Prof. Montarop Yamabhai

Year	Title	Journal
2018	Bioconversion of chitosan into chito-oligosaccharides (CHOS) using family 46 chitosanase from Bacillus subtilis (BsCsn46A) Pechsrichuang P., Lorentzen S.B., Aam B.B., Tuveng T.R., Hamre A.G., Eijsink V.G.H., Yamabhai M.	Carbohydrate Polymers
Abstract: BsCsn46A, a GH family 46 chitosanase from Bacillus subtilis had been previously shown to have potential for bioconversion of chitosan to chito-oligosaccharides (CHOS). However, so far, in-depth analysis of both the mode of action of this enzyme and the composition of its products were lacking. In this study, we have employed size exclusion chromatography, 1H NMR, and mass spectrometry to reveal that BsCsn46A can rapidly cleave chitosans with a wide-variety of acetylation degrees, using a non-processive endo-mode of action. The composition of the product mixtures can be tailored by varying the degree of acetylation of the chitosan and the reaction time. Detailed analysis of product profiles revealed differences compared to other chitosanases. Importantly, BsCsn46A seems to be one of the fastest chitosanases described so far. The detailed analysis of preferred endo-binding modes using H218O showed that a hexameric substrate has three productive binding modes occurring with similar frequencies. © 2018 Elsevier Ltd Keyword: Bacillus; Bioconversion; Chito-oligosaccharides; Chitosan; Chitosanase; GH46 Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85041519108&doi=10.1016%2fj.carbpol.2018.01.059&partnerID=40&md5=596fbaac8c6045d62e4d769d30e17244 DOI: 10.1016/j.carbpol.2018.01.059
2017	Immobilization of β-Galactosidases from Lactobacillus on Chitin Using a Chitin-Binding Domain Pham M.-L., Leister T., Nguyen H.A., Do B.-C., Pham A.-T., Haltrich D., Yamabhai M., Nguyen T.-H., Nguyen T.-T.	Journal of Agricultural and Food Chemistry
Abstract: Two β-galactosidases from Lactobacillus, including a heterodimeric LacLM type enzyme from Lactobacillus reuteri L103 and a homodimeric LacZ type β-galactosidase from Lactobacillus bulgaricus DSM 20081, were studied for immobilization on chitin using a carbohydrate-binding domain (chitin-binding domain, ChBD) from a chitinolytic enzyme. Three recombinant enzymes, namely, LacLM-ChBD, ChBD-LacLM, and LacZ-ChBD, were constructed and successfully expressed in Lactobacillus plantarum WCFS1. Depending on the structure of the enzymes, either homodimeric or heterodimeric, as well as the positioning of the chitin-binding domain in relation to the catalytic domains, that is, upstream or downstream of the main protein, the expression in the host strain and the immobilization on chitin beads were different. Most constructs showed a high specificity for the chitin in immobilization studies; thus, a one-step immobilizing procedure could be performed to achieve up to 100% yield of immobilization without the requirement of prior purification of the enzyme. The immobilized-on-chitin enzymes were shown to be more stable than the corresponding native enzymes; especially the immobilized LacZ from L. bulgaricus DSM20081 could retain 50% of its activity when incubated at 37 °C for 48 days. Furthermore, the immobilized enzymes could be recycled for conversion up to eight times with the converting ability maintained at 80%. These results show the high potential for application of these immobilized enzymes in lactose conversion on an industrial scale. © 2017 American Chemical Society. Keyword: chitin-binding domain; immobilization; Lactobacillus; β-galactosidase Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85040371014&doi=10.1021%2facs.jafc.6b04982&partnerID=40&md5=8bc70262f7c4de6138ec1be9aab81a2c DOI: 10.1021/acs.jafc.6b04982
2016	OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system Pechsrichuang P., Songsiriritthigul C., Haltrich D., Roytrakul S., Namvijtr P., Bonaparte N., Yamabhai M.	SpringerPlus
Abstract: The production of secreted recombinant proteins from E. coli is pivotal to the biotechnological industry because it reduces the cost of downstream processing. Proteins destined for secretion contain an N-terminal signal peptide that is cleaved by secretion machinery in the plasma membrane. The resulting protein is released in an active mature form. In this study, Bacillus subtilis chitosanase (Csn) was used as a model protein to compare the effect of two signal peptides on the secretion of heterologous recombinant protein. The results showed that the E. coli secretion machinery could recognize both native bacillus and E. coli signal peptides. However, only the native bacillus signal peptide could generate the same N-terminal sequence as in the wild type bacteria. When the recombinant Csn constructs contained the E. coli OmpA signal peptide, the secreted enzymes were heterogeneous, comprising a mixed population of secreted enzymes with different N-terminal sequences. Nevertheless, the E. coli OmpA signal peptide was found to be more efficient for high expression and secretion of bacillus Csn. These findings may be used to help engineer other recombinant proteins for secretory production in E. coli. © 2016, The Author(s). Keyword: Bacillus; Chitosanase; E. coli; Expression; OmpA; Recombinant; Secretion; Signal peptide Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-84979783863&doi=10.1186%2fs40064-016-2893-y&partnerID=40&md5=284b108c9927826444ece65d059817d1 DOI: 10.1186/s40064-016-2893-y
2016	Display of a β-mannanase and a chitosanase on the cell surface of Lactobacillus plantarum towards the development of whole-cell biocatalysts Nguyen H.-M., Mathiesen G., Stelzer E.M., Pham M.L., Kuczkowska K., Mackenzie A., Agger J.W., Eijsink V.G.H., Yamabhai M., Peterbauer C.K., Haltrich D., Nguyen T.-H.	Microbial Cell Factories
Abstract: Background: Lactobacillus plantarum is considered as a potential cell factory because of its GRAS (generally recognized as safe) status and long history of use in food applications. Its possible applications include in situ delivery of proteins to a host, based on its ability to persist at mucosal surfaces of the human intestine, and the production of food-related enzymes. By displaying different enzymes on the surface of L. plantarum cells these could be used as whole-cell biocatalysts for the production of oligosaccharides. In this present study, we aimed to express and display a mannanase and a chitosanase on the cell surface of L. plantarum. Results: ManB, a mannanase from Bacillus licheniformis DSM13, and CsnA, a chitosanase from Bacillus subtilis ATCC 23857 were fused to different anchoring motifs of L. plantarum for covalent attachment to the cell surface, either via an N-terminal lipoprotein anchor (Lp_1261) or a C-terminal cell wall anchor (Lp_2578), and the resulting fusion proteins were expressed in L. plantarum WCFS1. The localization of the recombinant proteins on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest mannanase and chitosanase activities obtained for displaying L. plantarum cells were 890 U and 1360 U g dry cell weight, respectively. In reactions with chitosan and galactomannans, L. plantarum CsnA- and ManB-displaying cells produced chito- and manno-oligosaccharides, respectively, as analyzed by high performance anion exchange chromatography (HPAEC) and mass spectrometry (MS). Surface-displayed ManB is able to break down galactomannan (LBG) into smaller manno-oligosaccharides, which can support growth of L. plantarum. Conclusion: This study shows that mannanolytic and chitinolytic enzymes can be anchored to the cell surface of L. plantarum in active forms. L. plantarum chitosanase- and mannanase-displaying cells should be of interest for the production of potentially 'prebiotic' oligosaccharides. This approach, with the enzyme of interest being displayed on the cell surface of a food-grade organism, may also be applied in production processes relevant for food industry. © 2016 The Author(s). Keyword: Cell wall anchor; Cell-surface display; Chitosanase; Lactobacillus plantarum; Lipoprotein anchor; Mannanase; Whole-cell biocatalyst Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-84992145732&doi=10.1186%2fs12934-016-0570-z&partnerID=40&md5=970a6d9e2d6ea7844ece57b84ff806b3 DOI: 10.1186/s12934-016-0570-z
2016	Mannan biotechnology: From biofuels to health Yamabhai M., Sak-Ubol S., Srila W., Haltrich D.	Critical Reviews in Biotechnology
Abstract: Mannans of different structure and composition are renewable bioresources that can be widely found as components of lignocellulosic biomass in softwood and agricultural wastes, as non-starch reserve polysaccharides in endosperms and vacuoles of a wide variety of plants, as well as a major component of yeast cell walls. Enzymatic hydrolysis of mannans using mannanases is essential in the pre-treatment step during the production of second-generation biofuels and for the production of potentially health-promoting manno-oligosaccharides (MOS). In addition, mannan-degrading enzymes can be employed in various biotechnological applications, such as cleansing and food industries. In this review, fundamental knowledge of mannan structures, sources and functions will be summarized. An update on various aspects of mannan-degrading enzymes as well as the current status of their production, and a critical analysis of the potential application of MOS in food and feed industries will be given. Finally, emerging areas of research on mannan biotechnology will be highlighted. © 2014 Informa Healthcare USA, Inc. Keyword: Beta-mannanase; biofuels; biorefinery; hemicelluloses; lignocellulose; mannan; mannooligosaccharides; prebiotic Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-84949320507&doi=10.3109%2f07388551.2014.923372&partnerID=40&md5=5d830809c81fa3891746c9e4786b67da DOI: 10.3109/07388551.2014.923372
2016	Secretory production of a beta-mannanase and a chitosanase using a Lactobacillus plantarum expression system Sak-Ubol S., Namvijitr P., Pechsrichuang P., Haltrich D., Nguyen T.-H., Mathiesen G., Eijsink V.G.H., Yamabhai M.	Microbial Cell Factories
Abstract: Background: Heterologous production of hydrolytic enzymes is important for green and white biotechnology since these enzymes serve as efficient biocatalysts for the conversion of a wide variety of raw materials into value-added products. Lactic acid bacteria are interesting cell factories for the expression of hydrolytic enzymes as many of them are generally recognized as safe and require only a simple cultivation process. We are studying a potentially foodgrade expression system for secretion of hydrolytic enzymes into the culture medium, since this enables easy harvesting and purification, while allowing direct use of the enzymes in food applications. Results: We studied overexpression of a chitosanase (CsnA) and a β-mannanase (ManB), from Bacillus licheniformis and Bacillus subtilis, respectively, in Lactobacillus plantarum, using the pSIP system for inducible expression. The enzymes were over-expressed in three forms: without a signal peptide, with their natural signal peptide and with the well-known OmpA signal peptide from Escherichia coli. The total production levels and secretion efficiencies of CsnA and ManB were highest when using the native signal peptides, and both were reduced considerably when using the OmpA signal. At 20 h after induction with 12.5 ng/mL of inducing peptide in MRS media containing 20 g/L glucose, the yields and secretion efficiencies of the proteins with their native signal peptides were 50 kU/L and 84 % for ManB, and 79 kU/L and 56 % for CsnA, respectively. In addition, to avoid using antibiotics, the erythromycin resistance gene was replaced on the expression plasmid with the alanine racemase (alr) gene, which led to comparable levels of protein production and secretion efficiency in a suitable, alr-deficient L. plantarum host. Conclusions: ManB and CsnA were efficiently produced and secreted in L. plantarum using pSIP-based expression vectors containing either an erythromycin resistance or the alr gene as selection marker. � 2016 The Author(s). Keyword: Alanine racemase; Bacillus; Chitosanase; Food-grade; L. plantarum; OmpA; PSIP; Secretion; Signal peptide; β-Mannanase Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85007467172&doi=10.1186%2fs12934-016-0481-z&partnerID=40&md5=cc8d58eb64b97b46d7e2f5df50dcf94e DOI: 10.1186/s12934-016-0481-z
2014	Characterization of recombinant malarial RecQ DNA helicase Suntornthiticharoen P., Srila W., Chavalitshewinkoon-Petmitr P., Limudomporn P., Yamabhai M.	Molecular and Biochemical Parasitology
Abstract: RecQ DNA gene of multi-drug resistant Plasmodium falciparum K1 (PfRecQ1) was cloned, and the recombinant C-terminal-decahistidine-tagged PfRecQ1 was expressed in Escherichia coli. The purified enzyme could efficiently unwind partial duplex DNA substrate in a 3′ to 5′ direction. The malarial RecQ1 could not unwind substrates with both 5′ and 3′ overhangs, those with a 5′ overhang, or blunt-ended DNA duplexes. Unwinding of DNA helicase activity was driven by the hydrolysis of ATP. The drug inhibitory effects of six compounds indicated that only doxorubicin and daunorubicin could inhibit the unwinding activity. © 2014 Elsevier B.V. Keyword: Cloning; DNA helicase; Expression; Malaria; Plasmodium falciparum; RecQ Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-84906190012&doi=10.1016%2fj.molbiopara.2014.07.013&partnerID=40&md5=beb927267bb5d967e47ed3364b8a4cb2 DOI: 10.1016/j.molbiopara.2014.07.013
2014	Diverse biological effects of electromagnetic-treated water Yamabhai M., Chumseng S., Yoohat K., Srila W.	Homeopathy
Abstract: The effects of water treated with an electromagnetic field (EMF) were investigated on two biological systems, humans and plants. Purified de-ionised water was treated by (1) boiling, (2) exposure to microwave radiation, and (3) low frequency electromagnetic oscillation molecular resonance effect technology(MRET), before being used to prepare media for culturing human peripheral blood mononuclear cells (PBMC) from three healthy females. Our results indicated that PBMC culture in MRET-activated medium showed significantly less oxidative metabolism when compared to media prepared from other types of water. As for the effects on soybean, our results indicated that both MRET- and microwave-treated water greatly enhanced the length of the root. These results suggested that electromagnetic-treated water can have diverse biological effects on both animal and plant cells. Since these effects are related to the 'Memory of Water', hypothesis which has been suggested as an explanation of the action of high homeopathic dilutions, our finding warrant afurther investigation on the mechanisms of various types of physically conditioned water on specific cellular activities. © 2013 The Faculty of Homeopathy. Keyword: Biological effect; Molecular resonance effect technology; PBMC; Resazurin; Soybean; Water Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-84902283650&doi=10.1016%2fj.homp.2013.11.004&partnerID=40&md5=a9cc47762726c712e81c160555e8e26a DOI: 10.1016/j.homp.2013.11.004
2013	Identification of amino acid residues responsible for the binding to anti-FLAG™ M2 antibody using a phage display combinatorial peptide library Srila W., Yamabhai M.	Applied Biochemistry and Biotechnology
Abstract: FLAG, a short hydrophilic peptide consisting of eight amino acids (DYKDDDDK), has been widely used as a fusion tag for the purification and detection of a wide variety of recombinant proteins. One of the monoclonal antibodies against this peptide, anti-FLAG M2, recognises a FLAG peptide sequence at the N terminus, Met-N terminus, C terminus, or internal site of a fusion protein and has been extremely useful for the detection, identification, and purification of recombinant proteins. Nevertheless, detailed binding specificity of anti-FLAG M2 has yet to be determined. In the current study, a phage display combinatorial peptide library was used to determine that the motif DYKxxD encompasses the critical amino acid residues responsible for the binding of FLAG peptide to this antibody. This study demonstrates the utility of phage display technology and helps to elucidate the mode of action of this detection system. © 2013 Springer Science+Business Media New York. Keyword: Anti-FLAG M2; Combinatorial peptide; Epitope tag; FLAG; Mapping; Monoclonal antibody; Phage display Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-84885172106&doi=10.1007%2fs12010-013-0326-8&partnerID=40&md5=0fd7b29e2a3c7a369c7d18427a3093db DOI: 10.1007/s12010-013-0326-8
2013	Production of recombinant Bacillus subtilis chitosanase, suitable for biosynthesis of chitosan-oligosaccharides Pechsrichuang P., Yoohat K., Yamabhai M.	Bioresource Technology
Abstract: Chitosanases are enzymes that catalyse the hydrolysis of the β-1,4 glycosidic bond of chitosan. One of the most promising applications of this enzyme is for the bioconversion of chitosan into value-added chitosan-oligosaccharides (COS). GH46 chitosanase (Csn) from Bacillus subtilis 168 was expressed in Escherichia coli by fusing the gene encoding mature Csn to the E. coli OmpA signal peptide sequence. The recombinant enzyme was secreted into the culture supernatant. The recombinant Csn showed high specific activity and stability over a wide range of pH. The enzyme was >100 times more thermostable in the presence of the substrate, with a half-life time of activity (τ1/2) of approximately 20h at 50°C and pH 5.5. Efficient bioconversion of chitosan into different mixtures of COS, using crude culture supernatant containing secreted enzyme was demonstrated. © 2012 Elsevier Ltd. Keyword: Bacillus subtilis; Chitin waste; Chitosan-oligosaccharide production; Recombinant chitosanase; Secretion Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-84868325951&doi=10.1016%2fj.biortech.2012.09.130&partnerID=40&md5=1d8442b96bb372b89eb11a1e0f78fc47 DOI: 10.1016/j.biortech.2012.09.130