Characterization of poly(L-lactide)-degrading enzyme produced by thermophilic filamentous bacteria Laceyella sacchari LP175

Abstract

on an emulsified PLLA agar plate at 50 C. Among the isolates, strain LP175 showed the highest PLLAdegrading ability. It was closely related to Laceyella sacchari, with 99.9% similarity based on the 16S rRNA gene sequence. The PLLA-degrading enzyme produced by the strain was purified to homogeneity by 48.1% yield and specific activity of 328 U·mg-protein-1 with a 15.3-fold purity increase. The purified enzyme was strongly active against specific substrates such as casein and gelatin and weakly active against Suc-(Ala) 3 -pNA. Optimum enzyme activity was exhibited at a temperature of 60 C with thermal stability up to 50 C and a pH of 9.0 with pH stability in a range of 8.5 10.5. Molecular weight of the enzyme was approximately 28.0 kDa, as determined by gel filtration and SDS-PAGE. The inhibitors phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetate (EDTA), and ethylene glycolbis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) strongly inhibited enzyme activity, but the activity was not inhibited by 1 mM 1,10-phenanthroline (1,10-phen). The N-terminal amino acid sequences had 100% homology with thermostable serine protease (thermitase) from Thermoactinomyces vulgaris. The results obtained suggest that the PLLA-degrading enzyme produced by L. sacchari strain LP175 is serine protease.Key words: Laceyella sacchari; PLLA-degrading enzyme; Thermoactinomycetaceae; thermophilic filamentous bacteria IntroductionPoly(L-lactide) (PLLA); (C 3 H 4 O 2 )n is one of the aliphatic biodegradable polyesters derived from renewable resources such as corn, cassava, sugar cane, rice and potato through lactic acid fermentation, and it is also fully degradable by both microbial and enzymatic processes (Tokiwa et al., 2009;Gupta et al., 2007;Jarerat et al., 2006;Tokiwa and Calabia, 2006;Tomita et al., 2004). PLLA, as an environmentally friendly (eco-friendly or green ) product, has been developed on a large scale and is currently used for a wide range of applications, including packaging materials (Bhalla et al., 2007;Nolan-Itu Pty Ltd., 2002), medical applications (Jalil, 1990), agricultural products (Gross and Kalra, 2002;Sakai et al., 2001) and textiles.Most PLLA-degrading microorganisms are found in the bacterial order Actinomycetales, e.g. the families Pseudonocardiaceae (Pranamuda and Tokiwa, 1999;Pranamuda et al., 1997), Thermomonosporaceae (Sangwan and Wu, 2008;Sukkhum et al., 2009b), and Streptosporangiaceae (Sukkhum et al., 2009b). Apart from that, thermophilic bacteria in the families Thermoactinomycetaceae (Sukkhum et al., 2009b) and Firmicutes (Oda et al., 2000;Sakai et al., 2001), filamentous fungi in the genera Tritirachium and Paecilomyces (Sangwan and Wu, 2008), and yeast in the genus Cryptococcus have also been reported to be PLLA-degrading microorganisms.Many different types of enzymes are found in PLLAdegrading microorganisms, such as: protease from Amycola- Full PaperCharacterization of poly(L-lactide)-degrading enzyme produced by thermophilic filamentous bacteria Laceyella sacchari LP175 (Received F...

Production of raw starch degrading enzyme by the thermophilic filamentous bacterium Laceyella sacchari LP175 and its application for ethanol production from dried cassava chips

“…Each microorganism has different physicochemical conditions for enzyme production . Aeration rate at 0.5 vvm was the optimum condition for aeration supplied on growth and enzyme production by the aerobic filamentous bacterium L. sacchari LP175, requires oxygen for energy production . The results showed that aeration at lower and higher than 0.5 vvm caused lower the enzyme production and growth of L. sacchari LP175 (supplementary Fig.…”

“…Laceyella sacchari LP175, a strain of thermophilic filamentous bacterium previously isolated from soil in Thailand which has demonstrated the ability to produce raw starch degrading enzyme , was kept at the Department of Microbiology, Kasetsart University, Bangkok, Thailand, and at the Thailand Institute of Scientific and Technological Research (TISTR) culture collection ( L. sacchari TISTR 2280). A loop of the strain grown on a nutrient agar slant (3 g/L beef extract, 5 g/L peptone, and 15 g/L agar) was inoculated in 100 mL of nutrient broth at 50°C and 150 rpm for 12 h. The cell suspension was then centrifuged (10 000 rpm) at 4°C for 10 min.…”

A review on degradation mechanisms of polylactic acid: Hydrolytic, photodegradative, microbial, and enzymatic degradation

“…Thus, modern molecular biological techniques must be used to purify and characterize extracellular PLA-degrading enzymes from microorganisms using UV-vis spectrophotometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 16S rRNA analyses. [143,144,148] In general PLA-degrading enzymes within microorganisms are proteases (serine proteases) [158] and, to a lesser degree, lipases (esterase) [155,156] and cutinases. [142] Figure 9A shows the enzymatic degradation of PLA and Figure 9B shows the biodegradation process used by PLA-degrading enzymes to degrade PLA films.…”

Polyester‐based biodegradable plastics: an approach towards sustainable development

“…Jarerat et al (2006) isolated a PLA‐degrading mesophillic actinomycete, Amycolatopsis orientalis , exhibiting considerably good enzymatic activity when stimulated by different nitrogen sources such as proteins, peptides or amino acids. A PLA‐degrading protease has reported from thermophillic actinomycetes strains Laceyella sacchari LP175 (Hanphakphoom et al 2014) isolated from forest soil and Actinomadura sp. T16‐1 (Sukkhum et al 2009).…”