• Journal of Microbiology and Biotechnology
• /
• v.18 no.5
• /
• pp.901-907
• /
• 2008

Thermotoga neapolitana $\beta$-glucosidase (BglA) was subjected to site-directed mutagenesis in an effort to increase its ability to synthesize arbutin derivatives by transglycosylation. The transglycosylation reaction of the wild-type enzyme displays major ${\beta}(1,6)$ and minor ${\beta}(1,3)$ or ${\beta}(1,4)$ regioselectivity. The three mutants, N291T, F412S, and N291T/F412S, increased the ratio of transglycosylation/hydrolysis compared with the wild-type enzyme when pNPG and arbutin were used as a substrate and an acceptor, respectively. N291T and N219T/F412S had transglycosylation/hydrolysis ratios about 3- and 8-fold higher, respectively, than that of the wild-type enzyme. This is due to the decreased hydrolytic activity of the mutant rather than increased transglycosylation activity. Interestingly, N291T showed altered regioselectivity, as well as increased transglycosylation products. TLC analysis of the transglycosylation products indicated that N291T retained its ${\beta}(1,3)$ regioselectivity, but lost its ${\beta}(1,4)$ and ${\beta}(1,6)$ regioselectivity. The altered regioselectivity of N291T using two other acceptors, esculin and salicin, was also confirmed by TLC. The major transglycosylation products of the wild type and N291T mutant were clearly different. This result suggests that Asn-291 is highly involved in the catalytic mechanism by controlling the transglycosylation reaction.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.12
• /
• pp.1547-1556
• /
• 2009

A novel thermostable $\alpha$-glucosidase (TtGluA) from Thermoanaerobacter tengcongensis MB4 was successfully expressed in E. coli and characterized. The TtgluA gene contained 2,253 bp, which encodes 750 amino acids. The native TtGluA was a trimer with monomer molecular mass of 89 kDa shown by SDS-PAGE. The purified recombinant enzyme showed hydrolytic activity on maltooligosaccharides, p-nitrophenyl-$\alpha$-D-glucopyranide, and dextrin with an exotype cleavage manner. TtGluA showed preference for short-chain maltooligosaccharides and the highest specific activity for maltose of 3.26 units/mg. Maximal activity was observed at $60^{\circ}C$ and pH 5.5. The half-life was 2 h at $60^{\circ}C$. The enzyme showed good tolerance to urea and SDS but was inhibited by Tris. When maltose with the concentration over 50 mM was used as substrate, TtGluA was also capable of catalyzing transglycosylation to produce $\alpha$-1,4-linked maltotriose and $\alpha$-1,6-linked isomaltooligosaccharides. More importantly, TtGluA showed exclusive regiospecificity with high yield to produce $\alpha$-1,6-linked isomaltooligosaccharides when the reaction time extended to more than 10 h.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.6
• /
• pp.771-780
• /
• 2014

A putative lipolytic enzyme gene, named as est9x, was obtained from a marine microbial metagenome of the South China Sea. Sequence analysis showed that Est9X shares lower than 27% sequence identities with the characterized lipolytic enzymes, but possesses a catalytic triad highly conserved in lipolytic enzymes of the ${\alpha}/{\beta}$ hydrolase superfamily. By phylogenetic tree construction, Est9X was grouped into a new lipase/esterase family. To understand Est9X protein in depth, it was recombinantly expressed, purified, and biochemically characterized. Within potential hydrolytic activities, only lipase/esterase activity was detected for Est9X, confirming its identity as a lipolytic enzyme. When using p-nitrophenol esters with varying lengths of fatty acid as substrates, Est9X exhibited the highest activity to the C2 substrate, indicating it is an esterase. The optimal activity of Est9X occurred at a temperature of $65^{\cric}C$, and Est9X was pretty stable below the optimum temperature. Distinguished from other salt-tolerant esterases, Est9X's activity was tolerant to and even promoted by as high as 4 M NaCl. Our results imply that Est9X is a unique esterase and could be a potential candidate for industrial application under extreme conditions.

• Journal of the Korea Academia-Industrial cooperation Society
• /
• v.10 no.1
• /
• pp.194-199
• /
• 2009

An efficient production method of food-grade heamatococcus extract was developed based on stepwise enzymatic hydrolysis. In the first step, Haematococcus pluvialis cells hydrolysis carried out with commercially available exopeptidase(Flavourzyme) and endopeptidase (Alcalase), resulted in increased astaxanthin content. In the second step, proteolytic hydrolyzed H. pluvialis cells treated with hetero-polysaccharides hydrolytic enzyme (Viscozyme). By two-stage treatments using Alcalase and Flavourzyme and Viscozyme, the highest astaxanthin content was obtained. The astaxanthin content was remarkably enhanced by 320% $(529{\mu}g/g\rightarrow2,256{\mu}g/g)$ than that of the non-treated extract. And then, antioxidative activities determined by DPPH method were increased with increasing the astaxanthin content in haematococcus extract prepared by enzymatic hydrolysis.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.10
• /
• pp.1403-1412
• /
• 2013

Ethanol fuel production from lignocellulosic biomass is emerging as one of the most important technologies for sustainable development. To use this biomass, it is necessary to circumvent the physical and chemical barriers presented by the cohesive combination of the main biomass components, which hinders the hydrolysis of cellulose and hemicellulose into fermentable sugars. This study evaluated the hydrolytic capacity of enzymes produced by yeasts, isolated from the soils of the Brazilian Cerrado biome (savannah) and the Amazon region, on sugarcane bagasse pre-treated with $H_2SO_4$. Among the 103 and 214 yeast isolates from the Minas Gerais Cerrado and the Amazon regions, 18 (17.47%) and 11 (5.14%) isolates, respectively, were cellulase-producing. Cryptococcus laurentii was prevalent and produced significant ${\beta}$-glucosidase levels, which were higher than the endo- and exoglucanase activities. In natura sugarcane bagasse was pre-treated with 2% $H_2SO_4$ for 30 min at $150^{\circ}C$. Subsequently, the obtained fibrous residue was subjected to hydrolysis using the Cryptococcus laurentii yeast enzyme extract for 72 h. This enzyme extract promoted the conversion of approximately 32% of the cellulose, of which 2.4% was glucose, after the enzymatic hydrolysis reaction, suggesting that C. laurentii is a good ${\beta}$-glucosidase producer. The results presented in this study highlight the importance of isolating microbial strains that produce enzymes of biotechnological interest, given their extensive application in biofuel production.

• The Korean Journal of Mycology
• /
• v.39 no.2
• /
• pp.126-130
• /
• 2011

In the course of search for multifunctional microbial inoculants, three Bacillus strains (BS11-1,BS11-2,BS11-3) with biological control and biofertilizing effects were selected. In this study, their ability for solubilization of insoluble phosphate, production of indole-3-acetic acid (IAA), siderophore, and hydrolytic enzymes, and antagonism against phytopathogenic fungi were estimated. All strains produced IAA and siderophore depending on culture time and produced a visible clear zone on agar plate containing 0.5% carboxylmethyl cellulose as a carbon source. Also, these strains exhibited antifungal activities against phytopathogenic fungi, Botrytis cinerea, Cylindrocarpon destructans, Fusarium oxysporum, Rhizoctonia solani, and Phytophthora capsici.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.1
• /
• pp.19-25
• /
• 2014

An extracellular agarase was purified from Bacillus sp. BI-3, a thermophilic agar-degrading bacterium isolated from a hot spring in Indonesia. The purified agarase revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 58 kDa. The optimum pH and temperature of the agarase were 6.4 and $70^{\circ}C$, respectively. The activity of the agarase was stable at high temperatures, and more than 50% activity was retained at $80^{\circ}C$ for 15 min. Furthermore, the enzyme was stable in the pH range of 5.8-8.0, and more than 60% of the residual activity was retained. Significant activation of the agarase was observed in the presence of $K^+$, $Na^+$, $Ca^{2+}$, $Mg^{2+}$, and $Sr^{2+}$; on the other hand, $Ba^{2+}$, $Zn^{2+}$, $Cu^{2+}$, $Mn^{2+}$, $Co^{2+}$, $Fe^{2+}$, and EDTA inhibited or inactivated the enzyme activity. The components of the hydrolytic product analyzed by thin-layer chromatography showed that the agarase mainly produced neoagarobiose. This study is the first to present evidence of agarolytic activity in aerobic thermophilic bacteria.

• Applied Biological Chemistry
• /
• v.40 no.5
• /
• pp.395-399
• /
• 1997

Lintnerization properties and yield of enzyme-resistant starch in maize starches with different amylose content were investigated. Hydrolytic patterns of starches showed two distinct stages. With decreasing the amylose content, hydrolysis extents of starch increased. X-ray diffraction patterns of Amloca, PFP and commercial maize starch were the A crystalline type, and those of Amaizo 5 and Amylomaize VII were the B crystalline type. As acid hydrolysis time increased, the relative crystallinity of starches increased. After heating-cooling treatment was repeated up to 4 times, the yield of RS increased with increasing the amylose content. The yields of RS in lintnerzed maize starches decreased with acid treatment.

• Korean Journal of Food Science and Technology
• /
• v.53 no.6
• /
• pp.695-700
• /
• 2021

The purpose of this study was to prepare low-molecular weight collagen using a commercial proteolytic enzyme (Protamex) from collagen extracted from feet of Korean native chicken and to investigate the quality characteristics of this collagen. The collagen content of Korean native chicken feet was 13.9 g/100 g, which was higher than the 6.21 g/100 g of general broilers. It was found that the content of low molecular weight collagen increased as the concentration of proteolytic enzymes and reaction time increased. In particular, reaction with 1% Protamex for 7 h resulted in 55.6% of low molecular weight (1,000-5,000 Da) collagen content, and the average molecular weight was 5,390 Da. Regarding the texture of the enzyme-treated collagen, the collagen with high molecular weight peptides decomposed into low molecular weight peptides, and the gel type could not be formed, whereas the sol type was maintained.

• Journal of Microbiology and Biotechnology
• /
• v.32 no.4
• /
• pp.447-457
• /
• 2022

Notoginsenoside R1 and ginsenoside Rg1 are the main active ingredients of Panax notoginseng, exhibiting anti-fatigue, anti-tumor, anti-inflammatory, and other activities. In a previous study, a GH39 β-xylosidase Xln-DT was responsible for the bioconversion of saponin, a natural active substance with a xylose group, with high selectivity for cleaving the outer xylose moiety of notoginsenoside R1 at the C-6 position, producing ginsenoside Rg1 with potent anti-fatigue activity. The optimal bioconversion temperature, pH, and enzyme dosage were obtained by optimizing the transformation conditions. Under optimal conditions (pH 6.0, 75℃, enzyme dosage 1.0 U/ml), 1.0 g/l of notoginsenoside R1 was converted into 0.86 g/l of ginsenoside Rg1 within 30 min, with a molar conversion rate of approximately 100%. Furthermore, the in vivo anti-fatigue activity of notoginsenoside R1 and ginsenoside Rg1 were compared using a suitable rat model. Compared with the control group, the forced swimming time to exhaustion was prolonged in mice by 17.3% in the Rg1 high group (20 mg/kg·d). Additionally, the levels of hepatic glycogen (69.9-83.3% increase) and muscle glycogen (36.9-93.6% increase) were increased. In the Rg1 group, hemoglobin levels were also distinctly increased by treatment concentrations. Our findings indicate that treatment with ginsenoside Rg1 enhances the anti-fatigue effects. In this study, we reveal a GH39 β-xylosidase displaying excellent hydrolytic activity to produce ginsenoside Rg1 in the pharmaceutical and food industries.