Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.
Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., Von Schantz, L., Marcus, S. E., Andersen, M.C. F., Field, R., Ohlin, M., Knox, J. P., Clausen, M. H. & Willats, W. G. T. (2012). Journal of Biological Chemistry, 287(47), 39429-39438.
Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.
Operational and storage stability of neutral β-mannanase from Bacillus licheniformis.
Zhang, J., He, M. & He, Z. (2002). Biotechnology Letters, 24(19), 1611-1613.
The stability of neutral β-mannanase from Bacillus licheniformis during operation and storage was investigated. The enzyme activity decreased by 70% with a hydrolysate of glucomannan at 20 g l-1 over 30 min at 25°C. In an enzymatic membrane reactor operated at 50°C after 24 h, the loss of enzyme activities were 23% and 9% in the absence/presence of the substrate. The residual activities of the enzyme were 21% and 90%, respectively, when stored in 30% (v/v) glycerol solution and in solid state at 4°C after one year.
Model for random hydrolysis and end degradation of linear polysaccharides: Application to the thermal treatment of mannan in solution.
Nattorp, A., Graf, M., Spühler, C. & Renken, A. (1999). Industrial & Engineering Chemistry Research, 38(8), 2919-2926.
The kinetics for homogeneous hydrolysis of mannan is studied in a batch reactor at temperatures from 160 to 220°C. A formate buffer ensures a pH of 3.8−4.0, measured at 25°C. Samples are analyzed for oligosaccharides up to a degree of polymerization of 6 and also for the total amount of mannose after acid hydrolysis. A mathematical model with two reactions (1, random hydrolysis of the glucosidic bonds; 2, degradation of the reducing end of the molecule) describes accurately the time course of oligosaccharides. Optimized rate constants follow closely an Arrhenius relationship, with the degradation having a higher activation energy (140 kJ/mol) than the hydrolysis (113 kJ/mol). The mathematical model has the advantage that production of small molecules is independent of the initial chain-length distribution as long as the average initial chain length is some 5 times longer than the largest species measured. It can be applied to first-order depolymerization of other linear polymers with one link type in order to determine reaction rate constants or make predictions about molecular weight distribution on the base of known reaction rate constants.
The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation.
Hogg, D., Pell, G., Dupree, P., Goubet, F., Martin-Orue, S., Armand, S. & Gilbert, H. (2003). Biochem. J, 371(3), 1027-1043.
β-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.
Determination of galactose and mannose residues in natural galactomannans using a fast and efficient high-performance liquid chromatography/UV detection.
Tapie, N., Malhiac, C., Hucher, N. & Grisel, M. (2008). Journal of Chromatography A, 1181(1), 45-50.
The present work describes the validation of an easy, fast and efficient precolumn derivatization method for the quantification of oligosides, mannose and galactose obtained by degradation of galactomannans. This work combines an acid hydrolysis and an enzymatic degradation of natural galactomannans with the quantification of released residues by reversed-phase HPLC–UV, the most usual HPLC system in laboratories. In case of enzymatic degradation, mannotetraose has been detected and quantified for the first time, and an application to the evaluation of the galactosyl distribution in galactomannans is proposed. After an acidic hydrolysis, this method also allowed to obtain the mannose/galactose (M/G) ratio.
Cloning and biochemical characterization of an endo-1,4-β-mannanase from the coffee berry borer hypothenemus hampei.
Aguilera-Gálvez, C., Vásquez-Ospina, J. J., Gutiérrez-Sanchez, P. & Acuña-Zornosa, R. (2013). BMC Research Notes, 6(1), 333.
Background: The study of coffee polysaccharides-degrading enzymes from the coffee berry borer Hypothenemus hampei, has become an important alternative in the identification for enzymatic inhibitors that can be used as an alternative control of this dangerous insect. We report the cloning, expression and biochemical characterization of a mannanase gene that was identified in the midgut of the coffee berry borer and is responsible for the degradation of the most abundant polysaccharide in the coffee bean. Methods: The amino acid sequence of HhMan was analyzed by multiple sequence alignment comparisons with BLAST (Basic Local Alignment Search Tool) and CLUSTALW. A Pichia pastoris expression system was used to express the recombinant form of the enzyme. The mannanase activity was quantified by the 3,5-dinitrosalicylic (DNS) and the hydrolitic properties were detected by TLC. Results: An endo-1,4-β-mannanase from the digestive tract of the insect Hypothenemus hampei was cloned and expressed as a recombinant protein in the Pichia pastoris system. This enzyme is 56% identical to the sequence of an endo-β-mannanase from Bacillus circulans that belongs to the glycosyl hydrolase 5 (GH5) family. The purified recombinant protein (rHhMan) exhibited a single band (35.5 kDa) by SDS-PAGE, and its activity was confirmed by zymography. rHhMan displays optimal activity levels at pH 5.5 and 30°C and can hydrolyze galactomannans of varying mannose:galactose ratios, suggesting that the enzymatic activity is independent of the presence of side chains such as galactose residues. The enzyme cannot hydrolyze manno-oligosaccharides such as mannobiose and mannotriose; however, it can degrade mannotetraose, likely through a transglycosylation reaction. The Km and Kcat values of this enzyme on guar gum were 2.074 mg ml-1 and 50.87 s-1 respectively, which is similar to other mannanases. Conclusion: This work is the first study of an endo-1,4-β-mannanase from an insect using this expression system. Due to this enzyme’s importance in the digestive processes of the coffee berry borer, this study may enable the design of inhibitors against endo-1,4-β-mannanase to decrease the economic losses stemming from this insect.
Influence of a mannan binding family 32 carbohydrate binding module on the activity of the appended mannanase.
Mizutani, K., Fernandes, V. O., Karita, S., Luís, A. S., Sakka, M., Kimura, T., Jackson, A., Zhang, X., Fontes, C. M. G. A., Gilbert, H. J. & Sakka, K. (2012). Applied and Environmental Microbiology, 78(14), 4781-4787.
In general, cellulases and hemicellulases are modular enzymes in which the catalytic domain is appended to one or more noncatalytic carbohydrate binding modules (CBMs). CBMs, by concentrating the parental enzyme at their target polysaccharide, increase the capacity of the catalytic module to bind the substrate, leading to a potentiation in catalysis. Clostridium thermocellum hypothetical protein Cthe_0821, defined here as C. thermocellum Man5A, is a modular protein comprising an N-terminal signal peptide, a family 5 glycoside hydrolase (GH5) catalytic module, a family 32 CBM (CBM32), and a C-terminal type I dockerin module. Recent proteomic studies revealed that Cthe_0821 is one of the major cellulosomal enzymes when C. thermocellum is cultured on cellulose. Here we show that the GH5 catalytic module of Cthe_0821 displays endomannanase activity. C. thermocellum Man5A hydrolyzes soluble konjac glucomannan, soluble carob galactomannan, and insoluble ivory nut mannan but does not attack the highly galactosylated mannan from guar gum, suggesting that the enzyme prefers unsubstituted β-1,4-mannoside linkages. The CBM32 of C. thermocellum Man5A displays a preference for the nonreducing ends of mannooligosaccharides, although the protein module exhibits measurable affinity for the termini of β-1,4-linked glucooligosaccharides such as cellobiose. CBM32 potentiates the activity of C. thermocellum Man5A against insoluble mannans but has no significant effect on the capacity of the enzyme to hydrolyze soluble galactomannans and glucomannans. The product profile of C. thermocellum Man5A is affected by the presence of CBM32.
Acidic β-mannanase from Penicillium pinophilum C1: Cloning, characterization and assessment of its potential for animal feed application.
Cai, H., Shi, P., Luo, H., Bai, Y., Huang, H., Yang, P. & Yao, B. (2011). Journal of Bioscience and Bioengineering, 112(6), 551-557.
The β-mannanase gene, man5C1, was cloned from Penicillium pinophilum C1, a strain isolated from the acidic wastewater of a tin mine in Yunnan, China, and expressed in Pichia pastoris. The sequence analysis displayed the gene consists of a 1221-bp open reading frame encoding a protein of 406 amino acids (Man5C1). The deduced amino acid sequence of Man5C1 showed the highest homology of 57.8% (identity) with a characterized β-mannanase from Aspergillus aculeatus belonging to glycoside hydrolase family 5. The purified rMan5C1 had a high specific activity of 1035 U mg-1 towards locust bean gum (LBG) and showed highest activity at pH 4.0 and 70°C. rMan5C1 was adaptable to a wide range of acidity, retaining > 60% of its maximum activity at pH 3.0–7.0. The enzyme was stable over a broad pH range (3.0 to 10.0) and exhibited good thermostability at 50°C. The Km and Vmax values were 5.6 and 4.8 mg mL-1, and 2785 and 1608 μmol min-1 mg-1, respectively, when LBG and konjac flour were used as substrates. The enzyme had strong resistance to most metal ions and proteases (pepsin and trypsin), and released 8.96 mg g-1 reducing sugars from LBG in the simulated gastric fluid. All these favorable properties make rMan5C1 a promising candidate for use in animal feed.
Ligands of thermophilic ABC transporters encoded in a newly sequenced genomic region of Thermotoga maritima MSB8 screened by differential scanning fluorimetry.
Boucher, N. & Noll, K. M. (2011). Applied and Environmental Microbiology, 77(18), 6395-6399.
The chromosome of Thermotoga maritima strain MSB8 was found to have an 8,870-bp region that is not present in its published sequence. The isolate that was sequenced by The Institute for Genomic Research (TIGR) in 1999 is apparently a laboratory variant of the isolate deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM 3109) in 1986. This newly sequenced region from the DSMZ culture was located between TM1848 (cbp, cellobiose phosphorylase) and TM1847 (the 3′ end of a truncated ROK regulator). The new region contained seven genes: a beta glucosidase gene (bglA), three trehalose ABC transporter genes (treEFG), three xylose ABC transporter genes (xylE2F2K2), and the 5′ end of a gene encoding the ROK regulator TM1847. We present a new differential scanning fluorimetry method using a low pH that was necessary to screen potential ligands of these exceptionally thermostable periplasmic substrate-binding proteins. This method showed that trehalose, sucrose, and glucose stabilized TreE, and their binding was confirmed by measuring changes in intrinsic fluorescence upon ligand binding. Binding constants of 0.024 µM, 0.300 µM, and 56.78 µM at 60°C, respectively, were measured. XylE2 ligands were similarly determined and xylose, glucose, and fucose bound with Kd (dissociation constant) values of 0.042 µM, 0.059 µM, and 1.436 µM, respectively. Since there is no discernible phenotypic difference between the TIGR isolate and the DSMZ isolate despite the variance in their genomes, we propose that they be called genomovars: T. maritima MSB8 genomovar TIGR and T. maritima MSB8 genomovar DSM 3109, respectively.
Fractionation of extracted hemicellulosic saccharides from Pinus pinaster wood by multistep membrane processing.
González-Muñoz, M. J., Rivas, S., Santos, V. & Parajó, J. C. (2013). Journal of Membrane Science, 428, 281-289.
Hemicelluloses of Pinus pinaster wood were selectively separated from cellulose and lignin by reaction with hot, compressed water (autohydrolysis) under optimized conditions. The reaction liquor contained polymeric or oligomeric hemicellulose saccharides (POHS, accounting jointly for 69.6% of the dissolved wood fraction), followed by monosaccharides (accounting for 20.0% of the non-volatile compounds), and non-saccharide compounds. For concentration, purification and fractionation purposes, liquors from hydrothermal processing were subjected to consecutive steps of diafiltration and concentration using membranes of 10, 5, 3, 1 and 0.3 kDa molar mass cut-off. Samples from selected process streams were characterized by chromatographic and spectrometric methods. The experimental results provided information on the separation and refining effects achieved by the various membrane processing steps, which affect the technological properties of products.
Two-stage hot-water extraction of galactoglucomannans from spruce wood.
Pranovich, A., Holmbom, B. & Willför, S. (2016). Journal of Wood Chemistry and Technology, 36(2), 140-156.
In order to preserve the polymeric structure and the acetylation degree of extracted galactoglucomannans and, at the same time, achieve high yield, ground spruce wood was subjected to a series of sequential two-stage extractions with an Accelerated Solvent Extraction (ASE) apparatus using plain water at 170 deg;C. The total combined extraction time was one hour in all the extractions. The total yield of the dissolved material after 1 h extraction was almost the same, about 25% of the wood, irrespective of the time ratios between the first and the second extractions. The yield of hemicellulose high polymers with the weight average molar mass of 8-10 kDa during the first extraction had a maximum at 20 min extraction time, amounting to about 7% on dry wood basis, and comprising about half of the total extract. Along with the progress of the extraction, the molar mass of the hemicelluloses decreased and hemicellulose-derived low polymers with the weight average molar mass of 6-2 kDa became dominating. The extracted substances were fractionated, mainly according to their molar mass, by sequential precipitation with ethanol, acetone, and methyl tert-butyl ether (MTBE). The hemicelluloses with some amount of pectins comprised 83-90% of the precipitated polymeric material and the content of galactoglucomannans was about 80%.
A comparison between a yeast cell wall extract (Bio-Mos®) and palm kernel expeller as mannan-oligosac-charides sources on the performance and ileal microbial population of broiler chickens.
Navidshad, B., Liang, J. B., Jahromi, M. F., Akhlaghi, A. & Abdullah, N. (2015). Italian Journal of Animal Science, 14(1), 3452.
The present study was conducted to determine the effect of a yeast cell wall extract (Bio-Mos) and palm kernel expeller (PKE) on the performance, nutrient digestibility, and ileal bacteria population of broiler chickens. A total of 60 1-d-old male broiler chicks (Cobb 500) were fed one of the 3 isonitrogenous and isocaloric diet including a control diet, or a control diet supplemented with 2 g/kg Bio-Mos (1-42 d), and for the third group, the control diet at 1-28 d following a diet containing 200 g/kg of an enzymatically-treated PKE at 29-42 d. The weight gains of birds fed the PKE containing diet (96.17 g/d) were less than other groups (109.10 and 104.42 g/d for the Bio-Mos and control diet, respectively) (P<0.05). Dietary inclusion of PKE increased bird’s feed intake (214.45 g/d) and feed conversion ratio (FCR) (2.23) than the Bio-Mos diet (194.87 and 1.79 g/d for feed intake and FCR, respectively) (P<0.05). The PKE diet had lower digestibility coefficients for dry matter (83.37%), ash and crude protein (78.63%) than the PKE free diets (P<0.05). As a ratio of the ileal total bacteria, there were no differences in the ileal population of Lactobacilli and Enterococcus genus or Enterobacteriaceae among the experimental groups (P>0.05), but the birds fed PKE or Bio-Mos containing diets had a lower population of Escherichia colithan the control group (P<0.05). The results showed that PKE potentially has a prebiotic property for chicken; however, a 200 g/kg dietary inclusion rate of PKE is not commercially recommendable because of its negative effects on the nutrients digestibility.
A Novel β-1, 4-mannanase Isolated from Paenibacillus polymyxa KT551.
Hori, K., Kawabata, Y., Nakazawa, Y., Nishizawa, M. & Toeda, K. (2014). Food Science and Technology Research, 20(6), 1261-1265.
A β-1,4-mannanase producing bacterium was isolated from soil collected in Akita Prefecture, Japan. The bacterium was identified as Paenibacillus polymyxa KT551 and was shown to produce a novel β-1,4-mannanase. The novelty of the enzyme was established by its N-terminal amino acid sequence, molecular weight and isoelectric point. The isolated β-1,4-mannanase showed activity against mannotetraose, mannopentaose and mannohexaose to produce mannobiose, mannotriose and mannotetraose. However, the enzyme exhibited no activity against mannobiose and mannotriose. Moreover, the crude enzyme preparation of the bacterium had no or minimal β-mannosidase or α-galactosidase activity. Therefore, the enzyme preparation from P. polymyxa KT551 holds potential for the efficient production of mannooligosaccharides from natural resources of galactomannans.
Biochemical characterization of an acidophilic β-mannanase from Gloeophyllum trabeum CBS900. 73 with significant transglycosylation activity and feed digesting ability.
Wang, C., Zhang, J., Wang, Y., Niu, C., Ma, R., Wang, Y., Bai, Y., Luo, H. & Yao, B. (2016). Food Chemistry, 197, 474-481.
Acidophilic β-mannanases have been attracting much attention due to their excellent activity under extreme acidic conditions and significant industrial applications. In this study, a β-mannanase gene of glycoside hydrolase family 5, man5A, was cloned from Gloeophyllum trabeum CBS900.73, and successfully expressed in Pichia pastoris. Purified recombinant Man5A was acidophilic with a pH optimum of 2.5 and exhibited great pH adaptability and stability (>80% activity over pH 2.0-6.0 and pH 2.0-10.0, respectively). It had a high specific activity (1356 U/mg) against locust bean gum, was able to degrade galactomannan and glucomannan in a classical four-site binding mode, and catalyzed the transglycosylation of mannotetrose to mannooligosaccharides with higher degree of polymerization. Besides, it had great resistance to pepsin and trypsin and digested corn-soybean meal based diet in a comparable way with a commercial β-mannanase under the simulated gastrointestinal conditions of pigs. This acidophilic β-mannanase represents a valuable candidate for wide use in various industries, especially in the feed.
An Aspergillus nidulans GH26 endo-β-mannanase with a novel degradation pattern on highly substituted galactomannans.
von Freiesleben, P., Spodsberg, N., Blicher, T. H., Anderson, L., Jørgensen, H., Stålbrand, H., Meyer, A. S. & Krogh, K. B. (2016). Enzyme and Microbial Technology, 83, 68-77.
The activity and substrate degradation pattern of a novel Aspergillus nidulans GH26 endo-β-mannanase (AnMan26A) was investigated using two galactomannan substrates with varying amounts of galactopyranosyl residues. The AnMan26A was characterized in parallel with the GH26 endomannanase from Podospora anserina (PaMan26A) and three GH5 endomannanases from A. nidulans and Trichoderma reesei (AnMan5A, AnMan5C and TrMan5A). The initial rates and the maximal degree of enzymatically catalyzed conversion of locust bean gum and guar gum galactomannans were determined. The hydrolysis product profile at maximal degree of conversion was determined using DNA sequencer-Assisted Saccharide analysis in High throughput (DASH). This is the first reported use of this method for analyzing galactomannooligosaccharides. AnMan26A and PaMan26A were found to have a novel substrate degradation pattern on the two galactomannan substrates. On the highly substituted guar gum AnMan26A and PaMan26A reached 35-40% as their maximal degree of conversion whereas the three tested GH5 endomannanases only reached 8-10% as their maximal degree of conversion. α-Galactosyl-mannose was identified as the dominant degradation product resulting from AnMan26A and PaMan26A action on guar gum, strongly indicating that these two enzymes can accommodate galactopyranosyl residues in the -1 and in the +1 subsite. The degradation of α-64-63-di-galactosyl-mannopentaose by AnMan26A revealed accommodation of galactopyranosyl residues in the -2, -1 and +1 subsite of the enzyme. Accommodation of galactopyranosyl residues in subsites -2 and +1 has not been observed for other characterized endomannanases to date. Docking analysis of galactomannooligosaccharides in available crystal structures and homology models supported the conclusions drawn from the experimental results. This newly discovered diversity of substrate degradation patterns demonstrates an expanded functionality of fungal endomannanases, than hitherto reported.
A novel thermostable GH5_7 β-mannanase from Bacillus pumilus GBSW19 and its application in manno-oligosaccharides (MOS) production.
Zang, H., Xie, S., Wu, H., Wang, W., Shao, X., Wu, L., Rajer, F. U. & Gao, X. (2015). Enzyme and microbial technology, 78, 1-9.
A novel thermostable mannanase from a newly isolated Bacillus pumilus GBSW19 has been identified, expressed, purified and characterized. The enzyme shows a structure comprising a 28 amino acid signal peptide, a glycoside hydrolase family 5 (GH5) catalytic domain and no carbohydrate-binding module. The recombinant mannanase has molecular weight of 45 kDa with an optimal pH around 6.5 and is stable in the range from pH 5-11. Meanwhile, the optimal temperature is around 65°C, and it retains 50% relative activity at 60°C for 12 h. In addition, the purified enzyme can be activated by several ions and organic solvents and is resistant to detergents. Bpman5 can efficiently convert locus bean gum to mainly M2, M3 and M5, and hydrolyze manno-oligosaccharides with a minimum DP of 3. Further exploration of the optimum condition using HPLC to prepare oligosaccharides from locust bean gum was obtained as 10 mg/ml locust bean gum incubated with 10 U/mg enzyme at 50°C for 24 h. By using this enzyme, locust bean gum can be utilized to generate high value-added oligosaccharides with a DP of 2-6.
High-level expression and characterization of a thermophilic β-mannanase from Aspergillus niger in Pichia pastoris.
Yu, S., Li, Z., Wang, Y., Chen, W., Fu, L., Tang, W., Chen, C., Liu, Y., Zhang, X. & Ma, L. (2015). Biotechnology Letters, 37(9), 1853-1859.
Objectives: A novel, high-level expression, thermostable mannan endo-1,4-β-mannosidase is urgently needed for industrial applications. Results: The mannan endo-1,4-β-mannosidase gene (MAN) from Aspergillus niger CBS 513.88 was optimized based on the codon usage bias in Pichia pastoris and synthesized by overlapping PCR to produce MAN-P. It was expressed in P. pastoris GS115 from a constitutive expression vector pHBM-905 M. MAN-P reached 594 mg/l in shake-flasks after 192 h induction. On production in a 5 l fermenter, the yield of MAN-P reached ~3.5 mg/ml and the enzyme activity was 1612 U/ml. The enzyme exhibited a maximum activity of 3049 U/ml at 80°C and retained 60 % enzyme activity at 80°C for 2 h. The pH optimum was 4.5 and the enzyme was stable over the pH range 1.5-11. Conclusion: The thermostability of MAN-P is higher than other known fungal mannanases and the expression and thermophilic properties make MAN-P useful for industrial applications.
From native malt to pure starch-Development and characterization of a purification procedure for modified starch.
Rittenauer, M., Kolesnik, L., Gastl, M. & Becker, T. (2016). Food Hydrocolloids, 56, 50-57.
Starch characteristics influence the gelatinization process, which is an important prerequisite for the saccharification required in many industrial processes. In order to determine these characteristics in barley malt, an adapted purification procedure allowing to preserve the native starch composition and simultaneously segregating the amylolytic enzymes which were formed during the germination is indispensable. Therefore, this research aimed to develop a method based on a combination of dry milling, micro-sieving and density gradient centrifugation. The impact on the starch characteristics was evaluated for three germinated barley varieties. The purified starches showed starch contents greater than 90% and proteins contents less than 0.4%. Yields ranged from 40.3 to 48.6%, depending on the variety. Considering the starch properties, the amylose/amylopectin ratio was not modified during the purification. The circularity of the granules as well as the ratio of A- and B-type granules remained constant. The particle size distribution of A-granules was not shifted, B-granules with a specific diameter of 5-10 µm were slightly reduced in dependency of the native granule composition. The highest impact could be observed on the amylolytic enzymes, which were completely segregated regardless of their initial value. The standard deviation of repeatability was less than 5%, except for the determination of B-type particle size distribution (7%). The newly developed procedure supplements existing isolation methods of unmalted grains by enabling the purification of germinated barley in a reproducible manner, without altering the native starch properties and by providing pure starch free of amylolytic activity.
Mannan endo-1, 4-β-mannosidase from Kitasatospora sp. isolated in Indonesia and its potential for production of mannooligosaccharides from mannan polymers.;
Rahmani, N., Kashiwagi, N., Lee, J., Niimi-Nakamura, S., Matsumoto, H., Kahar, P., Lisdiyanti, P., Yopi, Prasetya, B., Ogino, C. & Kondo, A. (2017). AMB Express, 7(1), 100.
Mannan endo-1,4-β-mannosidase (commonly known as β-mannanase) catalyzes a random cleavage of the β-D-1,4-mannopyranosyl linkage in mannan polymers. The enzyme has been utilized in biofuel production from lignocellulose biomass, as well as in production of mannooligosaccharides (MOS) for applications in feed and food industries. We aimed to obtain a β-mannanase, for such mannan polymer utilization, from actinomycetes strains isolated in Indonesia. Strains exhibiting high mannanase activity were screened, and one strain belonging to the genus Kitasatospora was selected. We obtained a β-mannanase from this strain, and an amino acid sequence of this Kitasatospora β-mannanase showed a 58-71% similarity with the amino acid sequences of Streptomyces β-mannanases. The Kitasatospora β-mannanase showed a significant level of activity (944 U/mg) against locust bean gum (0.5% w/v) and a potential for oligosaccharide production from various mannan polymers. The β-mannanase might be beneficial particularly in the enzymatic production of MOS for applications of mannan utilization.
High level expression of β-mannanase (RmMan5A) in Pichia pastoris for partially hydrolyzed guar gum production.
Li, Y. X., Yi, P., Wang, N. N., Liu, J., Liu, X. Q., Yan, Q. J. & Jiang, Z. Q. (2017). International Journal of Biological Macromolecules, 105, 1171-1179.
Partially hydrolyzed guar gum (PHGG), an important supplemental dietary fiber, has been used as food ingredient in many industries. In this study, a novel β-mannanase gene (RmMan5A) from Rhizomucor miehei was successfully expressed in Pichia pastoris and subjected for PHGG production. Enzyme activity of fermentation supernatant reached 85,200 U mL-1 after 168 h high cell density fermentation. The purified RmMan5A exhibited the highest enzyme activity at pH 7.0 and 65°C. RmMan5A was then employed for GG hydrolysis and PHGG obtained demonstrated a weight-average molecular weight (Mw) of 2.5 × 104 Da. Total dietary fiber accounted 90.6% of PHGG and 24.9% (w/w) of PHGG were identified as manno-oligosaccharides with degree of polymerization < 7. PHGG was further fractionated (F1-F4) by gradual ethanol precipitation. PHGG F1 with an Mw value of 3.6 × 104 Da and a mannose/galactose (M/G) ratio of 1.47 was precipitated initially, followed by PHGG F2 and F3 which showed lower Mw and higher M/G ratio. According to the structure analysis, the distribution of α-D-galactose of PHGG F1 was compact and regular, and that of other fractions was more random. A suitable β-mannanase for PHGG production and some useful information of PHGG are provided in this paper.