Mannanase Research Articles

Heterologous expression of MirMAN enhances root development and salt tolerance in Arabidopsis.

β-Mannanase is a plant cell wall remodeling enzyme involved in the breakdown of hemicellulose and plays an important role in growth by hydrolyzing the mannan-like polysaccharide, but its function in adaptation to salt stress has been less studied. Based on cloned the mannanase (MAN) gene from Mirabilis jalapa L., the study was carried out by heterologously expressing the gene in Arabidopsis thaliana, and then observing the plant phenotypes and measuring relevant physiological and biochemical indicators under 150 mM salt treatment. The results indicate that MirMAN is a protein with a glycohydrolase-specific structural domain located in the cell wall. We first found that MirMAN reduced the susceptibility of transgenic Arabidopsis thaliana to high salt stress and increased the survival rate of plants by 38%. This was corroborated by the following significant changes, including the reduction in reactive oxygen species (ROS) levels, increase in antioxidant enzyme activity, accumulation of soluble sugars and increase of the expression level of RD29 in transgenic plants. We also found thatthe heterologous expression of MirMAN promoted root growth mainly by elongating the primary roots and increasing the density of lateral roots. Meanwhile, the expression of ARF7, ARF19, LBD16 and LBD29 was up-regulated in the transgenic plants, and the concentration of IAA in the roots was increased. Those results indicate that MirMAN is involved in the initiation of lateral root primordia in transgenic plants through the IAA-ARF signalling pathway. In conclusion, MirMAN improves plant salt tolerance not only by regulating ROS homeostasis, but also by promoting the development of lateral roots. Reflecting the potential of the MirMAN to promote root plastic development in adaptation to salt stress adversity.

Effects of nonantibiotic growth promoter combinations on growth performance, nutrient utilization, digestive enzymes, intestinal morphology, and cecal microflora of broilers.

Given the ban on antibiotic growth promoters, the effects of nonantibiotic alternative growth promoter combinations (NAGPCs) on the growth performance, nutrient utilization, digestive enzyme activity, intestinal morphology, and cecal microflora of broilers were evaluated. All birds were fed pellets of two basal diets-starter (0-21 d) and grower (22-42 d)-with either enramycin (ENR) or NAGPC supplemented. 1) control + ENR; 2) control diet (CON, basal diet); 3) control + mannose oligosaccharide (MOS) + mannanase (MAN) + sodium butyrate (SB) (MMS); 4) control + MOS + MAN + Bacillus subtilis (BS) (MMB); 5) control + MOS + fruit oligosaccharide (FOS) + SB (MFS); 6) control + FOS + BS (MFB); 7) control + MOS + FOS + MAN (MFM); 8) control + MOS + BS + phytase (PT) (MBP). ENR, MOS, FOS, SB, MAN, PT, and BS were added at 100, 2,000, 9,000, 1,500, 300, 37, and 500 mg/kg, respectively. The experiment used a completely random block design with six replicates per group: 2400 Ross 308 broilers in the starter phase and 768 in the grower phase. All NAGPCs significantly improved body weight gain (P < 0.01), utilization of dry matter, organic matter, and crude protein (P < 0.05), villus height and villus height/crypt depth in the jejunum and ileum (P < 0.01), and decreased the feed conversion ratio (P < 0.01) at d 21 and 42. MMS, MMB, MFB, and MFM duodenum trypsin, lipase, and amylase activities increased significantly (P < 0.05) at d 21 and 42. On d 21 and 42, MMS, MMB, and MBP increased the abundance of Firmicutes and Bacteroides whereas MMB, MFB, and MBP decreased the abundance of Proteobacteria, compared to ENR and CON. Overall, the NAGPCs were found to have some beneficial effects and may be used as effective antibiotic replacements in broilers.

Preservation of Paenibacillus polymyxa BTK01 and Bacillus subtilis BTK07 as lignocellulolytic bacterial starters for industrial applications: Physicochemical conditions, enzyme stability, freeze-drying processes and cryoprotection

In a previous study, Paenibacillus polymyxa BTK01 (BTK01) and Bacillus subtilis BTK07 (BTK07) were identified as lignocellulolytic strains that produced xylanase (XLN) and mannanase (MNN), respectively. As a biocontrol agent and a biofertilizer, these bacteria can be beneficial for organic agriculture, and they could be developed by upscaling as a bacterial starter. Moreover, the enzymes produced can be useful in the wood pulp industry. Therefore, this study focused on determining the physicochemical conditions for enzyme catalysis and for preserving BTK01 and BTK07 using a freeze-drying process. We show that the optimum physicochemical conditions for XLN and MNN produced by these strains were clearly defined by pH 7–8 and 40–60 °C, in which the lignocellulolytic enzymes of BTK01 were thermally stable. The XLN from BTK01 was stable at 40 °C but became unstable at lower temperatures. As expected, freeze-drying without cryoprotectant reduced bacterial survival rather than enzymatic activity, but the 30-day-storage of the freeze-dried bacteria primarily affected enzyme catalysis. For added value, the fermentation broth could be divided into sediment and supernatant to obtain freeze-dried products as a bacterial starter and a crude enzyme, respectively. Additionally, formulating skimmed milk and glycerol as a cryoprotectants could improve bacterial survival and enzymatic activity of freeze-dried bacteria, particularly BTK07. Accordingly, the results indicate that the freeze-dried products of BTK01 and BTK07 are suitable for industrial-scale production to supply the agricultural sector and other related fields.

Lignocellulolytic bacteria isolated from organic rice field soils for enzyme production using agricultural wastes: Screening, medium optimization, and co-culture

Lignocellulolytic bacteria can degrade agricultural biomass and fungal cell walls. Organic agriculture intensively employs these microbes as biofertilizers and biocontrol agents. This study focused on isolating, screening, identifying, and optimizing enzyme production of the lignocellulolytic bacteria obtained from organic rice field soils for utilizing in organic farming. Here we have shown that the farming periods can alter the bacterial biodiversity in organic rice field soil by providing different nutrients. Paenibacillus polymyxa BTK01 and Bacillus subtilis BTK07, which were isolated from the preharvest soil, exhibited a high activity of mannanase (MNN) and xylanase (XLN). By optimizing the enzyme production, BTK01 and BTK07 were specifically productive in xylanase and mannanase, respectively, because of the induction of substrate-like carbon sources. Varying the type and concentration of nitrogen source revealed that mannanase production positively correlated to bacterial growth of both strains. Throughout the study, we used soybean meal as a nitrogen source with the nitrogen concentration of 0.144% (w/v), BTK01 was productive in xylanase when cultured in the medium comprised of 1.5% (w/v) rice straw (0.905 ± 0.016 U/mL), and BTK07 was efficient in mannanase production when cultured in the medium consisted of 1.0% (w/v) copra meal (0.440 ± 0.000 U/mL). The co-culture of BTK01 and BTK07 using the volume ratio of 1:10 could yield MNN and XLN to be 2.470 ± 0.013 and 0.906 ± 0.015 U/mL, respectively. This result demonstrates that Paenibacillus polymyxa BTK01 and Bacillus subtilis BTK07 can be useful and applicable for organic agriculture as biocontrol agents and biofertilizers.

Bacillus subtilis 분리균 2주 유래 mannanases의 특성 비교

우리나라의 전통 발효 된장으로부터 균체외 효소로 mannanase를 생산하는 세균 2주가 분리되었다. 분리균 WL-6과 WL-11은 형태적 특성, 생화학적 성질 및 16S rDNA의 염기서열에 따라 Bacillus subtilis로 확인되었다. 이들 두 균주로부터 각각 mannanase 유전자를 대장균에 클로닝하여 염기서열을 결정한 결과 mannanase 유전자는 362 아미노산으로 구성된 단백질을 코드하며 1,086 뉴클레오티드로 동일하게 이루어졌다. WL-6과 WL-11 mannanase (Man6, Man11)의 아미노산 잔기 배열은 서로 8개 잔기가 다르며 GH family 26에 속하는 B. subtilis의 mannanases와 매우 상동성이 높았다. Man6과 Man11의 아미노 말단의 26개 아미노 잔기가 signal peptide로 예측되었다. 재조합 대장균로부터 각각 생산된 Man6과 Man11은 94~95% 정도가 균체내에 존재하였고, mannotriose, mannotetraose, mannopentaose, mannohexaose와 같은 만노올리고당과 locust bean gum을 유사하게 분해하여 주된 반응산물로 mannobiose와 mannotriose를 생성하였다. Man6는 55&#x2103;와 pH 6.0, Man11은 60&#x2103;와 pH 5.5에서 각각 최대 반응활성을 보였으며, Man11이 Man6에 비해 열안정성이 높았다. Two bacterial strains producing extracellular man nanase were isolated from doenjang, a traditionally fermented soybean paste in Korea. The isolates, WL-6 and WL-11, were identified as Bacillus subtiis on the basis of their 16S rRNA gene sequences, morphological, and biochemical properties. Two genes encoding the mannanase of both B. subtilis WL-6 and B. subtilis WL-11 were each cloned into Escherichia coli, and their nucleotide sequences were determined. Both mannanase genes consisted of 1,086 nucleotides, encoding polypeptides of 362 amino acid residues. The deduced amino acid sequences of the two WL-6 and WL-11 mannanases, designated Man6 and Man11, respectively, differed from each other by eight amino acid residues, and they were highly homologous to those of mannanases belonging to the glycosyl hydrolase family 26. The 26 amino acid stretch in the N-terminus of Man6 and Man11 was a predicted signal peptide. Both Man6 and Man11 were localized at the level of 94&#x2013;95% in an intracellular fraction of recombinant E. coli cells. The enzymes hydrolyzed both locust bean gum and mannooligosaccharides, including mannotriose, mannotetraose, mannopentaose, and mannohexaose, forming mannobiose and mannotriose as predominant products. The optimal reaction conditions were 55&#xB0;C and pH 6.0 for Man6, and 60&#xB0;C and pH 5.5 for Man11. Man11 was more stable than Man6 at high temperatures.

Effect of different dietary levels of low-glucosinolate rapeseed (canola) meal and non-starch polysaccharide-degrading enzymes on growth performance and gut physiology of growing turkeys

Zdunczyk, Z., Jankowski, J., Juskiewicz, J., Mikulski, D. and Slominski, B. A. 2013. Effect of different dietary levels of low-glucosinolate rapeseed (canola) meal and non-starch polysaccharide-degrading enzymes on growth performance and gut physiology of growing turkeys. Can. J. Anim. Sci. 93: 353-362. One-day-old male turkey poults were randomly assigned to eight dietary treatments and were fed diets containing 0, 60, 120, or 180 g kg-1 of low-glucosinolate rapeseed meal (RSM) from 1 to 56 d of age without or with a commercial non-starch polysaccharide-degrading enzyme product containing pectinase, cellulase, xylanase, glucanase, mannanase and galactanase activities. There were no significant differences (P>0.05) in body weight and feed conversion ratio between the Control soybean meal-based diet and the diets containing RSM. Overall, the inclusion of up to 180 g kg-1 of RSM did not affect the growth performance of turkeys. When compared with the Control treatment, the diet with the lowest RSM content i...

Studies on the Mannanase of Streptomyces

Studies on the Mannanase of Streptomyces

Cloning and characterization of a novel mannanase from Paenibacillus sp. BME-14.

A mannanase gene (man26B) was obtained from a sea bacterium, Paenibacillus sp. BME-14, through the constructed genomic library and inverse PCR. The gene of man26B had an open reading frame of 1,428 bp that encoded a peptide of 475- amino acid residues with a calculated molecular mass of 53 kDa. Man26B possessed two domains, a carbohydrate binding module (CBM) belonging to family 6 and a family 26 catalytic domain (CD) of glycosyl hydrolases, which showed the highest homology to Cel44C of P. polymyxa (60% identity). The optimum pH and temperature for enzymatic activity of Man26B were 4.5 and 60 degrees C, respectively. The activity of Man26B was not affected by Mg(2+) and Co(2+), but was inhibited by Hg(2+), Ca(2+), Cu(2+), Mn(2+), K(+), Na(+), and beta-mercaptoethanol, and slightly enhanced by Pb(2+) and Zn(2+). EDTA did not affect the activity of Man26B, which indicates that it does not require divalent ions to function. Man26B showed a high specific activity for LBG and konjac glucomannan, with K(m), V(max), and k(cat) values of 3.80 mg/ml, 91.70 micromol/min/mg protein, and 77.08/s, respectively, being observed when LBG was the substrate. Furthermore, deletion of the CBM6 domain increased the enzyme stability while enabling it to retain 80% and 60% of its initial activity after treatment at 80 degrees C and 90 degrees C for 30 min, respectively. This finding will be useful in industrial applications of Man26B, because of the harsh circumstances associated with such processes.

Purification and characterization of endo-beta-1,4 mannanase from Aspergillus niger gr for application in food processing industry.

A thermostable extracellular beta-mannanase from the culture supernatant of a fungus Aspergillus niger gr was purified to homogeneity. SDS-PAGE of the purified enzyme showed a single protein band of molecular mass 66 kDa. The beta- mannanase exhibited optimum catalytic activity at pH 5.5 and 55 degrees C. It was thermostable at 55 degrees C, and retained 50% activity after 6 h at 55 degrees C. The enzyme was stable at a pH range of 3.0 to 7.0. The metal ions Hg(2+), Cu(2+), and Ag(2+) inhibited complete enzyme activity. The inhibitors tested, EDTA, PMSF, and 1,10-phenanthroline, did not inhibit the enzyme activity. N-Bromosuccinimide completely inhibited enzyme activity. The relative substrate specificity of enzyme towards the various mannans is in the order of locust bean gum>guar gum>copra mannan, with K(m) of 0.11, 0.28, and 0.33 mg/ml, respectively. Since the enzyme is active over a wide range of pH and temperature, it could find potential use in the food-processing industry.

Embryo-endosperm interactions in the hydrolysis of lettuce seed reserves.

Red light- and gibberellin-induced seeds of lettuce ( Lactuca sativa L., cv. Grand Rapids) produce copious amounts of endo-β-mannanase after germination is completed. This enzyme is produced and secreted by the endosperm itself and participates in the hydrolysis of its cell walls, which are largely mannans. The resulting oligomannans are further hydrolysed by β-mannosidase, an enzyme produced within the cotyledons. Degradation of the endosperm is completed prior to mobilization of the main storage reserves within the cotyledons. Isolated endosperms produce and release mannanase. This can be prevented by placing the endosperms in a small incubation volume, probably due to insufficient dilution of endogenous leachable inhibitor(s). The inhibition is lifted during germination: it also can be relieved by benzyladenine and gibberellic acid when the cotyledons are present. Abscisic acid can prevent mannanase production by isolated endosperms and it may contribute to the endogenous inhibitory regulation of this enzyme. By using dissection experiments it has been demonstrated that mannanase production by the endosperm is under the control of the embryo, and that both the axis and cotyledons play a role. The cotyledons are capable of lifting the inhibition, but depend upon an axial event early in germination, apparently involving the transmission of diffusible substances.

細菌マンナン分解酵素による蒟蒻糊抜に就て

細菌マンナン分解酵素による蒟蒻糊抜に就て