ABSTRACT
Aims@#Lytic polysaccharide monooxygenase (LPMO) is an enzyme capable of cleaving glycoside bonds of recalcitrant polysaccharides through an oxidative mechanism. LPMO activity, in synergy with hydrolytic enzymes, increases the production of monomer sugars from the biodegradation of lignocellulose. This study was aimed at evaluating actinomycete S2 strain LPMO activity based on the release of xylose as one of reducing sugar and hydrogen peroxide (H2O2) in the course of lignocellulosic biodegradation. @*Methodology and results@#The oxidation activity of LPMO from actinomycete S2 strain was measured by using the substrate of Avicel supplemented with ascorbic acid and copper ions (Cu2+) to identify its effect on the release of xylose as one of reducing sugar. The optimum incubation time for the LPMO production was also conducted. Further, H2O2 quantitative analysis was performed as by-product of LPMO activity and 16S rRNA gene sequence of actinomycete S2 strain were subsequently determined. We found that supplementation of 1 mM ascorbic acid and 0.2 mM Cu2+ increased xylose as one of reducing sugar production by up to 5-fold from 255.03 to 1290 μg/mL after an optimal incubation period of 6 days. Based on H2O2 production, the LPMO activity of actinomycete S2 strain was 0.019 ± 0.001 U/mL. There is likelihood that LPMO activity derived from actinomycete S2 strain has a synergistic effect with the activity of other lignocellulose-degrading enzymes. This actinomycete showed 99% similarity to the 16S rRNA gene sequence of Streptomyces avermitilis strain EAAG80. @*Conclusion, significance, and impact of study@#LPMO enzyme activity from actinomycete S2 strain as determined by the production of reducing sugar and H2O2 was greatly increased by supplementation with ascorbic acid as an electron donor and Cu2+ ions. To the best of our knowledge, this is the first elucidation of LPMO activity from an indigenous Indonesian actinomycete.
ABSTRACT
Streptomyces avermitilis produces clinically useful drugs such as avermectins and oligomycins. Its genome contains approximately 33 cytochrome P450 genes and they seem to play important roles in the biosynthesis of many secondary metabolites. The SAV_7130 gene from S. avermitilis encodes CYP158A3. The amino acid sequence of this enzyme has high similarity with that of CYP158A2, a biflaviolin synthase from S. coelicolor A3(2). Recombinant S. avermitilis CYP158A3 was heterologously expressed and purified. It exhibited the typical P450 Soret peak at 447 nm in the reduced CO-bound form. Type I binding spectral changes were observed when CYP158A3 was titrated with myristic acid; however, no oxidative product was formed. An analog of flaviolin, 2-hydroxynaphthoquinone (2-OH NQ) displayed similar type I binding upon titration with purified CYP158A3. It underwent an enzymatic reaction forming dimerized product. A homology model of CYP158A3 was superimposed with the structure of CYP158A2, and the majority of structural elements aligned. These results suggest that CYP158A3 might be an orthologue of biflaviolin synthase, catalyzing C-C coupling reactions during pigment biosynthesis in S. avermitilis.
Subject(s)
Amino Acid Sequence , Cytochrome P-450 Enzyme System , Genome , Myristic Acid , Oligomycins , StreptomycesABSTRACT
Gene deletion vector pLJ04(pKC1139∷bkdF+bkdH)was used to disrupt bkdFGH in Streptomyces avermitilis 76-02-e,an industrial producer of anthelmintic avermectin.The disruptants were confirmed by PCR.Shake flask experiment and HPLC analysis showed that the mutant lost the ability to produce avermectins.As it is expected,the mutant,named S.avermitilis bkd76-3,could restore the ability of producing avermectins when the feeding of methylbutyric acid and isobutyric acid to its fermentations was carried out.The addition of cyclohexanecarboxylic acid(CHC)into fermentations of the S.avermitilis bkd76-3 allowed for production of four components,two of which was confirmed as CHC-B1 and CHC-A2 by LC/MS analysis,respectively.