The clinical significance of 1,3-beta-D glucanase detection in plasma to the diagnosis of fungal rhinosinusitis / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To investigate the clinical significance of 1,3-beta-D glucanase detection in plasma to the diagnosis of fungal rhinosinusitis.@*METHOD@#MB-80 rapid microorganism detection system was used to detect preoperative and postoperative 1,3-beta-D glucanase in plasma of 37 patients of fungal rhinosinusitis which were treated by endoscopic sinus surgery and confirmed by postoperative pathology. Blood samples of 47 patients who underwent endoscopic surgery(fungal rhinosinusitis excluded) were taken as control group.@*RESULT@#1,3-beta-D glucanase content is more than 10 pg/ml in 34 cases of the fungal rhinosinusitis group,and every 1,3-beta-D glucanase content of 47 control cases is less than 10 pg/ml. Significant difference is found between case and control groups(chi2 = 72.6, P < 0.01).@*CONCLUSION@#1,3-beta-D glucanase detection in plasma is a simple,rapid diagnostic method of fungal rhinosinusitis.

Improvement of catalytic capability of Paecilomyces thermophila J18 thermostable beta-1,3-1,4-glucanase under acidic condition by directed evolution / 生物工程学报

Directed evolution was used to improve the performance of beta-1,3-1,4-glucanase (designated as PtLicl6A) from Paecilomyces thermophila J18 under acidic condition. A mutant library was constructed by error-prone PCR and DNA shuffling, and positive clones were screened by Congo red staining. More than 1 500 mutants were selected. One mutant (PtLic16AM1) exhibited an optimal activity at pH 5.5, while the optimal pH of the wild-type enzyme was 7.0. The mutant PtLic16AM1 kept the high specific activity and thermotolerence of the wild-type enzyme. Sequence analysis revealed that the mutant enzyme has four sense substitutions which caused four amino acid substitutions - namely T58S, Y110N, G195E and D221G.. Homology modeling showed that among the four amino acid substitutions, Y110N was near the active site of the enzyme, while the other three was distant. T58S and G195E may play key roles in the change of optimal pH. This study provided a new perspective of obtaining applicable 3-1,3-1,4-glucanase for industrial use.

Molecular cloning, purification and characterization of thermostable -1,3-1,4 glucanase from Bacillus subtilis A8-8.

A gene encoding a -1,3-1,4-glucanase (CelA) belonging to family 5 of glycoside hydrolases was cloned and sequenced from the Bacillus subtilis A8-8. The open-reading-frame of celA comprised 1499 base pairs and the enzyme was composed of 500 amino acids with a molecular mass of 55 kDa. The recombinant -1,3-1,4 glucanase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 8.0 and 60oC, respectively. The enzyme was stable within pH 6.0-9.0. It was stable up to 60oC and retained 30% of its original activity at 70oC for 60 min. It hydrolyzed lichenan, CMC, xylan, laminarin, avicel and pNPC, but was inactive towards cellobiose. The enzyme activity was markedly activated by Co2+ and Mn2+, but was strongly inactivated by Fe3+. The truncated gene, devoid of cellulose-binding domain (CBD) showed 60% of activity and bound to avicel.

Improvement of thermostability of beta-1,3-1,4-glucanase from Bacillus amyloliquefaciens BS5582 through in vitro evolution / 生物工程学报

In vitro evolution methods are often used to modify protein with improved characteristics. We developed a directed evolution protocol to enhance the thermostability of the beta-1,3-1,4-glucanase. The thermostability of the enzyme was significantly improved after two rounds of directed evolution. Three variants with higher thermostability were obtained. The mutant enzymes were further analyzed by their melting temperature, halftime and kinetic parameters. Comparing to intact enzyme, the T50 of mutant enzymes 2-JF-01, 2-JF-02 and 2-JF-03 were increased by 2.2 degrees C, 5.5 degrees C and 3.5 degrees C, respectively, the halftime (t1/2, 60 degrees C) of mutant enzymes 2-JF-01, 2-JF-02 and 2-JF-03 were shortened by 4,13 and 17 min, respectively, the V(max) of mutant enzymes were decreased by 8.3%, 2.6% and 10.6%, respectively, while K(m) of mutant enzymes were nearly unchanged. Sequence analysis revealed seven single amino acid mutant happened among three mutant enzymes, such as 2-JF-01 (N36S, G213R), 2-JF-02 (C86R, S115I, N150G) and 2-JF-03 (E156V, K105R). Homology-modeling showed that five of seven substituted amino acids were located on the surface of or in hole of protein. 42.8% of substituted amino acids were arginine, which indicated that arginine may play a role in the improvement of the thermostability of the beta-1,3-1,4-glucanase.This study provide some intresting results of the structural basis of the thermostability of beta-1,3-1,4-glucanase,and provide some new point of view in modifying enzyme for future industrial use.

Functional cell surface display of endo-beta-1, 3-1, 4-glucanase in Lactococcus lactis using N-acetylmuraminidase as the anchoring motif / 生物工程学报

In this report, we utilized N-Acetylmuraminidase (AcmA) to develop a whole-cell catalyst of endo-beta-1, 3-1, 4-glucanase in Lactococcus lactis. The PCR-amplified full-length acmA gene from L. lactis MB191 was fused with the green fluorescent gene (gfp), followed by ligating the chimeric acmA-gfp into the Escherichia coli-L. lactis shuttle expression vector pMG36k, yielding the recombinant plasmid pMB137. SDS-PAGE analysis showed that the constitutive expression of AcmA-GFP fusion protein in the L. lactis AS1.2829 construct harboring pMB137 (named MB137), with the predicted Mr of 74 kD. Western blotting, GFP specific fluorescence intensity assays and flow cytometry analysis confirmed that AcmA-GFP was immobilized on the outer membrane, which constituted approx. 35% of the total intracellular fusion protein. Furthermore, acmA was fused with a PCR-amplified encoding fragment of the endo-beta-1, 3-1, 4-glucanase gene (gls) from Bacillus sublitis BF7658, resulting in the recombinant plasmid pMB138. By transferring pMB138 into L. lactis AS1.2829, the derived L. lactis MB138 expressing the AcmA-GLS fusion enzyme exhibited a distinct whole-cell glucanase activity (by 12 U/mL) compared to the control strain, indicating AcmA had served as a functional anchoring motif to immobilize the heterologous enzyme on the cell surface of L. lactis.

Optimization of cloning and expression of beta-glucanase gene from Bacillus amyloliquefaciens / 生物工程学报

To compare of performance of beta-1,3-1,4-glucanase gene (bgl) in different expression systems, the beta-1,3-1,4-glucanase gene (GenBank Accession No. EU623974) was amplified from Bacillus amyloliquefaciens BS5582 by PCR and was cloned into three vectors pEGX-4T-1, pET20b(+) and pET28a(+) to construct pEGX-4T-1-bgl, pET20b(+)-bgl and pET28a(+)-bgl recombinant plasmids. The pEGX-4T-1-bgl was transformed into three different Escherichia coli host strains. The pET20b (+)-bgl and pET28a (+)-bgl were transformed into E. coli BL21 (DE3) respectively. Recombinant beta-glucanase was expressed by IPTG inducement in these recombinants. E. coli BL21 (DE3)-pET28a (+)-bgl had the highest enzyme activity. In Luria-Bertani medium, the total enzyme activity was (322.0 +/- 8.8) U/mL, which was 40.1% of original strain in optimal shaking flask condition. This recombinant's performance was studied in Terrific Broth medium under inducement of IPTG and lactose at the same time., and the highest total enzyme activity could reach (1883.3 +/- 45.8) U/mL (818.8% of the original), which indicate that the recombinant strain has a good value in industry application.

Identification of novel catalytic features of endo-beta-1,4-glucanase produced by mulberry longicorn beetle Apriona germari / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Mulberry longicorn beetle, Apriona germari, has been reported to produce two endo-beta-1,4-glucanases or AgEGases (accession Nos. Q6SS52 and Q5XQD1). AgEGase sequence contains catalytic motif (amino acid residues 37-48), which is the characteristic of family Glycohydrolase 45 and is identified as the substrate binding site. The application of bioinformatics approaches includes sequence analysis, structural modeling and inhibitor docking to relate the structure and function of AgEGases. We have dissected the sequence and structure of AgEGase catalytic motif and compared it with crystal structure of Humicola insolens endoglucanases V. The results show an involvement of sulfur containing amino acid residues in the active site of the enzyme. Cys residues and position of disulfide bonds are highly conserved between the two structures of endoglucanases of A. germari. Surface calculation of AgEGase structure in the absence of Cys residues reveals greater accessibility of the catalytic site to the substrate involving Asp42, a highly conserved residue. For the inhibition study, tannin-based structure was docked into the catalytic site of AgEGase using ArgusLab 4.0 and it resulted in a stable complex formation. It is suggested that the inhibition could occur through formation of a stable transition state analog-enzyme complex with the tannin-based inhibitor, as observed with other insect cellulases in our laboratory.

Partitioning and purification of extracellular beta-1,3-1,4-glucanase in aqueous two-phase systems / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The partition behaviors of beta-1,3-1,4-glucanase, alpha-amylase and neutral proteases from clarified and whole fermentation broths of Bacillus subtilis ZJF-1A5 were investigated. An aqueous two-phase system (polyethylene glycol (PEG)/MgSO(4)) was examined with regard to the effects of PEG molecular weight (MW) and concentration, MgSO(4) concentration, pH and NaCl concentration on enzyme partition and extraction. The MW and concentration of PEG were found to have significant effects on enzyme partition and extraction with low MW PEG showing the greatest benefit in the partition and extraction of beta-glucanase with the PEG/MgSO(4) system. MgSO(4) concentration influenced the partition and extraction of beta-glucanase significantly. pH had little effect on beta-glucanase or proteases partition but affected alpha-amylase partition when pH was over 7.0. The addition of NaCl had little effect on the partition behavior of beta-glucanase but had very significant effects on the partitioning of alpha-amylase and on the neutral proteases. The partition behaviors of beta-glucanase, alpha-amylase and proteases in whole broth were also investigated and results were similar to those obtained with clarified fermentation broth. A two-step process for purifying beta-glucanase was developed, which achieved beta-glucanase recovery of 65.3% and specific activity of 14027 U/mg, 6.6 times improvement over the whole broth.