Chartchai Khanongnuch

β-Mannanase and xylanase of Bacillus subtilis 5H active for bleaching of crude pulp

A bacterial strain, 5H was isolated as a utilizer of locust bean gum (LBG) from soil in Chiang Mai, Thailand, and identified as Bacillus subtilis. Strain 5H produced β-mannanase and xylanase, which were purified to homogeneity from the culture filtrate by ion exchange chromatography and gel filtration. The molecular weights of β-mannanase and xylanase were found to be 37,000 and 26,000 by sodium dodecyl sulfonate polyacrylamide gel electrophoresis, respectively. The enzymatic properties of both were determined. Mannanase hydrolyzed LBG and konjak mannan endwisely, to a final hydrolytic degree of 15% and 21%, respectively, and the main products of both were disaccharides. Xylanase hydrolyzed larch wood xylan and oat spelt xylan, to a final hydrolytic degree of 19% and 38%, respectively, and produced mainly disaccharides. This culture filtrate (4.9 units/ml β-mannanase and 3.2 units/ml xylanase), a mixture of purified β-mannanase (4.9 units/ml) and xylanase (3.2 units/ml), and purified xylanase (3.2 units/ml) bleached crude paper pulp from 30% to 38% brightness, and xylanase was found to be more effective for paper-bleaching than β-mannanase.

Purification and characterization of the extracellular laccase produced by Trametes polyzona WR710-1 under solid-state fermentation

Laccase from Trametes polyzona WR710–1 was produced under solid‐state fermentation using the peel from the Tangerine orange (Citrus reticulata Blanco) as substrate, and purified to homogeneity. This laccase was found to be a monomeric protein with a molecular mass of about 71 kDa estimated by SDS–PAGE. The optimum pH was 2.0 for ABTS, 4.0 for L‐DOPA, guaiacol, and catechol, and 5.0 for 2,6‐DMP. The Km value of the enzyme for the substrate ABTS was 0.15 mM, its corresponding Vmax value was 1.84 mM min−1, and the kcat/Km value was about 3960 s−1 mM−1. The enzyme activity was stable between pH 6.0 and 8.0, at temperatures of up to 40 °C. The laccase was inhibited by more than 50% in the presence of 20 mM NaCl, by 95% at 5 mM of Fe2+, and it was completely inhibited by 0.1 mM NaN3. The N‐terminal amino acid sequence of this laccase is AVTPVADLQISNAGISPDTF, which is highly similar to those of laccases from other white‐rot basidiomycetes.

Use of Bacillus subtilis isolates from Tua-nao towards nutritional improvement of soya bean hull for monogastric feed application

Soya bean hull (SBH) is a cheap and high‐fibre content feed ingredient that obtained after soya bean oil extraction. Microbial fermentation was expected to improve SBH qualities before applying to animals, especially monogastric animals. Two bacterial strains, Bacillus subtilis MR10 and TK8 that were isolated from Tua‐nao, a traditional fermented soya bean in northern Thailand, were used for fermented soya bean hull (FSBH) production. Both could easily grow at 37°C in SBH as the sole substrate. MR10 produced the highest β‐mannanase activity (400 U g−1 SBH) on day 2, while TK8 produced the highest cellulase activity (14·5 U g−1 SBH) on day 3. After fermentation, the nutritional quality of SBH was obviously improved by an increase in soluble sugars, soluble proteins, crude protein and crude lipid, and a decrease in the content of raffinose family oligosaccharides. Scavenging activity (%) of SBH against ABTS radical cation was also increased from 14 to 27 and 20% by MR10 and TK8 fermentation, respectively. According to the GRAS property of these both strains and various improvements of nutritional values, the fermented SBH proved to be a potential feed ingredient, especially for the monogastric animals.

High efficacy bioconversion of starch to lactic acid using an amylolytic lactic acid bacterium isolated from Thai indigenous fermented rice noodles

Amylolytic lactic acid bacterium (ALAB) strain S21 was isolated from Thai indigenous fermented rice noodles and identified as Lactobacillus plantarum, based on 16S rDNA sequence and recA gene analysis. L. plantarum S21 exhibited a specific growth rate (μ) of 0.24 1/h in modified MRS broth containing 10 g/L of starch as the sole carbon source, and a high efficacy in producing lactic acid (9.41, 24.48, 41.84, 74.33, and 94.04 g/L from 10, 25, 50, 75, and 100 g/L of cassava starch, respectively), which are higher values than previously reported for ALAB. Crude amylase from L. plantarum S21 had broad pH stability (3.5–8.0), and hydrolyzed starch to maltose and glucose as the major and minor products. L. plantarum S21 should be considered useful for industrial bioconversion of starch to lactic acid.

A selected probiotic strain of Lactobacillus fermentum CM33 isolated from breast-fed infants as a potential source of β-galactosidase for prebiotic oligosaccharide synthesis

Lactic acid bacteria from healthy breast-fed infants were isolated and screened for β-galactosidase production in MRS broth. Among 49 isolates that exhibited the yellow clear zone on MRS agar supplemented with bromocresol blue, the isolate CM33 was selected as being the highest β-galactosidase producer and was identified as Lactobacillus fermentum based on its morphological characteristics and 16S rDNA nucleotide sequence. L. fermentum CM33 exhibited a good survival rate under the simulated stomach passage model, comparable to known probiotic strains L. gallinarum JCM2011 and L. agilis JCM1187. L. fermentum CM33 was antagonistic to pathogenic bacteria Listeria monocytogenes, Escherichia coli 0157:H7, Salmonella typhi, and Salmonella enteriditis, using the well diffusion method. In addition, the selected lactobacilli exhibited a high growth rate when cultivated in modified MRS containing commercial galactooligosaccharide (GOS) as a sole carbon source, as well as in glucose. A preliminary study on the enzymatic synthesis of oligosaccharide using crude β-galactosidase revealed the capability for oligosaccharide synthesis by the transgalactosylation activity.

Screening and selection of Bacillus spp. for fermented corticate soybean meal production

To screen and select the Bacillus spp. from Tua‐nao of northern Thailand for fermented corticate soybean meal (FCSBM) production.

Distribution of tannin-'tolerant yeasts isolated from Miang, a traditional fermented tea leaf (Camellia sinensis var. assamica) in northern Thailand.

Miang is a fermented food product prepared from the tea leaves of Camellia sinensis var. assamica, and is traditionally produced in mountainous areas of northern Thailand. Although Miang has a long history and reveals deep-rooted cultural involvement with local people in northern Thailand, little is known regarding its microbial diversity. Yeasts were isolated from 47 Miang samples collected from 28 sampling sites, including eight provinces in upper northern Thailand. A hundred and seven yeast isolates were recovered and identified within 14 species based on the comparison of the D1/D2 sequence of the large subunit (LSU) rRNA gene. Candida ethanolica was determined to be the dominant species that was frequently found in Miang together with minor resident yeast species. All yeast isolates demonstrated their tannin-tolerant capability when cultivated on yeast malt agar (YMA) containing 50g/l tannin, but nine isolates displayed clear zones forming around their colonies, e.g., Debaryomyces hansenii, Cyberlindnera rhodanensis, and Sporidiobolus ruineniae. The results obtained from a visual reading method of tannase revealed that all yeast isolates were positive for methyl gallate, indicating that they possess tannase activity. It is assumed that a tannin-tolerant ability is one of the most important factors for developing a yeast community in Miang. This research study is the first report to describe tannin-tolerant yeasts and yeast communities in traditionally fermented tea leaves.

Medium component improvement for β-galactosidase production by a probiotic strain Lactobacillus fermentum CM33

Plackett and Burman statistical design was applied to screen the nutritional factors affecting β-galactosidase production by Lactobacillus fermentum CM33. Accordingly, lactose, tryptone and Tween80 were found to be the positive effective factors at the significant level above 80%. Central composite design (CCD) and response surface plot predicted that the maximum enzyme activity of 49.81 U/100 ml would be obtained at 3.23 (w/v) lactose, 4.91% (w/v) tryptone and 0.62% (w/v) Tween80 and a trial experiment validated the activity total at 114% at 24 h of cultivation. However, the maximum activity of 63.31 U/100 ml culture was found at 16 h. The initial pH range 6.0 to 6.5 was found to be the most suitable for enzyme production. The final stage of optimization increased the productivity by 14.60 folds over that obtained with the non-optimized medium. The replacing of the expensive ingredients such as yeast extract, peptone and beef extract composed in the optimized medium by skimmed milk, did not significantly alter the enzyme productivity. Keywords: Medium optimization, Lactobacillus sp., β-galactosidase, experimental design

Cloning and nucleotide sequence of β-mannanase and cellulase genes from Bacillus sp. 5H

The two genes for β-mannanase and cellulase of Bacillus sp. 5H have been cloned in Escherichia coli JM 109 by a shotgun method, though the cellulase gene was not expressed in Bacillus sp. 5H. The nucleotide sequences of the β-mannanase gene and the cellulase gene revealed open reading frames of 1,086 and 1,503 base pairs, respectively, coding for a proteins of Mr 40,803 Da (β-mannanase) and 55,420 Da (cellulase). The deduced primary structure of β-mannanase comprised 362 amino acids which had a mature protein of 336 amino acids and a signal peptide of 26 amino acids and that of cellulase comprised 501 amino acid residues.

Molecular structure of cyclomaltodextrinase derived from amylolytic lactic acid bacterium Enterococcus faecium K-1 and properties of recombinant enzymes expressed in Escherichia coli and Lactobacillus plantarum

Gene encoding cyclomaltodextrinase (Cdx) from amylolytic lactic acid bacterium Enterococcus faecium K-1 was cloned and nucleotide sequence was analyzed. The open-reading frame consisted of 1767bp encoding 588 deduced amino acids. Consequently, four typically conserved regions of the glycoside hydrolase family 13 were revealed; however, nine exceeding amino acids (DSYQMTDVP) were found at the 282-290 position in comparison to previously reported cyclomaltodextrinases. This difference is believed to have an influence on the substrate specificity of this enzyme. The recombinant CDases expressed in Escherichia coli BL21 (CDX_E) and Lactobacillus plantarum WCFS1 (CDX_L) with high expression levels of 8041 and 5511U/L were purified by Ni-NTA affinity chromatography. The active form CDX is a dimeric protein with two identical subunits of 62kDa, approximately. Both CDX_E and CDX_L revealed nearly similar properties, but the thermostability of CDX_L was slightly higher. Mn2+ and Co2+ at a concentration of 1mM stimulated the enzyme activity, while the Ag+, Cu2+ and SDS solution completely inhibited enzyme activity. CDX exhibited the highest activity with α-cyclodextrin and β-cyclodextrin, but lower toward pullulan and starch. Importantly, this is the first report describing genes, the molecular structure and properties of cyclomaltodextrinase derived from lactic acid bacteria E. faecium.