Prof. Montarop Yamabhai

Year	Title	Journal
2010	Efficient recombinant expression and secretion of a thermostable GH26 mannan endo-1,4-β-mannosidase from Bacillus licheniformis in Escherichia coli Songsiriritthigul C., Buranabanyat B., Haltrich D., Yamabhai M.	Microbial Cell Factories
Abstract: Background: Mannans are one of the key polymers in hemicellulose, a major component of lignocellulose. The Mannan endo-1,4-β-mannosidase or 1,4-β-D-mannanase (EC 3.2.1.78), commonly named β-mannanase, is an enzyme that can catalyze random hydrolysis of β-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans. The enzyme has found a number of applications in different industries, including food, feed, pharmaceutical, pulp/paper industries, as well as gas well stimulation and pretreatment of lignocellulosic biomass for the production of second generation biofuel. Bacillus licheniformis is a Gram-positive endospore-forming microorganism that is generally non-pathogenic and has been used extensively for large-scale industrial production of various enzymes; however, there has been no previous report on the cloning and expression of mannan endo-1,4-β-mannosidase gene (manB) from B. licheniformis.Results: The mannan endo-1,4-β-mannosidase gene (manB), commonly known as β-mannanase, from Bacillus licheniformis strain DSM13 was cloned and overexpressed in Escherichia coli. The enzyme can be harvested from the cell lysate, periplasmic extract, or culture supernatant when using the pFLAG expression system. A total activity of approximately 50,000 units could be obtained from 1-l shake flask cultures. The recombinant enzyme was 6 × His-tagged at its C-terminus, and could be purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The specific activity of the purified enzyme when using locust bean gum as substrate was 1672 ± 96 units/mg. The optimal pH of the enzyme was between pH 6.0 - 7.0; whereas the optimal temperature was at 50 - 60°C. The recombinant β-mannanase was stable within pH 5 - 12 after incubation for 30 min at 50°C, and within pH 6 - 9 after incubation at 50°C for 24 h. The enzyme was stable at temperatures up to 50°C with a half-life time of activity (τ1/2) of approximately 80 h at 50°C and pH 6.0. Analysis of hydrolytic products by thin layer chromatography revealed that the main products from the bioconversion of locus bean gum and mannan were various manno-oligosaccharide products (M2 - M6) and mannose.Conclusion: Our study demonstrates an efficient expression and secretion system for the production of a relatively thermo- and alkali-stable recombinant β-mannanase from B. licheniformis strain DSM13, suitable for various biotechnological applications. © 2010 Songsiriritthigul et al; licensee BioMed Central Ltd. Keyword: Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-77950682492&doi=10.1186%2f1475-2859-9-20&partnerID=40&md5=6e0fb1f144c23dc769c470d0dcd89b15 DOI: 10.1186/1475-2859-9-20
2010	Chitin research revisited Khoushab F., Yamabhai M.	Marine Drugs
Abstract: Two centuries after the discovery of chitin, it is widely accepted that this biopolymer is an important biomaterial in many aspects. Numerous studies on chitin have focused on its biomedical applications. In this review, various aspects of chitin research including sources, structure, biosynthesis, chitinolytic enzyme, chitin binding protein, genetic engineering approach to produce chitin, chitin and evolution, and a wide range of applications in bio- and nanotechnology will be dealt with. © 2010 by the authors. Keyword: Application; Biotechnology; Chitin; Chito-oligosaccharide; Chitosan; Nanobiotechnology; Nanotechnology Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-77955288971&doi=10.3390%2fmd8071988&partnerID=40&md5=38ee29d489bc20a6fcf5c04c0550e341 DOI: 10.3390/md8071988
2009	Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01 Bien-Cuong D., Thi-Thu D., Berrin J.-G., Haltrich D., Kim-Anh T., Sigoillot J.-C., Yamabhai M.	Microbial Cell Factories
Abstract: Background: Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-β-mannosidases (1,4-β-D-mannanases) catalyze the random hydrolysis of β-1,4-mannosidic linkages in the main chain of β-mannans. Biodegradation of β-mannans by the action of thermostable mannan endo-1,4-β-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS). Results: A gene encoding mannan endo-1,4-β-mannosidase or 1,4-β-D-mannan mannanohydrolase (E.C. 3.2.1.78), commonly termed β-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5), was cloned and successfully expressed heterologously (up to 243 μg of active recombinant protein per mL) in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant β-mannanase is highly thermostable with a half-life time of approximately 56 h at 70°C and pH 4.0. The optimal temperature (10-min assay) and pH value for activity are 80°C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Kmvalues of the enzyme for konjac glucomannan (low viscosity), locust bean gum galactomannan, carob galactomannan (low viscosity), and 1,4-β-D-mannan (from carob) are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1and 1.5 mg mL-1, respectively, while the kcatvalues for these substrates are 215 s-1, 330 s-1, 292 s-1and 148 s-1, respectively. Judged from the specificity constants kcat/Km, glucomannan is the preferred substrate of the A. niger β -mannanase. Analysis by thin layer chromatography showed that the main product from enzymatic hydrolysis of locust bean gum is mannobiose, with only low amounts of mannotriose and higher manno-oligosaccharides formed. Conclusion: This study is the first report on the cloning and expression of a thermostable mannan endo-1,4-β-mannosidase from A. niger in Pichia pastoris. The efficient expression and ease of purification will significantly decrease the production costs of this enzyme. Taking advantage of its acidic pH optimum and high thermostability, this recombinant β-mannanase will be valuable in various biotechnological applications. © 2009 Bien-Cuong et al; licensee BioMed Central Ltd. Keyword: Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-71049124032&doi=10.1186%2f1475-2859-8-59&partnerID=40&md5=9253d7657631a687ee46dee10425f5e6 DOI: 10.1186/1475-2859-8-59
2009	A thermostable triple mutant of pyranose 2-oxidase from Trametes multicolor with improved properties for biotechnological applications Spadiut O., Radakovits K., Pisanelli I., Salaheddin C., Yamabhai M., Tan T.-C., Divne C., Haltrich D.	Biotechnology Journal
Abstract: In order to increase the thermal stability and the catalytic properties of pyranose oxidase (P2Ox) from Trametes multicolor toward its poor substrate D-galactose and the alternative electron acceptor 1, 4-benzoquinone (1, 4-BQ), we designed the triple-mutant T169G/E542K/ V546C. Whereas the wild-type enzyme clearly favors D-glucose as its substrate over D-galactose [substrate selectivity (kcat/KM)Glc/(kcat/ KM)Gal = 172], the variant oxidizes both sugars equally well [(kcat/KM)Glc/ (kcat/KM)Gal = 0.69], which is of interest for food biotechnology. Furthermore, the variant showed lower KM values and approximately ten-fold higher kcat values for 1, 4-BQ when D-galactose was used as the saturating sugar substrate, which makes this enzyme particularly attractive for use in biofuel cells and enzyme-based biosensors. In addition to the altered substrate specificity and reactivity, this mutant also shows significantly improved thermal stability. The half life time at 60°C was approximately 10 h, compared to 7.6 min for the wild-type enzyme. We performed successfully small-scale bioreactor pilot conversion experiments of D-glucose/D-galactose mixtures at both 30 and 50°C, showing the usefulness of this P2Ox variant in biocatalysis as well as the enhanced thermal stability of the enzyme. Moreover, we determined the crystal structure of the mutant in its unligated form at 1.55 Å resolution. Modeling D-galactose in position for oxidation at C2 into the mutant active site shows that substituting Thr for Gly at position 169 favorably accommodates the axial C4 hydroxyl group that would otherwise clash with Thr169 in the wild-type. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Keyword: Biofuel cell; Enzymatic batch conversion; Enzyme engineering; Flavoprotein; Rational protein design Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-65549159161&doi=10.1002%2fbiot.200800260&partnerID=40&md5=56469f4ff50491312bc528942a683f00 DOI: 10.1002/biot.200800260
2009	Sticky PCR: A PCR-based protocol for targeted protein engineering Yamabhai M.	Biotechnology Journal
Abstract: This article describes a simple but powerful PCR-based protocol for the generation of cohesive ends on linear DNA fragments, permitting the precise engineering of DNA constructs for a variety of applications. These include the introduction of deletion mutations, domain swapping, creating hybrid DNA fusions, or targeted protein engineering. This novel method can also facilitate the cloning of large or complex DNA fragments into a relevant cloning vector independent of the use of internal restriction endonuclease sites. The protocol involves the amplification of the required fragments by polymerase chain reaction through the use of two sets of overlapping desalted oligonucleotide primers. The subsequent mixing, denaturation and re-annealing of these products present correct cohesive terminal ends for ligation. There is no requirement for special vectors, enzymes or bases, suggesting that this protocol provides a unique way of engineering constructs in a rapid and cost-effective way for specific applications, such as precise deletion or swapping of various domains of the epidermal growth factor receptor to determine their role in membrane localization. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Keyword: Cloning; Mutagenesis; PCR; Protein engineering Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-65549144980&doi=10.1002%2fbiot.200800198&partnerID=40&md5=b8ac972a72b5be169609f2ca22e37323 DOI: 10.1002/biot.200800198
2009	Directed evolution of a Bacillus chitinase Songsiriritthigul C., Pesatcha P., Eijsink V.G.H., Yamabhai M.	Biotechnology Journal
Abstract: Chitinases have potential in various industrial applications including bioconversion of chitin waste from crustacean shells into chito-oligosaccharide-based value-added products. For industrial applications, obtaining suitable chitinases for efficient bioconversion processes will be beneficial. In this study, we established a straightforward directed evolution method for creating chitinase variants with improved properties. A library of mutant chitinases was constructed by error-prone PCR and DNA shuffling of two highly similar (99% identical) chitinase genes from Bacillus licheniformis. Activity screening was done in two steps: first, activity towards colloidal chitin was screened for on culturing plates (halo formation). This was followed by screening activity towards the chitotriose analogue p-nitrophenyl-β-1, 4-N, N′-diacetyl-chitobiose at various pH in microtiter plates. From a medium-throughput screening (517 colonies), we were able to isolate one mutant that demonstrated improved catalytic activity. When using p-nitrophenyl-β-1, 4-N, N′-diacetyl-chitobiose as substrate, the overall catalytic efficiency, kcat/Km of the improved chitinase was 2.7- and 2.3-fold higher than the average kcat/Km of wild types at pH 3.0 and 6.0, respectively. The mutant contained four residues that did not occur in either of the wild types. The approach presented here can easily be adopted for directed evolution of suitable chitinases for various applications. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Keyword: Bacillus licheniformis; Chitinase; Directed evolution; DNA shuffling; Error-prone PCR Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-65549108312&doi=10.1002%2fbiot.200800258&partnerID=40&md5=b2b5f93b9e7faebcc99d2698ce48fec5 DOI: 10.1002/biot.200800258
2009	A compact phage display human scFv library for selection of antibodies to a wide variety of antigens Pansri P., Jaruseranee N., Rangnoi K., Kristensen P., Yamabhai M.	BMC Biotechnology
Abstract: Background: Phage display technology is a powerful new tool for making antibodies outside the immune system, thus avoiding the use of experimental animals. In the early days, it was postulated that this technique would eventually replace hybridoma technology and animal immunisations. However, since this technology emerged more than 20 years ago, there have only been a handful reports on the construction and application of phage display antibody libraries world-wide. Results: Here we report the simplest and highly efficient method for the construction of a highly useful human single chain variable fragment (scFv) library. The least number of oligonucleotide primers, electroporations and ligation reactions were used to generate a library of 1.5 × 108 individual clones, without generation of sub-libraries. All possible combinations of heavy and light chains, among all immunoglobulin isotypes, were included by using a mixture of primers and overlapping extension PCR. The key difference from other similar libraries was the highest diversity of variable gene repertoires, which was derived from 140 non-immunized human donors. A wide variety of antigens were successfully used to affinity select specific binders. These included pure recombinant proteins, a hapten and complex antigens such as viral coat proteins, crude snake venom and cancer cell surface antigens. In particular, we were able to use standard bio-panning method to isolate antibody that can bind to soluble Aflatoxin B1, when using BSA-conjugated toxin as a target, as demonstrated by inhibition ELISA. Conclusion: These results suggested that by using an optimized protocol and very high repertoire diversity, a compact and efficient phage antibody library can be generated. This advanced method could be adopted by any molecular biology laboratory to generate both naïve or immunized libraries for particular targets as well as for high-throughput applications. © 2009 Pansri et al; licensee BioMed Central Ltd. Keyword: Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-60549116209&doi=10.1186%2f1472-6750-9-6&partnerID=40&md5=b681b0410e37fe76e4bf920f5298a952 DOI: 10.1186/1472-6750-9-6
2009	Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-beta-mannosidase from Aspergillus niger BK01. Do B.C., Dang T.T., Berrin J.G., Haltrich D., To K.A., Sigoillot J.C., Yamabhai M.	Microbial cell factories
Abstract: BACKGROUND: Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-beta-mannosidases (1,4-beta-D-mannanases) catalyze the random hydrolysis of beta-1,4-mannosidic linkages in the main chain of beta-mannans. Biodegradation of beta-mannans by the action of thermostable mannan endo-1,4-beta-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS). RESULTS: A gene encoding mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannan mannanohydrolase (E.C. 3.2.1.78), commonly termed beta-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5), was cloned and successfully expressed heterologously (up to 243 microg of active recombinant protein per mL) in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant beta-mannanase is highly thermostable with a half-life time of approximately 56 h at 70 degrees C and pH 4.0. The optimal temperature (10-min assay) and pH value for activity are 80 degrees C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity), locust bean gum galactomannan, carob galactomannan (low viscosity), and 1,4-beta-D-mannan (from carob) are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these substrates are 215 s-1, 330 s-1, 292 s-1 and 148 s-1, respectively. Judged from the specificity constants kcat/Km, glucomannan is the preferred substrate of the A. niger beta -mannanase. Analysis by thin layer chromatography showed that the main product from enzymatic hydrolysis of locust bean gum is mannobiose, with only low amounts of mannotriose and higher manno-oligosaccharides formed. CONCLUSION: This study is the first report on the cloning and expression of a thermostable mannan endo-1,4-beta-mannosidase from A. niger in Pichia pastoris. The efficient expression and ease of purification will significantly decrease the production costs of this enzyme. Taking advantage of its acidic pH optimum and high thermostability, this recombinant beta-mannanase will be valuable in various biotechnological applications. Keyword: Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-75649143355&partnerID=40&md5=c9eac748c7d9ea763e76a3dbe7401d04 DOI:
2008	Mimotope identification from monoclonal antibodies of Burkholderia pseudomallei using random peptide phage libraries Na-ngam N., Kalambaheti T., Ekpo P., Pitaksajjakul P., Jamornthanyawat N., Chantratita N., Sirisinha S., Yamabhai M., Thamlikitkul V., Ramasoota P.	Transactions of the Royal Society of Tropical Medicine and Hygiene
Abstract: Summary: This study used random peptide libraries, displayed by bacteriophage T7 and M13, to identify mimotopes from four monoclonal antibodies (mAbs) specific to Burkholderia pseudomallei. Bound phages, selected from fourth-round panning with each mAb, were tested for binding specificity with each mAb using ELISA, before being further amplified and checked for phage peptide sequence using PCR and DNA sequencing. Overall, 75 of 90 phages (83.3%) were ELISA-positive with each mAb. Mimotopes from all 75 phages (100%) were found to match protein sequences of Burkholderia spp. from GenBank. The predominant mimotopes were TP-GRTRVT found in 13.3%, LTPCGRTxD (8%), AREVTLL (6.7%), NxVxKVVSR (5.3%), PCAPRSS (4%), LGRVLAN (4%), RNPKKA (2.7%) and CPYPR (2.7%). The following GenBank-matched proteins (i.e. the hypothetical proteins) were located at the outer membrane of Burkholderia spp.: BPSL2046 of B. pseudomallei K96243 (matched with mimotope CGRTxD), BpseP_02000035 (matched with LGRVLAN), BPSS0784 of B. pseudomallei K96243 (matched with CPYPR), BURPS1710b_1104 of B. pseudomallei 1710b (matched with CARQY) and TonB-dependent siderophore receptor of B. cenocepacia H12424 (matched with CVRxxLTPC and TPCRxRT). These phage mimotopes and matched proteins may have the potential for further use as diagnostic reagent and immunogen against melioidosis. These results demonstrate that phage-display technique has the potential for rapidly identifying phage mimotopes that interact with B. pseudomallei mAbs. © 2008 Elsevier Ltd. All rights reserved. Keyword: Burkholderia pseudomallei; Melioidosis; Mimotope; Monoclonal antibody; Phage display Random peptide library Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-58049143318&doi=10.1016%2fS0035-9203%2808%2970014-2&partnerID=40&md5=847d5ec966c1f7f36299ed83918c623e DOI: 10.1016/S0035-9203(08)70014-2
2008	BAP-fusion: A versatile molecular probe for biotechnology research Yamabhai M.	Biotechnology: Research, Technology and Applications
Abstract: Bacterial alkaline phosphatase (BAP) is a useful enzyme for detection in biotechnological researches. There are vast arrays of commercial available substrates, which can be converted to soluble or precipitated products for either colorimetric or chemiluminescent detection. This research article describes the application of bacterial alkaline phosphatase fusion protein as a convenient and versatile molecular probe for direct detection of different molecular interactions. Short peptide, protein binding domain, or single chain variable fragment (scFv) of monoclonal antibody were fused to bacterial alkaline phosphatase and used as one step detection probe for the study proteinprotein or antibody-antigen interactions. The BAP-fusion could be generated by cloning a gene of interest in frame of BAP gene in an Escherichia coli expression vector. The fusion protein contained N-terminal signal peptide for extracellular secretion and could be induced for over-expression with isopropyl-β-D-thiogalactopyranoside (IPTG), allowing simple harvesting from culture broth or periplasmic extract. The BAP-fusion that was tagged with poly-histidine could be further purified by nickel affinity chromatography. This one step detection probe generated a specific and robust signal, suitable for detection in various formats as demonstrated on microtiter plate, dot blot, or western blot. In addition, it could also be used for an estimation of binding affinity by competitive inhibition with soluble ligand. © 2008 Nova Science Publishers, Inc. All rights reserved. Keyword: Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-84891972760&partnerID=40&md5=7fc927e473480af4756d964aa04bba95 DOI: