High salt-tolerant protease from a potential biocontrol agent<Emphasis Type="Italic">Bacillus pumilus</Emphasis> M3-16

In this paper, we investigate the characterization and evaluation of the antifungal protease activity from a halotolerant strain M3-16 ofBacillus pumilus, earlier isolated from a shallow salt lake in Tunisia. Protease enzyme was highly induced by the pathogen testedin vitro (27.4 U/ml). This is the first report on high salt-tolerant protease fromB. Pumilus, since it was active at high salinity (from 5 to 30% NaCl, w/v) as well as in the absence of salinity. This enzyme showed optimal activity at 60 °C and pH 8. At 80 °C and 30 min, the enzyme retained up to 91% and it showed stability over a wide pH range (from pH 5 to 11). The enzyme was found to be monomer with an estimated molecular mass of 31 kDa. The amino acid sequence showed high similarity (94%) to ATP-dependent protease fromB. Pumilus strain ATCC 7061. Thus, our alkaline thermostable and high salt-tolerant protease induced by a phytopathogenic fungus, could be useful for application in diverse areas such as biotechnology alimentary and agronomy industries.     

Possible correlation between levansucrase production and probiotic activity of <Emphasis Type="Italic">Bacillus</Emphasis> sp. isolated from honey and honey bee

Five bacterial isolates from honey and bee gut were selected based on their high levansucrase activity and levan yield which were strongly positively correlated. All isolates showed good tolerance to temperature up to 70?°C, to NaCl up to 3 M and to 0.1% H₂O₂. They maintained over 59 and 64% survival at pH 9.0 and 2.0 respectively, but showed varying tolerance to 0.1% bile salts and pancreatic enzymes. Most isolates were susceptible to widely used antibiotics, but demonstrated diverse antimicrobial activity. Non hemolytic isolates were identified on the basis of 16S rRNA sequencing as Bacillus subtilis HMNig-2 and B. subtilis MENO2 with 97% homology. They exhibited promising probiotic characteristics and achieved highest levansucrase activity of 94.1 and 81.5 U/mL respectively. Both exhibited highest biofilm formation ability in static microtiter plate assay. Also, they achieved 34 and 26% adhesion respectively to Caco-2cells and had highest free radical scavenging activity of 30.8 and 26.2% respectively. The levans of the two isolates showed good antimicrobial activity against some pathogens and exhibited positive prebiotic effect (prebiotic index >1) with Lactobacillus casei and Lactobacillus reuteri. Results suggest a correlation between levansucrase production, levan yield and pre-probiotic activities of the studied strains.     

Purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, <Emphasis Type="Italic">Bacillus iranensis (</Emphasis>X5B)

This study reports the purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, Bacillus iranensis, strain X5B. The enzyme was purified to homogeneity by acetone precipitation, ultrafiltration and carboxymethyl (CM) cation exchange chromatography, respectively. The purified protease was a monomeric enzyme with a relative molecular mass of 48–50 kDa and it was inhibited by PMSF indicating that it is a serine-protease. The optimum pH, temperature and NaCl concentration were 9.5, 35 °C and 0.98 M, respectively. The enzyme showed a significant tolerance to salt and alkaline pH. It retained approximately 50 % of activity at 2.5 M NaCl and about 70 % of activity at highly alkaline pH of 11.0; therefore, it was a moderately halophilic and also can be activated by metals, especially by Ca²⁺. The specific activity of the purified protease was measured to be 425.23 μmol of tyrosine/min per mg of protein using casein as a substrate. The apparent K _m and V _max values were 0.126 mM and 0.523 mM/min, respectively and the accurate value of k _cat was obtained as 3.284 × 10^?2 s^?1. These special and important characteristics make this serine protease as valuable tool for industrial applications.     

Isolation of hydroquinone (benzene-1,4-diol) metabolite from halotolerant <Emphasis Type="Italic">Bacillus methylotrophicus</Emphasis> MHC10 and its inhibitory activity towards bacterial pathogens

A halotolerant bacterial isolate-MHC10 with broad spectrum antibacterial activity against clinical pathogens was isolated from saltpans located in Tuticorin and Chennai (India). 16S rRNA gene analysis of MHC10 revealed close similarity to that of Bacillus methylotrophicus. The culture conditions of B. methylotrophicus MHC10 strain were optimized for antibacterial production using different carbon and nitrogen sources, as well as varying temperature, pH, sodium chloride (NaCl) concentrations and incubation periods. The maximum antibacterial activity of B. methylotrophicus MHC10 was attained when ZMB was optimized with 1 % (w/v) glucose, 0.1 % (w/v) soybean meal which corresponded to a C/N ratio of 38.83, temperature at 37 °C, pH 7.0 and 8 % NaCl. The activity remained stable between 72 and 96 h and then drastically decreased after 96 h. Solvent extraction followed by chromatographic purification steps led to the isolation of hydroquinone (benzene-1,4-diol). The structure of the purified compound was elucidated based on FTIR, ¹H NMR, and ¹³C NMR spectroscopy. The compound exhibited efficient antibacterial activity against both Gram-positive and Gram-negative bacterial pathogens. The minimum inhibitory concentration (MIC) for Gram-positive pathogens ranged from 15.625 to 62.5 µg/mL^?1, while it was between 7.81 and 250 µg/mL^?1 for Gram-negative bacterial pathogens. This is the first report of hydroquinone produced by halotolerant B. methylotrophicus exhibiting promising antibacterial activity.     

Extraction,purification and characterization of a novel cysteine protease from the latex of plant <Emphasis Type="Italic">Vallaris solanacea</Emphasis>

Plant proteases with excellent catalytical properties perform many functions in biological systems. A novel plant protease Vallaris solanacea, was identified. Its proteolytic activity was screened using the substrate casein. This protein activity was specifically inhibited by p-chloromercuribenzoate, which showed that it is a cysteine protease. Preliminary investigations such as pH effect and temperature dependence on the caseinolytic activity of crude protease were done. Stability towards temperature and pH were also evaluated. The activity curves drawn in relation to pH, temperature and stability suggested the presence of one protease in the latex of Vallaris solanacea. In the present study, separation and purification of the latex cysteine protease solanain from Vallaris solanacea to a state of near homogeneity was also done using ion exchange and size exclusion chromatography. SDS PAGE was used to determine molecular weight of the solanain (28–29 kDa). The molecular weight was confirmed as 28.9 kDa using MALDI-TOF. Purified protease was named solanain and it was further characterized. An internal tryptic fragment was identified by MALDI-TOF, and this peptide showed a homology (66% sequence similarity) with target sequence of cysteine endopeptidase from Ricinus communis.     

Isolation and characterization of a serine protease-producing marine bacterium <Emphasis Type="Italic">Marinomonas arctica</Emphasis> PT-1

A serine protease-producing marine bacterial strain named as PT-1 was isolated and identified as a family of Marinomonas arctica, based on molecular characterization of 16S rRNA gene sequence, phylogenetic tree, and fatty acid composition analyses. Optimized culture conditions for growth of the bacterium PT-1 and production of protease (ProA) were determined to be pH 8.0 in the presence of 5 % NaCl, at 37 °C during 24 h of incubation in the presence of 1.0 % skim milk. The molecular weight of the purified ProA was estimated to be 63-kDa as a major band by SDS-PAGE. We were intrigued to find that the activity of ProA was not inhibited by pepstatin A, chymostatin, and leupeptin known as inhibitors for cysteine protease. However, phenylmethylsulfonyl fluoride (PMSF) completely inhibited protease activity, suggesting that the ProA is like a serine protease. To the best of our knowledge, this is the first report on serine protease of Marinomonas species.     

Optimization of Microbial Flocculant-Producing Medium for <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

This study aimed to improve microbial flocculant production by optimizing the components of a Bacillus subtilis CZ1003 culture medium. Using the flocculation rate as the dependent variable, single-factor experiments were performed and beef extract at a concentration of 9 g/L was found to be the optimal nitrogen source, while glucose at a concentration of 20 g/L was the optimal carbon source. KCl, MgCl₂, NaCl, and CaCl₂ at concentrations of 0.75, 2.5, 0.5, and 5.0 g/L, respectively, were the optimum inorganic salts, in order of flocculant production activity. Orthogonal experimental demonstrated that KCl played a dominant role for Bacillus subtilis production of bioflocculants, followed by NaCl and CaCl₂. Optimization experiments demonstrated that the optimal combination of the two salts was 0.75 g/L KCl and 0.5 g/L NaCl, resulting in a flocculation rate of 36.2% when included together at these concentrations. The final optimized medium consisted of 20 g/L glucose, 9 g/L beef extract, 0.75 g/L KCl, and 0.5 g/L NaCl. Compared with the initial medium, the optimized medium enhanced the flocculation activity from 12.1 to 36.2%, which equates to an increase of 199.17%. Meanwhile, the flocculant yield was increased from 0.058 g/L to 0.134/L, an increase of 131.03%. The optimized medium could be used to improve microbial flocculant production and provides a basis for further exploration.     

Screening and genetic identification of acidic and neutral protease-producing yeasts strains by 26S rRNA gene sequencing

Protease enzymes (proteases), particularly those produced by microorganisms, play very important roles in industry, due to their diverse applications. Considering the richness of microbial diversity in nature, a good chance always exists that proteases more suitable, with better properties for commercial application, may be discovered while screening novel microorganisms from local environments. In this study, 94 yeasts were isolated from different natural sources collected from the Abha region, Kingdom of Saudi Arabia, to determine extracellular protease production and activity. Among them, 23 isolates (24.46%) showed protease activity using a casein hydrolysis test. Of these, five isolates (21.74%) were selected and identified as the best protease producers by exhibiting the largest clearance zones around colonies. A 26S rRNA gene D1/D2 domain sequence alignment, comparison, and phylogenetic analysis of our study yeasts to published D1/D2 domain rRNA gene sequences from GenBank, identifies the isolates as Rhodotorula mucilaginosa KKU-M12c, Cryptococcus albidus KKU-M13c, Pichia membranifaciens KKU-M18c, Hanseniaspora uvarum KKU-M19c, and Candida californica KKU-M20c. The influence of varying pH (4.0–9.0) on the yield and activity of the proteases was investigated using 0.5% (w/v) casein as a substrate, to detect optimum pH values for yeast extracellular protease production. Enzyme activity was measured using qualitative and quantitative assays. Results show all of the study yeasts secreting protease enzyme at all tested pH levels, with the exception of pH 9.0. This indicates that none of the five yeasts are alkaline protease producers. Maximum protease activity (187 U/mL) was observed in strain H. uvarum KKU-M19c at pH 6.0 (only), indicating that strain KKU-M19c only produces neutral protease. The other four yeast isolates, R. mucilaginosa KKU-M12c, C. albidus KKU-M13c, P. membranifaciens KKU-M18c, and C. californica KKU-M20c, produced both acidic (at pH 4.0) and neutral (at pH 6.0 and 7.0) proteases. Strain C. californica KKU-M20c was found to be the best acidic and neutral protease producer (138 U/mL at pH 4.0, and 185 U/mL at pH 7.0). This is the first report of the discovery and isolation of local, powerful yeasts producing acidic and neutral protease enzymes from the Abha region, Kingdom of Saudi Arabia.     

Heterologous expression and characterization of a new heme-catalase in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> 168

Reactive oxygen species (ROS) is an inherent consequence to all aerobically living organisms that might lead to the cells being lethal and susceptible to oxidative stress. Bacillus pumilus is characterized by high-resistance oxidative stress that stimulated our interest to investigate the heterologous expression and characterization of heme-catalase as potential biocatalyst. Results indicated that recombinant enzyme significantly exhibited the high catalytic activity of 55,784 U/mg expressed in Bacillus subtilis 168 and 98.097 µmol/min/mg peroxidatic activity, the apparent K _m of catalytic activity was 59.6 ± 13 mM with higher turnover rate (K _cat = 322.651 × 10³ s^?1). The pH dependence of catalatic and peroxidatic activity was pH 7.0 and pH 4.5 respectively with temperature dependence of 40 °C and the recombinant heme-catalase exhibited a strong Fe²⁺ preference. It was further revealed that catalase KatX2 improved the resistance oxidative stress of B. subtilis. These findings suggest that this B. pumilus heme-catalase can be considered among the industrially relevant biocatalysts due to its exceptional catalytic rate and high stability and it can be a potential candidate for the improvement of oxidative resistance of industrially produced strains.     

The purification and characterization of a novel alkali-stable pectate lyase produced by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> PB1

Pectinase is an important kind of enzyme with many industrial applications, among which pectinases produced by bacteria were scarce compared with fungal sources. In this study, a novel bacterium which produced extracellular pectinase was firstly isolated from flue-cured tobacco leaves and identified as Bacillus subtilis PB1 according to its 16S rRNA gene. The pectinolytic enzyme was purified by ammonium sulfate precipitation, ion-exchange and gel filtration chromatography, after which molecular weight was determined as 43.1?±?0.5 kDa by SDS–PAGE. Peptide mass fingerprinting of the pectinase by MALDI-TOF MS showed that the purified enzyme shared homology with pectate lyase and was designated as BsPel-PB1. The optimal temperature for BsPel-PB1 was 50 °C. The optimal pH was pH 9.5 for BsPel-PB1 while it had a broad pH stability from 5 to 11. The values of K _m and V _max were 0.312 mg/mL and 1248 U/mL, respectively. Accordingly, the BsPel-PB1 was a novel alkaline pectate lyase which could find potential application as a commercial candidate in the pectinolytic related industries.     

A new <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> mutant strain producing serine protease efficient for hydrolysis of soy meal proteins

Induced mutagenesis with γ-irradiation of the industrial strain Bacillus licheniformis-60 VKM B-2366 D was used to obtain a new highly active producer of an extracellular serine protease, Bacillus licheniformis-145. Samples of dry concentrated preparations of serine protease produced by the original and mutant strains were obtained, and identity of their protein composition was established. Alkaline serine protease subtilisin DY was the main component of the preparations. The biochemical and physicochemical properties of the Protolicheterm-145 enzyme preparation obtained from the mutant strain were studied. It exhibited proteolytic activity (1.5 times higher than the preparation from the initial strain) within broad ranges of pH (5–11) and temperature (30–70°C). Efficient hydrolysis of extruded soybean meal protein at high concentrations (20 to 50%) in the reaction mixture was the main advantage of the Protolicheterm-145 preparation. Compared to the preparation obtained using the initial strain, the new preparation with increased proteolytic activity provided for more complete hydrolysis of the main non-nutritious anti-nutritional soy proteins (glycinin and β-conglycinin) with the yield of soluble protein increased by 19–28%, which decreased the cost of bioconversion of the proteinaceous material and indicated promise of the new preparation in resource-saving technologies for processing soybean meals and cakes.     

Heterologous expression and characterization of a novel halotolerant,thermostable, and alkali-stable GH6 endoglucanase from <Emphasis Type="Italic">Thermobifida halotolerans</Emphasis>

A novel endoglucanase gene was cloned from Thermobifida halotolerans YIM 90462^T, designated as thcel6A for being a member of glycoside hydrolase family 6. The gene was 1332 bp long and encoded a 443-amino-acid protein with a molecular mass of 45.9 kDa. The purified recombinant endoglucanase had optimal activity at 55 °C and pH 8.5. Thcel6A showed high hydrolytic activities at 25–55 °C and retained 58 % of initial activity after incubation at 90 °C for 1 h. It retained more than 80 % of activity after incubation for 12 h at pH values from 4 to 12. Thcel6A displayed higher hydrolytic activities in 5–15 % NaCl (w/v) than at 0 % NaCl. Activity increased 2.5-fold after incubation with 20 % (w/v) NaCl at 37 °C for 10 min. These properties suggest that this novel endoglucanase has potential for specific industrial application.     

Optimization of culture conditions for the production of halothermophilic protease from halophilic bacterium <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TVSP101

An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease. Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl₂ concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease.     

Gene cloning and characterization of recombinant L-Asparaginase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain R5

L-asparaginase gene from Bacillus subtilis strain R5 (Asn-R5), comprising 990 nucleotides corresponding to a polypeptide of 329 amino acids, was cloned and expressed in Escherichia coli. Recombinant Asn-R5 was produced in soluble and active form exhibiting a specific activity of 223 μmol min^?1 mg^?1. The optimal temperature and pH for L-asparaginase activity of Asn-R5 were 35 °C and 9.0, respectively. Asn-R5 displayed a 50% activity with D-asparagine and 2% with L-glutamine compared to 100% with L-asparagine. No activity could be detected when D-glutamine was used as substrate. Half-life of the enzyme was 180 min at 35 °C and 40 min at 50 °C. There was no effect of metal ions and EDTA on the activity indicating that Asn-R5 enzyme activity is not metal ion dependent. The K_m and V_max values were 2.4 mM and 265 μmol min^?1 mg^?1, respectively. Activation energy for reaction catalyzed by Asn-R5 was 28 kJ mol^?1. High L-asparaginase activity and thermostability of recombinant Asn-R5 may be beneficial for industrial production and application.     

Surface Display of Bacterial Laccase CotA on <Emphasis Type="Italic">Escherichia coli</Emphasis> Cells and its Application in Industrial Dye Decolorization

Laccase CotA from Bacillus subtilis 168 was successfully displayed on the membrane of Escherichia coli cells using poly-γ-glutamate synthetase A protein (PgsA) from B. subtilis as an anchoring matrix. Further analyses demonstrated that the fusion protein PgsA/CotA efficiently translocates to the cell surface of E. coli with an enzymatic activity of 65 U/10⁸ cells. Surface-displayed CotA was shown to possess improved enzymatic properties compared with those of the wild-type CotA, including higher thermal stability (above 90% activity at 70 °C and nearly 40% activity at 90 °C after 5-h incubation) and stronger inhibitor tolerance (approximately 80 and 65% activity when incubated with 200 and 400 mM NaCl, respectively). Furthermore, the whole-cell system was demonstrated to have high enzymatic activity against anthraquinone dye, Acid Blue 62, triphenylmethane dye, Malachite Green, and azo dye, Methyl Orange with the decolorization percentages of 91, 45, and 75%, after 5-h incubation, respectively.     

Structural Changes in Halophilic and Non-halophilic Proteases in Response to Chaotropic Reagents

Halophilic enzymes have been established for their stability and catalytic abilities under harsh operational conditions. These have been documented to withstand denaturation at high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. The present study targets an important aspect in understanding protein–urea/GdmCl interactions using proteases from halophilic Bacillus sp. EMB9 and non-halophilic subtilisin (Carlsberg) from Bacillus licheniformis as model systems. While, halophilic protease containing 1 % (w/v) NaCl (0.17 M) retained full activity towards urea (8 M), non-halophilic protease lost about 90 % activity under similar conditions. The secondary and tertiary structure were lost in non-halophilic but preserved for halophilic protein. This effect could be due to the possible charge screening and shielding of the protein surface by Ca²⁺ and Na⁺ ions rendering it stable against denaturation. The dialyzed halophilic protease almost behaved like the non-halophilic counterpart. Incorporation of NaCl (up to 5 %, w/v or 0.85 M) in dialyzed EMB9 protease containing urea/GdmCl, not only helped regain of proteolytic activity but also evaded denaturing action. Deciphering the basis of this salt modulated stability amidst a denaturing milieu will provide guidelines and templates for engineering stable proteins/enzymes for biotechnological applications.     

Isolation of biosurfactant-producing <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> RS29 from oil-contaminated soil and evaluation of different nitrogen sources in biosurfactant production

An efficient biosurfactant-producing native Pseudomonas aeruginosa RS29 has been isolated from crude oil contaminated soil. Isolation was followed by optimization of different factors to achieve maximum production of biosurfactant in terms of surface tension reduction (STR) and emulsification index (E24). The isolated strain produced highest biosurfactant in the presence of glycerol after 48 h of incubation at 37.5°C, with pH range of 7–8 and at salinity <0.8% (w/v). The extent of STR and the E24 of medium with different nitrogen sources were investigated and found to be maximal for sodium nitrate (26.3 mN/m, E24?=?80%) and potassium nitrate (26.4 mN/m, E24?=?79%). The production of biomass by the designated strain was found to be maximal in ammonium-nitrate-containing medium as compared to the other nitrogen sources. A kinetic study revealed that biosurfactant production is positively correlated with growth of P. aeruginosa, and highest STR was achieved (27.0 mN/m) after 44 h of growth. The biosurfactant was produced as a primary metabolite and 6 g/L crude biosurfactant was extracted by chloroform:methanol (2:1). The critical micelle concentration of the biosurfactant was 90 mg/L. The absorption bands of the FTIR spectra confirmed the rhamnolipid nature of the biosurfactant. The biosurfactant was thermostable (up to 121°C for 15 min) and could withstand a wide range of pH (2–10) and NaCl concentration (2%–10% w/v). The extracted biosurfactant had good foaming and emulsifying activities and was of satisfactory quality in terms of stability (temperature, pH and salinity) and foaming activity.     

Properties of an ionic liquid-tolerant <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">amyloliquefaciens</Emphasis> CMW1 and its extracellular protease

An ionic liquid-tolerant bacterium, Bacillus amyloliquefaciens CMW1, was isolated from a Japanese fermented soybean paste. Strain CMW1 grew in the presence of 10 % (v/v) 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), a commonly used ionic liquid. Additionally, strain CMW1 grew adequately in the presence of the hydrophilic ionic liquids 10 % (v/v) 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([EMIM]CF₃SO₃) or 2.5 % (v/v) 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([BMIM]CF₃SO₃). Strain CMW1 produced an extracellular protease (BapIL) in the culture medium. BapIL was stable in the presence of 80 % (v/v) ionic liquids, [EMIM]CF₃SO₃, [BMIM]Cl, [BMIM]CF₃SO₃, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, and 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, and functioned in 10 % (v/v) these ionic liquids. BapIL was stable at pH 4.0–12.6 or in 4004 mM NaCl solution, and exhibited activity in the presence of 50 % (v/v) hydrophilic or hydrophobic organic solvents. BapIL was completely inhibited by 1 mM PMSF and partially by 5 mM EDTA. BapIL belongs to the true subtilisins according to analysis of the deduced amino acid sequence. We showed that BapIL from the ionic liquid-tolerant B. amyloliquefaciens CMW1 exhibited tolerance to ionic liquid and halo, alkaline, and organic solvents.     

Surface display of lipolytic enzyme,Lipase A and Lipase B of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> on the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> spore

For the enhancement of lipase stability in organic solvent containing reaction, live immobilization method, using Bacillus subtilis spore as a display vehicle was attempted. Bacillus subtilis coat protein cotE was used as an anchoring motif for the display of lipA and lipB of Bacillus subtilis. Using this motif, lipolytic enzyme Lipase A and Lipase B were functionally displayed on the surface of Bacillus subtilis spore. Purified spore displaying CotE-LipB fusion protein showed higher lipolytic activity compared to that of CotE-LipA fusion protein. The surface localization of Lipase B was verified with flow cytometry and protease accessibility experiment. Spore displayed lipase retained its activity against acetone and benzene which completely deactivated free soluble lipase in the same reaction condition.     

Production of pectinolytic enzymes pectinase and pectin lyase by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> SAV-21 in solid state fermentation

Pectin-degrading enzymes (pectinase and pectin lyase) were produced in solid state fermentation by Bacillus subtilis SAV-21 isolated from fruit and vegetable market waste soil of Yamuna Nagar, Haryana, India, and identified by 16S rDNA sequencing. Under optimized conditions, maximum production of pectinase (3315 U/gds) and pectin lyase (10.5 U/gds) was recorded in the presence of a combination of orange peel and coconut fiber (4:1), with a moisture content of 60% at 35 °C and pH 4.0 after 4 days and 8 days of incubation, respectively. Pectinase yield was enhanced upon supplementation with galactose and yeast extract, whereas pectin lyase production was unaffected by adding carbon and nitrogen source to the basal medium. Thus, B. subtilis SAV-21 can be exploited for cost-effective production of pectinase and pectin lyase using agro-residues.