Alkaline lipase from a novel strain Burkholderia multivorans: Statistical medium optimization and production in a bioreactor

An alkaline lipase from Burkholderia multivorans was produced within 15 h of growth in a 14 L bioreactor. An overall 12-fold enhanced production (58 U mL⁻¹ and 36 U mg⁻¹ protein) was achieved after medium optimization following the “one-variable-at-a-time” and the statistical approaches. The optimal composition of the lipase production medium was determined to be (% w/v or v/v): KH₂PO₄ 0.1; K₂HPO₄ 0.3; NH₄Cl 0.5; MgSO₄·7H₂O 0.01; yeast extract 0.36; glucose 0.1; olive oil 3.0; CaCl₂ 0.4 mM; pH 7.0; inoculum density 3% (v/v) and incubation time 36 h in shake flasks. Lipase production was maximally influenced by olive oil/oleic acid as the inducer and yeast extract as the additive nitrogen. Plackett–Burman screening suggested catabolite repression by glucose. Amongst the divalent cations, Ca²⁺ was a positive signal while Mg²⁺ was a negative signal for lipase production. RSM predicted that incubation time, inoculum density and oil were required at their higher levels (36 h, 3% (v/v) and 3% (v/v), respectively) while glucose and yeast extract were required at their minimal levels for maximum lipase production in shake flasks. The production conditions were validated in a 14 L bioreactor where the incubation time was reduced to 15 h.     

Influence of phosphorus nutrition on growth and metabolism of Duo grass (Duo festulolium)

Use of suitable plants that can extract and concentrate excess P from contaminated soil serves as an attractive method of phytoremediation. Plants vary in their potential to assimilate different organic and inorganic P-substrates. In this study, the response of Duo grass (Duo festulolium) to variable rates of soil-applied potassium dihydrogen phosphate (KH₂PO₄) on biomass yield and P uptake were studied. Duo grown for 5 weeks in soil with 2.5, 5 and 7.5 g KH₂PO₄ kg^?1 soil showed a significantly higher biomass and shoot P content of 8.3, 11.4 and 12.3 g P kg^?1 dry weight respectively compared to plants that received no soil added P. Also, the ability of Duo to metabolize different forms of P-substrates was determined by growing them in sterile Hoagland's agar media with different organic and inorganic P-substrates, viz. KH₂PO₄, glucose-1-phosphate (G1P), inositiol hexaphosphate (IHP), adenosine triphosphate (ATP) and adenosine monophosphate (AMP) for 2 weeks. Plants on agar media with different P-substrates also showed enhanced biomass yield and shoot P relative to no P control and the P uptake was in the order of ATP > KH₂PO₄ > G1P > IHP = AMP > no P control. The activities of both phytase (E.C.3.1.3.26) and acid phosphatases (E.C.3.1.3.2) were higher in all the P received plants than the control. Duo grass is capable of extracting P from the soil and also from the agar media and thus it can serve as possible candidate for phytoextraction of high P-soil.     

Application of experimental designs to optimize medium composition for production of thermostable lipase/esterase by Geobacillus thermodenitrificans AZ1

Thirty two morphologically different bacterial were isolated from different soil samples and screened for their ability to produce lipolytic enzymes. Among all isolates, the isolate coded AZ1 was selected due to its high potency to produce lipase at elevated temperature up to 65 °C. Phylogenetic analysis based on 16SrDNA sequence revealed its close relationship to Geobacillus thermodenitrificans. The effect of ten culture variable on lipase production was evaluated by implementing Plackett–Burman statistical design. d-sucrose, peptone and soy bean flour were the most significant variables affecting lipase production. A pre-optimized medium based on this experiment yielded an enzyme activity of 260 U min^?1 ml^?1. For further optimization, a fourteen trials’ multi-factorial Box–Behnken experimental design was applied to find out the optimum level of each of the significant variables. The tested variables, namely: d-sucrose (X₁); peptone (X₂) and soy bean flour (X₃) were examined, each at three different levels coded ?1, 0, +1. The optimal levels of the three components were founded to be (g/L): d-sucrose, 6.56; peptone, 6.35; and soy bean flour, 6.92, with a predicted activity of approximately 610 U min^?1 ml^?1. According to the results of the Plackett–Burman and Box–Behnken designs the following medium composition is expected to be optimum (g/L): d-sucrose 6.56, peptone 6.35, soy bean flour 6.92, CaCl₂ 0.02, Y.E. 2.5, K₂HPO₄ 1.0, MgSO₄.7H₂O 0.2 and Fe₂ (SO₄)₃ 0.02; pH, 8; cultivation temperature 55 °C and incubation time 24 h, the enzyme activity measured in the medium was approximately 593 U min^?1 ml^?1.     

Improvement of halophilic cellulase production from locally isolated fungal strain

Halophilic cellulases from the newly isolated fungus, Aspergillus terreus UniMAP AA-6 were found to be useful for in situ saccharification of ionic liquids treated lignocelluloses. Efforts have been taken to improve the enzyme production through statistical optimization approach namely Plackett–Burman design and the Face Centered Central Composite Design (FCCCD). Plackett–Burman experimental design was used to screen the medium components and process conditions. It was found that carboxymethylcellulose (CMC), FeSO₄·7H₂O, NaCl, MgSO₄·7H₂O, peptone, agitation speed and inoculum size significantly influence the production of halophilic cellulase. On the other hand, KH₂PO₄, KOH, yeast extract and temperature had a negative effect on enzyme production. Further optimization through FCCCD revealed that the optimization approach improved halophilic cellulase production from 0.029 U/ml to 0.0625 U/ml, which was approximately 2.2-times greater than before optimization.     

Application of statistical experimental design for optimization of keratinases production by Bacillus pumilus A1 grown on chicken feather and some biochemical properties

A new keratinolytic enzyme-producing bacterium was isolated from slaughter house polluted water and identified as Bacillus pumilus A1. Medium composition and culture conditions for the keratinases production by B. pumilus A1 were optimized using two statistical methods: Plackett–Burman design applied to find the key ingredients and conditions for the best yield of enzyme production and central composite design used to optimize the concentration of the five significant variables: feathers meal, soy peptone, NaCl, KCl, and KH₂PO₄. The medium optimization resulted in a 3.4-fold increase in keratinase production (87.73 U/ml) compared to that of the initial medium (25.9 U/ml). The zymography analysis shows the presence of at least five keratinolytic enzymes. The keratinolytic activity of the extracellular proteinases was examined by incubation with non-autoclaved chicken feathers. Complete solubilisation of whole feathers was observed after a 6-h incubation at temperatures ranging from 45 °C to 60 °C. The crude enzyme exhibited maximal activity at 60 °C and pH 8.5 or 55 °C and pH 9.0 using casein or keratin as substrates, respectively.     

Bioactive pigment production by Pseudomonas spp. MCC 3145: Statistical media optimization,biochemical characterization,fungicidal and DNA intercalation-based cytostatic activity

Multifunctional redox-active pyocyanin (PYC) produced by Pseudomonas aeruginosa has diverse biotechnological applications, but no efforts have been made to improve its yield. The yield obtained in initial study using Pseudomonas spp. MCC 3145 was 24.21 mg L⁻¹ PYC in pigment production medium D; hence, optimization of the media components using statistical tools for more production of PYC was undertaken. Of the 11 medium constituents screened for PYC production using Plackett–Burman design (PBD), glycerol, peptone, and CuSO₄ were recognized as the most significant variables. The optimal concentration of the variables for maximum PYC production was evaluated using a five-level three-factor central composite design (CCD). Optimal concentration of the three variables, glycerol, peptone and CuSO₄ showed 313.94 ± 10.09 mg L⁻¹ the PYC production, with an 18-fold increase. Fine structural details of PYC were verified by chromatographic and various spectroscopic analyses. In vitro bioactivity studies demonstrated significant antifungal activity of PYC against fungal phytopathogens and substantial cytostatic activity against four major cancer cell lines. Furthermore, PYC displayed nonspecific DNA intercalation, which may be the reason for proliferation arrest in cancer cells. Thus, the study rigorously improved PYC production through medium optimization and further demonstrated its agricultural and therapeutic applications.     

Efficient production of α-arbutin by whole-cell biocatalysis using immobilized hydroquinone as a glucosyl acceptor

The aim of the present study is to develop an efficient and cost-effective method for α-arbutin production by using whole-cell of Xanthomonas maltophilia BT-112 as a biocatalyst. Hydroquinone (HQ), substrate for the bioconversion as glucosyl acceptor, was immobilized on H107 macroporous resin to reduce its toxic effect on the cells, and the optimal reaction conditions for α-arbutin synthesis were investigated. When 350 g/L H107 resin (254.5 mM HQ) and 20 g/L (4.2 U/g) of cells were shaken in 10 mL Na₂HPO₄–KH₂PO₄ buffer (50 mM, pH 6.5) containing 509 mM sucrose at 35 °C with 150 rpm for 48 h, the final yield of α-arbutin reached 65.9 g/L with a conversion yield of 95.2% based on the amount of HQ supplied. The α-arbutin production was 202% higher than that of the control (free HQ) and the cells maintained its full activity for almost six consecutive batch reactions, indicating a potential for reducing production costs. Additionally, the product was one-step isolated and identified as α-arbutin by ¹³C NMR and ¹H NMR analysis. In conclusion, the combination of whole cells and immobilized hydroquinone (IMHQ) is a promising approach for economical and industrial-scale production of α-arbutin.     

Characterization of the extracellular biodemulsifier of Bacillus mojavensis XH1 and the enhancement of demulsifying efficiency by optimization of the production medium composition

The demulsifying bacterium XH1 was identified as a Bacillus mojavensis by the 16S rDNA gene. The extracellular biodemulsifier produced by this species was purified by ethanol extraction and column chromatography through a sephadex and silicon gel column. Preliminary investigation using UV–vis and TLC indicated that the biodemulsifier had two components a protein and a lipopeptide. All major components of the medium, including the sources of soluble and insoluble carbon, nitrogen, phosphate, and metal ions were investigated to improve the biosynthesis and efficiency of the biodemulsifier. The optimal carbon sources were glucose and liquid paraffin. Glucose participated in the biosynthesis of the demulsifier, while liquid paraffin promoted the lipophilicity and secretion of biosurfactants. The absence of yeast extract, ammonium chloride or phosphate (K₂HPO₄/KH₂PO₄) had a negative effect on the production of the biodemulsifier and significantly inhibited its activity. To further enhance the biodemulsifier efficiency, the optimal medium composition was determined using the response surface methodology (RSM) based on the central composite rotation design (CCRD). Using the optimized biodemulsifier production medium: 8.5 g/l glucose; 3% (v/v) liquid paraffin; 1.5 g/l yeast extract; 3.36 g/l NH₄Cl and15 g/l phosphate, the demulsifying ratio increased 35.5% and biodemulsifier yield increased to 2.07 g/l.     

Extracellular cold-active lipase of Microbacterium luteolum isolated from Gangotri glacier,western Himalaya: Isolation,partial purification and characterization

A psychrophilic bacterium producing cold-active lipase upon growth at low temperature was isolated from the soil samples of Gangotri glacier and identified as Microbacterium luteolum. The bacterial strain produced maximum lipase at 15 °C, at a pH of 8.0. Beef extract served as the best organic nitrogen source and ammonium nitrate as inorganic for maximum lipase production. Castor oil served as an inducer and glucose served as an additional carbon source for production of cold-active lipase. Ferric chloride as additional mineral salt in the medium, highly influenced the lipase production with an activity of 8.01 U ml^?1. The cold-active lipase was purified to 35.64-fold by DEAE-cellulose column chromatography. It showed maximum activity at 5 °C and thermostability up to 35 °C. The purified lipase was stable between pH 5 and 9 and the optimal pH for enzymatic hydrolysis was 8.0. Lipase activity was stimulated in presence of all the solvents (5%) tested except with acetonitrile. Lipase activity was inhibited in presence of Mn²⁺, Cu²⁺, and Hg²⁺; whereas Fe⁺, Na⁺ did not have any inhibitory effect on the enzyme activity. The purified lipase was stable in the presence of SDS; however, EDTA and dithiothreitol inhibited enzyme activity. Presence of Ca²⁺ along with inhibitors stabilized lipase activity. The cold active lipase thus exhibiting activity and stability at a low temperature and alkaline pH appears to be practically useful in industrial applications especially in detergent formulations.     

l-Lactic acid production from liquefied cassava starch by thermotolerant Rhizopus microsporus: Characterization and optimization

The thermotolerant Rhizopus microsporus DMKU 33 capable of producing l-lactic acid from liquefied cassava starch was isolated and characterized for its phylogenetic relationship and growth temperature and pH ranges. The concentrations of (NH₄)₂SO_4, KH₂PO₄, MgSO₄ and ZnSO₄·7H₂O in the fermentation medium was optimized for lactic acid production from liquefied cassava starch by Rhizopus microsporus DMKU 33 in shake-flasks at 40 °C. The fermentation was then studied in a stirred-tank bioreactor with aeration at 0.75 vvm and agitation at 200 rpm, achieving the highest lactic acid production of 84 g/L with a yield of 0.84 g/g at pH 5.5 in 3 days. Lactic acid production was further increased to 105–118 g/L with a yield of 0.93 g/g and productivity of 1.25 g/L/h in fed-batch fermentation. R. microsporus DMKU 33 is thus advantageous to use in simultaneous saccharification and fermentation for l-lactic acid production from low-cost starchy substrates.     

Statistical media optimization for growth and PHB production from methanol by a methylotrophic bacterium

Media components were optimized by statistical design for cell growth and PHB production of Methylobacterium extorquens DSMZ 1340. Four important components of growth media were optimized by central composite design. The growth increased from an OD = 1.35 for Choi medium as control to an OD = 2.15 for optimal medium. Then media components for PHB production were optimized. Optimization of five important factors was conducted by response surface method. The optimal composition of PHB production medium was found to be at 7.8 (g/L) Na₂HPO₄ · 12H₂O, and surprisingly at zero concentration of (NH₄)₂SO₄, KH₂PO₄, MgSO₄ and MnSO₄. The PHB production was found to be 2.95 (g/L) at this medium. RSM results indicated that a deficiency of nitrogen and magnesium is crucial for PHB accumulation in this microorganism. Also, PHB production was carried out in a 5 L fermentor at the optimum condition which resulted in 9.5 g/L PHB and 15.4 g/L cell dry weight with 62.3% polymer content.     

Bioprocess development for kefiran production by Lactobacillus kefiranofaciens in semi industrial scale bioreactor

Lactobacillus kefiranofaciens is non-pathogenic gram positive bacteria isolated from kefir grains and able to produce extracellular exopolysaccharides named kefiran. This polysaccharide contains approximately equal amounts of glucose and galactose. Kefiran has wide applications in pharmaceutical industries. Therefore, an approach has been extensively studied to increase kefiran production for pharmaceutical application in industrial scale. The present work aims to maximize kefiran production through the optimization of medium composition and production in semi industrial scale bioreactor. The composition of the optimal medium for kefiran production contained sucrose, yeast extract and K₂HPO₄ at 20.0, 6.0, 0.25 g L⁻¹, respectively. The optimized medium significantly increased both cell growth and kefiran production by about 170.56% and 58.02%, respectively, in comparison with the unoptimized medium. Furthermore, the kinetics of cell growth and kefiran production in batch culture of L. kefiranofaciens was investigated under un-controlled pH conditions in 16-L scale bioreactor. The maximal cell mass in bioreactor culture reached 2.76 g L⁻¹ concomitant with kefiran production of 1.91 g L⁻¹.     

Production of polysaccharide–peptide complexes by submerged mycelial culture of an entomopathogenic fungus Cordyceps sphecocephala

The effects of medium components and environmental factors on the production of mycelial biomass and polysaccharide–peptide complexes (exobiopolymers) by Cordyceps sphecocephala J-201 were investigated in submerged cultures. The optimal temperature and initial pH for the production of both mycelial biomass and exobiopolymers in flask cultures were found to be 25 °C and pH 4–5, respectively. The optimal combination of the media constituents was as follows (g l⁻¹): sucrose 40, yeast extract 6, polypepton 2, KH₂PO₄ 0.46, K₂HPO₄ 1, and MgSO₄·7H₂O 0.5. The results of bioreactor culture revealed that the maximum concentration of mycelial biomass (28.2 g l⁻¹) was obtained at an agitation speed of 300 rpm and at an aeration rate of 2 vvm, whereas maximum exobiopolymer production (2.5 g l⁻¹) was achieved at a milder agitation speed (150 rpm). There was a significant variance in mycelial morphology between different aeration conditions. Looser mycelial pellets were developed, and their size and hairiness increased as the aeration rate increased from 0.5 to 2.0 vvm, resulting in enhanced exobiopolymer production. The apparent viscosities of fermentation broth increased rapidly towards the end of fermentations at the conditions of high aeration rate and agitation speed, which were mainly due to high amount of mycelial biomass rather than exobiopolymers at the later stages of fermentation. The three different exobiopolymers (FR-I, -II, and -III) were fractionated by a gel filtration chromatography on Sepharose CL-6B. The carbohydrate and protein contents in each fraction were significantly different and the molecular weights of FR-I, FR-II, and FR-III were determined to be 1831, 27, and 2.2 kDa, respectively. The compositional analysis revealed that the three fractions of crude exobiopolymers consisted of acidic and nonpolar amino acids, such as aspartic acid, glutamic acid, glycine, and valine in protein moiety, and of mainly mannose and galactose in sugar moiety.     

Optimization of date syrup for enhancement of the production of citric acid using immobilized cells of Aspergillus niger

Date syrup as an economical source of carbohydrates and immobilized Aspergillus niger J4, which was entrapped in calcium alginate pellets, were employed for enhancing the production of citric acid. Maximum production was achieved by pre-treating date syrup with 1.5% tricalcium phosphate to remove heavy metals. The production of citric acid using a pretreated medium was 38.87% higher than an untreated one that consumed sugar. The appropriate presence of nitrogen, phosphate and magnesium appeared to be important in order for citric acid to accumulate. The production of citric acid and the consumed sugar was higher when using 0.1% ammonium nitrate as the best source of nitrogen. The production of citric acid increased significantly when 0.1 g/l of KH₂PO₄ was added to the medium of date syrup. The addition of magnesium sulfate at the rate of 0.20 g/l had a stimulating effect on the production of citric acid. Maximum production of citric acid was obtained when calcium chloride was absent. One of the most important benefits of immobilized cells is their ability and stability to produce citric acid under a repeated batch culture. Over four repeated batches, the production of citric acid production was maintained for 24 days when each cycle continued for 144 h. The results obtained in the repeated batch cultivation using date syrup confirmed that date syrup could be used as a medium for the industrial production of citric acid.     

Production,characterization and application of a keratinase from Chryseobacterium L99 sp. nov.

Keratins are important bioresources for apparels and feedstuffs, but recalcitrant to common enzymes. Now, it is popular and essential to develop keratinolytic enzymes for environmental prevention and improvement of keratin product quality. In the study, the medium optimization, purification, characterization and application of the keratinase from a newly isolated Chryseobacterium L99 sp. nov. were conducted. Exogenous sucrose, malt sugar, glucose, starch, tryptone, Mg²⁺, Zn²⁺, Ca²⁺ and Cu²⁺ could promote the keratinase production, while exogenous urea, NH₄Cl and yeast extract exhibited strong inhibition effects. Response surface methodology predicted a maximum keratinase yield of 213.8 U mL⁻¹, at (g L⁻¹) sucrose 16.8, MgCl₂·6H₂O 1.9, feather keratin 40.0, NaH₂PO₄·2H₂O 6.0 and K₂HPO₄·6H₂O 1.0, where dry cell weight nearly had a minimum 8.58 g L⁻¹. Then, a serine keratinase about 33 kDa was purified, and its optimal activity was acquired at 40 °C and pH 8.0 with K⁺, Zn²⁺or Co²⁺. Compared with Savinase 16 L and transglutaminase, the L99 keratinase could efficient prevent shrinkage and eliminate directional frictional effect of wool, indicating it as a promising prospect in the biotreatment of wool fibres.     

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus for acetic acid production

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus was carried out for high yield of acetic acid. Acetic acid production process was divided into three stages. The first stage was the growth of S. cerevisiae and ethanol production, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. The second stage was the co-culture of S. cerevisiae and A. pasteurianus, fermentation temperature and aeration rate were maintained at 34 °C and 0.4 vvm, respectively. The third stage was the growth of A. pasteurianus and production of acetic acid, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. Inoculation volume of A. pasteurianus and S. cerevisiae was 16% and 0.06%, respectively. The average acetic acid concentration was 52.51 g/L under these optimum conditions. To enhance acetic acid production, a glucose feeding strategy was subsequently employed. When initial glucose concentration was 90 g/L and 120 g/L glucose was fed twice during fermentation, acetic acid concentration reached 66.0 g/L.     

Manganese ion concentration affects production of human core 3 O-glycan in Saccharomyces cerevisiae

BackgroundProduction of various mucin-like glycoproteins could be useful for development of antibodies specific to disease-related glycoproteins as well as for the biosynthesis of clinically useful glycoproteins. A Saccharomyces cerevisiae strain capable of in vivo production of mucin-type core 1 structure (Galβ1-3GalNAcα1-O-Ser/Thr) has been reported, but a strain producing core 3 structure (GlcNAcβ1-3GalNAcα1-O-Ser/Thr) has not been constructed.MethodsTo generate core 3-producing strain, genes encoding uridine diphosphate (UDP)-Gal-4-epimerase, UDP-GalNAc transporter, UDP-GlcNAc transporter, and two glycosyltransferases were integrated into the genome. A Mucin-1-derived acceptor peptide (MUC1ap) was expressed as an acceptor. The amount of the resulting modified peptide was analyzed by HPLC.ResultsIntroduction of a codon-optimized UDP-GlcNAc:βGal β-1,3-N-acetylglucosaminyltransferase 6 (β3Gn-T6) gene yielded increases in β3Gn-T6 activity but did not alter the level of core 3 production. The highest in vitro activity of β3Gn-T6 was observed at Mn^2 + concentrations of 10 mM and above. Supplementation of MnCl₂ to the culture medium yielded increases of up to 25% in the accumulation of core 3 on the MUC1ap. The yeast invertase from the core 3-producing strain was less extensively N-glycosylated; however, it was partially restored by the addition of MnCl₂ to the medium.ConclusionsPhysiological Mn^2 + concentration in S. cerevisiae was insufficient to facilitate optimal synthesis of core 3. Mn^2 + supplementation led to up-regulation of reaction of glycosylation in the Golgi, resulting in increases of core 3 production.General significanceThis study reveals that control of Mn^2 + concentration is important for production of specific mammalian-type glycans in S. cerevisiae.     

Lactobacillus plantarum isolated from molasses produces bacteriocins active against Gram-negative bacteria

Two bacteriocins, ST28MS and ST26MS, produced by Lactobacillus plantarum isolated from molasses, inhibited the growth of Lactobacillus casei, Lactobacillus sakei, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli and Acinetobacter baumanii. The mode of activity of the bacteriocins is bacteriostatic, as observed against L. casei and P. aeruginosa. Reduction in antimicrobial activity was recorded after treatment with Proteinase K, papain, trypsin, chymotrypsin, pronase, pepsin and protease. Both peptides remained active after 20 min at 121 °C. Bacteriocin ST28MS was produced at much higher levels (12,800 AU/mL) compared to bacteriocin ST26MS (6400 AU/mL) with glucose as carbon source. The activity of bacteriocin ST28MS decreased by 50% at pH below 4.0. Bacteriocin ST26MS, on the other hand, is more stable at this pH. Production of both bacteriocins is stimulated by tryptone. Potassium (KH₂PO₄ and K₂HPO₄) at 5 and 10 g/L stimulated the production of bacteriocin ST28MS, but not bacteriocin ST26MS. MRS supplemented with glycerol (1–5 g/L) did not result in any changes in the activity levels of the two bacteriocins. Ascorbic acid and Vitamins B₁ and B₁₂ are required for bacteriocin ST28MS production, but only Vitamin B₁₂ for bacteriocin ST26MS production. No plasmids were recorded for strains ST28MS and ST26MS, suggesting that the genes encoding production of the two bacteriocins are located on the genomes.     

Impact of NH4+ nitrogen source on the production of Rhizopus oryzae lipase in Pichia pastoris

BackgroundPichia pastoris is a highly successful system for heterologous expression. During the induction stage, the ammonium ion released into the fermentation broth has a deep impact on cell growth and protein expression. The impact of NH₄⁺ concentration on the expression of the Rhizopus oryzae lipase proAROL in P. pastoris was investigated.ResultsThe lipase activity under the optimum NH₄⁺ concentration of 440 mmol/L reached 12,019 U/mL. Increased concentrations of NH₄⁺ in the broth prevented the protease production, resulting in higher specific lipase activity in the supernatant. Furthermore, analysis of carbon metabolism and energy regeneration pattern revealed that under the definite NH₄⁺ concentrations more carbon source (methanol) was consumed with surged AOX activity and then the higher energy and amino acid precursors demand for recombinant protein synthesis is compensated for by the TCA cycle.ConclusionsIn this study, the R. oryzae lipase activity reaches the highest level ever reported under optimized NH₄⁺ concentration and the analysis of the carbon metabolism provides useful information for future optimization of protein production by P. pastoris in a molecular level.     

Homologous expression,purification and characterization of a novel high-alkaline and thermal stable lipase from Burkholderia cepacia ATCC 25416

The lipase secreted by Burkholderia cepacia ATCC 25416 was particularly attractive in detergent and leather industry due to its specific characteristics of high alkaline and thermal stability. The lipase gene (lipA), lipase chaperone gene (lipB), and native promoter upstream of lipA were cloned. The lipA was composed of 1095 bp, corresponding to 364 amino acid residues. The lipB located immediately downstream of lipA was composed of 1035 bp, corresponding to 344 amino acid residues. The lipase operon was inserted into broad host vector pBBRMCS1 and electroporated into original strain. The homologous expression of recombinant strain showed a significant increase in the lipase activity. LipA was purified by three-step procedure of ammonium sulfate precipitation, phenyl-sepharose FF and DEAE-sepharose FF. SDS-PAGE showed the molecular mass of the lipase was 33 kDa. The enzyme optimal temperature and pH were 60 °C and 11.0, respectively. The enzyme was stable at 30–70 °C. After incubated in 70 °C for 1 h, enzyme remained 72% of its maximal activity. The enzyme exhibited a good stability at pH 9.0–11.5. The lipase preferentially hydrolyzed medium-chain fatty acid esters. The enzyme was strongly activated by Mg²⁺, Ca²⁺, Cu²⁺, Zn²⁺, Co²⁺, and apparently inhibited by PMSF, EDTA and also DTT with SDS. The enzyme was compatible with various ionic and non-ionic surfactants as well as oxidant H₂O₂. The enzyme had good stability in the low- and non-polar solvents.