Β-mannanase Activity Research Articles

English

The present study was carried out to investigate the optimal culture conditions for β-mannanase production by Trichosporonoides oedocephalis using cheap and easily available agro-wastes as the carbon source in solid state cultivation. The effect of process parameters like incubation time, carbon source, incubation temperature, pH, percentage moisture content and inoculum concentration were optimized for enhanced mannanase production. Maximum β-mannanase activity was observed after 96 h of incubation. Different agricultural wastes were screened as substrates for β-mannanase production in comparison with locust bean gum (LBG). Among tested carbon sources, orange peels proved to the best for β-mannanase production. Initial pH of the culture medium was also optimized and a pH of 8.0 was found to be the optimum pH value for β-mannanase production. The optimum temperature, percentage moisture content and inoculum concentration were 30°C, 60% and 1×107 (spores/ml), respectively. Key words: Beta-mannanase, process parameters, solid state fermentation, agricultural wastes, Trichosporonoides oedocephalis.

Influence of Cultural and Nutritional Factors on β-Mannanase Production by Penicillium italicum under Submerged State Fermentation

Aims: The process parameters influencing enzyme production were optimized to ascertain the optimal cultural and nutritional conditions for β-mannanase production by Penicillium italicum in submerged state fermentation. Study Design: Five stages of experimental processes were designed for this study. The first stage, samples were withdrawn after 24, 48, 72, 96, 120, 144,168 and 192 h incubation. The second experiment, different agricultural wastes were screened as substitute to Locust Bean Gum. In third experiment, the effect of different pH values on β-mannanase production was evaluated, while the fermentation media were incubated at different temperatures in the fourth experiment. In fifth experiment, the effect of different surfactants on β-mannanase activity was evaluated. Place and Duration of Study: Microbiology Research Laboratory, Federal University of Technology, Akure Nigeria between September 2011 and March 2012. Methodology: Beta-Mannanase production was conducted in mineral salt medium and enzyme Original Research Article Olaniyi et al.; BMRJ, 5(6): 481-489, 2015; Article no.BMRJ.2015.051 482 activity determined by dinitrosalicylic acid method, while protein content was determined by Lowry method. Results: Maximum β-mannanase activity (41.667 U/ml) was observed after 120 h of incubation. Different agricultural wastes were screened as carbon substrates for β-mannanase production. Among tested carbon sources, orange peels proved to the best for β-mannanase production with an activity of 50.000 U/ml. Initial pH of the culture medium was optimized and a pH of 6.0 (130.556 U/ml) was found to be the best pH for β-mannanase activity. The optimum temperature was 30°C with an activity of 136.418 U/ml. Of all surfactants screened, Tween 20 (67.500 U/ml) gave the highest β-mannanase activity. Conclusion: The nutritional and cultural factors obtained to be optimal from this study will help to design an experiment for maximum mannanase biosynthesis using cheaper substrates in place of commercial substrates known to be expensive for enzyme production.

Engineering glycolysis branch pathways of Escherichia coli to improve heterologous protein expression

Escherichia coli expression systems are still preferred to other bacterial expression systems. However, by-product formation via glycolytic pathways inhibits protein production efficiency. In this paper, by-product-forming pathways were engineered to evaluate their effect on foreign protein production. Elimination of d-lactate dehydrogenase (encoded by ldhA) resulted in enhanced cell performance and 17.8% increase in recombinant β-mannanase production. Single deletions of pflB (encoding pyruvate formate lyase), pps (encoding phoenolpyruvate synthase) or poxB (encoding pyruvate oxidase) also had an affirmative impact on recombinant protein production. Furthermore, simultaneous deletions of ldhA, pflB, pps and poxB increased cell mass by 29% and β-mannanase production by 56% under shake-flasks cultivation. Meanwhile, overall acetate concentration showed a decrease of 33%. This quadruple mutant showed the best performance under bioreactor process, in which volume and specific activity of β-mannanase increased by 1.9 and 1.46 fold compared to the control strain respectively. The approach shown here indicated that rational engineering of glycolytic pathways can efficiently improve foreign protein production in E. coli.

Synthesis of galactosyl glycerol from guar gum by transglycosylation of α-galactosidase from Aspergillus sp. MK14

A guar gum-hydrolyzing strain, Aspergillus sp. MK14, secreted α-galactosidase selectively in liquid culture. Its α-galactosidase activity (0.820U/ml) was much higher than its β-mannosidase and β-mannanase activities (0.027 and 0.050U/ml, respectively). The molecular weight was estimated to be 59,000Da by SDS–PAGE. The optimal pH was 5 and it was active from pH 2.2 to 6.2. The optimal temperature was 60°C and the activity was stable below 50°C. Enzyme activity toward melibiose was much lower than that with pNP-α-d-galactopyranoside. The activities toward 61-α-d-galactosyl-mannobiose and 63,64-α-d-galactosyl-mannopentaose were relatively high (86.2% and 48.4% relative to pNP-α-d-galactopyranoside, respectively). MK14 crude enzyme released only the monosaccharides, galactose and mannose (Gal/Man: 0.64) from guar gum. When glycerol was added to the reaction mixture, the transglycosylation proceeded efficiently, and the synthesis of galactosyl glycerol was 76.6mg/g of guar gum. MK14 α-galactosidase could use guar gum as a good substrate (donor) in the transglycosylation.

Cloning, secretory expression and characterization of recombinant β-mannanase from Bacillus circulans NT 6.7.

The mannanase gene of B. circulans NT 6.7 was cloned and expressed in an Escherichia coli expression system. The B. circulans NT 6.7 mannanase gene consists of 1,083 nucleotides encoding a 360-amino acid residue long polypeptide, belonging to glycoside hydrolase family 26. The full-length mannanase gene including its native signal sequence was cloned into the vector pET21d and expressed in E. coli BL21 (DE3). β-Mannanase activities in the culture supernatant and crude cell extract were 37.10 and 515 U per ml, respectively, with most of the activity in the cell extract attributed to the periplasmic fraction. In contrast, expression of mannanase was much lower when using the B. circulans NT 6.7 mannanase gene without its signal sequence. The optimum temperature of recombinant β-mannanase activity was 50°C and the optimum pH was 6.0. The enzyme was very specific for β-mannan substrates with a preference for galactomannan. Hydrolysis products of locust bean gum were various mannooligosaccharides including mannohexaose, mannopentaose, mannotetraose, mannotriose and mannobiose, while mannose could not be detected. In conclusion, this expression system is efficient for the secretory production of recombinant β-mannanase from B. circulans NT 6.7, which shows good characteristics for various applications.

Improving the specific activity of β-mannanase from Aspergillus niger BK01 by structure-based rational design

β-Mannanase has found various biotechnological applications because it is capable of degrading mannans into smaller sugar components. A highly potent example is the thermophilic β-mannanase from Aspergillus niger BK01 (ManBK), which can be efficiently expressed in industrial yeast strains and is thus an attractive candidate for commercial utilizations. In order to understand the molecular mechanism, which helps in strategies to improve the enzyme's performance that would meet industrial demands, 3D-structural information is a great asset. Here, we present the 1.57Å crystal structure of ManBK. The protein adopts a typical (β/α)8 fold that resembles the other GH5 family members. Polysaccharides were subsequently modeled into the substrate binding groove to identify the residues and structural features that may be involved in the catalytic reaction. Based on the structure, rational design was conducted to engineer ManBK in an attempt to enhance its enzymatic activity. Among the 23 mutants that we constructed, the most promising Y216W showed an 18±2.7% increase in specific activity by comparison with the wild type enzyme. The optimal temperature and heat tolerance profiles of Y216W were similar to those of the wild type, manifesting a preserved thermostability. Kinetic studies showed that Y216W has higher kcat values than the wild type enzyme, suggesting a faster turnover rate of catalysis. In this study we applied rational design to ManBK by using its crystal structure as a basis and identified the Y216W mutant that shows great potentials in industrial applications.

A comparative analysis of β-mannanases of bacteria from Antarctica and Malaysia

β-mannanase is an enzyme that is commonly expressed in environmental bacteria. It degrades hemicellulose found in plant material and recycles nutrients back into the environment. Because this enzyme significantly contributes to biodegradation and has recently been applied in industry, we conducted a comparative analysis of bacterial isolates found in soil samples from Schirmacher Oasis, Antarctica, and Sabah, Malaysia that were capable of degrading mannan. A total of 9 bacterial isolates from Antarctica and 30 bacterial isolates from Malaysia exhibited β-mannanase activity. These bacteria were differentiated and clustered using their random amplified polymorphic DNA (RAPD) profiles, and the β-mannanase activity of these isolates was tested at different temperatures and pH. Five out of 9 Antarctica isolates and seven out of 30 Malaysian isolates were identified based on their 16S rDNA sequences. Identified bacterial isolates from Antarctica were: MP1 ( Bacillus amyloliquefaciens ), MP2 ( Bacillus pumilus ), MP5 ( Bacillus pumilus ), A40 ( Arthrobacter sp.), and C27 ( Arthrobacter oxydans ). Identified bacterial isolates from Malaysia were: Y1 ( Paenibacillus sp.), Y2 ( Bacillus sp.), Y16 ( Paenibacillus sp.), Y18 ( Paenibacillus sp.), A7 ( Paenibacillus sp.), B26 ( Streptomyces sp.), and D4 ( Paenibacillus amylolyticus ). β-mannanases produced by the Antarctica bacterial isolates MP1 ( Bacillus amyloliquefaciens ) and A40 ( Arthrobacter sp.) were active at 5℃ and 20℃, respectively, while the β-mannanase produced by the bacterial isolate from Malaysia, A7 ( Paenibacillus sp.), was active at 35℃.

Enhancing expression level of an acidophilic β-mannanase in Pichia pastoris by double vector system

An acidophilic β-mannanase-encoding gene (Auman5A) from Aspergillus usamii YL-01-78 was amplified and inserted into pPIC9K and pPICZαA vectors. The resulting recombinant vector, pPIC9K-Auman5A, was transformed into Pichia pastoris GS115. One strain having the highest recombinant β-mannanase activity of 54.6 U/ml, labeled GSKM4-8, was chosen from the first-batch P. pastoris transformants. Then, the pPICZαA-Auman5A was transformed into GSKM4-8 again. From the second-batch transformants, one strain (GSKZαM4-2) with the highest β-mannanase activity of 78.1 U/ml was obtained, and used to optimize expression conditions. As GSKZαM4-2 was induced under the optimized conditions (initial pH value 6.5, induction period 120 h, methanol concentration 1.5 %, and induction temperature 32 °C), β-mannanase activity reached 162.8 U/ml. Protein and carbohydrate assays showed that the β-mannanase, a glycoprotein with an apparent molecular weight of 49.8 kDa and a carbohydrate content of 21.3 %, was extracellularly expressed. It displayed the maximum activity at pH 3.0 and 70 °C, and was stable at a pH range of 3.0–7.0 and at 60 °C. Its activity was not significantly affected by metal ions tested and EDTA, but inhibited by Ag+ and Hg2+. Its most favorable substrate was locust bean gum, followed by konjac flour and guar gum. The K m and V max towards locust bean gum were 1.36 mg/ml and 415.8 U/mg, respectively. These results suggested that the β-mannanase can be expressed with higher level and possesses superior enzymatic properties, making it a good candidate in industrial processes.

Purification, characterization, and production of β-mannanase from Bacillus subtilis TJ-102 and its application in gluco-mannooligosaccharides preparation

A novel β-mannanase-producing strain, Bacillus subtilis TJ-102, was identified and characterized. Response surface method was applied to improving and enhancing the enzyme production. The optimized media components were obtained: 45.25 g/L konjac, 9.29 g/L Na2HPO4·12H2O, 2.60 g/L CaCO3, 1.0 g/L (NH4)2SO4, 0.3 g/L KH2PO4, 1.0 g/L NaCl, 1.0 g/L MgCl2·6H2O, and 0.01 g/L FeSO4. Under these conditions, the β-mannanase activity could achieve 205.3 U/mL in a 7-L fermentor. Then, β-mannanase was 7.39-fold purified by salting out, ultrafiltration, anion-exchange, and size-exclusion preparative chromatography with a recovery of 21.41 % and a specificity of 125.36 U/mg proteins. β-Mannanase was stable below 65 °C and pH 5.0–8.0, which exhibited excellently enzymatic efficiency in the preparation of gluco-mannooligosaccharides (GMOS) by hydrolyzing konjac flour. The GMOS yield of 57.76 % has been achieved with 8.71 % of mannose and 14.49 % of glucose, demonstrating the potential use of β-mannanase in food industry.

Overexpression of a fungal β-mannanase from Bispora sp. MEY-1 in maize seeds and enzyme characterization.

BackgroundMannans and heteromannans are widespread in plants cell walls and are well-known as anti-nutritional factors in animal feed. To remove these factors, it is common practice to incorporate endo-β-mannanase into feed for efficient nutrition absorption. The objective of this study was to overexpress a β-mannanase gene directly in maize, the main ingredient of animal feed, to simplify the process of feed production.Methodology/Principal FindingsThe man5A gene encoding an excellent β-mannanase from acidophilic Bispora sp. MEY-1 was selected for heterologous overexpression. Expression of the modified gene (man5As) was driven by the embryo-specific promoter ZM-leg1A, and the transgene was transferred to three generations by backcrossing with commercial inbred Zheng58. Its exogenous integration into the maize embryonic genome and tissue specific expression in seeds were confirmed by PCR and Southern blot and Western blot analysis, respectively. Transgenic plants at BC3 generation showed agronomic traits statistically similar to Zheng58 except for less plant height (154.0 cm vs 158.3 cm). The expression level of MAN5AS reached up to 26,860 units per kilogram of maize seeds. Compared with its counterpart produced in Pichia pastoris, seed-derived MAN5AS had higher temperature optimum (90°C), and remained more β-mannanase activities after pelleting at 80°C, 100°C or 120°C.Conclusion/SignificanceThis study shows the genetically stable overexpression of a fungal β-mannanase in maize and offers an effective and economic approach for transgene containment in maize for direct utilization without any purification or supplementation procedures.

ManR, a novel Zn(II)2Cys6 transcriptional activator, controls the β-mannan utilization system in Aspergillus oryzae

Fungal endo-β-mannanases (β-mannanases) are widely used as industrial enzymes; however, no transcriptional regulator of β-mannanases has been identified in fungi or other eukaryotic cells to date. To identify a transcriptional regulator of β-mannanases in Aspergillus oryzae, a gene-disruptant library of transcriptional regulators was screened for mutants exhibiting reduced β-mannanase activity by using konjac glucomannan as the substrate, and ManR, a Zn(II)2Cys6 type DNA binding protein was identified. Moreover, a manR-overexpressing strain showed significantly increased β-mannanase activity. DNA microarray analysis of the manR-disruptant strain further indicated that when konjac glucomannan is used as the carbon source, ManR positively regulates the gene expression of not only β-mannanase, but also the enzymes involved in the degradation of galactomannans and glucomannans such as α-galactosidase, β-mannosidase, acetylmannan esterase, and β-glucosidase. Furthermore, we demonstrated that the presence of 1,4-β-d-mannobiose increased the expression of the endo-β-mannanase gene (manG, AO090010000122), and that ManR plays a key role in the inducible expression of manG in A. oryzae. Therefore, we conclude that ManR is a positive regulator of the β-mannan utilization system in A. oryzae. This is the first study to identify a transcriptional regulator of this system in eukaryotic cells.

Optimization of production conditions for β-mannanase using apple pomace as raw material in solid-state fermentation

Apple pomace is a wasted resource produced in China in large quantities, disposal of which has caused serious environmental problems. In order to make the best of this residue, apple pomace together with cottonseed powder was used as a raw material to produce β-mannanase in solid-state fermentation (SSF) by Aspergillus niger SN-09. Optimization of fermentation conditions for maximizing β-mannanase production was carried out using Plackett-Burman and Central Composite designs. A mixture of apple pomace and cottonseed powder (3:2, w/w) with 59.2 % (w/w) initial moisture, together with certain ionic compounds and salts, proved to be the optimal medium. The test fungi were inoculated in the optimized medium and incubated at 30°C for 48 h. The activity of β-mannanase reached 561.3 U/g, an increase of 45.7 % compared with that in basal medium, and reached the same level of production as that achieved using wheat bran and soybean meal as raw materials as in most factories in China. This is the first report of the use of apple pomace as a raw material to produce β-mannanase in SSF. This will not only reduce the production cost of β-mannanase, but also represents a new and effective way to make the best use of apple pomace, which can consequently help to reduce the environmental pollution caused by this waste.

PRODUCTION, PURIFICATION AND PROPERTIES OF β-MANNANASE FROM SOIL BACTERIUM BACILLUS CIRCULANS M-21

A β-mannanase-producing bacterial strain Bacillus circulans M-21 was isolated from soil. The strain grew well when mannan (konjac gum, guar gum or locust bean gum) was used as sole carbon source. The optimum fermentation conditions were determined as follows: 4 g/L guar gum as carbon source, 20 g/L soybean powder and 5 g/L (NH4)2HPO4 as nitrogen source, initial pH 8.0, and 32C. An extracellular β-mannanase was purified by anion-exchange chromatography on Q-Sepharose Fast Flow. The purified protein exhibited a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a molecular mass of 33.4 kDa. Kinetic analysis showed that the enzyme exhibited the strongest binding affinity to konjac gum. The β-mannanase activity reached the maximum at 50C and pH 7.0, and was completely suppressed by Hg2+ and Ag+. The β-mannanase degrading products were mainly composed by disaccharide, trisaccharide and tetrasaccharide, which were all neutral hexose polymers. PRACTICAL APPLICATIONS Microbial β-mannanases are applied broadly in industrial processes, such as improving the quality of food and feed, biobleaching of softwood pulps in the paper and pulp industries, and reducing the viscosity of coffee extracts. They are also found application in oil drilling and detergent industry. β-Mannanase is an ideal tool for the preparation of manno-oligosaccharides, which have been proved to be excellent prebiotics stimulating growth of human-beneficial intestinal microflora. Potential applications of manno-oligosaccharides are found in foods, beverages and confectionary, especially in functional food additives. In the current study a β-1,4-mannanase was extracted and purified from Bacillus circulans M-21. The research results showed that this enzyme is suitable for the preparation of glucomanno- and galactomanno-oligosaccharide.

Bimutation breeding of Aspergillus niger strain for enhancing β-mannanase production by solid-state fermentation

A parent strain Aspergillus niger LW-1 was mutated by the compound mutagenesis of vacuum microwave (VMW) and ethyl methane sulfonate (EMS). A mutant strain, designated as A. niger E-30, with high- and stable-yield β-mannanase was obtained through a series of screening. The β-mannanase activity of the mutant strain E-30, cultivated on the basic fermentation medium at 32 °C for 96 h, reached 36,675 U/g dried koji, being 1.98-fold higher than that (18,501 U/g dried koji) of the parent strain LW-1. The purified E-30 β-mannanase, a glycoprotein with a carbohydrate content of 19.6%, had an apparent molecular weight of about 42.0 kDa by SDS–PAGE. Its optimal pH and temperature were 3.5 and 65 °C, respectively. It was highly stable at a pH range of 3.5–7.0 and at a temperature of 60 °C and below. The kinetic parameters K m and V max, toward locust bean gum and at pH 4.8 and 50 °C, were 3.68 mg/mL and 1067.5 U/mg, respectively. The β-mannanase activity was not significantly affected by an array of metal ions and EDTA, but strongly inhibited by Ag + and Hg 2+. In addition, the hydrolytic conditions of konjak glucomannan using the purified E-30 β-mannanase were optimized as follows: konjak gum solution 240 g/L (dissolved in deionized water), hydrolytic temperature 50 °C, β-mannanase dosage 120 U/g konjak gum, and hydrolytic time 8 h.

Production of Mannan-degrading enzyme by Aspergillus niger

Three local fungal isolates (Aspergillus flavus, A. niger and A.ochraceous) were screened for their ability to secrete digesting enzymes (endoglucanase; β-galactosidase; β-mannanase; β-mannosidase and xylanase). The results revealed that the highest activity detected in the culture filtrate was for the β-mannanase enzyme followed by xylanase, whereas endoglucanase and β-galactosidase activities were very low. On the other hand, the culture filtrate did not contain any activity for β-mannosidase. The most potent mannanase producer was A. niger, which produced the highest extracellular mannanase activity (2.90U/ml), followed by Aspergillus flavus (2.54 U/ml) and A. ochraceous (2.16 U/ml). The optimal operating conditions for β-mannanase activity by A. niger arising from this study are as follows: temperature of 30°C, 6 days incubation period, initial pH 5.0 and inoculum size of 3 × 106 spore/ml.

Improved mannan-degrading enzymes’ production by Aspergillus niger through medium optimization

The effect of different carbon and nitrogen sources on the production of mannan-degrading enzymes, focussing on β-mannanase, by Aspergillus niger was investigated using shake flask culture. The β-mannanase activity obtained during growth of A. niger on guar gum (GG, 1495 nkat mL(-1)) was much higher than those observed on other carbon substrates, locust bean gum (1148 nkat mL(-1)), α-cellulose (10.7 nkat mL(-1)), glucose (8.8 nkat mL(-1)) and carboxymethylcellulose (4.6 nkat mL(-1)). For fermentation using GG as a carbon source, bacteriological peptone gave the highest β-mannanase activity (1744 nkat mL(-1)) followed by peptone from meat (1168 nkat mL(-1)), yeast extract (817 nkat mL(-1)), ammonium sulphate (241 nkat mL(-1)), ammonium nitrate (113 nkat mL(-1)) and ammonium chloride (99 nkat mL(-1)) when used as a nitrogen source. The composition of bacteriological peptone and initial pH of the medium were further optimized using response surface methodology (RSM). Medium consisted of 21.3 g L(-1) GG and 57 g L(-1) peptone with initial culture pH of 5.5 was optimum for β-mannanase production (2063 nkat mL(-1)) by A. niger. The β-mannanase production obtained in this study using A. niger was significantly higher than those reported in the literature.

Optimal conditions for enhanced β-mannanase production by recombinant Aspergillus sojae

Optimization of the growth conditions for maximum β-mannanase production in shake flasks by using recombinant Aspergillus sojae ATCC11906 (AsT1) was carried out by Box–Behnken design of response surface methodology. The highest β-mannanase activity on the fourth day of cultivation at 30 °C was obtained as 363 U/ml in the optimized medium consisting of 7% sugar beet molasses, 0.43% NH 4NO 3, 0.1% K 2HPO 4 and 0.05% MgSO 4 (by weight per volume) at 207 rpm. On the sixth day of cultivation under the optimized conditions, the highest β-mannanase activity was achieved as 482 U/ml which is 1.4-fold of 352 U/ml activity found on glucose medium previously.

A comparative study of cellulase and hemicellulase activities of brackish water clam Corbicula japonica with those of other marine Veneroida bivalves

Corbicula japonica is a typical brackish water bivalve species belonging to the order Veneroida, and it is the most important inland fishery resource in Japan. Corbicula japonica has been suggested to assimilate organic matter from terrestrial plants, unlike Ruditapes philippinarum and Mactra veneriformis, which selectively assimilate organic matter of marine origin. This led us to hypothesize that C. japonica, despite being a suspension feeder, could assimilate cellulosic materials derived from terrestrial plants. In the present study, we measured cellulase and hemicellulase activities in the crystalline styles of C. japonica and other commercially important Veneroida bivalve species in Japan: Ruditapes philippinarum, Meretrix lamarckii and Meretrix lusoria. Corbicula japonica demonstrated notably higher cellulase, xylanase and beta-mannanase activities than the other marine bivalves, suggesting that this species possesses a far greater biochemical capacity to break down the structural polysaccharides of plant cell walls than the other species. In contrast, the beta-1,3-glucanase and pectinase activities of C. japonica were similar to or even lower than those of the others. This is possibly due to the presence of these polysaccharides in the cell walls of diatoms, a principal food of most marine bivalves. Although direct evidence is lacking, the high cellulase, xylanase and beta-mannanase activities of C. japonica may result from adaptation to an upstream estuarine environment where phytoplankton and diatoms are scarce, but plant-derived substances are abundant.

After-ripening alters the gene expression pattern of oxidases involved in the ethylene and gibberellin pathways during early imbibition of Sisymbrium officinale L. seeds

After-ripening (AR) in Sisymbrium officinale seeds altered SoACS7, SoACO2, SoGA20ox2, SoGA3ox2, and SoGA2ox6 gene expression. Except for SoGA20ox2 expression, which sharply diminished, the expression of the other genes rose during development, particularly that of SoACS7. In contrast, only the SoACO2 and SoGA2ox6 transcripts increased with seed desiccation; the others decreased. AR increased the SoGA3ox2 transcript in dry seed, but dramatically decreased the SoACS7 transcript. At the onset of imbibition, AR inhibited SoACS7 and SoACO2 expression and stimulated that of SoGA20ox2, SoGA3ox2, and SoGA2ox6, demonstrating that the participation of ethylene (ET) and gibberellins (GAs) differs in after-ripened and non-after-ripened seeds. The inhibition of SoACO2 expression in the presence of GA4+7, paclobutrazol (PB), inhibitors of ET synthesis and signalling (IESS), and notably ET+GA4+7 indicated ET–GA cross-talk in non-after-ripened seeds. A positive effect of AR in reversing this inhibition was found. The idea of ET–GA cross-talk is also supported by the effect of ET on SoGA3ox2 expression, notably induced by the AR process. In contrast, SoGA20ox2 expression did not appear to be susceptible to AR. SoGA2ox6 expression, poorly known in seeds, suggests that AR prompted an up-regulation under all treatments studied, whereas in non-after-ripened seeds expression was down-regulated. On the other hand, the β-mannanase (MAN) activity dramatically increased in dry after-ripened seed, being significantly boosted by ET. The absence of MAN inhibition by IESS suggests that although ET seems to be one of the factors controlling MAN, its presence did not appear to be essential. GA4+7 only increased MAN in seeds wich were after-ripened. Here, it is proposed that ET and GAs participate actively in establishing the AR process.

Inducible and constitutive expression of a novel thermostable alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5 in Pichia pastoris and characterization of the recombinant enzyme

A novel thermostable alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5 was expressed successfully in Pichia pastoris GS115. The combined usage of inducible and constitutive promoters ( AOX1 and GAP) enhanced the expression of β-mannanase. Among the parameters investigated in shaking flask cultures, the pH value of medium had significant influence on the production of β-mannanase by recombinant P. pastoris. β-Mannanase produced at pH 7.0 was 6.7 times of that at pH value of 6.0. The highest β-mannanase activity of 32.2 IU/ml in culture supernatant was achieved at 120 h of cultivation in BMGY medium (pH 7.0). The recombinant β-mannanase was purified and characterized. The purified β-mannanase produced by P. pastoris has optimum pH of 10.0 and optimum temperature of 70 °C, which are very close to those of the native enzyme from alkaliphilic Bacillus sp. N16-5. However, much higher thermal stability and pH stability were observed in recombinant β-mannanase. These properties make the recombinant β-mannanase more useful in the detergent industries, the pulp and paper processing and other industrial processes.