰素(5 mM)和硫酸铜(10 mM)有助于漆酶的合成增强。营养参数优化后,酶活性提高了4.8倍。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示该酶的表观分子量为43 kDa。该漆酶在pH 3和35℃时表现出最佳活性,孵育1小时后残留活性为82.8%。在ABTS上的Km和Vmax值分别为0.52 mM和9.33 U/g干物质。铜可增强酶活性,而钡、锰·、铅`、镁、钙和铁对其无影响。然而,该酶与叠氮化钠、十二烷基硫酸钠和2-巯基乙醇等试剂预孵育后其活性受到抑制。在35±2℃孵育20小时后,向刚果红染料溶液中添加粗漆酶导致脱色36.84%。本研究讨论了一株具有偶氮染料脱色潜力的菌株所产漆酶的生产及特性。