Renukaradhya K. Math

Novel multiplex PCR for the detection of lactic acid bacteria during kimchi fermentation

We developed a multiplex PCR assay for the detection of lactic acid bacteria (LAB) species, and used it to examine the LAB species involved in kimchi fermentation. The LAB profile during kimchi fermentation varied with pH and acidity. Leuconostoc mesenteroides was observed during early fermentation (pH 5.64-4.27 and acidity 0.48-0.89%), and Lactobacillus sakei become dominant later in fermentation (pH <or=4.15 and acidity >or=0.98%). The efficiency of the multiplex PCR ranged from 86.5% at day 0 (pH 6.17 and acidity 0.24%) to 100% at day 96 (pH 4.16 and acidity 1.14). This multiplex PCR assay will facilitate study of the microbial ecosystem of kimchi and its impact on kimchi fermentation.

Organophosphorus hydrolase (OpdB) of Lactobacillus brevis WCP902 from kimchi is able to degrade organophosphorus pesticides.

Lactobacillus brevis WCP902 that is capable of biodegrading chlorpyrifos was isolated from kimchi. The opdB gene cloned from this strain revealed 825 bp, encoding 274 aa, and an enzyme molecular weight of about 27 kDa. OpdB contains the same Gly-X-Ser-X-Gly motif found in most bacterial and eukaryotic esterase, lipase, and serine hydrolases, yet it is a novel member of the GDSVG family of esterolytic enzymes. Its conserved serine residue, Ser82, is significantly involved with enzyme activity that may have application for removing some pesticides. Optimum organophosphorus hydrolase (OpdB) activity appeared at pH 6.0 and 35 degrees C and during degradation of chlorpyrifos, coumaphos, diazinon, methylparathion, and parathion.

Changes in the activity of the multifunctional β-glycosyl hydrolase (Cel44C-Man26A) from Paenibacillus polymyxa by removal of the C-terminal region to minimum size

Paenibacillus polymyxa GS01 secretes Cel44C-Man26A as a multifunctional enzyme with cellulase, xylanase, lichenase, and mannanase activities. Cel44C-Man26A consists of 1,352 amino acids in which present a catalytic domain (CD) of the glycosyl hydrolase family 44 (GH44), fibronectin domain type 3 (Fn3), catalytic domain of glycosyl hydrolase family 26 (GH26), and a cellulose-binding module type 3 (CBM3). A truncated Cel44C-Man26A protein, consisting of 549 amino acid residues, reacted as a multifunctional mature enzyme despite the absence of the 10 amino acids containing GH44, Fn3, GH26, and CBM3. However, the multifunctional activity was not found in the mature Cel44C-Man26A protein truncated to less than 548 amino acids. The truncated Cel44C-Man26A proteins showed the optimum pH for the lichenase activity was pH 7.0, pH 6.0 for the xylanase and mannanase, and pH 5.0 for the cellulase. The truncated Cel44C-Man26A proteins exhibited enzymatic activity 40–120% higher than the full-length Cel44C.

Metagenomic characterization of oyster shell dump reveals predominance of Firmicutes bacteria

Metagenomic analyses were conducted to evaluate the biodiversity of oyster shell bacteria, under storage conditions, on the basis of 16s rDNA sequences. Temperature was recorded during a one year storage period, and the highest temperature (about 60°C) was observed after five months of storage. Bacterial diversity was greatest in the initial stage sample, with 33 different phylotypes classified under seven phyla (Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, Planctomycetes, Verrucomicrobia and unclassified bacteria), with 42.22% of phylotypes belonging to Proteobacteria. The lowest diversity was found in the high temperature (fermentation) stage sample, with 10 different phylotypes belonging to Firmicutes (78.57%) and Bacteroidetes. In the final stage sample, bacteria were found belonging to Proteobacteria, Bacteroidetes, and Firmicutes, and some were unclassified bacteria. Of the bacteria constituting the final stage metagenome, 69.70% belonged to Firmicutes. Our results show that bacteria belonging to phylum Firmicutes were predominant during fermentation, and during the final stages of oyster shell storage, which suggests that these bacteria supposed to be the key players for oyster shell biodegradation.

Comparative Analysis of the glg Operons of Pectobacterium chrysanthemi PY35 and Other Prokaryotes

A chromosomal region of Pectobacterium chrysanthemi PY35 that contains of genes for glycogen synthesis was isolated from a cosmid library. The operon consists of glycogen branching enzyme (glgB), glycogen debranching enzyme (glgX), ADP-glucose pyrophosphorylase (glgC), glycogen synthase (glgA), and glycogen phosphorylase (glgP) genes. Gene organization is similar to that of Escherichia coli. The purified ADP-glucose pyrophosphorylase (GlgC) was activated by fructose 1,6-bisphosphate and inhibited by AMP. The constructed glgX::Ω mutant failed to integrate into the chromosome of P. chrysanthemi by marker exchange. Phylogenetic analysis based on the 16S rDNA and the amino acid sequence of Glg enzymes showed correlation with other bacteria. γ-Proteobacteria have the glgX gene instead of the bacilli glgD gene in the glg operon. The possible evolutionary implications of the results among the prokaryotes are discussed.

Composted Oyster Shell as Lime Fertilizer Is More Effective Than Fresh Oyster Shell

Physio-chemical changes in oyster shell were examined, and fresh and composted oyster shell meals were compared as lime fertilizers in soybean cultivation. Structural changes in oyster shell were observed by AFM and FE-SEM. We found that grains of the oyster shell surface became smoother and smaller over time. FT-IR analysis indicated the degradation of a chitin-like compound of oyster shell. In chemical analysis, pH (12.3±0.24), electrical conductivity (4.1±0.24 dS m−1), and alkaline powder (53.3±1.12%) were highest in commercial lime. Besides, pH was higher in composted oyster shell meal (9.9±0.53) than in fresh oyster shell meal (8.4±0.32). The highest organic matter (1.1±0.08%), NaCl (0.54±0.03%), and moisture (15.1±1.95%) contents were found in fresh oyster shell meal. A significant higher yield of soybean (1.33 t ha−1) was obtained by applying composted oyster shell meal (a 21% higher yield than with fresh oyster shell meal). Thus composting of oyster shell increases the utility of oyster shell as a liming material for crop cultivation.

Cloning and Characterization of a Novel Carboxylesterase Gene from Cow Rumen Metagenomic Library

The gene encoding esterase enzyme was cloned from a metagenomic library of cow rumen bacteria. The esterase gene (est1R) was 2,465 bp in length, encoding a protein of 366 amino acid residues, and the molecular weight of the enzyme was 61,166 Da. Est1R of rumen cosmid library shared 5.9% amino acid identity with Est1R (P37967) of PNB carboxylesterase, 6.1% with Est1R (1EEAA) of acetylcholinesterase and 6.1% with Est1R (1H23A) of chain A. BlastP in NCBI database analysis of Est1R revealed that it was not homologous to previous known lipases and esterases. Est1R showed optimum activity at pH 7.0 and 40℃. On the other hand, the enzyme was found to be most active without organic solvent, followed by 95% activity with methanol, and the enzyme activity was highly affected by hexane (lost 51% activity). Therefore, the novel esterase gene est1R is likely obtainable from cow rumen metagenome and may be utilized for industrial purposes.

Cloning of Isoamylase Gene of Pectobacterium carotovorum subsp. carotovorum LY34 and Identification of Essential Residues of Enzyme

The gene encoding for isoamylase of the Pectobacterium carotovorum subsp. carotovorum (Pcc) LY34 was cloned and expressed into Escherichia coli DH5α. Isoamylase catalyzes the hydrolysis of α-1,6-glycosidic linkages specifically in amylopectin, glycogen, and derived oligosaccharides, while the enzyme did not hydrolyze α-1,4-glycosidic linkages of amylose. The isoamylase gene (glgX) had an open reading frame of 1,977 bp encoding 658 amino acid residues with a calculated molecular weight of 74,188 Da. The molecular weight of the enzyme was also estimated to be 74 kDa by activity staining of a SDS-PA gel. The mature GlgX had a calculated pI of 4.91. Isoamylase from Pcc LY34 had 70% amino acid identity with isoamylase from Pectobacterium chrysanthemi and contained the four regions conserved among all amylolytic enzymes. The isoamylase was optimally active at pH 7.0 and 40℃. GlgX was Ca²?-dependent. The changes of Asp-335, Glu-370, and Asp-442 into Ala, respectively, using site-directed mutagenesis techniques showed that three residues are essential to isolamyalse (GlgX) activity. The sequences around those residues were highly conserved in isoamylase of different origins and GlgX of the glg operon in glycongen biosynthesis.