Construction Of Recombinant Escherichia Coli Bad85 For The Production Of High Purity L-Lactic Acid

Lactate dehydrogenase (LDH, EC 1.1.1.27) catalyzes the oxidation of pyruvate to lactate in facultative anaerobes. Two forms of lactate dehydrogenase with different substrate specificities have been identified namely the L-lactate dehydrogenase (EC 1.1.1.27) and D-lactate dehydrogenase (EC 1.1.1.28). The L-lactate dehydrogenase is involved in the reduction of pyruvate into L-lactic acid whilst the D-lactate dehydrogenase is responsible for the formation of D-lactic acid. L-lactic acid is more preferable to D-lactic acid in the production of bioplastics since it is metabolizable in human and animals. The objective of this study is to isolate and clone the L-lactate dehydrogenase (L-ldh) gene from Enterococcus faecalis KK1 and express in Escherichia coli SZ85 for the production of L-lactic acid. E. coli SZ85 strain has five chromosomal deletions (pflB, ackA, adhE, ldhA, and frdBC), namely D-lactate dehydrogenase, pyruvate formate lyase, acetate kinase, alcohol/aldehyde dehydrogenase and fumarate reductase and a chromosomally integrated L-ldh gene from Pediococcus acidilactici. The 954 bp gene was isolated by using the polymerase chain reaction (PCR). Primers EF.f (forward) and EF.r (reverse) were designed based on published gene sequence of L-ldh, and the PCR amplified L-ldh gene was cloned into TOPO TA cloning vector. The DNA sequencing results revealed 99% homology with published sequence in the database. The gene was subcloned into E. coli expression vector (pBAD) using the restriction enzymes Eco R1 and Xho 1. The pBAD-ldh gene was later transformed into E. coli SZ85 using electroporation. Sodium dodecyl sulfide-polyacrylamide gel electrophoresis (SDS-PAGE) analyses indicated that L-lactate dehydrogenase recombinant protein was successfully expressed in E. coli SZ85 with the expected size of 40 kDa. Western blot analysis revealed an immunoreactive band at 40 kDa size which further confirmed the expression of L-ldh gene. In this study, the mouse monoclonal antibody acted as the primary antibody and horse radish peroxidase (HRP), conjugated to the secondary antibody (anti-goat antibody) was used as a probe to confirm the recombinant protein. The recombinant E. coli BAD85 underwent fermentation using shake flasks to establish the optimum pH and temperature conditions for lactic acid production from fructose and was conducted at pH between 5.0–7.0 and temperature 30-37ºC. The best condition was later selected to.investigate the effect of temperature and pH on the production of lactic acid using E. coli BAD85 in a 2-L bioreactor system. Batch cultivations in 2-L stirred tank fermenter were carried out using the conditions determined during shake flask fermentation to further improve L-lactic acid production by recombinant E. coli BAD85. Cultivation of E. coli BAD85 at pH 7.0 and incubation temperature of 37ºC was found to be the best condition for producing L-lactic acid. These conditions were able to produce 7.04 gL-1 L-lactic acid with a high purity of 98%, 0.70 gg-1 yield and productivity of 0.029 gg-1 h-1. The recombinant was able to achieve a 98% plasmid stability indicating that the cells were fairly stable for fermentation process

Engineering of E. coli for increased production of L-lactic acid

An over-expressed L-ldh gene derivative of Escherichia coli BAD-ldh was developed. L-ldh gene from Enterococcus facelis KK1 consisted of an open reading frame of 954 bp encoding 316 amino acids. L-ldh gene was cloned into pBAD vector and transformed into E. coli SZ85 by electroporation. SDS-page and western blotting method confirmed the presence of recombinant L-LDH enzyme with the approximate size of 40 kD. The activity of L-lactate dehydrogenase was achieved at 170 U ml-1. E. coli BAD85 was found to produce 0.62 g l-1 of lactic acid from 1 g l-1of fructose in 24 h. L-ldh gene from was successfully transformed into E. coli SZ85 with the maximum production of L-lactic acid at 0.62 g l-1

FTIR spectral changes in Candida albicans biofilm following exposure to antifungals

Candida albicans is a microbial fungus that exists as a commensal member of the human microbiome and an opportunistic pathogen. Biofilm formation by this fungal pathogen occurs mostly in the mucosa or endothelium associated with candidiasis and colonizes medical devices. The present work was performed to determine the efficacy of the antifungal creams on the viability and biochemical composition of C. albicans biofilm. Four commercial antifungal creams were used herein namely econazole nitrate, miconazole nitrate, ketoconazole and tolnaftate. Resazurin assay and Fourier transform infrared (FTIR) spectroscopy were performed to determine the viability and biochemical composition of C. albicans biofilm, respectively. Results demonstrated that the antifungal creams inhibited C. albicans biofilm. The highest percent inhibition shown by econazole nitrate, miconazole nitrate, ketoconazole, and tolnaftate were 16.5%, 17.1%, 15.8%, and 6.9%, respectively. Econazole nitrate with the lowest IC50 value of 43.42 μg/mL caused changes in the FTIR spectral peak shape at 1377 cm-1 and 1736 cm-1. On the other hand, miconazole nitrate with the second lowest IC50 value of 118.26 μg/mL caused spectral peak shifting from 1237 cm-1 to 1228 cm-1. In conclusion, the inhibition of C. albicans biofilm may be mediated by the changes in protein, lipid, and nucleic acid compositions

FTIR spectral changes in Candida albicans biofilm following exposure to antifungals

Candida albicans is a microbial fungus that exists as a commensal member of the human microbiome and an opportunistic pathogen. Biofilm formation by this fungal pathogen occurs mostly in the mucosa or endothelium associated with candidiasis and colonizes medical devices. The present work was performed to determine the efficacy of the antifungal creams on the viability and biochemical composition of C. albicans biofilm. Four commercial antifungal creams were used herein namely econazole nitrate, miconazole nitrate, ketoconazole and tolnaftate. Resazurin assay and Fourier transform infrared (FTIR) spectroscopy were performed to determine the viability and biochemical composition of C. albicans biofilm, respectively. Results demonstrated that the antifungal creams inhibited C. albicans biofilm. The highest percent inhibition shown by econazole nitrate, miconazole nitrate, ketoconazole, and tolnaftate were 16.5%, 17.1%, 15.8%, and 6.9%, respectively. Econazole nitrate with the lowest IC50 value of 43.42 μg/mL caused changes in the FTIR spectral peak shape at 1377 cm-1 and 1736 cm-1. On the other hand, miconazole nitrate with the second lowest IC50 value of 118.26 μg/mL caused spectral peak shifting from 1237 cm-1 to 1228 cm-1. In conclusion, the inhibition of C. albicans biofilm may be mediated by the changes in protein, lipid, and nucleic acid compositions

Screening of Aloe vera medium with different carbon and nitrogen sources for Lactobacillus acidophilus cultivation using fractional factorial design (FFD)

The aim of this research was to optimize the cultivation medium for economic production of a probiotic bacterium, Lactobacillus acidophilus using Aloe vera medium with different carbon (glucose, fructose and sucrose) and nitrogen (yeast extract, meat peptone, ammonium sulphate and urea) sources. Screening step was performed using 28 1/16 fractional factorial design (FFD) to investigate the significant effect of 8 factors used in this study on the biomass production expressed in log10 cfu/ mL. Biomass production was measured based on total plate count method for enumeration of viable cells. In the process of screening, the concentration range of Aloe vera, carbon and nitrogen used were from 1-2% (w/v), 1-2% (w/v) and 0.5-1% (w/v), respectively. The maximum biomass production was obtained with 11.816 log10 cfu/mL. It was shown that glucose, Aloe vera gel, combination of glucose and fructose and combination of glucose and ammonium sulphate were resulted significant (p <; 0.05) effect towards to the response, biomass production

Engineering of E. coli for increased production of L-lactic acid

An over-expressed L-ldh gene derivative of Escherichia coli BAD-ldh was developed. L-ldh gene from Enterococcus facelis KK1 consisted of an open reading frame of 954 bp encoding 316 amino acids. L-ldh gene was cloned into pBAD vector and transformed into E.coli SZ85 by electroporation. SDS-page and western blotting method confirmed the presence of recombinant L-LDH enzyme with the approximate size of 40kD. The activity of L-lactate dehydrogenase was achieved at 170 U ml¯¹. E.coli BAD85 was found to produce 0.62 g l¯¹ of lactic acid from 1 g 1¯¹ of fructose in 24 h. L-ldh gene from was successfully transformed into E.coli SZ85 with the maximum production of L-lactic acid at 0.62 g l¯¹