Mannanase Production Research Articles

Improving mannanase production in Bacillus subtilis for fibre hydrolysis during solid-state fermentation of palm kernel meal

The primary challenge in utilizing palm kernel meal (PKM, an agricultural by-product) as non-ruminant livestock feed is its high fibre content, predominantly in the form of mannan. Microbial fermentation offers an economically favourable alternative to enzyme supplementation for breaking down fibre in lignocellulosic biomass. In a recent study, our group isolated a B. subtilis strain F6 with a fast response time for mannanase production upon exposure to PKM. This work focuses on improving the mannanase production of the B. subtilis strain to achieve greater fibre hydrolysis of PKM without extending fermentation time. Mannanase GmuG, sourced from B. subtilis F6 and verified for its hydrolytic activity on PKM fibre, was homologously expressed using a replicative plasmid (pBE-S). Enzyme production was systematically improved by optimizing various regulatory elements, including the promoter, ribosome binding site, and signal peptide. Consequently, the neutral detergent fibre content of PKM was substantially reduced by 36.4 % in 22 h of solid-state fermentation using the engineered strain. Lastly, the highest mannanase-producing strain was examined for scaled-up fermentation. The impacts of fermentation on fibre and protein contents, as well as the surface morphology of PKM, were analysed. The outcomes of this study offer an efficient method for robust mannanase expression in B. subtilis and its potential application in the biotransformation of PKM and other mannan-rich bioresources for improved feed utilization.

Thermophilic β-mannanases from bacteria: production, resources, structural features and bioengineering strategies.

β-mannanases are pivotal enzymes that cleave the mannan backbone to release short chain mannooligosaccharides, which have tremendous biotechnological applications including food/feed, prebiotics and biofuel production. Due to the high temperature conditions in many industrial applications, thermophilic mannanases seem to have great potential to overcome the thermal impediments. Thus, structural analysis of thermostable β-mannanases is extremely important, as it could open up new avenues for genetic engineering, and protein engineering of these enzymes with enhanced properties and catalytic efficiencies. Under this scope, the present review provides a state-of-the-art discussion on the thermophilic β-mannanases from bacterial origin, their production, engineering and structural characterization. It covers broad insights into various molecular biology techniques such as gene mutagenesis, heterologous gene expression, and protein engineering, that are employed to improve the catalytic efficiency and thermostability of bacterial mannanases for potential industrial applications. Further, the bottlenecks associated with mannanase production and process optimization are also discussed. Finally, future research related to bioengineering of mannanases with novel protein expression systems for commercial applications are also elaborated.

Exploiting Systematic Engineering of the Expression Cassette as a Powerful Tool to Enhance Heterologous Gene Expression in Trichoderma reesei.

Many endeavors in expressing a heterologous gene in microbial hosts rely on simply placing the gene of interest between a selected pair of promoters and terminator. However, although the expression efficiency could be improved by engineering the host cell, how modifying the expression cassette itself systematically would affect heterologous gene expression remains largely unknown. As the promoter and terminator bear plentiful cis-elements, herein using the Aspergillus niger mannanase with high application value in animal feeds and the eukaryotic filamentous fungus workhorse Trichoderma reesei as a model gene/host, systematic engineering of an expression cassette was investigated to decipher the effect of its mutagenesis on heterologous gene expression. Modifying the promoter, signal peptide, the eukaryotic-specific Kozak sequence, and the 3'-UTR could stepwise improve extracellular mannanase production from 17 U/mL to an ultimate 471 U/mL, representing a 27.7-fold increase in expression. The strategies can be generally applied in improving the production of heterologous proteins in eukaryotic microbial hosts.

Production and purification of high activity β-mannanase from Streptomyces rochei and enzymatic hydrolysis of sugar palm fruit for galactomannan oligosaccharide (GMOS) production

The high activity of β-mannanase produced by Streptomyces rochei was achieved by enhancing the activity using a combination of the OFAT and the CCD of RSM method.The activity of β-mannanase produced by Streptomyces rochei was obtained to be 201.63 U/mL using OFAT method. Furthermore, the CCD of RSM optimized several process parameters such as nitrogen, carbon, and (KH2PO4) concentration. The maximum β-mannanase yield was nearly 7.5 times (460 U/mL) higher than the unoptimized condition (61.09 U/mL). The purified β-mannanase had a molecular weight of approximately 35.0 kDa and showed optimal activity at a pH of 6.0 and a temperature of 60 °C. Manannase could hydrolyze galactomannan-rich mannan polymers such as sugar palm fruit and other commercial mannans to create GMOS. For further research, developing a recombinant mannanase, scaling up the production of mannanase and also the study of GMOS activity for the health industrial applications are necessary. Additionally, it is important to conduct in vivo and in vitro assays to examine the activity of GMOS for potential application in the health industry.

The transcriptional activator ClrB is crucial for the degradation of soybean hulls and guar gum in Aspergillus niger

Low-cost plant substrates, such as soybean hulls, are used for various industrial applications. Filamentous fungi are important producers of Carbohydrate Active enZymes (CAZymes) required for the degradation of these plant biomass substrates. CAZyme production is tightly regulated by several transcriptional activators and repressors. One such transcriptional activator is CLR-2/ClrB/ManR, which has been identified as a regulator of cellulase and mannanase production in several fungi. However, the regulatory network governing the expression of cellulase and mannanase encoding genes has been reported to differ between fungal species. Previous studies showed that Aspergillus niger ClrB is involved in the regulation of (hemi-)cellulose degradation, although its regulon has not yet been identified. To reveal its regulon, we cultivated an A. niger ΔclrB mutant and control strain on guar gum (a galactomannan-rich substrate) and soybean hulls (containing galactomannan, xylan, xyloglucan, pectin and cellulose) to identify the genes that are regulated by ClrB. Gene expression data and growth profiling showed that ClrB is indispensable for growth on cellulose and galactomannan and highly contributes to growth on xyloglucan in this fungus. Therefore, we show that A. niger ClrB is crucial for the utilization of guar gum and the agricultural substrate, soybean hulls. Moreover, we show that mannobiose is most likely the physiological inducer of ClrB in A. niger and not cellobiose, which is considered to be the inducer of N. crassa CLR-2 and A. nidulans ClrB.

Enzymatic Activities of Halotolerant and Halophilic Fungi Isolated from Iko River Estuary, South-South Nigeria

Aim: Fungi that can tolerate unfavorable conditions are a potential source of stable bioactive metabolites that can be used for various industrial processes. Therefore this study aimed to isolate halotolerant and halophilic fungi from the Iko river estuary and screen them for three industrially important enzymes: amylase, cellulase, and mannanase.
 Place and Duration of Study: Department of Microbiology, Akwa Ibom State University, Nigeria, between September 2021 and August 2022.
 Methodology: Five sediment-dwelling fungi isolated from three locations along the Iko river estuary were assayed for their halotolerance on PDA containing different NaCl concentrations while their amylolytic, cellulolytic, and mannolytic activities were evaluated on agar plates containing 10% NaCl. The highest producer of the screened enzymes was identified using molecular techniques and further subjected to various submerged fermentation conditions to optimize the production of salt tolerant amylase, cellulase, and mannanase.
 Results: All isolates were halotolerant, best adapted to a salinity of 5% NaCl (w/v), and capable of secreting at least one of the three tested enzymes in 10% NaCl (w/v). Aspergillus niger isolate L2S1 had the highest enzymatic index among other isolates thus requiring optimization. The optimum production of amylase, cellulase, and mannanase was obtained at an incubation temperature of 30oC, initial pH of 6.0, and NaCl concentration of 5% (w/v). Out of the tested agro-wastes, wheat bran was most suited for the production of amylase, corncob was optimum for cellulase production, and maximum mannanase yield was obtained using copra meal. Amylase and cellulase production was optimal at 96 hours of incubation, whereas mannanase required 144 hours of fermentation to reach its maximum activity.
 Conclusion: Aspergillus niger isolate L2S1 was capable of utilizing different carbon sources including cheap agro-residues as substrates for the production of halostable amylase, cellulase, and mannanase.

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

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. In this study, a putative mannanase gene (ManBs31) from the genome of the marine bacterium Alteromonadaceae Bs31 was characterized. 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. 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. ManBs31 was a novel thermophilic endo-mannanase and it provided a new alternative for the biodegradation of mannans, especially for preparation of probiotic mannooligosaccharides.

β‐Mannanase production by Penicillium citrinum through solid‐state fermentation using açaí residual biomass (Euterpe oleracea)

AbstractBACKGROUNDAçaí core (Euterpe oleracea) is a residual biomass with high mannan content; thus it can become a good inducer for mannanase production by solid‐state fermentation (SSF). β‐Mannanase production from Penicillium citrinum was investigated using açaí seed as inducer and carbon source through SSF, as well as the properties of β‐mannanase in the crude extract.RESULTSIn this study, a two‐stage screening was performed. The P. citrinum strain exhibited high specific enzymatic activity (29 ± 0.2 U mg−1 protein). Nitrogen addition was necessary to improve β‐mannanase activity (an increase of 389% with sodium nitrate). The effect of pH and temperature on β‐mannanase activity in the crude extract was evaluated, with the highest activity found at pH 4.6 and a temperature of 40–60 °C. The enzymes exhibited good thermal stability with 5 h of incubation at 40, 50 and 60 °C, retaining 90%, 55% and 60% of the initial activity, respectively. Furthermore, P. citrinum β‐mannanase exhibited high specificity against ivory nut mannan (234% relative activity), good activity in the presence of a range of metal ions and the ability to hydrolyze locust bean gum, producing mannose and mannooligosaccharides.CONCLUSIONBased on such data, açaí seed can be used as an inducer of β‐mannanase. The P. citrinum crude enzymatic extract, rich in β‐mannanase, has potential industrial applications, especially in the food and feed industries.

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.

Purification and Characterization of Mannanase from Aspergillus awamori for Fruit Juice Clarification.

Fruit juice clarification is a challenging aspect of beverage industry which needs to be addressed for economical and hygienic production of fruit juices. Current study is focused on the complete purification, characterization and thermodynamic analysis of an efficient mannanase enzyme to analyze its applicability in biological clarification fruit juice. Mannanase production using Aspergillus awamori IIB037 in a 25 L stirred fermenter at pre optimized reaction conditions was carried out. Enzyme purification was carried out via series of steps. Characterization of enzyme along with kinetics and thermodynamic studies was conducted. Purified and characterized enzyme was assessed for its applicability in fruit juice clarification through clarification experiments on fresh apple juice. Purification fold of 3.98 was obtained along with 86.80% purification yield of mannanase with specific activity of 158.16 U/mg. The molecular size of purified enzyme was determined as 66 kDa. The enzyme depicted 56% residual activity at 60°C after 8 hrs. Thermodynamic studies of an enzyme revealed enthalpy of activation (ΔH) and activation energy (Ea) as 30.53KJ/mol, 27.76KJ/mol, respectively. The enzyme activity increased in the presence of ß-mercaptoethanol surprisingly. On the other hand, methyl alcohol, ethanol, Hg2+ and Cu2+ inhibited enzyme activity. The enzyme showed Km and Vmax values of 11.07 mM and 19.08 μM min-1 for Locust Bean Gum (LBG) under optimal conditions. Juice treated with mannanase showed decrease in absorbance and increase in reducing sugar content. The current study demonstrated that mannanase from Aspergillus awamori in its purified form has significant characteristics to be employed industrially for juice clarification.

Characterization of a thermotolerant and acidophilic mannanase producing Microbacterium sp. CIAB417 for mannooligosachharide production from agro-residues and dye decolorization

Mannanases are ubiquitous enzymes and are being explored for diverse industrial applications. In this study, a novel bacterial strain Microbacterium sp. CIAB417 was identified and characterized for extracellular production of mannanase. Microbacterium sp. CIAB417 was found to produce maximum mannanase after 36 h of incubation at 37 °C. Mannanase produced by the isolate was observed for maximum activity at optimum pH of 6 and optimum temperature of 50 °C. Crude mannanase was found to be capable of producing mannooligosachharides (MOS) by hydrolyzing hemicellulose from locust bean gum and Aloe vera. The produced MOS was characterized and found to be mixture of mannobiose to mannohexose units. Mannanase was also explored for decolorization of dyes. Bromophenol blue and coomassie blue R-250 were observed to be decolorized to the extent of 45.40 and 42.75%, respectively. Hence, the identified bacterial strain producing mannanase could be of great significance for applications in food and textile industry.

Production, Optimization, Purification and Characterization of Mannanase Produced by Bacillus Subtilis

Endo-β-1,4-D-mannanase (β-mannanase; EC 3.2.1.78) catalyses the random hydrolysis of mannoglycosidic bonds in mannan-based polysaccharides. These enyzmes are commonly found in nature and are located within the structure of mannans and heteromannans (galactomannan, glucomannan and galactoglucomannan) in the hemicellulose fraction of trees with soft tissues and hard tissues, locust bean seed. Most β-mannanases degrade mannooligosaccharides down to a degree of polymerization of four. In this study mannanase was produced using a submerged fermentation method from Bacillus subtilis. The effect of growth of the organism and processing parameters on the production of mannanase were determined after which optimization studies were carried out. The enzyme was partially purified using ammonium sulphate, dialysis and gel filtration. The partially purified enzyme was characterized. The result of the study showed that mannanase enzyme from Bacillus subtilis was optimally produced in a medium comprising of galactose, peptone, sugarcane bagasse at a pH of 6.0 and a temperature of 45 oC. The enzyme was most stable and active at pH 10.0 and at a temperature of 40 oC. and 60 respectively.

Functional Characterization and Transcriptional Analysis of clpP of Xanthomonas campestris pv. campestris.

The caseinolytic protease (Clp) system is essential for survival under stress conditions and for virulence in several pathogenic bacteria. Xanthomonas campestris pv. campestris (Xcc) is a plant pathogen which causes black rot disease in crucifers. In this study, the Xcc clpP gene which is annotated to encode the proteolytic core of Clp was characterized. Mutation of clpP resulted in susceptibility to high temperature and puromycin stresses. Site-directed mutagenesis revealed that S105, H130, and D179 are critical amino acid residues for ClpP function in puromycin tolerance. Inactivation of clpP also revealed an attenuation of virulence on the host plant and a reduction in the production of extracellular cellulase, mannanase, pectinase, and protease. The affected phenotypes of the clpP mutant could be complemented to wild-type levels by the intact clpP gene. Transcriptional analysis revealed that expression of clpP is induced under heat shock condition.

Halotolerant Bacillus sp. for Mannan Degradation Isolated from Mangrove Ecosystem at Hanura Beach Lampung

Mannose and mannooligosaccharide acted as prebiotic that consumed by probiotic bacteria. Mannanase were the second most important enzymes for the hydrolysis of hemicelluloses, beside xylanase. The objective research was to obtain eminent strains of Bacillus sp. in mannan degradation which can potentially be a probiotic candidate. The study employed a completely randomized design using four concentration levels repeated six times. Halotolerant Bacteria were isolated from mangrove ecosystem at Hanura beach, Teluk Pandan, Pesawaran District in Lampung Province. They are grown on the sea water complete agar media. Mannan degradation isolates were then characterized to determine their character with a variety of tests, including resistance to pH, salt, and metal ions, pathogenicity, and determination of mannanase production duration. Thirty strains are found to grow at 3-6% salt content, and 9 of them have mannanolytic activity. They grow optimally at pH 7-10. Seven isolates were proven to be positively hydrolyzed blood agar in the pathogenicity test. The addition of Iron (III) Chloride increased the enzyme activity by 11.12% in IBK3 isolates at 96 hours of cultivation period which was 0.05 UmL-1. It acted as cofactors of enzymatic reactions. Strains Bacillus sp., were able to degrade mannan substrate. It quantified using Index of mannanolytic. Strain IBK3 has the highest index of mannanolytic activity as much as 10.74. Their ability to grow in salt media indicated that they were halotolerant. They were more likely to live at base rather than acid habitat. Only IBK3 and ID2K1 showed non-pathogenic isolates. Only FeCl3 addition has proven to rise up enzymatic activity.

Mannanase and Cellulase Enzyme Production from the Agricultural Wastes by the Bacillus subtilis P2-5 Strain

Mannanase and Cellulase Enzyme Production from the Agricultural Wastes by the Bacillus subtilis P2-5 Strain

Comparative kinetic analysis of alginate lyase and mannanase co-produced via solid-state fermentation of cow dung supplemented with seaweed wastes by a novel Streptomyces sp. Alg-S23

The global industrial interest in the application of biocatalysts to manufacture green and sustainable chemicals necessitates cost-effective production of microbial enzymes via screening out potent microbial isolates from different environmental niches and identifying cheap feedstocks for their propagation. In this study, the biocatalytic efficacy of a novel Streptomyces sp. Alg-S23 (GenBank accession no. MK098188) isolated from putrefying Sargassum seaweed waste was assessed for the first time for co-production of alginate lyase and mannanase via solid-state fermentation of cow dung supplemented with decomposed Sargassum as the feedstocks. The optimization of the SSF feedstock components by using a 22-factorial central composite statistical design resulted in the enhanced co-production of alginate lyase and mannanase at 15 g of cow dung and 4.5 g of decomposed Sargassum biomass. Maximal activities of extracted alginate lyase and mannanase were recorded at pH 7.5 and at temperature 50 °C and 55 °C, respectively. The co-produced alginate lyase and mannanase had strong affinity toward the substrate by displaying Km and Vmax values as 0.72 mg/mL and 10.2 U/mg-protein, and 1.83 mg/mL and 78.7 U/mg-protein, respectively. Thermal denaturation kinetic analysis of the co-produced alginate lyase and mannanase at 40-60 °C suggested that both the enzymes were thermodynamically efficient by exhibiting higher values of activation, enthalpy, and free energies of denaturation. These promising enzymatic traits highlight the novel potential of cow dung supplemented with decomposed Sargassum seaweed waste as the inexpensive feedstocks for sustainable production of alginate lyase and mannanase.

Optimization of conditions for the production of lignocellulolytic enzymes by Sphingobacterium sp. ksn-11 utilizing agro-wastes under submerged condition

The present work was aimed at studying the production of lignocellulolytic enzymes, namely cellulase, xylanase, pectinase, mannanase, and laccase by a newly isolated bacterium Sphingobacterium sp. ksn-11, utilizing various agro-residues as a substrate under submerged conditions. The production of lignocellulolytic enzymes was found to be maximum at the loading of 10%(w/v) agro-residues. The enzyme secretion was enhanced by two-fold at 2 mM CaCO3, optimum pH 7, and temperature 40°. The Field Emission Gun-Scanning Electron Microscope (FEG-SEM) results have shown the degradative effect of lignocellulases; cellulase, xylanase, mannanase, pectinase, and laccase on corn husk with 3.55 U/ml, 79.22 U/ml, 12.43 U/ml, 64.66 U/ml, and 21.12 U/ml of activity, respectively. The hydrolyzed corn husk found to be good adsorbent for polyphenols released during hydrolysis of corn husk providing suitable conditions for stability of lignocellulases. Sphingobacterium sp. ksn is proved to be a promising candidate for lignocellulolytic enzymes in view of demand for enzymes in the biofuel industry.

Studies of the Nutritional, Environmental Effects and Repressive Nature of Simple Sugars on the Production of endo-β-mannanase by Aspergillus flavus PT7 on Solid State Fermentation

Aims: The importance of nutritional and environmental factors in the production of microbial enzymes cannot be overemphasized. Hence, endo-β-mannanase production was systematically studied in a step-wise approach of building up on the experimentally observed conditions favouring the production of this enzyme in Aspergillus flavus PT7.
 Place and Duration of Study: Department of Microbiology, University of Ibadan, Nigeria, between January 2018 and December 2018.
 Methodology: Thirty-eight (38) fungal isolates obtained were screened for mannolytic ability using standard method. The highest producer of endo-β-mannasase was subjected to various production conditions by adjusting the nutritional and environmental factors in view of optimizing the production of this enzyme in the isolate Aspergillus flavus PT7.
 Results: Copra meal was the highest inducer of mannanase production in the isolate at enzyme activity of 85.86±3.93 U/gds. Production increased to 94.54±0.42 when all forms of extraneous nitrogen sources were excluded from the production medium. pH 5.0, temperature 30°C, moisture content at 100% v/w and inoculum size of 8.0% v/w led to the increase in production by 44% (enzyme activity of 153.24±5.69 U/gds) in 5 days of incubation. Allowing the production set up additional two (2) days led to production increase with a recorded enzyme activity of 170.34±4.35 U/gds. Production of endo-β-mannanase in A. flavus PT7 was observed to be inductive as the presence of simple sugars like glucose, galactose, arabinose and xylose led to extended lag period in the production of the enzymes by the isolate.
 Conclusion: Production of endo-β-mannanase by Aspergillus flavus PT7 was successfully optimized in a step-wise and systematic experimental study of the nutritional and environmental growth conditions of the isolate.

Biochemical characterization and thermodynamic study of β-mannanase from Enterobacter asburiae

This study aimed for the partial purification, biochemical characterization, determination of bioprocess and thermodynamic parameters of mannanase from newly isolated strain Enterobacter asburiae SD26 under submerged culture. From different variables, locust bean gum and combination of peptone + yeast extract was found as best carbon and nitrogen sources respectively. The profiles of mannanases production and growth kinetics of E. asburiae SD26 were similar. The maximum specific growth rate (μ max ) and productivity ( γ Pmax ) of enzyme was 0.02 h -1 and 0.006 g l - 1 hr -1 . The highest mannanases activity was achieved at pH 6.0 and temperature 50°C. The kinetic parameters of mannanases was K m = 25 mg.ml -1 , V max = 2500 μmol.min -1 .ml -1 and thermodynamic parameters of enzyme, Δ H d ° , Δ G d ° , Δ S d ° were 67.74 KJmol -1 , 105.19 KJmol -1 , − 115.89 J.mol -1 .K -1 respectively, recorded for mathematical description of the mannanase action at different process conditions. • A bacterial strain Enterobacter asburiae SD26 as mannanase producer was identified. • Bioprocess parameters for growth of bacterial strain and mannanase production were estimated. • Optimum pH and temperature conditions for high mannanase activity were determined. • Kinetics and thermodynamics study of mannanase action were estimated.

TR OPTIMAL CONDITIONS FOR β-MANNANASE PRODUCTION BY BACILLUS CIRCULANS NT 6.7

Optimum medium for mannanase production is very important and producing medium has been shown effective for mannanase production from Bacillus circulans NT 6.7 but expensive. This work aim to find low cost medium and optimum condition for mannanase production from Bacillus circulans NT 6.7. 1.5% concentration of defatted copra meal as carbon source in shake flask resulted in highest growth and enzyme activities of 10.51±0.53 Log CFU/ml and 12.84±4.38 U/ml at 18 hours accordingly. Also, 2% defatted copra meal in fermenter produced highest growth of 7.94±0.41 Log CFU/ml and enzyme activities of 32.07±2.94 U/ml at 15 hours. Using taguchi design, optimal conditions for inoculum, agitation and aeration were 1%, 600 rpm and 0.75 vvm, respectively. Under this optimized condition, the mannanase experimental yield (29.57±4.81U/ml) closely matched the yield predicted by the statistical model (24.15U/ml) with R2 = 0.75. Using mineral salts medium, slightly increased in mannanase product was observed compare to previously used producing medium. Also, the cost of using mineral salts medium (cost per liter) is 27 time lower than the cost of producing medium. Suggesting that, mineral salt medium provides an economically feasible medium for producing mannanase from Bacillus circulans NT 6.7.