Montarop Yamabhai

Chitin Research Revisited

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.

A compact phage display human scFv library for selection of antibodies to a wide variety of antigens

BackgroundPhage 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.ResultsHere 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.ConclusionThese 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.

Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01

BackgroundMannans 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).ResultsA 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 Km values 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-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 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.ConclusionThis 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.

Molecular mechanism of NPF recognition by EH domains.

Eps15 homology (EH) domains are protein interaction modules that recognize Asn-Pro-Phe (NPF) motifs in their biological ligands to mediate critical events during endocytosis and signal transduction. To elucidate the structural basis of the EH–NPF interaction, the solution structures of two EH–NPF complexes were solved using NMR spectroscopy. The first complex contains a peptide representing the Hrb C-terminal NPFL motif; the second contains a peptide in which an Arg residue substitutes the C-terminal Leu. The NPF residues are almost completely embedded in a hydrophobic pocket on the EH domain surface and the backbone of NPFX adopts a conformation reminiscent of the Asx-Pro type I β-turn motif. The residue directly following NPF is crucial for recognition and is required to complete the β-turn. Five amino acids on the EH surface mediate specific recognition of this residue through hydrophobic and electrostatic contacts. The complexes explain the selectivity of the second EH domain of Eps15 for NPF over DPF motifs and reveal a critical aromatic interaction that provides a conserved anchor for the recognition of FW, WW, SWG and HTF ligands by other EH domains.

Efficient recombinant expression and secretion of a thermostable GH26 mannan endo-1,4-β-mannosidase from Bacillus licheniformis in Escherichia coli

BackgroundMannans 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.ResultsThe 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.ConclusionOur 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.

Expression and characterization of Bacillus licheniformis chitinase (ChiA), suitable for bioconversion of chitin waste

Chitinase (EC 3.2.1.14) is an enzyme with multiple industrial applications. These include bioconversion of chitin waste, a highly resistant and abundant biopolymer from crustacean food industry, into glucosamine and chito-oligosaccharide value-added products. This paper reports on the expression of endochitinase (ChiA) from Bacillus licheniformis strain DSM8785 in E. coli and characterization of the recombinant enzyme. Recombinant ChiA could efficiently convert colloidal chitin to N-acetyl glucosamine and chitobiose at pH 4.0, 6.0 and 9.0 at 50 degrees C and retained its activity up to 3days under these conditions, suggesting that this enzyme is suitable for bioconversion of chitin waste.

Mannan biotechnology: from biofuels to health

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.

β‐Galactosidase from Lactobacillus pentosus: Purification, characterization and formation of galacto‐oligosaccharides

A novel heterodimeric beta-galactosidase with a molecular mass of 105 kDa was purified from crude cell extracts of the soil isolate Lactobacillus pentosus KUB-ST10-1 using ammonium sulphate fractionation followed by hydrophobic interaction and affinity chromatography. The electrophoretically homogenous enzyme has a specific activity of 97 U(oNPG)/mg protein. The K(m), k(cat) and k(cat)/K(m) values for lactose and o-nitrophenyl-beta-D-galactopyranoside (oNPG) were 38 mM, 20 s(-1), 530 M(-1).s(-1) and 1.67 mM, 540 s(-1), 325 000 M(-1).s(-1), respectively. The temperature optimum of beta-galactosidase activity was 60-65 degrees C for a 10-min assay, which is considerably higher than the values reported for other lactobacillal beta-galactosidases. Mg(2+) ions enhanced both activity and stability significantly. L. pentosus beta-galactosidase was used for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. A maximum yield of 31% GOS of total sugars was obtained at 78% lactose conversion. The enzyme showed a strong preference for the formation of beta-(1-->3) and beta-(1-->6) linkages, and the main transgalactosylation products identified were the disaccharides beta-D-Galp-(1-->6)-D-Glc, beta-D-Galp-(1-->3)-D-Glc, beta-D-Galp-(1-->6)-D-Gal, beta-D-Galp-(1-->3)-D-Gal, and the trisaccharides beta-D-Galp-(1-->3)-D-Lac, beta-D-Galp-(1-->6)-D-Lac.

Production of recombinant Bacillus subtilis chitosanase, suitable for biosynthesis of chitosan-oligosaccharides

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 (τ(1/2)) of approximately 20 h 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.

Efficient expression and purification of recombinant glutaminase from Bacillus licheniformis (GlsA) in Escherichia coli

Glutaminase or L-glutamine aminohydrolase (EC 3.5.1.2) is an enzyme that catalyzes the formation of glutamic acid and ammonium ion from glutamine. This enzyme functions in cellular metabolism of every organism by supplying nitrogen required for the biosynthesis of a variety of metabolic intermediates, while glutamic acid plays a role in both sensory and nutritional properties of food. So far there have been only a few reports on cloning, expression and characterization of purified glutaminases. Microbial glutaminases are enzymes with emerging potential in both the food and the pharmaceutical industries. In this research a recombinant glutaminase from Bacillus licheniformis (GlsA) was expressed in Escherichia coli, under the control of a ptac promoter. The recombinant enzyme was tagged with decahistidine tag at its C-terminus and could be conveniently purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The enzyme could be induced for efficient expression with IPTG, yielding approximately 26,000 units from 1-l shake flask cultures. The enzyme was stable at 30°C and pH 7.5 for up to 6h, and could be used efficiently to increase glutamic acid content when protein hydrolysates from soy and anchovy were used as substrates. The study demonstrates an efficient expression system for the production and purification of bacterial glutaminase. In addition, its potential application for bioconversion of glutamine to flavor-enhancing glutamic acid has been demonstrated.