Biophysical characterization of thermophilic laccase from the xerophytes: Cereus pterogonus and Opuntia vulgaris

Biophysical characterization of laccase enzyme isoforms from two different xerophytic plants Cereus pterogonus and Opuntia vulgaris was carried out using EPR, fluorescence and circular dichroism (CD) spectroscopy while their thermal denaturation profiles were investigated employing differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). EPR analysis revealed the presence of endogenous copper. The hyperfine splitting of EPR spectrum reduced with increase in the complexity of enzyme protein. Raise in temperature did not alter the protein fluorescence emission suggestive of high temperature stability of the enzyme, causing the tryptophan and tyrosine residues to remain buried within the protein structure. Far-UV CD spectrum revealed existence of 60 % random coils in enzyme structure even at elevated temperatures and in presence of metal ions and protein denaturants. DSC analysis provided a Tm in the range 95–121 °C for the native and 158–199 °C for the metal associated laccase isoforms. Loss in weight of the enzyme protein by 10–18 % was noted up to 100 °C when determined through TGA. The thermostable plant xerophytic laccase enzyme isoforms will be of potential use in textile, dyeing, pulping and biotechnology applications.     

CO<Subscript>2</Subscript> Removal from Biogas by Cyanobacterium <Emphasis Type="Italic">Leptolyngbya</Emphasis> sp. CChF1 Isolated from the Lake Chapala,Mexico: Optimization of the Temperature and Light Intensity

(S)-N-Boc-3-hydroxypiperidine ((S)-NBHP) is a key pharmaceutical intermediate and the chiral source in synthesizing Imbruvica, which is a newly approved drug in lymphoma therapy by targeting Bruton’s tyrosine kinase (BTK). Current chemical synthesis of (S)-NBHP suffered from the need of noble metal catalyst and low yield. The single reported bioconversion of (S)-NBHP was achieved by using recombinant ketoreductase, but enzyme sequence was kept confidential and the catalytic process suffered from the thermodeactivation and substrate inhibition. In the current study, we presented a thermostable aldo-keto reductase (AKR)—AKR-43—which showed high activity toward N-Boc-3-piperidone (NBP) to produce (S)-NBHP, high enantioselectivity, and no substrate inhibition. The molecular simulations demonstrated the structural rationale for the enantioselectivity of AKR-43 toward NBP and supported the classic ordered two-step catalytic mechanism. The catalytic process was achieved by using glucose dehydrogenase (GDH) for cofactor recycling, and the optimal reaction conditions were determined to be 30 °C and pH 7.5. Within a reaction time of 16 h, the 16 % substrate concentration (w/w), over 99 % ee and under 3.5 % of enzyme loading (w/w) characterized a high efficiency process with promising industrial values.     

Molecular Cloning,Characterization, and Dye-Decolorizing Ability of a Temperature- and pH-Stable Laccase from Bacillus subtilis X1

Laccases from fungal origin are typically unstable at high temperatures and alkaline conditions. This characteristic limits their practical applications. In this study, a new bacterial strain exhibiting laccase activity was isolated from raw fennel honey samples and identified as Bacillus subtilis X1. The CotA-laccase gene was cloned from strain X1 and efficiently expressed in Escherichia coli in a biologically active form. The purified recombinant laccase demonstrated an extensive pH range for catalyzing substrates and high stability toward alkaline pH and high temperatures. No loss of laccase activity was observed at pH 9.0 after 10 days of incubation, and approximately 21 % of the initial activity was detected after 10 h at 80 °C. Two anthraquinonic dyes (reactive blue 4 and reactive yellow brown) and two azo dyes (reactive red 11 and reactive brilliant orange) could be partially decolorized by purified laccase in the absence of a mediator. The decolorization process was efficiently promoted when methylsyringate was present, with more than 90 % of color removal occurring in 3 h at pH 7.0 or 9.0. These unusual properties indicated a high potential of the novel CotA-laccase for industrial applications.     

Production and Characterization of a Novel Laccase with Cold Adaptation and High Thermal Stability from an Isolated Fungus

A new white-rot fungus SYBC-L1, which could produce an extracellular laccase, was isolated from a decayed Elaeocarpus sylvestris. The strain was identified as Pycnoporus sp. SYBC-L1 according to the morphological characteristics and ribosomal ITS1-5.8S-ITS2 RNA genomic sequence analysis. The highest laccase activity of 24.1 U ml⁻¹, which was approximately 40-fold than that in basal medium, was achieved in optimal culture medium in submerged fermentation. The laccase produced by Pycnoporus sp. SYBC-L1 was not only a cold adaptation enzyme with a relative catalytic activity of 30.2% at 0°C but also a high thermostable enzyme. The half-lives at 60, 70 and 80°C were 85.5, 37.2, and 2.6 h, respectively. The laccase could effectively decolorize weak acid blue AS and diamond black PV up to 88% and 74.7%, respectively, within 2 h in the absence of any redox mediators. The results suggested Pycnoporus sp. SYBC-L1 was a potential candidate for laccase production and industrial application.     

Essential Role of the N- and C-terminals of Laccase from Pleurotus florida on the Laccase Activity and Stability

POXA1b is the most thermostable laccase isoenzyme from Pleurotus ostreatus. POXA1b is remarkably stable at alkaline pH (the t_1/2 at pH 10 was 30 days), and its C-terminal affects its catalytic and stability properties. We cloned POXA1c from P. florida, which showed 99 % identity with POXA1b. POXA1c was functionally expressed in Pichia pastoris. The functions of the N and C termini of POXA1c were investigated using site-directed mutagenesis. Compared with POXA1c, the N-terminal R5V site effectively increased the specific activities for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and guaiacol by 2- and 3.5-fold, respectively. A C-terminal truncated mutant, POXA1c△13, also increased the specific activities for ABTS and guaiacol by 2.3- and 3.4-fold, respectively. A double mutant, POXA1cΔ13-R5V, combined the R5V and △13 effects. The specific activity of this double mutant for ABTS was 1,321 U/mg, which indicated a 4-fold increase compared with the wild type. The role of residue V5 on laccase catalytic properties was also observed for laccases from Trametes versicolor and Rigidoporus lignosus. The specific activities of the V5R of the laccases from T. versicolor and R. lignosus were half of that of the wild type. The pH and thermal stability analysis of POXA1c and its mutants showed that the enzymes were remarkably stable because they showed 63 % residual activity after incubation for 108 h at 30 °C over a pH range of 4.5 to 9.0. Similar results were observed for POXA1cΔ13-R5V. POXA1cΔ13-R5V can be widely used in industrial biotechnology because of its excellent catalytic properties.     

Effect of Synergistic Inducement on the Production of Laccase by a Novel Shiraia bambusicola Strain GZ11K2

In this study, an easily detectable method was employed for screening laccase-producing microorganisms by using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) as laccase secretion indicator. A novel laccase-producing strain was isolated and identified as Shiraia bambusicola Henn. strain GZ11K2 according to the morphological characteristics and the comparison of internal transcribed spacer ribosomal DNA gene sequences. In further investigation, the production of laccase by S. bambusicola GZ11K2 was greatly enhanced by the nontoxic inducers of copper sulfate and rhodamine B. Copper and rhodamine B were added into the cultivation medium at 24 and 12 h, respectively, and the maximum laccase production was obtained. Under the induction of 2.0 mM copper sulfate and 35 μM rhodamine B, an increment of about 80 times of laccase activity compared with that in the inducer-free medium and about 20 times compared with that in the single copper-supplemented medium was observed. Compared with other species, S. bambusicola GZ11K2 exhibits better laccase-producing characteristics with an activity of 16,400 U/L after 108 h, suggesting its potential ability for industrial application.     

Enhanced Lignin Biodegradation by a Laccase-Overexpressed White-Rot Fungus Polyporus brumalis in the Pretreatment of Wood Chips

The laccase gene of Polyporus brumalis was genetically transformed to overexpress its laccase. The transformants exhibited increased laccase activity and effective decolorization of the dye Remazol Brilliant Blue R than the wild type. When the transformants were pretreated with wood chips from a red pine (softwood) and a tulip tree (hardwood) for 15 and 45 days, they showed higher lignin-degradation activity as well as higher wood-chip weight loss than the wild type. When the wood chips treated with the transformant were enzymatically saccharified, the highest sugar yields were found to be 32.5 % for the red pine wood and 29.5 % for the tulip tree wood, on the basis of the dried wood weights, which were 1.6-folds higher than those for the wild type. These results suggested that overexpression of the laccase gene from P. brumalis significantly contributed to the pretreatment of lignocellulose for increasing sugar yields.     

Molecular Cloning and Heterologous Expression of Laccase from Aeromonas hydrophila NIU01 in Escherichia coli with Parameters Optimization in Production

Prior studies disclosed that Aeromonas hydrophila NIU01 was a biodecolorization and bioelectricity bacterium which was isolated from a cross-strait of Taiwan. However, enzymatic function, laccase, involved in this strain had never been reported. This first attempt is to explore its laccase activity, the molecular cloning and heterologous recombinant expression in Escherichia coli. A full-length novel gene of 1,647 bp, LacA, encoding of 549 amino acids was successfully cloned by polymerase chain reaction. The recombinant pET-15b(+)-NIU-LacA expression was compared in different E. coli strains. By applying Taguchi’s L9 in culture optimization, the soluble laccase increased to 22.7 %, in which the conditions were obtained at 22 °C with initial shaking speed at 200 rpm, addition of lactose of 0.2 mM and CuSO₄ of 0.5 mM to the medium, and shaking off while cell mass reached to OD_600nm of 1.5. NIU-LacA was strongly inhibited by chloride ion. The optimal temperature was 60 °C and the optimum pH for ABTS (2,2′-azino-bis (3-ethylbenzthiazolinesulfonic acid) and 2,6-DMP (2,6-dimethoxyphenol) were pH 2.1 and pH 7.5 which enzymatic activity was 274.6 and 44.8 U/L, respectively. Further study in structural modeling of NIU-LacA showed the C terminal domain was the major variance in the three most closely A. hydrophila strains.     

Purification, Characterization, and Heterologous Expression of a Thermostable β-1,3-1,4-Glucanase from Bacillus altitudinis YC-9

Purification, characterization, gene cloning, and heterologous expression in Escherichia coli of a thermostable β-1,3-1,4-glucanase from Bacillus altitudinis YC-9 have been investigated in this paper. The donor strain B. altitudinis YC-9 was isolated from spring silt. The native enzyme was purified by ammonium sulfate precipitation, diethylaminoethyl-cellulose anion exchange chromatography, and Sephadex G-100 gel filtration. The purified β-1,3-1,4-glucanase was observed to be stable at 60 °C and retain more than 90 % activity when incubated for 2 h at 60 °C and remain about 75 % and 44 % activity after incubating at 70 °C and 80 °C for 10 min, respectively. Acidity and temperature optimal for this enzyme was pH 6 and 65 °C. The open reading frame of the enzyme gene was measured to be 732 bp encoding 243 amino acids, with a predicted molecular weight of 27.47 kDa. The gene sequence of β-1,3-1,4-glucanase showed a homology of 98 % with that of Bacillus licheniformis. After being expressed in E. coli BL21, active recombinant enzyme was detected both in the supernatants of the culture and the cell lysate, with the activity of 102.7 and 216.7 U/mL, respectively. The supernatants of the culture were used to purify the recombinant enzyme. The purified recombinant enzyme was characterized to show almost the same properties to the wild enzyme, except that the specific activity of the recombinant enzyme reached 5392.7 U/mg, which was higher than those ever reported β-1,3-1,4-glucanase from Bacillus strains. The thermal stability and high activity make this enzyme broad prospect for industry application. This is the first report on β-1,3-1,4-glucanase produced by B. altitudinis.     

Production and Characterization of Extracellular α-Amylase Produced by Wickerhamia sp. X-Fep

A yeast isolate able to produce high levels of extracellular ??-amylase was selected from a collection of 385 yeasts and identified as Wickerhamia sp. by the sequence of the D1/D2 domain of the 26?S rDNA gene. Part of the nucleotide sequence of the amy1-W gene was cloned, and a sequence of 191 amino acids deduced from this gene was analyzed. The peptide contains three characteristic well-conserved regions in the active sites of ??-amylases (EC 3.2.1.1). The enzyme was purified and in situ activity showed only one band with amylolytic activity. The molecular mass of the ??-amylase was estimated at 54?kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Enzymatic activity on soluble starch as substrate was optimal at pH 5?C6 and 50 °C. This thermostable enzyme was inhibited by EDTA?CNa₂ and 1,10-phenanthroline; the activity of the dialyzed enzyme was reactivated with Ca²⁺ and Mg²⁺ cations, which indicates that the ??-amylase is a metalloenzyme. ??-Amylase production was induced by starch and maltose and repressed by glucose. The high yield and productivity found in this work makes this Wickerhamia sp. strain a promising candidate for the biotechnological production of ??-amylase.     

Thermostable and Alkalistable Endoxylanase of the Extremely Thermophilic Bacterium Geobacillus thermodenitrificans TSAA1: Cloning, Expression, Characteristics and Its Applicability in Generating Xylooligosaccharides and Fermentable Sugars

Xylanase encoding gene (1,224 bp) from Geobacillus thermodenitrificans was cloned in pET28a (+) vector and successfully expressed in Escherichia coli BL21 (DE3). The deduced amino acid sequence analysis revealed homology with that of glycosyl hydrolase (GH) 10 family with a high molecular mass (50 kDa). The purified recombinant xylanase is optimally active at pH 9.0 and 70 °C with T _1/2 of 10 min at 80 °C, and retains greater than 85 % activity after exposure to 70 °C for 180 min. The enzyme liberates xylose as well as xylooligosaccharides from birchwood xylan and agro-residues, and therefore, this is an endoxylanase. The xylan hydrolytic products (xylooligosaccharides, xylose, and xylobiose) find application as prebiotics and in the production of bioethanol. The xylanase being thermostable and alkalistable, it has released chromophores and phenolics from the residual lignin of pulps, suggesting its utility in mitigating chlorine requirement in pulp bleaching.     

A Recombinant Highly Thermostable β-Mannanase (ReTMan26) from Thermophilic <Emphasis Type="Italic">Bacillus subtilis</Emphasis> (TBS2) Expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and Its pH and Temperature Stability

A gene encoding a highly thermostable β-mannanase from a thermophilic Bacillus subtilis (TBS2) was successfully expressed in Pichia pastoris. The maximum activity of the recombinant thermostable β-mannanase (ReTMan26) was 5435 U/mL, which was obtained by high-density, fed-batch cultivation after 168-h induction with methanol in a 50-L bioreactor. The protein yield reached 3.29 mg/mL, and the protein had a molecular weight of ~42 kDa. After fermentation, ReTMan26 was purified using a 10-kDa cut-off membrane and Sephadex G-75 column. The pH and temperature optima of purified ReTMan26 were pH 6.0 and 60 °C, respectively, and the enzyme was stable at pH 2.0–8.0 and was active at 20–100 °C. HPLC analysis of the products of locust bean gum hydrolysis showed that the mannan-oligosaccharide content was 62.5%. ReTMan26 retained 58.6% of its maximum activity after treatment at 100 °C for 10 min, which was higher than any other β-mannanase reported up to now, suggesting its potential for industrial applications.     

Gene Cloning and Characterization of a Novel Salt-Tolerant and Glucose-Enhanced β-Glucosidase from a Marine Streptomycete

The gene BglNH encoding a β-glucosidase was cloned from a marine streptomycete. Sequence analysis revealed that BglNH encoded a 456-aa peptide with a calculated mass of 51 kDa. The deduced amino acid sequence of BglNH showed the highest identities of 61 % with known β-glucosidases and contained a catalytic domain which belonged to the glycoside hydrolase family 1. The gene BglNH was expressed in Escherichia coli and the recombinant enzyme (r-BglNH) was purified. The optimum pH and temperature of r-BglNH were pH?6.0 and 45 °C, respectively. The r-BglNH displayed the typical salt-tolerant and glucose-enhanced characteristics. Its activity was remarkably enhanced in the presence of 0.5 M NaCl (rose more than 1.6-fold) and 0.1 M glucose (rose more than 1.4-fold). Moreover, r-BglNH displayed good pH stability and metal tolerance. It remained stable after incubating with buffers from pH?4.0 to 10.0, and most metal ions had no significant inhibition on its activity. These properties indicate that r-BglNH is an ideal candidate for further research and industrial applications.     

Isolation,purification, and characterization of thermophilic laccase from the xerophyte <Emphasis Type="Italic">Cereus pterogonus</Emphasis>

Three laccase temperature isoforms were isolated and purified to homogeneity from the xerophyte plant species Cereus pterogonus. This catalytically active protein exhibited an apparent molecular mass of 137 kDa, 90 kDa, and 43 kDa. Under reducing conditions the enzyme yielded a subunit molecular mass of 43 kDa alone, suggesting that the enzyme is a multimer of its subunits. The enzyme exhibited an optimum pH of 10 with 2,6-dimethoxyphenol used as a substrate. The 137 and 90 kDa forms yielded optimum activity at 90°C; whereas the 43 kDa molecular form yielded optimum activity at 60°C. The enzyme kinetic constant Km remained closely similar for all three enzyme forms, whereas Vmax varied by 25 % overall. The catalytic activity remained above its t_1/2 value in excess of the 30 min denaturation assay period at 60°C and 90°C. These high-temperature isoforms of the plant laccase enzyme with alkaline pH optima can find great industrial use.     

Screening and Production of Ligninolytic Enzyme by a Marine-Derived Fungal Pestalotiopsis sp. J63

Marine-derived fungi are prone to produce structurally unique secondary metabolites, a considerable number of which display the promising biological properties and/or industrial applications. Among those, ligninolytic enzymes have attracted great interest in recent years. In this work, about 20 strains were isolated from sea mud samples collected in the East China Sea and then screened for their capacity to produce lignin-degrading enzymes. The results showed that a strain, named J63, had a great potential to secrete a considerable amount of laccase. Using molecular method, it was identified as an endophytic fungus, Pestalotiopsis sp. which was rarely reported as ligninolytic enzyme producer in the literature. The production of laccase by Pestalotiopsis sp. J63 was investigated under submerged fermentation (SF) and solid state fermentation (SSF) with various lignocellulosic by-products as substrates. The SSF of rice straw powder accumulated the highest level of laccase activity (10,700 IU/g substrate), whereas the SF of untreated sugarcane bagasse provided the maximum amount of laccase activity (2,000 IU/ml). The value was far higher than those reported by other reports. In addition, it produced 0.11 U/ml cellulase when alkaline-pretreated sugarcane bagasse was used as growth substrate under SF. Meanwhile, the growth of fungi and laccase production under different salinity conditions were also studied. It appeared to be a moderately halo-tolerant organism.     

Cloning,Expression, and Characterization of a Milk-Clotting Aspartic Protease Gene (Po-Asp) from Pleurotus ostreatus

An aspartic protease gene from Pleurotus ostreatus (Po-Asp) had been cloned based on the 3′ portion of cDNA in our previous work. The Po-Asp cDNA contained 1,324 nucleotides with an open reading frame (ORF) of 1,212 bp encoding 403 amino acid residues. The putative amino acid sequence included a signal peptide, an activation peptide, two most possible N-glycosylation sites and two conserved catalytic active site. The mature polypeptide with 327 amino acid residues had a calculated molecular mass of 35.3 kDa and a theoretical isoelectric point of 4.57. Basic Local Alignment Search Tool analysis showed 68–80 % amino acid sequence identical to other basidiomycetous aspartic proteases. Sequence comparison and evolutionary analysis revealed that Po-Asp is a member of fungal aspartic protease family. The DNA sequence of Po-Asp is 1,525 bp in length without untranslated region, consisting of seven exons and six introns. The Po-Asp cDNA without signal sequence was expressed in Pichia pastoris and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the molecular mass of recombinant Po-Asp was about 43 kDa. The crude recombinant aspartic protease had milk-clotting activity.     

Characterization of a New 1,3-1,4-β-Glucanase Gene from Bacillus tequilensis CGX5-1

1,3-1,4-β-Glucanase received great interest due to its application in brewing and feed industries. Application of 1,3-1,4-β-glucanase in brewing industry helps make up for the defect that plant-derived β-glucanases are heat-sensitive. A new strain, CGX5-1, exhibited remarkable 1,3-1,4-β-glucanase, was isolated from Asian giant hornet nest and identified Bacillus tequilensis. Moreover, a new 1,3-1,4-β-glucanase gene from B. tequilensis was cloned and measured to be 720 bp encoding 239 amino acids, with a predicted molecular weight of 26.9 kDa. After expressed in Escherichia coli BL21, active recombinant enzyme of 24 kDa was detected in the supernatant of cell culture, with the activity of 2,978.2 U/mL. The new enzyme was stable in the pH 5.0–7.5 with the highest activity measured at pH 6.0. Moreover, it is thermostable within 45 to 60 °C. The property of the new recombinant enzyme makes this enzyme a broad prospect in brewing industry. Moreover, this is the first report on 1,3-1,4-β-glucanase produced by B. tequilensis.     

Cotton Stalk Pretreatment Using <Emphasis Type="Italic">Daedalea flavida</Emphasis>, <Emphasis Type="Italic">Phlebia radiata</Emphasis>, and <Emphasis Type="Italic">Flavodon flavus</Emphasis>: Lignin Degradation,Cellulose Recovery,and Enzymatic Saccharification

Lignocellulolytic enzyme activities of selective fungi Daedalea flavida MTCC 145 (DF-2), Phlebia radiata MTCC 2791 (PR), and non-selective fungus Flavodon flavus MTCC 168 (FF) were studied for pretreatment of cotton stalks. Simultaneous productions of high LiP and laccase activities by DF-2 during early phase of growth were effective for lignin degradation 27.83 ± 1.25 % (w/w of lignin) in 20-day pretreatment. Production of high MnP activity without laccase in the early growth phase of PR was ineffective and delayed lignin degradation 24.93 ± 1.53 % in 25 days due to laccase production at later phase. With no LiP activity, low activities of MnP and laccase by FF yielded poor lignin degradation 15.09 ± 0.6 % in 20 days. Xylanase was predominant cellulolytic enzyme produced by DF-2, resulting hemicellulose as main carbon and energy source with 83 % of cellulose recovery after 40 days of pretreatment. The glucose yield improved more than two fold from 20-day DF-2 pretreated cotton stalks after enzymatic saccharification.     

Gene Cloning, Expression, and Characterization of a Thermostable Xylanase from Nesterenkonia xinjiangensis CCTCC AA001025

An endo-β-1,4-xylanase-encoding gene, xyn11NX, was cloned from Nesterenkonia xinjiangensis CCTCC AA001025 and expressed in Escherichia coli. The gene encoded a 192-amino acid polypeptide and a putative 50-amino acid signal peptide. The deduced amino acid sequence exhibited a high degree of similarity with the xylanases from Streptomyces thermocyaneoviolaceus (68%) and Thermobifida fusca (66%) belonging to glycoside hydrolase family 11. After purification to homogeneity, the recombinant Xyn11NX exhibited optimal activity at pH 7.0 and 55 °C and remained stable at weakly acidic to alkaline pH (pH 5.0–11.0). The enzyme was thermostable, retaining more than 80% of the initial activity after incubation at 60 °C for 1 h and more than 40% of the activity at 90 °C for 15 min. The K _m and V _max values for oat spelt xylan and birchwood xylan were 16.08 mg ml^?1 and 45.66 μmol min^?1 mg^?1 and 9.22 mg ml^?1 and 16.05 μmol min^?1 mg^?1, respectively. The predominant hydrolysis products were xylobiose and xylotriose when using oat spelt xylan or birchwood xylan as substrate.     

Production of Extremely Alkaliphilic, Halotolerent, Detergent, and Thermostable Mannanase by the Free and Immobilized Cells of Bacillus halodurans PPKS-2. Purification and Characterization

The alkaliphilic Bacillus halodurans strain PPKS-2 was shown to produce extracellular extreme alkaliphilic, halotolerent, detergent, and thermostable mannanase activity. The cultural conditions for the maximum enzyme production were optimized with respect to pH, temperature, NaCl, and inexpensive agro wastes as substrates. Mannanase production was enhanced more than 4-fold in the presence of 1 % defatted copra meal and 0.5 % peptone or feather hydrolysate at pH 11 and 40 °C. Mannanase was purified to 10.3-fold with 34.6 % yield by ion exchange and gel filtration chromatography methods. Its molecular mass was estimated to be 22 kDa by SDS-PAGE. The mannanase had maximal activity at pH 11 and 70 °C. This enzyme was active over a broad range of NaCl (0–16 %) and thermostable retaining 100 % of the original activity at 70 °C for 3 h. Immobilization of whole cells proved to be effective for continuous production of mannanase. Since the strain PPKS-2 grows on cheaper agro wastes such as defatted copra meal, corn husk, jowar bagasse, and wheat bran, these can be exploited for mannanase production on an industrial scale.