Β-glucanase Gene Research Articles

Pemodelan protein dan analisis molecular docking enzim β-glukanase solat Bacillus subtilis W3.15

The β-glucanase enzyme is an enzyme protein that can hydrolyze β-glucan, one of the main components of the fungal cell wall. This enzyme protein is produced by several bacteria, one of which is B. subtilis. The three-dimensional (3D) structure of proteins is necessary to understand their properties and functions of proteins. Enzyme proteins can be analyzed for their structure and function using in silico method. This study aims to detect the β-glucanase gene from B. subtilis W3.15 and analyze it using the in silico method. The methods in this research are homology modeling and molecular docking analyses. Modeling was carried out using the SWISS-MODEL server and docking analysis using the PLANTS 1.1 program. Modeling the β-glucanase enzyme is based on the template of the β-glucanase enzyme protein model with PDB code 3o5s. The results of sequence alignment and model visualization were quite good as indicated by the model having a Ramachandran Plot value in the favored area of 91.10 %, a MolProbity score of 0.95, and a QMEAN value of 0.90 ± 0.06. The β-glucanase enzyme model was then docked using the PLANTS1.1 program with native ligand B3P, 1,4-β-D-Glucan, D-glucose, β-D-Glucan from oats, and N-Acetyl glucosamine. The results of docking analysis showed that the β-glucan ligand (β-D-glucan from oats) used as a substrate in the cultivation of isolate B. subtilis W3.15 had a better binding energy prediction value compared to the B3P ligand, which is a natural ligand in the template proteins. [Keywords: β-Glucan, β-D-Glucan from oat, ligand, PLANTS 1.1, 3D structure, SWISS-MODEL]

Discovery and functional verification of endogenous glucanases for scleroglucan hydrolysis in Sclerotium rolfsii

Scleroglucan is a high-molecular water-soluble microbial exopolysaccharide and mainly applied in the fields of petroleum, food, medicine and cosmetics. The high molecular weight of scleroglucan produced by microbial fermentation leads to low solubility, high viscosity and poor dispersibility, thus bringing a series of difficulties to extraction, preservation and application. It is important to explore suitable degradation method to adjust the molecular weight of scleroglucan for expanding its industrial application. Taking Sclerotium rolfsii WSH-G01 as a model strain, in which functional annotations of the glucanase genes were conducted by whole genome sequencing. Based on design of culture system for culture system for differential expression of β-glucanase, endogenous β-glucanase genes in S. rolfsii WSH-G01 were excavated by transcriptomics analysis. Functions of these potential hydrolases were further verified. Finally, 14 potential endogenous hydrolase genes were obtained from S. rolfsii. After heterologous overexpression in Pichia pastoris, 10 soluble enzymes were obtained and 5 of them had the activity of laminarin hydrolysis by SDS-PAGE and enzyme activity analysis. Further investigation of the 5 endogenous hydrolases on scleroglucan degradation showed that enzyme GME9860 has positive hydrolysis effect. The obtained results provide references not only for obtaining low and medium molecular weight of scleroglucan with enzymatic hydrolysis, but also for producing different molecular weight of scleroglucan during S. rolfsii fermentation process with metabolic engineering.

Expression of the β-Glucanase Gene from Paenibacillus jamilae Bg1 in Pichia pastoris and Characteristics of the Recombinant Enzyme

The isolation, heterologous expression, and characterization of a new, thermostable β-glucanase from Paenibacillus jamilae are described. The bgl26 gene from the P. jamilae Bg1 VKPM B-13 093 strain, which consists of 714 nucleotides, encodes endo-1,3-1,4-β-glucanase (EC 3.2.1.73), which contains 213 amino acids and 24 residues of the putative signal peptide in the N-terminal region. The nucleotide sequence of the bgl26 gene and the amino acid sequence of the mature Bgl26 protein have the greatest homology with the sequences of the Paenibacillus macerans endo-1,3-1,4-β-glucanase (82 and 88%, respectively). A gene fragment encoding the mature protein was expressed in Pichia pastoris. The purified recombinant enzyme Bgl26 was active against barley β-glucan. The optimal pH for the enzyme activity was 7.0, and the optimum temperature range was 40–45°C. The specific β-glucanase activity was at the level of 6650 U/mg of protein; KM and Vmax were equal to 6.4 ± 0.3 mg/mL and 9450.1 ± 471.2 μmol/(min mg), respectively. The recombinant protein Bgl26 was characterized by a high pH and thermal stability, as well as resistance to digestive enzymes. It was also shown that Co2+ ions have a positive effect on enzyme activity.

A Strategic Production Improvement of Streptomyces Beta Glucanase Enzymes with Aid of Codon Optimization and Heterologous Expression

β-glucan rich cereals such as barley and oats serve as a raw material in breweries and also used as an animal feed. Digestion of β-glucan is often a major hurdle, thus providing exogenous enzyme β-glucanase serves as an option. The present study takes an effort for the expression and over production of β-glucanase genes from Streptomyces sp in E. coli. The exo-β-1,4-glucanase and endo-β-1,3-glucanase encoding genes were isolated and codon optimized and significant-high expression levels were obtained in E. coli strain. The expressed enzymes showed broad pH stability, good thermostability, and better affinity towards the barley β-glucan substrate. The study implies that heterologous expression with codon optimization strategy enhances the production of Streptomyces origin beta-glucanase enzymes with prominent physio-chemical properties for efficient beta-glucan degradation.

Directed mutation of β-glucanases from probiotics to enhance enzymatic activity, thermal and pH stability.

β-glucanases are widely applied in biological control, brewing and feed industries; however, there are seldom studies of β-glucanases in probiotics. Here, β-glucanase genes were cloned from Bacillus licheniformis, Lactobacillus fermentum and L. johnsonii. β-glucanase genes, as blg, lfg and ljg isolated from B. licheniformis, L. fermentum and L. johnsonii were prokaryotic expressed to obtain recombinant strains BL, LF and LJ, respectively. Directed mutations in these genes were introduced by sequential error-prone PCR. Results showed that β-glucanase activities in three mutants mblg, mlfg and mljg were 1.94-, 2.72- and 1.29-fold higher than the BL, LF and LJ, respectively. Mutation sites analysis showed substitutions at Ser370Gly and Leu395Phe in mblg; Arg169His and Asn302Ser in mlfg; Val132Met, Ser226Asn, and Asp355Gly in mljg. Spatial structural predictions revealed the numbers and positions of α-helices and β-strands in the three mutants were altered, which might result in β-glucanase activity increasement. Analysis of β-glucanase properties revealed no significant differences in the optimal temperatures and pH between mutant and wild-type strains. However, mlfg and mljg exhibited greater thermal stability at 30-50℃ than the wild-type strains, and mblg improved pH stability compared with wild-type strain. This is the first report about β-glucanase-encoding genes in L. fermentum and L. johnsonii. These findings provide an efficient way to improve the activity of β-glucanase.

Cloning and Expression of Bacillus pumilis Bg57 β-Glucanase Gene in Pichia pastoris: Purification and Characteristics of Recombinant Enzyme

The isolation, heterologous gene expression, and characteristics of a new β-glucanase from Bacillus pumilus is described. The bgl1 gene from the Bacillus pumilus Bg57 VKPM В-13195 strain, which consists of 729 nucleotides, encodes a secreted endo-β-l,3(4)-D-glucanase (EC 3.2.1.6) containing 214 amino acids and 28 residues of the putative signal peptide in the N-terminal area. The nucleotide sequence of bgl1 and amino acid sequence of the mature Bgl protein have the highest homology (89 and 95%, respectively) with the sequences for β-l,3-l,4-glucanase from Bacillus licheniformis. A fragment of the gene encoding the mature protein was expressed in Pichia pastoris. The purified recombinant enzyme of Bgl was active towards barley β-glucan and lichenin. With barley β-glucan, the optimal pH and temperature were shown to be 6.0 and 50°C, respectively; Km and Vmax were 2.2 mg/mL and 3036.4 μmol/(min mg), respectively. The recombinant protein Bgl showed a high specific activity, thermal stability, and resistance to digestive enzymes. Other characteristics of the recombinant protein, including pH stability and sensitivity to metal ions and chemical reagents, were also determined.

Cytological and Molecular Characteristics of Pollen Abortion in Lily with Dysplastic Tapetum

Lily was grown worldwide as a fresh cutting flower because of its colorful petals, but its anther contained a large number of pollen grains that cause serious pollen contamination, however, pollen abortion can effectively reduce the level of pollen pollution. Our analysis aims to use cytological observation to detect the critical stage when pollen abortion occurs and to provide comprehensive gene expression information at the transcriptional level. The result showed that pollen abortion in ‘Little Kiss’ began at the mononuclear stage and the callose that covers the microspores failed to degenerate when young pollens were released from the tetrads. In addition, compared with the normally developed one, the tapetum of ‘Little Kiss’ degraded in advance while the degradation of callose was delayed. Furthermore, 103 differentially expressed genes (DEGs) related to the advance degeneration of tapetum cells and callose were found in the expression levels, including 22 transcription factors (TFs). In particular, two β-glucanase genes (endo-1,3(4)-β-glucanase, exo-β-glucanase) responsible for callose degeneration were significantly down-regulated. These results suggested that pollen abortion may occur at mononuclear stage and that early degeneration of tapetum cells resulted in a significant down-regulation of β-glucanase genes. As a result, the callose to cover microspores impedes the formation of pollen walls, which may possibly lead to pollen abortion.

β-Glucanase specific expression in the intestine of transgenic pigs.

Producing heterologous enzymes in the animal digestive tract to improve feed utilization rate is a new research strategy by transgenic technology. In this study, transgenic pigs specifically expressing β-glucanase gene in the intestine were successfully produced by somatic cell nuclear transfer technology in order to improve digestibility of dietary β-glucan and absorption of nutrients. The β-glucanase activity in the intestinal juice of 4 transgenic pigs was found to be 8.59 ± 2.49U/mL. The feeding trial results showed that the crude protein digestion of 4 transgenic pigs was significantly increased compared with that of the non-transgenic pigs. In order to investigate the inheritance of the transgene, 7 G1 transgenic pigs were successfully obtained. The β-glucanase activity in the intestinal juice of 7 G1 transgenic pigs was found to be 2.35 ± 0.72U/mL. The feeding trial results showed the crude protein digestion and crude fat digestion were significantly higher in 7 G1 transgenic pigs than in non-transgenic pigs. Taken together, our study demonstrated that the foreign β-glucanase expressing in the intestine of the transgenic pigs could reduce the anti-nutritional effect of β-glucans in feed. In addition, β-glucanase gene could be inherited to the offsprings and maintain its physiological function. It is a promising approach to improve feed utilization by producing transgenic animals.

Isolation and characterization of halotolerant bacilli from chickpea (Cicer arietinum L.) rhizosphere for plant growth promotion and biocontrol traits

The present study describes the identification and polyphasic characterization of salt tolerant bacilli displaying plant growth promoting and broad spectrum antifungal activities. A total of 110 strains were isolated, out of which nine were selected on the basis of halotolerance, in vitro plant growth promoting attributes and antagonism against multiple phytopathogens. Preliminary identification of strains was done on the basis of phylogenetic analysis and comparison of 16S RNA sequences with type strains. The test strains were identified as Bacillus pumilus, B. subtilis, B. licheniformis, B. safensis and B. cereus. These strains possess ability to tolerate high salt (8–10% NaCl), form endospore, showed broad spectrum antifungal activity and therefore might cope with adverse environments. Scanning electron microscopy of promising test strains (CG18 and CM25) and pathogen interaction indicated destruction of fungal mycelia by halotolerant antagonists due to cytoplasmic extrusion. Moreover, PCR amplification of bacillomycin (bmyB), chitinase (ChiA), and β-glucanase genes and production of hydrolytic enzymes suggested that test strains could have a role to manage plant pathogens under saline conditions. Finally, the selected strains exhibited some plant growth promotion traits. Overall, this study demonstrated that the evaluated strains could be useful in developing microbial products to enhance tolerance to biotic and abiotic stresses and boost plant growth.

Activity of defense related enzymes and gene expression in pigeon pea (Cajanus cajan) due to feeding of Helicoverpa armigera larvae

ABSTRACTThe biochemical and molecular basis of the defense in a mild tolerant (ICPL-332) and susceptible (ICPL-87) cultivars of pigeon pea (Cajanus cajan) due to Helicoverpa. armigera infestation was studied. We found that feeding by the larvae generated H2O2 in a localized manner and activity was observed upto 12 h (hrs) of with a sharp decline within 24 h. Similarly, PPO activity was also detected till 12 h of treatment, which decreased after 24 h of feeding by larvae. The activity of trypsin inhibitor was detected in all the treatments when assayed at 12 and 24 h after larval feeding. The expression of defense genes like the Pre-hevein-like protein PR-4 precursor (PR-4), protease inhibitor/seed storage/LTP family protein (Ltp) were significantly up-regulated in ICPL-332 upon infestation after 12 h as compared to ICPL-87, whereas the endo 1, 4 -β-glucanase (Kor-1) gene was expressed in both the cultivars after 24 h of infestation. Both the cultivars varied with respect to the induction of defense-related genes during larval feeding, both the PR-4 and Ltp genes appeared to be important for defense against H. armigera in pigeon pea. Thus, the present study revealed an insight of herbivore-induced biochemical and molecular changes in pigeon pea.

Клонирование и экспрессия β-глюканазы Bacillus pumilus Bg57 в Pichia pastoris: очистка и характеристика рекомбинантного фермента

Клонирование и экспрессия β-глюканазы Bacillus pumilus Bg57 в Pichia pastoris: очистка и характеристика рекомбинантного фермента

Variation in barley (1→ 3, 1→4)-β-glucan endohydrolases reveals novel allozymes with increased thermostability.

Novel barley (1→3, 1→4)-β-glucan endohydrolases with increased thermostability. Rapid and reliable degradation of (1→3, 1→4)-β-glucan to produce low viscosity wort is an essential requirement for malting barley. The (1→3, 1→4)-β-glucan endohyrolases are responsible for the primary hydrolysis of cell wall β-glucan. The variation in β-glucanase genes HvGlb1 and HvGlb2 that encode EI and EII, respectively, were examined in elite and exotic germplasm. Six EI and 14 EII allozymes were identified, and significant variation was found in β-glucanase from Hordeum vulgare ssp. spontaneum (wild barley), the progenitor of modern cultivated barley. Allozymes were examined using prediction methods; the change in Gibbs free energy of the identified amino acid substitutions to predict changes in enzyme stability and homology modelling to examine the structure of the novel allozymes using the existing solved EII structure. Two EI and four EII allozymes in wild barley accessions were predicted to have improved barley β-glucanase thermostability. One novel EII candidate was identified in existing backcross lines with contrasting HvGlb2 alleles from wild barley and cv Flagship. The contrasting alleles in selected near isogenic lines were examined in β-glucanase thermostability analyses. The EII from wild barley exhibited a significant increase in β-glucanase thermostability conferred by the novel HvGlb2 allele. Increased β-glucanase thermostability is heritable and candidates identified in wild barley could improve malting and brewing quality in new varieties.

Improvement of anti-nutritional effect resulting from β-glucanase specific expression in the parotid gland of transgenic pigs.

β-Glucan is the predominant anti-nutritional factors in monogastric animal feed. Although β-glucanase supplementation in diet can help to eliminate the adverse effects, enzyme stability is substantially modified during the feed manufacturing process. To determine whether the expression of endogenous β-glucanase gene (GLU) in vivo can improve digestibility of dietary β-glucan and absorption of nutrients, we successfully produced transgenic pigs via nuclear transfer which express the GLU from Paenibacillus polymyxa CP7 in the parotid gland. In three live transgenic founders, β-glucanase activities in the saliva were 3.2, 0.07 and 0.03U/mL, respectively, and interestingly the enzyme activities increased in the pigs from 178days old to 789days old. From the feed the amount of gross energy, crude protein and crude fat absorbed by the transgenic pigs was significantly higher than the non-transgenic pigs. Meanwhile the moisture content of the feces was significantly reduced in transgenic pigs compared with the non-transgenic pigs. Furthermore, in all positive G1 pigs, β-glucanase activity was detectable and the highest enzyme activity reached 3.5U/mL in saliva. Also, crude protein digestion was significantly higher in G1 transgenic pigs than in control pigs. Taken together, our data showed that the transgenic β-glucanase exerted its biological catalytic function in vivo in the saliva, and the improved performance of the transgenic pigs could be accurately passed on to the offspring, indicating a promising alternative approach to improving nutrient availability was established to improve utilization of livestock feed through transgenic animals.

A novel efficient β-glucanase from a paddy soil microbial metagenome with versatile activities.

BackgroundCellulose, an abundant and renewable polysaccharides, constitutes the largest resource for bioconversion of biofuels. Plant polysaccharides hydrolysis is catalyzed by cellulases, which include endoglucanases, exoglucanases, and β-glucosidases. Converting cellulose and hemicellulose to short chains of oligosaccharides by endo-/exoglucanases is the key step for biofuel transformation. Intriguingly, β-glucanases with transglycosylation activity not only can relieve product inhibition of glucan hydrolysis but also has potential application as biocatalysts for functional materials.ResultsHere, a metagenomic fosmid library was constructed from a paddy soil for cellulase screening. One purified clone showing carboxymethylcellulase activity was isolated, and the complete β-glucanase gene (umcel9y-1) was cloned and overexpressed in Escherichia coli. Phylogenetic analysis indicated that β-glucanase Umcel9y-1 belonged to the theme C of glycoside hydrolase family 9. Amino acids sequence showed 58.4 % similarity between Umcel9y-1 and its closest characterized reference, cellulase Cel01. Biological characterization showed that Umcel9y-1 was an efficient endoglucanase and also exhibited high activities of exoglucanase and transglycosylation. The transglycosylation products of Umcel9y-1 including sophorose, laminaribiose, and gentiobiose, and transglycosylation was detected under all activated conditions. The order of catalytic efficiency for polysaccharides, cellooligosaccharides, and aryl-β-glycosides was p-nitrophenol-D-cellobioside, barley glucan, cellopentaose, cellotetraose, cellotriose, hydroxyethylcellulose, cellohexose, laminarin, and carboxymethylcellulose, respectively. The barley glucan was the optimal polysaccharides for Umcel9y-1 with Km and Kcat/Km values of 13.700 mM and 239.152 s−1 mM−1, respectively.ConclusionBiological characterizations of recombinant Umcel9y-1 showed that the versatile β-glucanase had efficient endoglucanase activity to barley glucan and also exhibited high activities of exoglucanase and transglycosylation. The optimum conditions of recombinant Umcel9y-1 was pH 6.5–7.0 at 37 °C with predominant halotolerance and high-thermal stability. These results indicate that the novel metagenomic-derived β-glucanase may be a potent candidate for industrial applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0449-6) contains supplementary material, which is available to authorized users.

Oligonucleotide treatment causes flax β-glucanase up-regulation via changes in gene-body methylation.

BackgroundNowadays, the challenge for biotechnology is to develop tools for agriculture and industry to provide plants characterized by productivity and quality that will satisfy the growing demand for different kinds of natural products. To meet the challenge, the generation and application of genetically modified plants is justified. However, the strong social resistance to genetically modified organisms and restrictive regulations in European Union countries necessitated the development of a new technology for new plant types generation which uses the knowledge resulting from analysis of genetically modified plants to generate favourably altered plants while omitting the introduction of heterologous genes to their genome. Four-year experiments led to the development of a technology inducing heritable epigenetic gene activation without transgenesis.ResultsThe method comprises the induction of changes in methylation/demethylation of the endogenous gene by the plant’s treatment with short oligodeoxynucleotides antisense to the coding region. In vitro cultured plants and F3 generation flax plants overproducing the β-1,3-glucanase gene (EMO-βGlu flax) were characterized by up-regulation of β-glucanase and chitinase genes, decreases in the methylation of CCGG sequences in the β-glucanase gene and in total DNA methylation and, more importantly, reasonable resistance against Fusarium infection. In addition, EMO-βGlu flax obtained by this technology showed similar features as those obtained by genetic engineering.ConclusionTo our best knowledge, this is the first report on plant gene activation by treatment with oligodeoxynucleotides homologous to the coding region of the gene. Apart from the evident effectiveness, the most important issue is that the EMO method allows generation of favourably altered plants, whose cultivation makes the plant producer independent from the complicated procedure of obtaining an agreement on GMO release into the environment and whose products might be more easily introduced to the global market.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0261-z) contains supplementary material, which is available to authorized users.

Intestine-specific expression of the β-glucanase in mice

Guan, L.-Z., Xi, Q.-Y., Sun, Y.-P., Wang, J.-L., Zhou, J.-Y., Shu, G., Jiang, Q.-Y. and Zhang, Y.-L. 2014. Intestine-specific expression of the β-glucanase in mice. Can. J. Anim. Sci. 94: 287-293. The β-glucanase gene (GLU, from Paenibacillus polymyxa CP7) was cloned into a specific expression plasmid (MUC2-GLU-LV). Transgenic mice were prepared by microinjection. Polymerase chain reaction and Southern blot analysis of genomic DNA extracted from the tail tissue of transgenic mice showed that the mice carried the β-glucanase gene. Northern blot analysis indicated that β-glucanase was specifically expressed in the intestine of the transgenic mice. The β-glucanase activity in the intestinal contents was found to be 1.23±0.32 U mL-1. The crude protein, crude fat digestibility of transgenic mice were increased by 9.32 and 5.09% (P<0.05), respectively, compared with that of the non-transgenic mice, while moisture in feces was reduced by 12.16% (P<0.05). These results suggest that the expression of β-glucanase i...

Intestine-specific expression of the β-glucanase in mice

Guan, L.-Z., Xi, Q.-Y., Sun, Y.-P., Wang, J.-L., Zhou, J.-Y., Shu, G., Jiang, Q.-Y. and Zhang, Y.-L. 2014. Intestine-specific expression of the β-glucanase in mice. Can. J. Anim. Sci. 94: 287–293. The β-glucanase gene (GLU, from Paenibacillus polymyxa CP7) was cloned into a specific expression plasmid (MUC2-GLU-LV). Transgenic mice were prepared by microinjection. Polymerase chain reaction and Southern blot analysis of genomic DNA extracted from the tail tissue of transgenic mice showed that the mice carried the β-glucanase gene. Northern blot analysis indicated that β-glucanase was specifically expressed in the intestine of the transgenic mice. The β-glucanase activity in the intestinal contents was found to be 1.23±0.32 U mL−1. The crude protein, crude fat digestibility of transgenic mice were increased by 9.32 and 5.09% (P&lt;0.05), respectively, compared with that of the non-transgenic mice, while moisture in feces was reduced by 12.16% (P&lt;0.05). These results suggest that the expression of β-glucanase in the intestine of animals offers a promising biological approach to reduce the anti-nutritional effect of β-glucans in feed.

Multifarious plant growth promoting characteristics of chickpea rhizosphere associated Bacilli help to suppress soil-borne pathogens

Wilt and root rot are the major constraints in chickpea production and very difficult to manage through agrochemicals. Hence, for an ecofriendly and biological management, 240 strains of Bacillus and Bacillus derived genera were isolated from chickpea rhizosphere, further narrowed down to 14 strains on the basis of in vitro production of indole acetic acid, siderophore, phosphate solubilization, hydrolytic enzymes and were evaluated for antagonism against chickpea pathogens (Fusarium oxysporum f. sp. ciceri race 1, F. solani and Macrophomina phaseolina). The strains were identified on the basis of physiological characters and 16S RNA gene sequencing. The genotypic comparisons of strains were determined by BOX-polymerase chain reaction profiles and amplified rDNA restriction analysis. These isolates were evaluated in greenhouse assay in which B. subtilis (B-CM191, B-CV235, B-CL-122) proved to be effective in reducing wilt incidence and significant enhancement in growth (root and shoot length) and dry matter of chickpea plants. PCR amplification of bacillomycin (bmyB) and β-glucanase genes suggests that amplified genes from the Bacillus could have a role to further define the diversity, ecology, and biocontrol activities in the suppression of soil-borne pathogens.

Regulation of three genes encoding cell-wall-degrading enzymes ofTrichoderma aggressivumduring interaction withAgaricus bisporus

Members of the genus Trichoderma are very effective competitors of a variety of fungi. Cell-wall-degrading enzymes, including proteinases, glucanases, and chitinases, are commonly secreted as part of the competitive process. Trichoderma aggressivum is the causative agent of green mould disease of the button mushroom, Agaricus bisporus. The structures of 3 T. aggressivum genes, prb1 encoding a proteinase, ech42 encoding an endochitinase, and a β-glucanase gene, were determined. Promoter elements in the prb1 and ech42 genes suggested that transcription is regulated by carbon and nitrogen levels and by stress. Both genes had mycoparasitism-related elements indicating potential roles for the protein products in competition. The promoter of the β-glucanase gene contained CreA and AreA binding sites indicative of catabolite regulation but contained no mycoparasitism elements. Transcription of the 3 genes was measured in mixed cultures of T. aggressivum and A. bisporus. Two A. bisporus strains, U1, which is sensitive to green mould disease, and SB65, which shows some resistance, were used in co-cultivation tests to assess possible roles of the genes in disease production and severity. prb1 and ech42 were coordinately upregulated after 5 days, whereas β-glucanase transcription was upregulated from day 0 with both Agaricus strains. Upregulation was much less pronounced in mixed cultures of T. aggressivum with the resistant strain, SB65, than with the sensitive strain, U1. These observations suggested that the proteins encoded by these genes have roles in both nutrition and in severity of green mould disease.

β-Glucanase specific expression in the parotid gland of transgenic mice

The feasibility of using the pig parotid secretory protein promoter to drive the β-glucanase transgene expression in mouse parotid glands was examined in this study. The parotid gland-specific vector expressing β-glucanase gene (GLU, from Paenibacillus polymyxa CP7) was constructed. Transgenic mice were produced by the pronuclear microinjection. Both PCR and Southern blot analysis showed that the mice carried the β-glucanase gene and the β-glucanase gene could be stably inherited. Furthermore, RT-PCR and northern blot analysis indicated that it was specifically expressed in the parotid. The β-glucanase activity in the saliva was found to be 0.18U/mL. After feeding a diet containing 2% β-glucan, the average daily gain of transgenic was significantly higher than non-transgenic mice. The crude protein and crude fat concentration in faeces of transgenic mice were significantly reduced compared with that of the non-transgenic mice. These results suggest that the successful expression of foreign β-glucanase in the animal parotid would offer a promising biological approach to reduce the anti-nutritional effect of β-glucans in feed.