Heterologous expression and enhanced production of ß-1, 4-glucanase of Bacillus halodurans C-125 in Escherichia coli

Background: Recombinant DNA technology enables us to produce proteins with desired properties and insubstantial amount for industrial applications. Endo-1, 4-ß-glucanases (Egl) is one of the major enzyme involved in degradation of cellulose, an important component of plant cell wall. The present study was aimed at enhancing the production of endo-1, 4-ß-glucanases (Egl) of Bacillus halodurans in Escherichia coli. Results: A putative Egl gene of Bacillus Halodurans was expressed in E. coli by cloning in pET 22b (+). On induction with isopropyl-b-D-1-thiogalactopyranoside, the enzyme expression reached upto ~20% of the cell protein producing 29.2 mg/liter culture. An increase in cell density to 12 in auto-inducing LB medium (absorbance at 600 nm) enhanced ß-glucanase production up to 5.4 fold. The molecular mass of the enzyme was determined to be 39 KDa, which is nearly the same as the calculated value. Protein sequence was analyzed by CDD, Pfam, I TASSER, COACH, PROCHECK Servers and putative amino acids involved in the formation of catalytic, substrate and metal binding domains were identified. Phylogenetic analysis of the ß-glucanases of B. halodurans was performed and position of Egl among other members of the genus Bacillus producing endo-glucanases was determined. Temperature and pH optima of the enzyme were found to be 60°C and 8.0, respectively, under the assay conditions. Conclusion: Production of endo-1, 4 ß-glucanase enzymes from B. halodurans increased several folds when cloned in pET vector and expressed in E. coli. To our knowledge, this is the first report of high-level expression and characterization of an endo-1, 4 ß-glucanases from B. halodurans.

Establishment of fingerprint of Astragali Radix polysaccharides based on endo-1,4-β-mannanase hydrolysis and identification of Astragali Radix of different germplasm resources / 中国中药杂志

The polysaccharides of different germplasm resources of Astragalus membranaceus var. mongholicus〓(cultured Astragalus Radix (RA) and natural RA) and A. membranaceus (MJ) (cultured RA and natural RA) were studied by using the optimal enzymatic conditions of endo-1,4-β-mannanase. Saccharide fingerprints were obtained for the identification and evaluation of the germplasm resources of RA by Fluorophore-assisted Carbohydrate Electrophoresis (FACE). The data were analyzed by principal component analysis to obtain the difference between RA of different germplasm resources. The results showed that trisaccharide, tetrasaccharide and pentasaccharide of endo-1,4-β-mannanase hydrolyzate could be used as the differential fragments to distinguish MG (cultured RA and natural RA); the pentasaccharide and hexasaccharide can be used as differentially expressed carbohydrate fragments that distinguish MJ (cultured RA and natural RA); the trisaccharide and tetrasaccharide can be used as the differential fragments to distinguish the cultured MG and cultured MJ. Studies have shown that polysaccharide products degraded by endo-1,4-β-mannanase can well distinguish RA species (MG and MJ), growth mode (cultured RA and natural RA). This study laid the foundation for the quality evaluation of Astragalus medicinal herbs and screening of active oligosaccharides.

Establishment of fingerprint of Astragali Radix polysaccharides based on endo-1,3-β-glucanasehydrolysis and identification of Astragali Radix of different germplasm resources / 中草药

Objective Enzymatic hydrolysis of Astragali Radix polysaccharides from different germplasm resources Astragalusmembranaceus var. mongholicus (MG) (cultured and natural) or Astragalusmembranaceus (MJ) (cultured and natural) was carried out by the best enzymolysis conditions of endo-1,3-β-glucanase. Saccharide fingerprints were obtained for the identification and evaluation of the germplasm resources of Astragali Radix by Fluorophore-assisted Carbohydrate Electrophoresis (FACE). Methods The data were analyzed by principal component analysis and t test using SMICA software to distinguishdifferential sugar segments among different germplasm resources of Astragali Radix. Results Pentasaccharide and hexasaccharide of endo-1,3-β-glucanasehydrolyzate could be used as differentiated saccharide fragments between natural MG and MJ.Trisaccharide, tetrasaccharide, and pentasaccharide could be used as differentiated saccharide fragments to distinguish the cultured MG and MJ.The pentasaccharide and hexasaccharide can be used as differential fragments to distinguish MJ (culturedandnatural). Conclusion Thepolysaccharide products degraded by endo-1,3-β-glucanase can well distinguish Astragali Radix species (MG and growth mode (cultured and natural Astragali Radix). This study laid the foundation for the quality evaluation of Astragali Radix and screening of active oligosaccharides.

Endo-and exoglucanase activities in bacteria from mangrove sediment

The mangrove ecosystem is an unexplored source for biotechnological applications. In this unique environment, endemic bacteria have the ability to thrive in the harsh environmental conditions (salinity and anaerobiosis), and act in the degradation of organic matter, promoting nutrient cycles. Thus, this study aimed to assess the cellulolytic activities of bacterial groups present in the sediment from a mangrove located in Ilha do Cardoso (SP, Brazil). To optimize the isolation of cellulolytic bacteria, enrichments in two types of culture media (tryptone broth and minimum salt medium), both supplemented with 5% NaCl and 1% of cellulose, were performed. Tests conducted with the obtained colonies showed a higher occurrence of endoglycolytic activity (33 isolates) than exoglycolytic (19 isolates), and the degradation activity was shown to be modulated by the presence of NaCl. The isolated bacteria were clustered by BOX-PCR and further classified on the basis of partial 16S rRNA sequences as Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes or Bacteroidetes. Therefore, this study highlights the importance of studies focusing on the endemic species found in mangroves to exploit them as novel biotechnological tools for the degradation of cellulose.

Xylanases: An Overview.

Endo-1, 4-β-xylanase (Endo-β-1, 4-xylan, xylanohydrolase; EC. 3.2.1.8, commonly called xylanase) is an industrially important enzyme which degrades xylan randomly and produces xylooligosaccharides, xylobiose and xylose. It is mainly present in microbes and plants but not in animals. Xylanases from fungal and bacterial sources have been extensively studied and produced commercially. Its potential use in paper industries has been discussed which is directly related to reduction in environmental pollution. It has role in bio-bleaching paper pulp and increasing pulp brightness. Besides, it can be exploited for ethanol production and as an additive in animal feedstock to improve its nutritional value. Endo-1, 4-β-xylanase can also be exploited in baking and fruit juice industries. Here, we reviewed its distribution, structural aspects and industrial/ biotechnological applications. Besides, we also discussed studies related to cloning of the gene encoding endo-1, 4-β-xylanase with the objectives of overproducing the enzyme and altering its properties to suit commercial applications.

A simple and efficient diffusion technique for assay of endo -1,4-xylanase activity

Endo--1, 4-xylanases is thought to be of great significance for several industries namely paper, pharmaceuticals, food, feed etc. in addition to better utilization of lignocellulosic biomass. The present investigation was aimed to develop an easy, simple and efficient assay technique for endo--1, 4-xylanases secreted by the aerobic fungi. Under the proposed protocol, 9 g/L xylan containing agar was prepared in 100 mM phosphate buffer at different pH (4.5, 5.5 and 6.5). The sterilized xylan agar was dispensed in 90 mm petri dishes. 100 µl of culture supernatant of 12 fungal isolates was added to the wells and left overnight at 31±1(0)C. The petri dishes were observed for zone of clearance by naked eye and diameter was measured. Congo red solution (1 g/L) was applied over the petri dishes as per the established protocol and thereafter plates were flooded with 1M Sodium chloride solution for the appearance of zone of clearance. The diameter for zone of clearance by the proposed method and the established protocol was almost identical and ranged from 21 to 42 mm at different pH depending upon the activity of endo--1, 4-xylanases. Change of pH towards alkaline side enabled similar or marginal decrease of diameter for the zone of clearance in most of the fungal isolates. The specific activities of these fungal isolates varied from 1.85 to 11.47 IU/mg protein. The present investigation revealed that the proposed simple diffusion technique gave similar results as compared to the established Congo red assay for endo--1, 4-xylanases. Moreover, the present technique avoided the cumbersome steps of staining by Congo red and de-staining by sodium chloride.

Isolation and characterization by asymmetric PCR of the ENDO1 gene for glucan endo-1, 3-β-D-glucosidase in Phytophthora cinnamomi associated with the ink disease of Castanea sativa Mill

Ink disease is one of the most destructive diseases in Castanea sativa. The most common symptoms are root necrosies and a reduction in root growth, which invariably lead to the death of the trees. Phytophthora cinnamomi is an oomycete associated with this disease whose life cycle develops integrally in the soil. In the present work, was a fragment with 1231bp of the glucan endo-1,3-β-D-glucosidase gene obtained by amplification, using conserved primers and the full-length gene sequence by flanking this known sequence by asymmetric PCR. This fragment was obtained from genomic DNA of Phytophthora cinnamomi isolated in the European Regions of Castilla-Leon (Spain) and Trás-os-Montes (Portugal) and associated with the ink disease of Castanea sativa Mill.

Doença da tinta é um das doenças mais destrutivas em Castanea sativa. Os sintomas mais comuns são necroses e uma redução em crescimento da raiz que invariavelmente leva à morte das plantas. Phytophthora cinnamomi é o oomycete associado a esta doença cujo ciclo de vida acontece integralmente no solo. Foi obtido um fragmento com 1231pb do gene glucan endo-1,3-β-D-glucosidase por amplificação usando oligonucleotidos conservados e a sequência completa do gene foi obtido flanqueando esta sequência conhecida por PCR assimétrico. Este fragmento foi obtido de ADN genómico de Phytophthora cinnamomi isolado por nós nas Regiões Europeias de Castilla-Léon (Espanha) e Trás-os-Montes (Portugal) e associado à doença da tinta da Castanea sativa Mill.

Clinical study of plasma(1→3)-β-D-glucan in invasive fungal infections / 中国医师进修杂志

Objective To study the clinical significance of plasma(1→3)-β-D-Oaten measurement in invasive fungal infections.Methods The levels of plasma(1→3)-β-D-glucan were measured bymicrobiology kinetic rapid reader MB-80 and GKT-5M set dymmic fungus detecting kit in 14 patients proven to suffer from invasive fungal infection and 13 healthIy voluntary persons.And the difference between them was compared.Results In 14 patients with invasive fungal infection,8 patients had fungal infection of lower respiratory tract and lung,6 patients had fungemia.There were 11 patients infected by monilia(1 patient combined infection),2 patients infected by aspergillus,and 2 patients infected by pneumocystis(1 Datient clinical diagnosis without aetiology proof).The levels of plasma(1→3)-β-D-slucan in invasive fungal infections patients were(105.02±82.22)ng/L,which were higherthan thosein healthy persons[(6.65±1.01)ng/L)J,P＜0.01.Conclusion The levels of plasma(1→3)-β-D-glucanisan ia an important index in diagnosis of invasive fungal infections.

Expression of the Trichoderma reesei Endoglucanse IV in Pichia pastoris / 微生物学通报

In this work,the Pichia pastoris expression system was applied to express the T.reesei EGⅣ.The eg4 gene was isolated from rice hull induced T.reesei culture through RT-PCR,and was ligated with the Pichia expression vector pPICZ?A,resulting in the recombinant plasmid pPICZ?A-eg4.The recombinant plasmid pPICZ?A-eg4 was then linearized and transformed into P.pastoris GS115,and the eg4 gene was in frame integrated into the Pichia genome through homologous recombination,resulting the recombinant strain P.pastoris-EGⅣ1.With methanol induction,the recombinant strain P.pastoris-EGⅣ1 expressed and secreated EGⅣ into the culture supernatant with CMC activity of 2.11U/mL.The SDS-PAGE analysis showed that the apparent molecular weight of expressed protein was about 50kD,slightly less than that produced by T.reesei.