• Microbiology and Biotechnology Letters
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• v.27 no.5
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• pp.399-403
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• 1999

For the production of trehalose, microorganisms capable of producing trehalose extracellularly were screened from the stock cultures in our laboratory. among them, Micrococcus luteus IFO 12708 showed the highest productivity of trehalose. For the increase of productivity, the mutant strai Hs-208 having higher trehalose production was selected with NTG(N-methyl-N'-nitrosoguanidine) mutagenesis, which led to the decrease of the specific activity of trehalose phosphorylase(3.2-fold) as compared to the wild strain. The optimum condition for the trehalose production was established as follows: 20g/l of glucose and 6g/l of tryptone were used as a sole carbon source and nitrogen source, respectively, and cultivations were carried out at 3$0^{\circ}C$ and pH 6.0. After 20hrs cultivation, addition of 20unit/ml penicillin G led to the higher conversion yield of trehalose. Under the optimum condition, 6.547g/l trehalose was produced with conversion yield of 32.7%.

• Journal of Microbiology and Biotechnology
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• v.32 no.8
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• pp.1054-1063
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• 2022

Trehalose is a non-conventional sugar with potent applications in the food, healthcare and biopharma industries. In this study, trehalose was synthesized from maltose using whole-cell Pseudomonas monteilii TBRC 1196 producing trehalose synthase (TreS) as the biocatalyst. The reaction condition was optimized using 1% Triton X-100 permeabilized cells. According to our central composite design (CCD) experiment, the optimal process was achieved at 35℃ and pH 8.0 for 24 h, resulting in the maximum trehalose yield of 51.60 g/g after 12 h using an initial cell loading of 94 g/l. Scale-up production in a lab-scale bioreactor led to the final trehalose concentration of 51.91 g/l with a yield of 51.60 g/g and productivity of 4.37 g/l/h together with 8.24 g/l glucose as a byproduct. A one-pot process integrating trehalose production and byproduct bioremoval showed 53.35% trehalose yield from 107.4 g/l after 15 h by permeabilized P. moteilii cells. The residual maltose and glucose were subsequently removed by Saccharomyces cerevisiae TBRC 12153, resulting in trehalose recovery of 99.23% with 24.85 g/l ethanol obtained as a co-product. The present work provides an integrated alternative process for trehalose production from maltose syrup in bio-industry.

• Journal of Microbiology and Biotechnology
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• v.31 no.10
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• pp.1455-1464
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• 2021

Trehalose is a non-reducing disaccharide in increasing demand for applications in food, nutraceutical, and pharmaceutical industries. Single-step trehalose production by trehalose synthase (TreS) using maltose as a starting material is a promising alternative process for industrial application due to its simplicity and cost advantage. Pseudomonas monteilii TBRC 1196 was identified using the developed screening method as a potent strain for TreS production. The TreS gene from P. monteilii TBRC 1196 was first cloned and expressed in Escherichia coli. Purified recombinant trehalose synthase (PmTreS) had a molecular weight of 76 kDa and showed optimal pH and temperature at 9.0 and 40℃, respectively. The enzyme exhibited >90% residual activity under mesophilic condition under a broad pH range of 7-10 for 6 h. Maximum trehalose yield by PmTreS was 68.1% with low yield of glucose (4%) as a byproduct under optimal conditions, equivalent to productivity of 4.5 g/l/h using enzyme loading of 2 mg/g substrate and high concentration maltose solution (100 g/l) in a lab-scale bioreactor. The enzyme represents a potent biocatalyst for energy-saving trehalose production with potential for inhibiting microbial contamination by alkaline condition.

• Microbiology and Biotechnology Letters
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• v.44 no.4
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• pp.497-503
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• 2016

This study aimed to optimize the culture conditions of recombinant Escherichia coli expressing otsBA using crude glycerol for the enhanced production of trehalose. The effects of culture temperature and isopropyl ${\beta}$-D-1-thiogalactopyranoside (IPTG)-induction were investigated. Trehalose production and cell growth were highest when cells were cultured at $37^{\circ}C$ and induced with IPTG. The concentrations of IPTG, validamycin A, and NaCl were optimized using Box-Behnken design. Statistical analyses of the experimental data revealed that the concentrations of IPTG and NaCl had significant effects on trehalose production, but that of validamycin A did not. Contour plot analysis and model calculation showed that the highest amount of trehalose could be produced at 298 mM NaCl and 0.1 mM IPTG. Under these optimal conditions, the optical density at 600 nm and trehalose production were $5.4{\pm}0.2$ and $304{\pm}15mg/l$, respectively.

• KSBB Journal
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• v.21 no.4
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• pp.298-301
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• 2006

A gene(GeneBank AF 135796) coding for a trehalose synthase from Thermus caldophilus GK24 was cloned into Escherichia coli K12 using five vector systems. The constitutive expression system(pHCETS) which shows the highest trehalose synthase activity from flask culture of recombinant E. coli was selected for the production of trehalose from maltose. For the shake flask culture, the final dry cell weight was 0.9 g/L and the trehalose synthase activity was 25 U/mL. Fed-batch culture of recombinant E. coli harboring plasmid pHCETS which uses the glycerolas a carbon source was performed in jar fermentor: the dry cell weight of 20 g/L and the trehalose synthase activity of 13.7 U/mL were attained in 48 h.

• Preventive Nutrition and Food Science
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• v.16 no.4
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• pp.357-363
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• 2011

Recently, we found that Arthrobacter crystallopoietes N-08 isolated from soil directly produces trehalose from maltose by a resting cell reaction. In this study, the optimal set of conditions and substrate specificity for the trehalose production using resting cells was investigated. Optimum temperature and pH of the resting cell reaction were $55^{\circ}C$ and pH 5.5, respectively, and the reaction was stable for two hours at $37{\sim}55^{\circ}C$ and for one hour at the wide pH ranges of 3~9. Various disaccharide substrates with different glycosidic linkages, such as maltose, isomaltose, cellobiose, nigerose, sophorose, and laminaribiose, were converted into trehalose-like spots in thin layer chromatography (TLC). These results indicated broad substrate specificity of this reaction and the possibility that cellobiose could be converted into other trehalose anomers such as ${\alpha},{\beta}$- and ${\beta},{\beta}$-trehalose. Therefore, the product after the resting cell reaction with cellobiose was purified by ${\beta}$-glucosidase treatment and Dowex-1 ($OH^-$) column chromatography and its structure was analyzed. Component sugar and methylation analyses indicated that this cellobiose-conversion product was composed of only non-reducing terminal glucopyranoside. MALDI-TOF and ESI-MS/MS analyses suggested that this oligosaccharide contained a non-reducing disaccharide unit with a 1,1-glucosidic linkage. When this disaccharide was analyzed by $^1H$-NMR and $^{13}C$-NMR, it gave the same signals with ${\alpha}$-D-glucopyranosyl-(1,1)-${\alpha}$-D-glucopyranoside. These results suggest that cellobiose can be converted to ${\alpha},{\alpha}$-trehalose by the resting cells of A. crystallopoietes N-08.

• Journal of Applied Biological Chemistry
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• v.43 no.4
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• pp.240-245
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• 2000

Two genes encoding maltooligosyltrehalose synthase (SaMTS) and maltooligosyltrehalose trehalohydrolase (SaMTH) were isolated from a hyperthermophilic microorganism, Sulfolobus acidocaldarius (ATCC 49462). ORFs of the SaMTS and SaMTH genes are 2,163 and 1,671 bp long and encode 720 and 556 amino acid residues, respectively. A bifunctional fusion enzyme (SaMTSH) was constructed through the gene fusion of SaMTS and SaMTH. Recombinant SaMTS, SaMTH, and SaMTSH fusion enzyme were overexpressed in E. coli BL21. SaMTS and SaMTH produced trehalose and maltotriose from maltopentaose in a sequential reaction. SaMTSH fusion enzyme catalyzed the sequential reaction in which the formation of maltotriosyltrehalose was followed by hydrolysis leading to the synthesis of trehalose and maltotriose. The SaMTSH fusion enzyme showed the highest activity at pH 5.0-5.5 and $70-75^{\circ}C$. SaMTS, SaMTH, and SaMTSH fusion enzyme were active in soluble starch, which resulted in the production of trehalose.

• Journal of Microbiology and Biotechnology
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• v.18 no.9
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• pp.1544-1549
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• 2008

MhMTS and MhMTH are trehalose ($\alpha$-D-glucopyranosyl-[1,1]-$\alpha$-D-glucopyranose) biosynthesis genes of the thermophilic microorganism Metallosphaera hakonensis, and encode a maltooligosyltrehalose synthase (MhMTS) and a maltooligosyltrehalose trehalohydrolase (MhMTH), respectively. In this study, the two genes were fused in-frame in a recombinant DNA, and expressed in Escherichia coli to produce a bifunctional fusion enzyme, MhMTSH. Similar to the two-step reactions with MhMTS and MhMTH, the fusion enzyme catalyzed the sequential reactions on maltopentaose, maltotriosyltrehalose formation, and following hydrolysis, producing trehalose and maltotriose. Optimum conditions for the fusion enzyme-catalyzed trehalose synthesis were around $70^{\circ}C$ and pH 5.0-6.0. The MhMTSH fusion enzyme exhibited a high degree of thermostability, retaining 80% of the activity when pre-incubated at $70^{\circ}C$ for 48 h. The stability was gradually abolished by incubating the fusion enzyme at above $80^{\circ}C$. The MhMTSH fusion enzyme was active on various sizes of maltooligosaccharides, extending its substrate specificity to soluble starch, the most abundant natural source of trehalose production.

• KSBB Journal
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• v.18 no.1
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• pp.8-13
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• 2003

Recombinant trehalose synthase from Thermus caldophilus GK24 showed an ability to produce trehalose from maltose. The activity of the partially purified enzyme was not influenced by most metal ions at 1 mM but was inhibited by 10 mM $Co^{2+}$, $Mn^{2+}$, and $Fe^{2+}$. Enzyme activity varied during prolonged reaction due to changes in the environmental conditions. Thus, the reaction was carried out for an extended time with optimized conditions of $45^{\circ}C$ and pH 7.0. An yield of 32.9% was reached at $60^{\circ}C$ after reaction for 22 h, and, maximum trehalose conversion (69.2%) was attained at $25^{\circ}C$. The yields obtained using enzyme dosages of 10, 25, and 50 U/g were 62.3, 62.3 and 59.0 %, respectively, though the initial conversion rate was higher when the higher dose was used. Similar profiles of trehalose production yields were observed with reaction working volumes of 10 ml to 2,000 ml.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.12
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• pp.1614-1619
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• 2009

The present study was conducted to observe the effect of increased osmolality of basic tris extender supplemented with trehalose and sucrose on post-thawing quality (motility, progressive motility, viability, the rate of acrosome abnormality, total abnormality and membrane integrity) of Markhoz goat spermatozoa. Fresh semen samples were evaluated for motility and sperm concentration. Only semen samples with motility more than 70% and sperm concentration higher than $3{\times}10^{9}$ sperm/ml were used for cryopreservation. In Exp. 1, trehalose (50, 75 or 100 mM) and sucrose (40, 60 or 80 mM) were added to a basic tris diluent. Based on the results of experiment 1, the goal of Exp. 2 was to investigate the combinational effects of the highest and lowest concentrations ($T_{100}+S_{80}$ or $T_{50}+S_{40}$) of trehalose and sucrose. As the control, semen was diluted and frozen in the tris diluent without trehalose or sucrose. The results in Exp. 1 showed that all evaluated spermatozoa characteristics improved significantly after freezing and thawing (p<0.05) and at the same time the increase of trehalose and sucrose concentrations in basic extenders was seen, with the best results obtained for extenders containing 70 and 100 mM trehalose and 80 mM sucrose. Comparing these results with those of control diluents, the effects of supplementation were significantly (p<0.05) better. In Exp. 2, the results showed no significant differences (p>0.05) between $T_{100}+S_{80}$ and $T_{50}+S_{40}$ extenders, but the results of $T_{50}+S_{40}$were slightly better than obtained with $T_{100}+S_{80}$ diluents. Furthermore, the results of this experiment indicated that the sperm characteristics in the isotonic control extender were significantly (p<0.05) lower than examined extenders. In conclusion, the results of this study indicated that goat sperm can tolerate hypertonic trehalose and sucrose solutions better than isotonic control diluents in the freezing period. In particular, these positive effects have been shown for acrosome integrity, which is very important for the fertilization capacity of sperm. The data indicated that addition of trehalose plus sucrose to the freezing extender can be recommended for cryopreservation of goat spermatozoa, but more data is needed on pregnancy rate, acrosome reaction and IVF to ascertain the real effect.