Cloning and Expression of Extremely Heat-Resistant Endo-β-1,4-Glucanase Gene from Thermotoga maritima in Bacillus subtilis

Gene encoding endo-β-1,4-glucanase(TM1525)is derived from Thermotoga maritima(T.maritima),which has an open reading frame of 825 bp and encodes a 274 amino acid endo-β-1,4-glucanase.This enzyme has the same high temperature resistance as thermophilic bacteria,which is an ideal property for industrial applications.By molecular biological means,TM1525 was cloned into pHT43 vector and introduced into Bacillus subtilis(B.subtilis)WB800N by electroporation.The results showed that the WB800N expression system was successfully constructed,and extracellular expression of the recombinant gene was achieved.Cellulose hydrolyzed activity of the protein was exhibited.

Partitioning and purification of extracellular β-1,3-1,4-glucanase in aqueous two-phase systems

The partition behaviors of β-1,3-1,4-glucanase, α-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）/MgSO4） was examined with regard to the effects of PEG molecular weight （MW） and concentration, MgSO4 concentration, pH and NaC1 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 β-glucanase with the PEG/MgSO4 system. MgSO4 concentration influenced the partition and extraction of β-glucanase significantly, pH had little effect on β-glucanase or proteases partition but affected a-amylase partition when pH was over 7.0. The addition of NaCl had little effect on the partition behavior of β-glucanase but had very significant effects on the partitioning of α-amylase and on the neutral proteases. The partition behaviors of β-glucanase, α-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 β-glucanase was developed, which achieved β-glucanase recovery of 65.3% and specific activity of 14027 U/mg, 6.6 times improvement over the whole broth.

Purification and Some Properties of Endo-1,4-β-Glucanases of Trichoderma harzianum UzCF-28

This work aimed at isolation, purification and study of biochemical features of cellulolytic enzymes synthesized by Trichoderma harzianum UzCF-28 strain. Strain UzCF-28 revealed a high cellulolytic activity during submerged cultivation in the liquid culture on modified Mandels nutrient medium, where wheat straw was used as a source of carbon. As a result of purification by precipitation with ammonium sulfate and further ion exchange chromatography, two isoforms of endo- 1,4-β-glucanase-EG II and EG III with molecular weight of 135 and 75 kDa respectively were revealed. The pH optimum for EG I and EG III was 4.5, while for EG II—4.7, irrespective of the applied substrates—either CMC or “Whatman filter” paper. Heating up to 40°C of EG III did not lead to its inactivation, and on the contrary, its activity increased by more than three times comparing to the initial activity of the enzyme, i.e. thermostability of EG III among tested enzymes significantly varied.

Improved extracellular endo-1,4-β-mannosidase activity of recombinant Pichia pastoris by optimizing signal peptide

In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A, αpre, HFBI) were chosen to be analyzed by Signal P 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector p GAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4 I, INU1 A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/m L, while those mediated by MF4 I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/m L, respectively. The maximum MAN activity reached 347.5 U/m L with 4 gene copies mediated by MF4 I. These results indicate that replacing the signal peptide α-factor with MF4 I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.

Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination

The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtil＆ was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone a-factor （MFals）, and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction （PCR）-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae （SC-βG） was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A （PrA） activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/（h·ml） after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.

Xyloglucans of Monocotyledons Have Diverse Structures

Except in the Poaceae, little is known about the structures of the xyloglucans in the primary walls of monocotyledons. Xyloglucan structures in a range of monocotyledon species were examined. Wall preparations were isolated, extracted with 6 M sodium hydroxide, and the extracts treated with a xyloglucan-specific endo-（1→4）-β-glucanase preparation. The oligosaccharides released were analyzed by high-performance anion-exchange chromatography and by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. Oligosaccharide profiles of the non-commelinid monocotyledons were similar to those of most eudicotyledons, indicating the xyloglucans were fucogalactoxyloglucans, with a XXXG a core motif and the fucosylated units XXFG and XLFG. An exception was Lemna minor （Araceae）, which yielded no fucosylated oligosaccharides and had both XXXG and XXGn core motifs. Except for the Arecales （palms） and the Dasypogonaceae, which had fucogalactoxyloglucans, the xyloglucans of the commelinid monocotyledons were structurally different. The Zingiberales and Commelinales had xyloglucans with both XXGn and XXXG core motifs; small proportions of XXFG units, but no XLFG units, were present. In the Poales, the Poaceae had xyloglucans with a XXGn core motif and no fucosylated units. In the other Poales families, some had both XXXG and XXGn core motifs, others had only XXXG; XXFG units were present, but XLFG units were not.