A catalogue of Clostridium thermocellum endoglucanase, β-glucosidase and xylanase genes cloned in Escherichia coli

Abstract Two independent collections of clones containing Clostridium thermocellum genes involved in cellulose have been previously obtained at IAPGR, Cambridge, and at the Pasteur Institute, Paris. The two collections were compared for cross-hybridization, restriction maps and enzyme phenotypes. Truly distinct genes were one β-glucosidase gene, two xylanase genes, and fifteen endogluconase genes. Two of the cloned fragments contained extraneous DNA which was absent from their respective counterparts isolated in the other collection. The dicrepancies resulted from in vivo rearrangements which had occurred in either of the C. thermocellum NCIB 10682 stocks used to generate the two gene banks.     

Isolation and characterization of a cellulase-free pectinolytic and hemicellulolytic thermophilic fungus

A thermophilic fungus belonging to the Deuteromyces, having pectinase and xylanase activities, was grown at its optimum temperature of 55°C. It grew over a wide pH range of 4 to 10, being optimal at 6. The fungus grew well on modified Mandels' medium in which cellulose was substituted either with hemicellulose or pectin. With citrus pectin as carbon source, 121 units/ml of pectinase activity were obtained and with larch wood xylan as carbon source, 83 units/ml of xylanase activity were obtained.     

Purification and characterization of barley-aleurone xylanase

Xylanase (-1,4-D-xylan xylanohydrolase; EC 3.2.1.8) from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was purified and characterized. Purification was by preparative isoelectric focusing and a Sephadex G-200 column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme showed a single protein band with an apparent molecular weight (Mr)=34000 daltons. The isoelectric point of the enzyme was 4.6. The enzyme had maximum activity on xylan at pH 5.5 and at 35° C. It was most stable between pH 5 and 6 and at temperatures between 0 and 4° C. The K_m was 0.86 mg xylan·ml^-1.Abbreviations GA₃ gibberellic acid - kDa kilodalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

微生物产生的木聚糖酶的功能和应用

术聚糖是一种异质多糖,主要由木糖和阿拉伯糖组成。微生物产生的木聚糖酶来源广泛,能将木聚糖水解为木寡糖和D-木糖。该酶具有极大的应用价值,如可用于纸浆的漂白以减少环境污染,也可将造纸工业及农业废料中的木聚糖转化为D-木糖。     

Expression and characterization of amylase encoded by a gene cloned from Cellulomonas sp. NCIM 2353

A Cellulomonas genomic fragment encoding extracellular amylase activity was isolated as a clone (ACs2) in Escherichia coli DH10B. The amylase was expressed in the absence of IPTG and in the presence of starch or maltose. This enzyme corresponded to the low mobility activity of Cellulomonas amylases as demonstrated on gel electrophoresis. Maltose, as well as lactose, xylose and xylan cross-induced the amylase of clone ACs2. Maltose-induced amylase was purified to homogeneity. ACs2-coded amylase is a 70kDa acidic protein, with a pH optimum of 7.0 at 45°C. This enzyme exhibited an endo mode of action, similar to the corresponding Cellulomonas enzyme.     

Optimization for xylanase and cellulase production from Aspergillus niger ATTC 6275 in palm oil mill wastes and its application

Optimization of enzyme production from Aspergillus niger ATCC 6275 under both submerged and solid-substrate cultivation was investigated. Results from submerged cultivation using palm oil mill effluent revealed that pretreatment of ground palm cake did not improve enzyme production. Addition of 0.60g NH4NO3/l generated maximum activity of xylanase and cellulase (CMCase). The optimum aeration rate was 1.2 v/v min. Under solid-substrate cultivation, the results indicated that heating and alkali treatment of the ground palm cake gave no further improvement in enzyme production. The optimal N-source was 2% urea. Optimal initial moisture contents for xylanase and CMCase activities were 60% and 50% respectively, with temperature optima of 30°C and 35°C, respectively. The optimal inoculum size was 1× 108 spores/g palm cake with an initial pH of 4.5–5.0. The maximum activities of xylanase (282.9U/g) and CMCase (23.8U/g) were obtained under the optimum conditions. Solid-substrate cultivation was a better method for the production of enzyme, particularly xylanase, from A. niger ATCC 6275. The application of these enzymes to decanter effluent showed the separation of oil and grease and suspended solids from the effluent. This is comparable to the result achieved from using the commercial xylase preparation Meicelase and superior to the effect of Sumyzyme.     

His22 of TLXI plays a critical role in the inhibition of glycoside hydrolase family 11 xylanases

Recently, a novel wheat thaumatin-like protein, TLXI, which inhibits microbial glycoside hydrolase family (GH) 11 xylanases has been identified. It is the first xylanase inhibitor that exerts its inhibition in a non-competitive way. In the present study we gained insight into the interaction between TLXI and xylanases via combined molecular modeling and mutagenic approaches. More specifically, site-specific mutation of His22, situated on a loop which distinguishes TLXI from other, non-inhibiting, thaumatin-like proteins, and subsequent expression of the mutant in Pichia pastoris resulted in a protein lacking inhibition capacity. The mutant protein was unable to form a complex with GH11 xylanases. Based on these findings, the interaction of TLXI with GH11 xylanases is discussed.     

Structural and functional analyses of catalytic domain of GH10 xylanase from Thermoanaerobacterium saccharolyticum JW/SL‐YS485

Xylanases are capable of decomposing xylans, the major components in plant cell wall, and releasing the constituent sugars for further applications. Because xylanase is widely used in various manufacturing processes, high specific activity, and thermostability are desirable. Here, the wild‐type and mutant (E146A and E251A) catalytic domain of xylanase from Thermoanaerobacterium saccharolyticum JW/SL‐YS485 (TsXylA) were expressed in Escherichia coli and purified subsequently. The recombinant protein showed optimal temperature and pH of 75°C and 6.5, respectively, and it remained fully active even after heat treatment at 75°C for 1 h. Furthermore, the crystal structures of apo‐form wild‐type TsXylA and the xylobiose‐, xylotriose‐, and xylotetraose‐bound E146A and E251A mutants were solved by X‐ray diffraction to high resolution (1.32–1.66 Å). The protein forms a classic (β/α)₈ folding of typical GH10 xylanases. The ligands in substrate‐binding groove as well as the interactions between sugars and active‐site residues were clearly elucidated by analyzing the complex structures. According to the structural analyses, TsXylA utilizes a double displacement catalytic machinery to carry out the enzymatic reactions. In conclusion, TsXylA is effective under industrially favored conditions, and our findings provide fundamental knowledge which may contribute to further enhancement of the enzyme performance through molecular engineering. Proteins 2013; 81:1256–1265. © 2013 Wiley Periodicals, Inc.     

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.