Monoacylglycerol lipase from marine Geobacillus sp. showing lysophospholipase activity and its application in efficient soybean oil degumming.

Enzymatic degumming is an essential refining process to improve oil quality. In this study, a monoacylglycerol lipase GMGL was derived from marine Geobacillus sp., and was found that not only took monoacylglycerol (MAG) as substrate, but also had activity toward lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE) and glycerolphosphatidylcholine (GPC). Binding free energy showed LPC and LPE could bind with enzyme stably as MAG. It presented great potential in the field of enzymatic degumming. The phosphorus content in crude soybean oil decreased from 680.50 to 2.01 mg/kg and the yield of oil reached to 98.80 % after treating with phospholipase A1 (Lecitase Ultra) combined with lipase GMGL. An ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was developed to identify 21 differential phospholipids between crude soybean oil and enzymatic treatment. This work might shed some light on understanding the catalytic mechanism of monoacylglycerol lipase and provide an effective strategy for enzymatic degumming.

Immobilized GDEst-95, GDEst-lip and GD-95RM lipolytic enzymes for continuous flow hydrolysis and transesterification reactions.

In this study lipolytic biocatalysts GD-95RM, GDEst-95 and GDEst-lip were immobilized by encapsulation in calcium alginate beads. All three immobilized biocatalysts demonstrated significantly increased thermal stability at 60-70 °C temperatures and the activity of GD-95RM lipase increased by 50% at 70-80 °C following the immobilization. Moreover, encapsulated GDEst-95 esterase retained higher than 50% lipolytic activity after 3 months of incubation with butanol (25%) and ethanol (50%); GDEst-lip enzyme possessed 50% activity after 2 months of treatment with ethanol (25%) and methanol (25%); and GD-95RM lipase displayed higher that 50% activity after two-week incubation with methanol (50%). All three immobilized enzymes displayed long-term storage capability (>50% activity) at least until 3 months at 4 °C. It was also detected that immobilized GD-95RM and GDEst-lip can perform flow hydrolysis of both avocado oil and p-NP dodecanoate in prototype packed-bed column reactor. The analysis of continuous transesterification of avocado or sunflower oil with ethanol or methanol as substrates confirmed that encapsulated GD-95RM and GDEst-lip enzymes is a useful approach to produce fatty acid alkyl esters.

High-Yield Synthesis of Transglycosylated Mogrosides Improves the Flavor Profile of Monk Fruit Extract Sweeteners.

Luo Han Guo fruit extract (Siraitia grosvenorii), mainly composed of mogroside V (50%), could be considered a suitable alternative to free sugars; however, its commercial applications are limited by its unpleasant off-notes. In the present work, a central composite design method was employed to optimize the transglycosylation of a mogroside extract using cyclodextrin glucosyltransferases (CGTases) from three different bacteriological sources (Paenibacillus macerans, Geobacillus sp., and Thermoanaerobacter sp.) considering various experimental parameters such as maltodextrin and mogroside concentration, temperature, time of reaction, enzymatic activity, and pH. Product structures were determined by liquid chromatography coupled to a diode-array detector (LC-DAD), liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Sensory analysis of glucosylated mogrosides showed an improvement in flavor attributes relevant to licorice flavor and aftereffect. Consequently, an optimum methodology was developed to produce new modified mogrosides more suitable when formulating food products as free sugar substitutes.

Electricity from lignocellulosic substrates by thermophilic Geobacillus species.

Given our vast lignocellulosic biomass reserves and the difficulty in bioprocessing them without expensive pretreatment and fuel separation steps, the conversion of lignocellulosic biomass directly into electricity would be beneficial. Here we report the previously unexplored capabilities of thermophilic Geobacillus sp. strain WSUCF1 to generate electricity directly from such complex substrates in microbial fuel cells. This process obviates the need for exogenous enzymes and redox mediator supplements. Cyclic voltammetry and chromatography studies revealed the electrochemical signatures of riboflavin molecules that reflect mediated electron transfer capabilities of strain WSUCF1. Proteomics and genomics analysis corroborated that WSUCF1 biofilms uses type-II NADH dehydrogenase and demethylmenaquinone methyltransferase to transfer the electrons to conducting anode via the redox active pheromone lipoproteins localized at the cell membrane.

Effects of volatile fatty acids in biohydrogen effluent on biohythane production from palm oil mill effluent under thermophilic condition

Background: Biohydrogen effluent contains a high concentration of volatile fatty acid (VFA) mainly as butyric, acetic, lactic and propionic acids. The presence of various VFAs (mixture VFAs) and their cooperative effects on two-stage biohythane production need to be further studied. The effect of VFA concentrations in biohydrogen effluent of palm oil mill effluent (POME) on methane yield in methane stage of biohythane production was investigated. Results: The methane yield obtained in low VFA loading (0.9 and 1.8 g/L) was 15­20% times greater than that of high VFA loading (3.6 and 4.7 g/L). Butyric acid at high concentrations (8 g/L) has the individual significantly negative effect the methane production process (P b 0.05). Lactic, acetic and butyric acid mixed with propionic acid at a concentration higher than 0.5 g/L has an interaction significantly negative effect on the methanogenesis process (P b 0.05). Inhibition condition had a negative effect on both bacteria and archaea with inhibited on Geobacillus sp., Thermoanaerobacterium thermosaccharolyticum, Methanoculleus thermophilus and Methanothermobacter delfuvii resulting in low methane yield. Conclusion: Preventing the high concentration of butyric acid, and propionic acid in the hydrogenic effluent could enhance methane production in two-stage anaerobic digestion for biohythane production.

Draft genome sequence of pectic polysaccharide-degrading moderate thermophilic bacterium Geobacillus thermodenitrificans DSM 101594

Abstract Geobacillus thermodenitrificans DSM 101594 was isolated as a producer of extracellular thermostable pectic polysaccharide degrading enzymes. The completely sequenced genome was 3.6 Mb in length with GC content of 48.86%. A number of genes encoding enzymatic active against the high molecular weight polysaccharides of potential biotechnological importance were identified in the genome.

Draft genome sequence of pectic polysaccharide-degrading moderate thermophilic bacterium Geobacillus thermodenitrificans DSM 101594.

Geobacillus thermodenitrificans DSM 101594 was isolated as a producer of extracellular thermostable pectic polysaccharide degrading enzymes. The completely sequenced genome was 3.6Mb in length with GC content of 48.86%. A number of genes encoding enzymatic active against the high molecular weight polysaccharides of potential biotechnological importance were identified in the genome.

Isolation and characterization of a thermostable lipase from Bacillus thermoamylovorans NB501.

Two thermophilic bacterial strains, Bacillus thermoamylovorans NB501 and NB502, were isolated from a high-temperature aerobic fermentation reactor system that processes tofu refuse (okara) in the presence of used soybean oil. We cloned a lipase gene from strain NB501, which secretes a thermophilic lipase. The biochemical characteristics of the recombinant enzyme (Lip501r) were elucidated. Lip501r is monomeric in solution with an apparent molecular mass of 38 kDa on SDS-PAGE. The optimal pH and apparent optimal temperature of Lip501r were 8 and 60°C, respectively. Supplementation of 5 mM Ca2+ enhanced the thermostability, and the enzyme retained 56% of its activity for 30 min at 50°C. Lip501r was active on a wide range of substrates with different lengths of p-nitrophenyl (pNP) esters, and showed a remarkably higher activity with pNP-myristate. The Km and Vmax values for pNP-butyrate in the presence of 5 mM CaCl2 were 1.8 mM and 220 units/mg, respectively. The possible industrial use of the thermophilic lipase in modifying edible oil was explored by examining the degradation of soybean oil. A TLC analysis of the degraded products indicated that Lip501r is an 1,3-position specific lipase. A homology modeling study revealed that helix α6 in the lid domain of NB501 lipase was shorter than that of lipases from the Geobacillus group.

Complete genome sequence of the crude oil-degrading thermophilic bacterium Geobacillus sp. JS12.

Here, we report the complete genome sequence of Geobacillus sp. JS12, isolated from composts located in Namhae, Korea, which shows extracellular lipolytic activities at high temperatures. An array of genes related to the utilization of lipids was identified by whole genome analysis. The genome sequence of the strain JS12 provides basic information for wider exploitation of thermostable industrial lipases.

Characteristics of Newly Isolated Geobacillus sp. ZY-10 Degrading Hydrocarbons in Crude Oil.

An obligately thermophilic strain ZY-10 was isolated from the crude oil in a high-temperature oilfield, which was capable of degrading heavy crude oil. Phenotypic and phylogenetic analysis demonstrated that the isolate should be grouped in the genus Geobacillus, which shared thd highest similarity (99%) of the 16S rDNA sequence to Geobacillus stearothermophilus. However, the major cellular fatty acid iso-15:0 (28.55%), iso-16:0 (24.93%), iso-17:0 (23.53%) and the characteristics including indole production, tolerance to NaN3 and carbohydrate fermentation showed some difference from the recognized species in the genus Geobacillus. The isolate could use tridecane, hexadecane, octacosane and hexatridecane as sole carbon source for cell growth, and the digesting rate of long-chain alkane was lower than that of short-chain alkane. When the isolate was cultured in the heavy crude oil supplement with inorganic salts and trace yeast extract, the concentration of short-chain alkane was significantly increased and the content of long-chain alkane was decreased, suggesting that the larger hydrocarbon components in crude oil were degraded into shorter-chain alkane. Strain ZY-10 would be useful for improving the mobility of crude oil and upgrading heavy crude oil in situ.

The effects of a thermophile metabolite, tryptophol, upon protecting shrimp against white spot syndrome virus.

White spot syndrome virus (WSSV) is a shrimp pathogen responsible for significant economic loss in commercial shrimp farms and until now, there has been no effective approach to control this disease. In this study, tryptophol (indole-3-ethanol) was identified as a metabolite involved in bacteriophage-thermophile interactions. The dietary addition of tryptophol reduced the mortality in shrimp Marsupenaeus japonicus when orally challenged with WSSV. Our results revealed that 50 mg/kg tryptophol has a better protective effect in shrimp than 10 or 100 mg/kg tryptophol. WSSV copies in shrimp were reduced significantly (P < 0.01) when supplemented with 50 mg/kg tryptophol, indicating that virus replication was inhibited by tryptophol. Consequently, tryptophol represents an effective antiviral dietary supplement for shrimp, and thus holds significant promise as a novel and efficient therapeutic approach to control WSSV in shrimp aquaculture.

Changes in Sodium, Calcium, and Magnesium Ion Concentrations That Inhibit Geobacillus Biofilms Have No Effect on Anoxybacillus flavithermus Biofilms.

This study investigated the effects of varied sodium, calcium, and magnesium concentrations in specialty milk formulations on biofilm formation by Geobacillus spp. and Anoxybacillus flavithermus. The numbers of attached viable cells (log CFU per square centimeter) after 6 to 18 h of biofilm formation by three dairy-derived strains of Geobacillus and three dairy-derived strains of A. flavithermus were compared in two commercial milk formulations. Milk formulation B had relatively high sodium and low calcium and magnesium concentrations compared with those of milk formulation A, but the two formulations had comparable fat, protein, and lactose concentrations. Biofilm formation by the three Geobacillus isolates was up to 4 log CFU cm(-2) lower in milk formulation B than in milk formulation A after 6 to 18 h, and the difference was often significant (P ≤ 0.05). However, no significant differences (P ≤ 0.05) were found when biofilm formations by the three A. flavithermus isolates were compared in milk formulations A and B. Supplementation of milk formulation A with 100 mM NaCl significantly decreased (P ≤ 0.05) Geobacillus biofilm formation after 6 to 10 h. Furthermore, supplementation of milk formulation B with 2 mM CaCl2 or 2 mM MgCl2 significantly increased (P ≤ 0.05) Geobacillus biofilm formation after 10 to 18 h. It was concluded that relatively high free Na(+) and low free Ca(2+) and Mg(2+) concentrations in milk formulations are collectively required to inhibit biofilm formation by Geobacillus spp., whereas biofilm formation by A. flavithermus is not impacted by typical cation concentration differences of milk formulations.

Synergistic action modes of arabinan degradation by exo- and endo-arabinosyl hydrolases.

Two recombinant arabinosyl hydrolases, α-L-arabinofuranosidase from Geobacillus sp. KCTC 3012 (GAFase) and endo-(1,5)-α-L-arabinanase from Bacillus licheniformis DSM13 (BlABNase), were overexpressed in Escherichia coli, and their synergistic modes of action against sugar beet (branched) arabinan were investigated. Whereas GAFase hydrolyzed 35.9% of L-arabinose residues from sugar beet (branched) arabinan, endo-action of BlABNase released only 0.5% of L-arabinose owing to its extremely low accessibility towards branched arabinan. Interestingly, the simultaneous treatment of GAFase and BlABNase could liberate approximately 91.2% of L-arabinose from arabinan, which was significantly higher than any single exo-enzyme treatment (35.9%) or even stepwise exo- after endo-enzyme treatment (75.5%). Based on their unique modes of action, both exo- and endo-arabinosyl hydrolases can work in concert to catalyze the hydrolysis of arabinan to L-arabinose. At the early stage in arabinan degradation, exo-acting GAFase could remove the terminal arabinose branches to generate debranched arabinan, which could be successively hydrolyzed into arabinooligosaccharides via the endoaction of BlABNase. At the final stage, the simultaneous actions of exo- and endo-hydrolases could synergistically accelerate the L-arabinose production with high conversion yield.

Genome shuffling enhances lipase production of thermophilic Geobacillus sp.

Thermostable lipases are potential enzymes for biocatalytic application. In this study, the lipase production of Geobacillus sp. CF03 (WT) was improved by genome shuffling. After two rounds of genome shuffling, one fusant strain (FB1) achieved increase lipase activity from the populations generated by ultraviolet irradiation and ethyl methylsulfonate (EMS) mutagenesis. The growth rate and lipase production of FB1 increased highest by 150 and 238 %, respectively, in comparison to the wild type. The fusant enzyme had a significant change in substrate specificity but still prefers the long-chain length substrates. It had an optimum activity at 60 °C, pH at 7.0-8.0, with p-nitrophenyl palmitate (C16) as a substrate and retained about 50 % of their activity after 15 min at 70 °C, pH 8.0. Furthermore, the fusant lipase showed the preference of sesame oil, waste palm oil, and canola oil. Therefore, the genome shuffling strategy has been successful to strain improvement and selecting strain with multiple desirable characteristics.

A DNA break inducer activates the anticodon nuclease RloC and the adaptive immunity in Acinetobacter baylyi ADP1.

Double-stranded DNA breaks (DSB) cause bacteria to augment expression of DNA repair and various stress response proteins. A puzzling exception educes the anticodon nuclease (ACNase) RloC, which resembles the DSB responder Rad50 and the antiviral, translation-disabling ACNase PrrC. While PrrC's ACNase is regulated by a DNA restriction-modification (R-M) protein and a phage anti-DNA restriction peptide, RloC has an internal ACNase switch comprising a putative DSB sensor and coupled ATPase. Further exploration of RloC's controls revealed, first, that its ACNase is stabilized by the activating DNA and hydrolysed nucleotide. Second, DSB inducers activated RloC's ACNase in heterologous contexts as well as in a natural host, even when R-M deficient. Third, the DSB-induced activation of the indigenous RloC led to partial and temporary disruption of tRNA(Glu) and tRNA(Gln). Lastly, accumulation of CRISPR-derived RNA that occurred in parallel raises the possibility that the adaptive immunity and RloC provide the genotoxicated host with complementary protection from impending infections.

A robust whole-cell biocatalyst that introduces a thermo- and solvent-tolerant lipase into Aspergillus oryzae cells: characterization and application to enzymatic biodiesel production.

To develop a robust whole-cell biocatalyst that works well at moderately high temperature (40-50°C) with organic solvents, a thermostable lipase from Geobacillus thermocatenulatus (BTL2) was introduced into an Aspergillus oryzae whole-cell biocatalyst. The lipase-hydrolytic activity of the immobilized A. oryzae (r-BTL) was highest at 50°C and was maintained even after an incubation of 24-h at 60°C. In addition, r-BTL was highly tolerant to 30% (v/v) organic solvents (dimethyl carbonate, ethanol, methanol, 2-propanol or acetone). The attractive characteristics of r-BTL also worked efficiently on palm oil methanolysis, resulting in a nearly 100% conversion at elevated temperature from 40 to 50°C. Moreover, r-BTL catalyzed methanolysis at a high methanol concentration without a significant loss of lipase activity. In particular, when 2 molar equivalents of methanol were added 2 times, a methyl ester content of more than 90% was achieved; the yield was higher than those of conventional whole-cell biocatalyst and commercial Candida antarctica lipase (Novozym 435). On the basis of the results regarding the excellent lipase characteristics and efficient biodiesel production, the developed whole-cell biocatalyst would be a promising biocatalyst in a broad range of applications including biodiesel production.

Isolation of lipase producing thermophilic bacteria: optimization of production and reaction conditions for lipase from Geobacillus sp.

Lipases catalyze the hydrolysis and the synthesis of esters formed from glycerol and long chain fatty acids. Lipases occur widely in nature, but only microbial lipases are commercially significant. In the present study, thirty-two bacterial strains, isolated from soil sample of a hot spring were screened for lipase production. The strain TS-4, which gave maximum activity, was identified as Geobacillus sp. at MTCC, IMTECH, Chandigarh. The isolated lipase producing bacteria were grown on minimal salt medium containing olive oil. Maximal quantities of lipase were produced when 30 h old inoculum was used at 10% (v/v) in production medium and incubated in shaking conditions (150 rpm) for 72 h. The optimal temperature and pH for the bacterial growth and lipase production were found to be 60°C and 9.5, respectively. Maximal enzyme production resulted when mustard oil was used as carbon source and yeast extract as sole nitrogen source at a concentration of 1% (v/v) and 0.15% (w/v), respectively. The different optimized reaction parameters were temperature 65°C, pH 8.5, incubation time 10 min and substrate p-nitrophenyl palmitate. The Km and Vmax values of enzyme were found to be 14 mM and 17.86 µmol ml-1min-1, respectively, with p-nitrophenyl palmitate as substrate. All metal ions studied (1 mM) increased the lipase activity.

Manipulation of the conformation and enzymatic properties of T1 lipase by site-directed mutagenesis of the protein core.

In silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues, Val, Met, Leu, Ile, Trp, and Phe into Gln 114 of T1 lipase. The in silico investigations accurately predicted the enzymatic characteristics of the mutants in the experimental studies and provided rationalization for some of the experimental observations. Substitution with Leu successfully improved the conformational stability and enzymatic characteristics of T1 lipase. However, replacement of Gln114 with Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability, diminished lipase activity and inferior enzymatic characteristics. These results suggested that the substitution of a larger residue in a densely packed area of the protein core can have considerable effects on the structure and function of an enzyme. This is especially true when the residue is next to the catalytic serine as demonstrated with the Phe and Trp mutation.

Hydrocarbon degradation and bioemulsifier production by thermophilic Geobacillus pallidus strains.

Geobacillus pallidus XS2 and XS3 were isolated from oil contaminated soil samples in Yumen oilfield, China, and were able to produce bioemulsifiers on different hydrocarbons. Biodegradation assays exhibited that approximately 70% of PAH (250 mg/L) or 85% of crude oil (500 mg/L) was removed by the thermophilic bacteria after 20 days. The bioemulsifiers of the two strains were isolated and obtained a productive yield of 4.24±0.08 and 3.82±0.11g/L, respectively. GPC analysis revealed that the number-average molecular weights (M(n)) of the two bioemulsifiers were 271,785 Da and 526,369 Da, with PDI values of 1.104 and 1.027, respectively. Chemical composition studies exhibited that the bioemulsifier XS2 consisted of carbohydrates (68.6%), lipids (22.7%) and proteins (8.7%) while the bioemulsifier XS3 was composed by carbohydrates (41.1%), lipids (47.6%) and proteins (11.3%). Emulsification assays approved the effectiveness of bioemulsifiers over a wide range of temperature, pH and salinity.