Background Lipases are promising biocatalysts for industrial applications and attract attention to be explored. A novel acidic lipase has been isolated from the lipolytic bacteria Micrococcus luteus EMP48-D (LipEMP48-D) screened from tempeh. The lipase gene had previously been overexpressed in Escherichia coli BL21, but the expression level obtained was relatively low. Here, to improve the expression level, the lipase gene was cloned to Pichia pastoris. We eliminated the native signal sequence of M. luteus and replaced it with α-mating factor (α-MF) signal sequence. We also optimized and synthesized the lipase gene based on codon preference in P. pastoris. Results LipEMP48-D lipase was expressed as an extracellular protein. Codon optimization has been conducted for 20 codons, with the codon adaption index reaching 0.995. The highest extracellular lipase activity obtained reached 145.4 ± 4.8 U/mg under AOX1 promoter in P. pastoris KM71 strain, which was 9.7-fold higher than the previous activity in E. coli. LipEMP48-D showed the highest specific activity at pH 5.0 and stable within the pH range 3.0–5.0 at 40 °C. LipEMP48-D also has the capability of hydrolyzing various long-chain triglycerides, particularly olive oil (100%) followed by sunflower oil (88.5%). LipEMP48-D exhibited high tolerance for various polar organic solvents with low log P, such as isopropanol (115.7%) and butanol (114.6%). The metal ions (Na+, K+, Ca2+, Mg2+, Mn+) decreased enzyme activity up to 43.1%, while Fe2+ increased relative activity of enzymes up to 200%. The conversion of free fatty acid (FFA) into fatty acid methyl ester (FAME) was low around 2.95%. Conclusions This study was the first to report overexpression of Micrococcus lipase in yeast. The extracellular expression of this acidic lipase could be potential for biocatalyst in industrial fields, especially organic synthesis, food industry, and production of biodiesel.
The research was aimed to evaluate the effects of substitution level of corn with palm kernel meal (PKM) untreated and palm kernel meal (PKM) treated by enzyme on production performance and carcass quality of broilers. The method used in this research was experiment using nested of completely randomized design with 2 factors, the main factor was type of palm kernel meal factors consisting of PKM without enzyme (B 1 ) and PKM by mannanase enzyme (B 2 ), and substitution corn level L 0 (without substitution), L 1 (12.5%), L 2 (25%), L 3 (37.5%) and L 4 (50%). The different among the treatments were tested by Duncan's multiple range test. The results showed that corn substitution with PKM untreated and PKM treated by enzymes was significantly effect (P<0.01) on feed conversion ratio (FCR), tenderness, meat cholesterol of broilers and income over feed cost (IOFC) and significant effect (P<0.05) on feed intake, but there is no significant difference on body weight, percentages of abdominal fat and water holding capacity (WHC). Furthermore, effect of level corn substitition with PKM nested to PKM untreated and PKM treated by enzyme was significantly effect (P<0.01) on feed intake, body weight, FCR, and tenderness and significant effect (P<0.05) on IOFC, but there is no effect on percentages of abdominal fat, WHC and meat cholesterol. It can be concluded that the corn substitution with PKM treated by enzymes was better than the PKM untreated on production performance and carcass quality of broilers. The optimal level of PKM untreated as corn substitution was 12.5%. While replacement with PKM treated by enzyme can be used up to 25%.
ABSTRACT Random mutagenesis technique is a powerful technique capable of producing enzymes with desired biocatalytic activity. This study aims to obtain a mutant lipase with improved hydrolytic activity on palm oil substrate using random mutagenesis technique. Random mutagenesis by error-prone PCR was used to generate mutant lipases. A total of 1101 mutants were obtained, out of which two mutants, Lip M14.25, and Lip M14.57, showed an increased relative hydrolytic activity. Lip M14.25 and Lip M14.57 demonstrated a 14% and 16% increased activity respectively. A comparison of the mutants' hydrolytic activities using p-nitrophenyl esters showed a significantly high preference for p-nitrophenyl palmitate. Furthermore, the mutant, Lip M14.25 showed its highest activity at pH 5, and Lip M14.57 exhibited a 10 oC decrease in optimum temperature. The two mutants' protein modelling showed the substitution of N44S/S202N on M14.25 and F154L/S265C on M14.57 lipase, which caused changes in conformation and active site residue distance of the lipase. The study found two mutants of lipase, M14.25 and M14.57, which showed improved hydrolytic activity on palm oil substrate.
The genotype of antibiotic resistance in natural isolates of Escherichia coli was determined through integron detection and characterization of the associated antibiotic resistance. E. coli SG2 isolated from Varanus salvator of Java demonstrated resistance to spectinomycin (50ng/ml) and streptomycin (SOng/ml). Integron detection indicated that eight isolates out of nine E. coli isolates possessed a conserved segment of the integron. Amplification of the inserted cassette of the integron in this SG2 isolate yielded a 1-kb DNA fragment. Sequence analyses indicated that this fragment was homologous with aad gene, which confirmed the resistance to spectinomycin/streptomycin. This is the first report on the presence of integron in the E. coli isolated from the environment.
This study aimed to obtain a functional lipase (LipRM) from Ralstonia pickettii BK6 through a co-expression involving its foldase. The ORF of the LipRM was 999 bp while the gene encoding of lipase-specific foldase (LifRM) was 1,030 bp. LipRM and LifRM genes were cloned into a plasmid and were successfully co-expressed in Escherichia coli strains to produce functional LipRM. Enzyme activity from partially purified enzymes showed quite surprising results, LipRM activity in E. coli BL21 (DE3) was 25.84 U/ml, while the other strains (DH5α, HB101, S17-1λpir) were 628.98 U/ml, 761 U/ml, and 1206.46 U/ml, respectively. The highest relative activity of LipRM was found at 50-55°C and pH 7-8 with pNP-laurate (C12) as the preferred substrate specificity. LipRM activity was enhanced sharply in the presence of 30% organic solvents (methanol and ethanol) but decreased by more than 50% in the presence of detergents. This study was the first to report heterologous expression of Ralstonia pickettii lipase employing its native foldase resulting in functional lipase from subfamily I.2.
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