Biotechnology of Microbial Xylanases: Enzymology,Molecular Biology,and Application

ABSTRACT:?

Xylanases are hydrolases depolymerizing the plant cell wall component xylan, the second most abundant polysaccharide. The molecular structure and hydrolytic pattern of xylanases have been reported extensively and the mechanism of hydrolysis has also been proposed. There are several models for the gene regulation of which this article could add to the wealth of knowledge. Future work on the application of these enzymes in the paper and pulp, food industry, in environmental science, that is, bio-fueling, effluent treatment, and agro-waste treatment, etc. require a complete understanding of the functional and genetic significance of the xylanases. However, the thrust area has been identified as the paper and pulp industry. The major problem in the field of paper bleaching is the removal of lignin and its derivatives, which are linked to cellulose and xylan. Xylanases are more suitable in the paper and pulp industry than lignin-degrading systems.     

Streptococcus pneumoniae NanC: STRUCTURAL INSIGHTS INTO THE SPECIFICITY AND MECHANISM OF A SIALIDASE THAT PRODUCES A SIALIDASE INHIBITOR*

Streptococcus pneumoniae is an important human pathogen that causes a range of disease states. Sialidases are important bacterial virulence factors. There are three pneumococcal sialidases: NanA, NanB, and NanC. NanC is an unusual sialidase in that its primary reaction product is 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en, also known as DANA), a nonspecific hydrolytic sialidase inhibitor. The production of Neu5Ac2en from α2–3-linked sialosides by the catalytic domain is confirmed within a crystal structure. A covalent complex with 3-fluoro-β-N-acetylneuraminic acid is also presented, suggesting a common mechanism with other sialidases up to the final step of product formation. A conformation change in an active site hydrophobic loop on ligand binding constricts the entrance to the active site. In addition, the distance between the catalytic acid/base (Asp-315) and the ligand anomeric carbon is unusually short. These features facilitate a novel sialidase reaction in which the final step of product formation is direct abstraction of the C3 proton by the active site aspartic acid, forming Neu5Ac2en. NanC also possesses a carbohydrate-binding module, which is shown to bind α2–3- and α2–6-linked sialosides, as well as N-acetylneuraminic acid, which is captured in the crystal structure following hydration of Neu5Ac2en by NanC. Overall, the pneumococcal sialidases show remarkable mechanistic diversity while maintaining a common structural scaffold.     

天童常绿阔叶树种栲树生殖个体大小及其生殖构件特征

对浙江天童木荷-栲树林内的常绿阔叶树种栲树(Castanopsis fargesii Franch.)的生殖个体大小、生殖构件的分布及其动态变化特征进行了研究。结果表明, 该地区栲树生殖个体的胸径在17~50 cm 间, 平均胸径为31. 2±8.0 cm, 平均年龄约36.3±6.6 年;林缘附近的生殖个体小于木荷-林内。相对稳定的群落和比较丰富的土壤养分条件有利于生殖枝数量和花序数量的增多。栲树生殖个体的数量在两年中变化较大, 部分栲树个体可以在连续年份中生殖。从枝系水平分析:在持续生殖的栲树个体上, 生殖枝数量有明显变化, 并非所有的生殖枝在两年中都可开花或结果, 保持连续生殖的枝系约占48.2%。栲树果序枝数量在连续年份有明显差异(p < 0.01), 而且果序枝上的幼蕾数、果实数量及结实率等都有明显差异(p < 0.05)。     

The evolution of putative starch-binding domains

The present bioinformatics analysis was focused on the starch-binding domains (SBDs) and SBD-like motifs sequentially related to carbohydrate-binding module (CBM) families CBM20 and CBM21. Originally, these SBDs were known from microbial amylases only. At present homologous starch- and glycogen-binding domains (or putative SBD sequences) have been recognised in various plant and animal proteins. The sequence comparison clearly showed that the SBD-like sequences in genethonin-1, starch synthase III and glucan branching enzyme should possess the real SBD function since the two tryptophans (or at least two aromatics) of the typical starch-binding site 1 are conserved in their sequences. The same should apply also for the sequences corresponding with the so-called KIS-domain of plant AKINbetagamma protein that is a homologue of the animal AMP-activated protein kinase (AMPK). The evolutionary tree classified the compared SBDs into three distinct groups: (i) the family CBM20 (the motifs from genethonins, laforins, starch excess 4 protein, beta-subunits of the animal AMPK and all plant and yeast homologues, and eventually from amylopullulanases); (ii) the family CBM21 (the motifs from regulatory subunits of protein phosphatase 1 together with those from starch synthase III); and (iii) the (CBM20+CBM21)-related group (the motifs from the pullulanase subfamily consisting of pullulanase, branching enzyme, isoamylase and maltooligosyl trehalohydrolase).     

Hydrolysis of Proteins by Successive Incubation with Proteinase and Aminopeptidases Mixture

1. A trial test was attempted of complete hydrolysis of peptides and proteins into amino acids by enzymes. “Neutral proteinase” of Bacillus subtilis or “Alkalophilic proteinase” of a Streptomyces sp. was used for preliminary digestion of substrate, and a mixture of three aminopeptidases of Bacillus subtilis was employed for subsequent hydrolysis of proteinase digest.

2. The oxidized insulin B chain was hydrolyzed completely by the method. Several proteins including enzymes which contained no or less cystine and cysteine were also hydrolyzed almost completely.

3. On the other hand, certain glycoproteins were hydrolyzed to leave a few glycopeptides in which all glycomoieties of the proteins were retained. The implications of the results are discussed.     

A secondary xylan-binding site enhances the catalytic activity of a single-domain family 11 glycoside hydrolase

Bacillus circulans xylanase (BcX) is a single-domain family 11 glycoside hydrolase. Using NMR-monitored titrations, we discovered that an inactive variant of this enzyme, E78Q-BcX, bound xylooligosaccharides not only within its pronounced active site (AS) cleft, but also at a distal surface region. Chemical shift perturbation mapping and affinity electrophoresis, combined with mutational studies, identified the xylan-specific secondary binding site (SBS) as a shallow groove lined by Asn, Ser, and Thr residues and with a Trp at one end. The AS and SBS bound short xylooligosaccharides with similar dissociation constants in the millimolar range. However, the on and off-rates to the SBS were at least tenfold faster than those of kon approximately 3x10(5) M(-1) s(-1) and koff approximately 1000 s(-1) measured for xylotetraose to the AS of E78Q-BcX. Consistent with their structural differences, this suggests that a conformational change in the enzyme and/or the substrate is required for association to and dissociation from the deep AS, but not the shallow SBS. In contrast to the independent binding of small xylooligosaccharides, high-affinity binding of soluble and insoluble xylan, as well as xylododecaose, occurred cooperatively to the two sites. This was evidenced by an approximately 100-fold increase in relative Kd values for these ligands upon mutation of the SBS. The SBS also enhances the activity of BcX towards soluble and insoluble xylan through a significant reduction in the Michaelis KM values for these polymeric substrates. This study provides an unexpected example of how a single domain family 11 xylanase overcomes the lack of a carbohydrate-binding module through the use of a secondary binding site to enhance substrate specificity and affinity.     

Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K

Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤ 50 °C) was 10 °C higher than that for CITase-T3040 (≤ 40 °C); the k_cat/K_M value of CITase-598K was approximately two times higher (32.2 s^− 1 mM^− 1) than that of CITase-T3040 (17.8 s^− 1 mM^− 1). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes.     

Purification and carbohydrate-binding specificity of Agrocybe cylindracea lectin

A lectin was isolated from fruiting bodies of Agrocybe cylindracea by two ion-exchange chromatographies and gel filtration on Toyopearl HW55F. The lectin was homogeneous on polyacrylamide gel electrophoresis and its molecular mass was determined to be 30 000 by gel filtration, and 15 000 by sodium dodecylsulfate polyacrylamide gel electrophoresis, signifying a dimeric protein. Its carbohydrate-binding specificity was investigated both by sugar-hapten inhibition of hemagglutination and by enzyme-linked immunosorbent assay. The inhibition tests showed the affinity of the lectin to be weakly directed toward sialic acid and lactose, and the enhanced affinity toward trisaccharides containing the NeuAcα2,3Galβ-structure. Importantly, the lectin strongly interacted with glycoconjugates containing NeuAcα2,3Galβ1,3GlcNAc-/GalNAc sequences. This revised version was published online in November 2006 with corrections to the Cover Date.     

Scalable In-Plane Directional Crystallization for The Printable Hole-Conductor-Free Perovskite Solar Cell Based on The Carbon Electrode

Popular solution-processed approaches for producing the active layer of perovskite solar cells (PSCs) generally have to make compromise between crystallinity and compactness by inducing a rapid crystallization process with explosive nucleation and limited growth via removing solvent quickly. Here, a practical growth-dominated in-plane directional crystallization technique (IPDC) with a deeply retarded crystallization process for the scalable preparation of PSCs are reported. During the low-temperature annealing, a tiny chamber with a small height is built atop the wet perovskite precursor film to restrain the vertical diffusion and removal of solvent vapor. The chamber eliminates the vertical solvent vapor gradient and induce a horizonal in-plane gradient of solvent vapor pressure (SVP) toward the preset exhaust port which allows the slow escape of solvent vapor to outer space. In this way, nucleation is induced preferentially near the port and the as-formed heterogeneous nuclei then grow continuously and directionally. With IPDC, sufficient filling of perovskite with high crystallinity and obvious growth orientation is realized in non-ordered mesoporous scaffolds. An encouraging power conversion efficiency of 19.35% and 16.53% is achieved respectively for the 0.1 and 52.3-cm² printable mesoscopic hole-conductor-free PSCs with carbon electrodes.     

Extra carbohydrate binding module contributes to the processivity and catalytic activity of a non-modular hydrolase family 5 endoglucanase from Fomitiporia mediterranea MF3/22

FmEG from Fomitiporia mediterranea is a non-modular endoglucanase composed of a 24-amino acids extension and 13-amino acids linker-like peptide at the N-terminus and a 312-amino acids GH5 catalytic domain (CD) at the C-terminus. In this study, six FmEG derivatives with deletion of N-terminal fragments or fusion with an extra family 1 carbohydrate-binding module (CBM1) was constructed in order to evaluate the contribution of CBM1 to FmEG processivity and catalytic activity. FmEG showed a weak processivity and released cellobiose (G2) and cellotriose (G3) as main end products, and cellotriose (G4) as minor end product from filter paper (FP), but more amount of G4 was released from regenerated amorphous cellulose (RAC). All derivatives had similar activity on carboxymethylcellulose (CMC) with the same optimal pH (7.0) and temperature (50 °C). However, fusing an extra CBM1 to FmEG△24 or FmEG△37 with flexible peptide significantly improved its processivity and catalytic activity to FP and RAC. Overall, 1.79- and 1.84-fold increases in the soluble/insoluble product ratio on FP, and 1.38- and 1.39-fold increases on RAC, compared to FmEG△24, were recorded for CBM1-FmEG△24 and CBM1-linker-FmEG△24, respectively. Meanwhile, they displayed 2.64- and 2.67-fold more activity on RAC, and 1.68- and 1.77-fold on FP, respectively. Similar improvement was also obtained for CBM1-linker-FmEG△37 as compared with FmEG△37. Interestingly, fusion of an extra CBM1 with FmEG also caused an alteration of cleavage pattern on insoluble celluloses. Our results suggest that such improvements in processivity and catalytic activity may arise from CBM1 binding affinity. The N-terminal 24- or 37-amino acids may serve as linker for sufficient spatial separation of the two domains required for processivity and catalytic activity. In addition, deletion of the N-terminal 24- or 37-amino acids led to significant reduction in thermostability but not the enzymatic activity.