Research and application progress of microbial ß-mannanases: a mini-review.

Mannan is a predominant constituent of cork hemicellulose and is widely distributed in various plant tissues. ß-Mannanase is the principal mannan-degrading enzyme, which breaks down the ß-1,4-linked mannosidic bonds in mannans in an endo-acting manner. Microorganisms are a valuable source of ß-mannanase, which exhibits catalytic activity in a wide range of pH and temperature, making it highly versatile and applicable in pharmaceuticals, feed, paper pulping, biorefinery, and other industries. Here, the origin, classification, enzymatic properties, molecular modification, immobilization, and practical applications of microbial ß-mannanases are reviewed, the future research directions for microbial ß-mannanases are also outlined.

Beta-mannosidosis in a domestic cat associated with a missense variant in MANBA.

A 6-month-old cat of unknown ancestry presented for a neurologic evaluation due to progressive motor impairment. Complete physical and neurologic examinations suggested the disorder was likely to be hereditary, although the signs were not consistent with any previously described inherited disorders in cats. Due to the progression of disease signs including severely impaired motor function and cognitive decline, the cat was euthanized at approximately 10.5 months of age. Whole genome sequence analysis identified a homozygous missense variant c.2506G > A in MANBA that predicts a p.Gly836Arg alteration in the encoded lysosomal enzyme ß -mannosidase. This variant was not present in the whole genome or whole exome sequences of any of the 424 cats represented in the 99 Lives Cat Genome dataset. ß -Mannosidase enzyme activity was undetectable in brain tissue homogenates from the affected cat, whereas α-mannosidase enzyme activities were elevated compared to an unaffected cat. Postmortem examination of brain and retinal tissues revealed massive accumulations of vacuolar inclusions in most cells, similar to those reported in animals of other species with hereditary ß -mannosidosis. Based on these findings, the cat likely suffered from ß -mannosidosis due to the abolition of ß -mannosidase activity associated with the p.Gly836Arg amino acid substitution. p.Gly836 is located in the C-terminal region of the protein and was not previously known to be involved in modulating enzyme activity. In addition to the vacuolar inclusions, some cells in the brain of the affected cat contained inclusions that exhibited lipofuscin-like autofluorescence. Electron microscopic examinations suggested these inclusions formed via an autophagy-like process.

Proteome-Wide Mendelian Randomization Analysis Identified Potential Drug Targets for Atrial Fibrillation.

Background Finding effective and safe therapeutic drugs for atrial fibrillation (AF) is an important concern for clinicians. Proteome-wide Mendelian randomization analysis provides new ideas for finding potential drug targets. Methods and Results Using a proteome-wide Mendelian randomization approach, we assessed the genetic predictive causality between thousands of proteins and AF risk and found that genetically predicted plasma levels of phosphomevalonate kinase, tumor necrosis factor ligand superfamily member 12, sulfhydryl oxidase 2, interleukin-6 receptor subunit alpha, and low-affinity immunoglobulin gamma Fc region receptor II-b might decrease AF risk, while genetically predicted plasma levels of beta-mannosidase, collagen alpha-1(XV) chain, ANXA4 (annexin A4), COF2 (cofilin-2), and RAB1A (Ras-related protein Rab-1A) might increase AF risk (P<3.4×10-5). By using different Mendelian randomization methods and instrumental variable selection thresholds, we performed sensitivity analyses in 30 scenarios to test the robustness of positive findings. Replication analyses were also performed in independent samples to further avoid false-positive findings. Drugs targeting tumor necrosis factor ligand superfamily member 12, interleukin-6 receptor subunit alpha, low-affinity immunoglobulin gamma Fc region receptor II-b, and annexin A4 are approved or in development. The results of the phenome-wide Mendelian randomization analysis showed that changing the plasma levels of phosphomevalonate kinase, cofilin-2, annexin A4, Ras-related protein Rab-1A, sulfhydryl oxidase 2, and collagen alpha-1(XV) chain did not increase the risk of other diseases while decreasing the risk of AF. Conclusions We found a significant causal association between genetically predicted levels of 10 plasma proteins and AF risk. Four of these proteins have drugs targeting them that are approved or in development, and our results suggest the potential for these drugs to treat AF or cause AF. Sulfhydryl oxidase 2, low-affinity immunoglobulin gamma Fc region receptor II-b, and beta-mannosidase have not been suggested by previous laboratory or epidemiological studies to be associated with AF and may reveal new pathophysiological pathways as well as therapeutic targets for AF.

CRYPTOCHROME 1a-mediated blue light perception regulates tomato seed germination via changes in hormonal balance and endosperm-degrading hydrolase dynamics.

MAIN CONCLUSION: Blue light exposure delays tomato seed germination by decreasing endosperm-degrading hydrolase activities, a process regulated by CRY1a-dependent signaling and the hormonal balance between ABA and GA. The germination of tomato seeds (Solanum lycopersicum L.) is tightly controlled by an internal hormonal balance, which is also influenced by environmental factors such as light. In this study, we investigated the blue light (BL)-mediated impacts on physiological, biochemical, and molecular processes during the germination of the blue light photoreceptor CRYPTOCHROME 1a loss-of-function mutant (cry1a) and of the hormonal tomato mutants notabilis (not, deficient in ABA) and procera (pro, displaying a GA-constitutive response). Seeds were germinated in a controlled chamber in the dark and under different intensities of continuous BL (ranging from 1 to 25 µmol m-2 s-1). In general, exposure to BL delayed tomato seed germination in a fluency rate-dependent way due to negative impacts on the activities of endosperm-degrading hydrolases, such as endo-ß-mannanase, ß-mannosidase, and α-galactosidase. However, not and pro mutants presented higher germination speed index (GSI) compared to WT despite the BL influence, associated with higher hydrolase activities, especially evident in pro, indicating that the ABA/GA hormonal balance is important to diminish BL inhibition over tomato germination. The cry1a germination percentage was higher than in WT in the dark but its GSI was lower under BL exposure, suggesting that functional CRY1a is required for BL-dependent germination. BL inhibits the expression of GA-biosynthetic genes, and induces GA-deactivating and ABA-biosynthetic genes. The magnitude of the BL influence over the hormone-related transcriptional profile is also dependent upon CRY1a, highlighting the complex interplay between light and hormonal pathways. These results contribute to a better understanding of BL-induced events behind the photoregulation of tomato seed germination.

Marine bacteroidetes use a conserved enzymatic cascade to digest diatom ß-mannan.

The polysaccharide ß-mannan, which is common in terrestrial plants but unknown in microalgae, was recently detected during diatom blooms. We identified a ß-mannan polysaccharide utilization locus (PUL) in the genome of the marine flavobacterium Muricauda sp. MAR_2010_75. Proteomics showed ß-mannan induced translation of 22 proteins encoded within the PUL. Biochemical and structural analyses deduced the enzymatic cascade for ß-mannan utilization. A conserved GH26 ß-mannanase with endo-activity depolymerized the ß-mannan. Consistent with the biochemistry, X-ray crystallography showed the typical TIM-barrel fold of related enzymes found in terrestrial ß-mannan degraders. Structural and biochemical analyses of a second GH26 allowed the prediction of an exo-activity on shorter manno-gluco oligosaccharides. Further analysis demonstrated exo-α-1,6-galactosidase- and endo-ß-1,4-glucanase activity of the PUL-encoded GH27 and GH5_26, respectively, indicating the target substrate is a galactoglucomannan. Epitope deletion assays with mannanases as analytic tools indicate the presence of ß-mannan in the diatoms Coscinodiscus wailesii and Chaetoceros affinis. Mannanases from the PUL were active on diatom ß-mannan and polysaccharide extracts sampled during a microalgal bloom at the North Sea. Together these results demonstrate that marine microorganisms use a conserved enzymatic cascade to degrade ß-mannans of marine and terrestrial origin and that this metabolic pathway plays a role in marine carbon cycling.

Molecular insight into Aspergillus oryzae ß-mannanase interacting with mannotriose revealed by molecular dynamic simulation study.

Fungal ß-mannanases hydrolyze ß-1, 4-glycosidic bonds of mannans and find application in the generation of mannose and prebiotic mannooligosaccharides (MOS). Previously, a MOS generating ß-mannanase from Aspergillus oryzae MTCC 1846 (ßManAo) was characterized and its structural and functional properties were unraveled through homology modeling and molecular dynamics in this study. The ßManAo model was validated with 92.9% and 6.5% of the residues found to be distributed in the most favorable and allowed regions of the Ramachandran plot. Glu244 was found to play a key role in the interaction with mannotriose, indicating conserved amino acids for the catalytic reaction. A detailed metadynamic analysis of the principal components revealed the presence of an α8-helix in the C-terminus which was very flexible in nature and energy landscapes suggested high conformation sub-states and the complex dynamic behavior of the protein. The binding of the M3 substrate stabilized the ß-mannanase and resulted in a reduction in the intermediate conformational sub-states evident from the free energy landscapes. The active site of the ß-mannanase is mostly hydrophilic in nature which is accordance with our results, where the major contribution in the binding energy of the substrate with the active site is from electrostatic interactions. Define Secondary Structure of Proteins (DSSP) analysis revealed a major transition of the protein from helix to ß-turn for binding with the mannotriose. The molecular dynamics of the ßManAo-mannotriose model, and the role and interactions of catalytic residues with ligand were also described. The substrate binding pocket of ßManAo was found to be highly dynamic and showed large, concerted movements. The outcomes of the present study can be exploited in further understanding the structural properties and functional dynamics of ßManAo.

Impact of Multiple Sclerosis Risk Polymorphism rs7665090 on MANBA Activity, Lysosomal Endocytosis, and Lymphocyte Activation.

Deficiencies in Mannosidase ß (MANBA) are associated with neurological abnormalities and recurrent infections. The single nucleotide polymorphism located in the 3'UTR of MANBA, rs7665090, was found to be associated with multiple sclerosis (MS) susceptibility. We aimed to study the functional impact of this polymorphism in lymphocytes isolated from MS patients and healthy controls. A total of 152 MS patients and 112 controls were genotyped for rs7665090. MANBA mRNA expression was quantified through qPCR and MANBA enzymatic activity was analyzed. Upon phytohemagglutinin stimulation, immune activation was evaluated by flow cytometry detection of CD69, endocytic function, and metabolic rates with Seahorse XFp Analyzer, and results were stratified by variation in rs7665090. A significantly reduced gene expression (p < 0.0001) and enzymatic activity (p = 0.018) of MANBA were found in lymphocytes of MS patients compared to those of controls. The rs7665090*GG genotype led to a significant ß-mannosidase enzymatic deficiency correlated with lysosomal dysfunction, as well as decreased metabolic activation in lymphocytes of MS patients compared to those of rs7665090*GG controls. In contrast, lymphocytes of MS patients and controls carrying the homozygous AA genotype behaved similarly. Our work provides new evidence highlighting the impact of the MS-risk variant, rs7665090, and the role of MANBA in the immunopathology of MS.

Production and Functional Characterization of a Novel Mannanase from Alteromonadaceae Bacterium Bs31.

BACKGROUND: Mannans are the main components of hemicellulose in nature and serve as the major storage polysaccharide in legume seeds. To mine new mannanase genes and identify their functional characteristics are an important basis for mannan biotechnological applications. OBJECTIVE: In this study, a putative mannanase gene (ManBs31) from the genome of the marine bacterium Alteromonadaceae Bs31 was characterized. METHODS: Amino acid sequence analysis and protein structural modeling were used to reveal the molecular features of ManBs31. The catalytic domain of ManBs31 was recombinantly produced using Escherichia coli and Pichia pastoris expression systems. The biochemical properties of the enzymes were determined by reducing sugar assay and thin-layer chromatography. RESULTS: Sequence analysis revealed that ManBs31 was a multidomain protein, consisting of a catalytic domain belonging to glycoside hydrolase family 5 (GH5) and two cellulose-binding domains. Recombinant ManBs31-GH5 exhibited the maximum hydrolytic performance at 70 ºC and pH 6. It showed the best hydrolysis capacity toward konjac glucomannan (specific enzyme activity up to 1070.84 U/mg) and poor hydrolysis ability toward galactomannan with high side-chain modifications (with a specific activity of 344.97 U/mg and 93.84 U/mg to locust bean gum and ivory nut mannan, respectively). The hydrolysis products of ManBs31-GH5 were mannooligosaccharides, and no monosaccharide was generated. Structural analysis suggested that ManBs31-GH5 had a noncanonical +2 subsite compared with other GH5 mannanases. CONCLUSION: ManBs31 was a novel thermophilic endo-mannanase and it provided a new alternative for the biodegradation of mannans, especially for preparation of probiotic mannooligosaccharides.

Enhanced extracellular ß-mannanase production by overexpressing PrsA lipoprotein in Bacillus subtilis and optimizing culture conditions.

In this study, first, ß-mannanase gene man derived from Bacillus amyloliquefaciens CGMCC1.857 was cloned and expressed in Bacillus subtilis 168 to generate B. subtilis M1. However, the extracellular ß-mannanase activity of B. subtilis M1 was not very high. To further increase extracellular ß-mannanase extracytoplasmic molecular chaperone, PrsA lipoprotein was tandem expressed with man gene in B. subtilis 168 to yield B. subtilis M2. The secretion of ß-mannanase of B. subtilis M2 was enhanced by 15.4%, compared with the control B. subtilis M1. Subsequently, process optimization strategies were also developed to enhance ß-mannanase production by B. subtilis 168 M2. It was noted that the optimal temperature for ß-mannanase production (25°C) was different from the optimal growth temperature (37°C) for B. subtilis. Based on these findings, a two-stage temperature control strategy was proposed where the bacterial culture was maintained at 37°C for the first 12 h to obtain a high rate of cell growth, followed by lowering the temperature to 25°C to enhance ß-mannanase production. Using this strategy, the extracellular ß-mannanase activity reached 5016 ± 167 U/ml at about 36 h, which was 19.1% greater than the best result obtained using a constant temperature (25°C). The result of this study showed that PrsA lipoprotein overexpression and two-stage temperature control strategy were more efficient for ß-mannanase fermentation in B. subtilis.

Genome sequencing of a novel Microbacterium camelliasinensis CIAB417 identified potential mannan hydrolysing enzymes.

Here, whole genome sequencing of Microbacterium sp. CIAB417 was conducted to determine its novelty at species level and identification of genes encoding for enzymes for mannan degradation. The draft genome was predicted to have 6.53 mbp length represented by 41 contigs and 6078 genes. However, only 82.35% genes were allocated for their functions. The whole genome phylogeny, ANI score (78.84%), GGDC (genome to genome distance calculations) show probability (DDH ≥ 70%) equal to 0% and difference in advanced biochemical properties among closely predicted species. The Microbacterium sp. CIAB417 was stipulated to be novel at species level. Isolate was named as Microbacterium camelliasinensis CIAB417 (accession no JAHZUT000000000) based on its isolation from a tea garden soil. Genome was predicted for three novel mannanase coding genes man1 (MZ702740), man2 (MZ702741), and man3 (MZ702737) that belong to the GH5 and GH113 family. Besides that, mannan side chain hydrolysing enzymes alpha-galactosidase (gla1; MZ702739) and beta-glucosidase (glu1; MZ702738) were also predicted.

A novel neutral thermophilic ß-mannanase from Malbranchea cinnamomea for controllable production of partially hydrolyzed konjac powder.

Partially hydrolyzed konjac powder (PHKP) can be used to increase the daily intake of dietary fibers of consumers. To produce PHKP by enzymatic hydrolysis, a novel ß-mannanase gene (McMan5B) from Malbranchea cinnamomea was expressed in Pichia pastoris. It showed a low identity of less than 52% with other GH family 5 ß-mannanases. Through high cell density fermentation, the highest ß-mannanase activity of 42200 U mL-1 was obtained. McMan5B showed the maximal activity at pH 7.5 and 75 °C, respectively. It exhibited excellent pH stability and thermostability. Due to the different residues (Phe214, Pro253, and His328) in catalytic groove and the change of ß2-α2 loop, McMan5B showed unique hydrolysis property as compared to other ß-mannanases. The enzyme was employed to hydrolyze konjac powder for controllable production of PHKP with a weight-average molecular weight of 22000 Da (average degree of polymerization 136). Furthermore, the influence of PHKP (1.0%-4.0%) on the qualities of steamed bread was evaluated. The steamed bread adding 3.0% PHKP had the maximum specific volume and the minimum hardness, which showed 11.0% increment and 25.4% decrement as compared to the control, respectively. Thus, a suitable ß-mannanase for PHKP controllable production and a fiber supplement for steamed bread preparation were provided in this study. KEY POINTS: • A novel ß-mannanase gene (McMan5B) was cloned from Malbranchea cinnamomea and expressed in Pichia pastoris at high level. • McMan5B hydrolyzed konjac powder to yield partially hydrolyzed konjac powder (PHKP) instead of manno-oligosaccharides. • PHKP showed more positive effect on the quality of steamed bread than many other dietary fibers including konjac powder.

The response surface optimization of ß-mannanase produced by Weissella cibaria F1 and its potential in juice clarification.

A ß-mannanase-producing lactic acid bacteria (LAB) was identified as Weissella cibaria F1 according to physiological and biochemical properties, morphological observations, partial sequence of 16S rRNA gene and API 50 CHL test. In order to improve the yield of ß-mannanase, the response surface methodology (RSM) was originally used to optimize the fermentation conditions. The optimization results showed that when the konjac powder, glucose, and initial pH were 9.46 g/L, 14.47 g/L and 5.67, respectively, the ß-mannanase activity increased to 38.81 ± 0.33 U/mL, which was 1.33 times compared to initial yield (29.28 ± 0.26 U/mL). This result was also supported by larger clearance on the konjac powder-MRS agar plate through Congo Red dyeing. The W. cibaria F1 ß-mannanase could improve the clarity of five fruits juice, i.e., apple, orange, peach, persimmon and blue honeysuckle. Among these, peach juice was the most obvious, clarity increasing by 12.8%. These results collectively indicated that W. cibaria F1 ß-mannanase had an applicable potential in food-level fields.

High surfactant-tolerant ß-mannanase isolated from Dynastes hercules larvae excrement, and identification of its hotspot using site-directed mutagenesis and molecular dynamics simulations.

The ß-mannanase from Bacillus subtilis HM7 (Man26HM7) isolated from Dynastes hercules larvae excrement was cloned and expressed in Escherichia coli. Biochemical characterization shows that optimal pH and temperature for catalysis are 6.0 and 50 °C, respectively. Man26HM7 displayed excellent surfactant stability by retaining 70% of initial activity in 1%(w/v) SDS, and more than 90% of initial activity in 1%(w/v) Triton X-100 and Tween 80. Results from amino acid sequence alignment and molecular modeling suggest residue 238 of ß-mannanase as a hotspot of SDS-tolerance. Mutagenesis at the equivalent residue of another homolog, ß-mannanase from Bacillus subtilis CAe24 (Man26CAe24), significantly enhanced the SDS stability of this enzyme. Comparative computational analysis, including molecular docking and molecular dynamics simulation, were then performed to compute the binding free energy of SDS to Man26HM7, Man26CAe24, and variant enzymes. The results suggest that residue 238 of Man26HM7 is involved in SDS binding to the hydrophobic surface of ß-mannanase. This study provides not only the promising application of Man26HM7 in detergent and cleaning products but also valuable information for enhancing the surfactant stability of ß-mannanase by enzyme engineering.

Biochemical and structural characterization of a novel thermophilic and acidophilic ß-mannanase from Aspergillus calidoustus.

ß-Mannanases hydrolyze lignocellulosic biomass with the release of mannan oligosaccharides, which are considered as renewable resource in higher plants. Here, we cloned, expressed and characterized a novel endo-ß-mannanase (ManAC) from Aspergillus calidoustus. Homology alignment analysis indicated that ManAC belonged to glycosyl hydrolase (GH) 5 family members. The analysis of structural homologous model revealed that five residues, Arg116, Asn231, His305, Tyr307, and Trp370, constituted the active site of ManAC. Glu232 and Glu340, proton donor and nucleophile, formed the catalytic residues of ManAC. The recombinant ManAC exhibited maximal activity at pH 2.5 and 70 °C, and it was acid tolerant at a pH range of 2.0-6.0 and thermostable under 60 °C. Meanwhile, the activity of ManAC was not significantly affected by various metal ions, except for Mg2+ and Ag2+. The recombinant ManAC exhibited the highest ß-mannanase activity towards locust bean gum (669.7 U/mg) with the Km and Vmax values of 3.4 mg/mL and 982.4 µmol/min/mg, respectively. These thermophilic and acidophilicc characteristics is better than most extreme ß-mannanase. As the first reported mannanse from Aspergillus calidoustus (ManAC), these excellent properties of ManAC strongly promote the synthesis of mannooligosaccharides which have potential for food and feed industrial applications.

Production and in vitro evaluation of prebiotic manno-oligosaccharides prepared with a recombinant Aspergillus niger endo-mannanase, Man26A.

In this study, a GH26 endo-mannanase (Man26A) from an Aspergillus niger ATCC 10864 strain, with a molecular mass of 47.8 kDa, was cloned in a yBBH1 vector and expressed in Saccharomyces cerevisiae Y294 strain cells. Upon fractionation by ultra-filtration, the substrate specificity and substrate degradation pattern of the endo-mannanase (Man26A) were investigated using ivory nut linear mannan and two galactomannan substrates with varying amounts of galactosyl substitutions, guar gum and locust bean gum. Man26A exhibited substrate specificity in the order: locust bean gum ≥ ivory nut mannan > guar gum; however, the enzyme generated more manno-oligosaccharides (MOS) from the galactomannans than from linear mannan during extended periods of mannan hydrolysis. MOS with a DP of 2-4 were the major products from mannan substrate hydrolysis, while guar gum also generated higher DP length MOS. All the Man26A generated MOS significantly improved the growth (approximately 3-fold) of the probiotic bacterial strains Streptococcus thermophilus and Bacillus subtilis in M9 minimal medium. Ivory nut mannan and locust bean gum derived MOS did not influence the auto-aggregation ability of the bacteria, while the guar gum derived MOS led to a 50 % reduction in bacterial auto-aggregation. On the other hand, all the MOS significantly improved bacterial biofilm formation (approximately 3-fold). This study suggests that the prebiotic characteristics exhibited by MOS may be dependent on their primary structure, i.e. galactose substitution and DP. Furthermore, the data suggests that the enzyme-generated MOS may be useful as potent additives to dietary foods.

Novel ß-mannanase/GLP-1 fusion peptide high effectively ameliorates obesity in a mouse model by modifying balance of gut microbiota.

We constructed a novel ß-mannanase/GLP-1 fusion peptide, termed MGLP_1, and evaluated its ability to ameliorate obesity in a high-fat/high-sugar diet (HFSD)-induced mouse model. Eight-wk MGLP_1 treatment notably reduced obesity, as reflected by significant changes of body weight, serum triglyceride level, fatty liver and adipose tissue distribution. Amelioration of HFSD-induced gut dysbiosis by MGLP_1 was evidenced by reduced abundance ratio of bacterial phyla Firmicutes to Bacteroidetes, enhanced abundance of beneficial probiotic genera (Bifidobacterium, Lachnospiraceae, Ileibacterium), and reduced abundance of harmful genera (Clostridium, Romboutsia). Mechanisms of weight loss were investigated by comparing effects of treatment with MGLP_1 vs. prebiotics manno-oligosaccharides (MOS). MGLP_1 ameliorated gut microbiota imbalance by enhancing carbohydrate catabolism, whereas MOS promoted glycan synthesis and metabolism. Our findings, taken together, indicate that MGLP_1 fusion peptide has strong potential for amelioration of obesity by modifying relationships between gut microbiota and lipid and glucose metabolism.

A MYB4-MAN3-Mannose-MNB1 signaling cascade regulates cadmium tolerance in Arabidopsis.

Our previous studies showed that MAN3-mediated mannose plays an important role in plant responses to cadmium (Cd) stress. However, the underlying mechanisms and signaling pathways involved are poorly understood. In this study, we showed that an Arabidopsis MYB4-MAN3-Mannose-MNB1 signaling cascade is involved in the regulation of plant Cd tolerance. Loss-of-function of MNB1 (mannose-binding-lectin 1) led to decreased Cd accumulation and tolerance, whereas overexpression of MNB1 significantly enhanced Cd accumulation and tolerance. Consistently, expression of the genes involved in the GSH-dependent phytochelatin (PC) synthesis pathway (such as GSH1, GSH2, PCS1, and PCS2) was significantly reduced in the mnb1 mutants but markedly increased in the MNB1-OE lines in the absence or presence of Cd stress, which was positively correlated with Cd-activated PC synthesis. Moreover, we found that mannose is able to bind to the GNA-related domain of MNB1, and that mannose binding to the GNA-related domain of MNB1 is required for MAN3-mediated Cd tolerance in Arabidopsis. Further analysis showed that MYB4 directly binds to the promoter of MAN3 to positively regulate the transcript of MAN3 and thus Cd tolerance via the GSH-dependent PC synthesis pathway. Consistent with these findings, overexpression of MAN3 rescued the Cd-sensitive phenotype of the myb4 mutant but not the mnb1 mutant, whereas overexpression of MNB1 rescued the Cd-sensitive phenotype of the myb4 mutant. Taken together, our results provide compelling evidence that a MYB4-MAN3-Mannose-MNB1 signaling cascade regulates cadmium tolerance in Arabidopsis through the GSH-dependent PC synthesis pathway.

A multi-functional genetic manipulation system and its use in high-level expression of a ß-mannanase mutant with high specific activity in Pichia pastoris.

To further extend the practical application of a thermostable and acidic resistance ß-mannanase (ManAK) in animal feed additives, an effective strategy that combined directed evolution and metabolic engineering was developed. Four positive mutants (P191M, P194E, S199G and S268Q) with enhanced specific activity (25.5%-60.9%) were obtained. The S199G mutant exhibited 56.7% enhancement of specific activity at 37°C and good thermostability, and this was selected for high-level expression in P. pastoris X33. A multi-functional and scarless genetic manipulation system was proposed and functionally verified (gene deletion, substitution/insertion and point mutation). This was then subjected to Rox1p (an oxygen related transcription regulator) deletion and Vitreoscilla haemoglobin (VHb) co-expression for high enzyme productivity in P. pastoris X33VIIManAKS199G . An excellent strain, named X33VIIManAKS199G ∆rox1::VHb, was achieved by combining these two factors, and then the maximum enzymatic activity was further increased to 3753 U ml-1 , which was nearly twice as much as the maximum production of ManAK in P. pastoris. This work provides a systematic and effective method to improve the enzymatic yield of ß-mannanase, promotes the application of ManAK in feed additives, and also demonstrated that a scarless genetic manipulation tool is useful in P. pastoris.

Functional and structural investigation of a novel ß-mannanase BaMan113A from Bacillus sp. N16-5.

Mannan is an important renewable resource whose backbone can be hydrolyzed by ß-mannanases to generate manno-oligosaccharides of various sizes. Only a few glycoside hydrolase (GH) 113 family ß-mannanases have been functionally and structurally characterize. Here, we report the function and structure of a novel GH113 ß-mannanase from Bacillus sp. N16-5 (BaMan113A). BaMan113A exhibits a substrate preference toward manno-oligosaccharides and releases mannose and mannobiose as main hydrolytic products. The crystal structure of BaMan113A suggest that the enzyme shows a semi-enclosed substrate-binding cleft and the amino acids surrounding the +2 subsite form a steric barrier to terminate the substrate-binding tunnel. Based on these structural features, we conducted mutagenesis to engineer BaMan113A to remove the steric hindrance of the substrate-binding tunnel. We found that F101E and N236Y variants exhibit increased specific activity toward mannans comparing to the wild-type enzyme. Meanwhile, the product profiles of these two variants toward polysaccharides changed from mannose to a series of manno-oligosaccharides. The crystal structure of variant N236Y was also determined to illustrate the molecular basis underlying the mutation. In conclusion, we report the functional and structural features of a novel GH113 ß-mannanase, and successfully improved its endo-acting activity by using structure-based engineering.

Functional diversity of three tandem C-terminal carbohydrate-binding modules of a ß-mannanase.

Carbohydrate active enzymes, such as those involved in plant cell wall and storage polysaccharide biosynthesis and deconstruction, often contain repeating noncatalytic carbohydrate-binding modules (CBMs) to compensate for low-affinity binding typical of protein-carbohydrate interactions. The bacterium Saccharophagus degradans produces an endo-ß-mannanase of glycoside hydrolase family 5 subfamily 8 with three phylogenetically distinct family 10 CBMs located C-terminally from the catalytic domain (SdGH5_8-CBM10x3). However, the functional roles and cooperativity of these CBM domains in polysaccharide binding are not clear. To learn more, we studied the full-length enzyme, three stepwise CBM family 10 (CBM10) truncations, and GFP fusions of the individual CBM10s and all three domains together by pull-down assays, affinity gel electrophoresis, and activity assays. Only the C-terminal CBM10-3 was found to bind strongly to microcrystalline cellulose (dissociation constant, Kd = 1.48 µM). CBM10-3 and CBM10-2 bound galactomannan with similar affinity (Kd = 0.2-0.4 mg/ml), but CBM10-1 had 20-fold lower affinity for this substrate. CBM10 truncations barely affected specific activity on carob galactomannan and konjac glucomannan. Full-length SdGH5_8-CBM10x3 was twofold more active on the highly galactose-decorated viscous guar gum galactomannan and crystalline ivory nut mannan at high enzyme concentrations, but the specific activity was fourfold to ninefold reduced at low enzyme and substrate concentrations compared with the enzyme lacking CBM10-2 and CBM10-3. Comparison of activity and binding data for the different enzyme forms indicates unproductive and productive polysaccharide binding to occur. We conclude that the C-terminal-most CBM10-3 secures firm binding, with contribution from CBM10-2, which with CBM10-1 also provides spatial flexibility.