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.

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 × 10⁸individual 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.

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 K_m 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 k_cat values for these substrates are 215 s^-1, 330 s^-1, 292 s^-1 and 148 s^-1, respectively. Judged from the specificity constants k_cat/K_m, 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.

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.

Several Zika virus (ZIKV) vaccines designed to elicit protective antibody (Ab) responses are currently under rapid development, but the underlying mechanisms that control the magnitude and quality of the Ab response remain unclear. Here, we investigated the CD4⁺ T cell response to primary intravenous and intravaginal infection with ZIKV. Using the LysMCre⁺Ifnar1^fl/fl (myeloid type I IFN receptor-deficient) C57BL/6 mouse models, we identified six I-A^b-restricted ZIKV epitopes that stimulated CD4⁺ T cells with a predominantly cytotoxic Th1 phenotype in mice primed with ZIKV. Intravenous and intravaginal infection with ZIKV effectively induced follicular helper and regulatory CD4⁺ T cells. Treatment of mice with a CD4⁺ T cell-depleting Ab reduced the plasma cell, germinal center B cell, and IgG responses to ZIKV without affecting the CD8⁺ T cell response. CD4⁺ T cells were required to protect mice from a lethal dose of ZIKV after infection intravaginally, but not intravenously. However, adoptive transfer and peptide immunization experiments showed a role for memory CD4⁺ T cells in ZIKV clearance in mice challenged intravenously. These results demonstrate that CD4⁺ T cells are required mainly for the generation of a ZIKV-specific humoral response but not for an efficient CD8⁺ T cell response. Thus, CD4⁺ T cells could be important mediators of protection against ZIKV, depending on the infection or vaccination context. Several vaccine candidates are currently under development for Zika virus (ZIKV) infection, which causes life-threatening neurologic diseases, including congenital Zika syndrome in neonates and Guillain-Barré syndrome in adults. However, the mechanisms by which the adaptive immune system contributes to protection against ZIKV have not been fully investigated. In this report, we used mouse models of systemic and genital mucosal infection with ZIKV to evaluate the role of CD4⁺ T cells in regulating antiviral humoral and CD8⁺ T cell responses and in controlling ZIKV infection. We first defined the antigen specificity and phenotype of the CD4⁺ T cell response to ZIKV and then demonstrated that CD4⁺ T cells are required for inducing humoral, but not CD8⁺ T cell, responses in ZIKV-primed mice. CD4⁺ T cells were also required for local control of ZIKV burden in mice infected intravaginally. Although CD4⁺ T cells are not necessary for the control of primary ZIKV infection via the intravenous route, CD4⁺ T cells can confer protection against lethal intravaginal ZIKV challenge. Our results support ZIKV vaccine strategies that induce a protective CD4⁺ T cell response to ZIKV.