Molecular cloning and characterization of a new and highly thermostable esterase from Geobacillus sp. JM6.

A new lipolytic enzyme gene was cloned from a thermophile Geobacillus sp. JM6. The gene contained 750 bp and encoded a 249-amino acid protein. The recombinant enzyme was expressed and purified from Escherichia coli BL21 (DE3) with a molecular mass of 33.6 kDa. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding the highest activity with p-nitrophenyl butyrate. When p-nitrophenyl butyrate was used as a substrate, the optimum reaction temperature and pH for the enzyme were 60 °C and pH 7.5, respectively. Geobacillus sp. JM6 esterase showed excellent thermostability with 68% residual activity after incubation at 100 °C for 18 h. A theoretical structural model of strain JM6 esterase was developed with a monoacylglycerol lipase from Bacillus sp. H-257 as a template. The predicted core structure exhibits an α/ß hydrolase fold, and a putative catalytic triad (Ser97, Asp196, and His226) was identified. Inhibition assays with PMSF indicated that serine residue is involved in the catalytic activity of strain JM6 esterase. The recombinant esterase showed a relatively good tolerance to the detected detergents and denaturants, such as SDS, Chaps, Tween 20, Tween 80, Triton X-100, sodium deoxycholate, urea, and guanidine hydrochloride.

Molecular cloning and characterization of a thermostable lipase from deep-sea thermophile Geobacillus sp. EPT9.

A gene (1,254 bp) encoding a lipase was identified from a deep-sea hydrothermal field thermophile Geobacillus sp. EPT9. The open reading frame of this gene encoded 417 amino acid residues. The gene was cloned, overexpressed in Escherichia coli, and the target protein was purified to homogeneity. The purified recombinant enzyme presented a molecular mass of 44.8 kDa. When p-nitrophenyl palmitate was used as a substrate, the recombinant lipase was optimally active at 55 °C and pH 8.5. The recombinant enzyme retained 44 % residual activity after incubation at 80 °C for 1 h, which indicated that Geobacillus sp. EPT9 lipase was thermostable. Homology modeling of strain EPT9 lipase was developed with the lipase from Bacillus sp. L2 as a template. The core structure exhibits an α/ß-hydrolase fold and the typical catalytic triad might consist of Ser142, Asp346, and His387. The enzymatic activity of EPT9 lipase was inhibited by addition of phenylmethylsulfonyl fluoride, indicating that it contains serine residue, which plays an important role in the catalytic mechanism.

[Improving thermal stability of Geobacillus sp. ZH1 carboxylesterase by error-prone PCR].

OBJECTIVE: This study was aimed to improve the thermal stability of carboxylesterase from Geobacillus sp. ZH1 by directed evolution. METHODS: A library of carboxylesterase mutants was constructed by introducing random mutagenesis using error-prone PCR to screen mutant enzymes with improved thermostability. After induction, expression and purification, the mutant enzyme was characterized. RESULTS: After screening, one mutant strain 65 was obtained with improved carboxylesterase thermal stability. Sequence analysis revealed two amino acid substitutions, including T113S and M160K. According to homologous modeling, T113S was located on the fifth ß-sheet. Another mutant site M160K was located on a loop between the fifth and the sixth α-helix, being on the surface of the mutant enzyme. The mutated Lys160 formed an extra hydrogen bond with nearby Thr162. The half-life of mutant enzyme 65 and the parent enzyme at 90 degrees C was 3.1 h and 1.9 h, respectively. The mutant enzyme 65 had a better thermal stability than the parent enzyme. CONCLUSION: Directed evolution by error-prone PCR of Geobacillus sp. ZH1 carboxylesterase gene is effective to improve the thermal stability.

[Isolation, identification of a κ-carrageenase-producing bacterium and κ-Carrageenase characterization].

OBJECTIVE: The aim of this study was to screen and identify carrageenase-producing strain from mangrove soil leaf and to characterize produced carrageenase. METHODS: The culture medium with κ-carrageenan as sole carbon source was used to isolate the strain exhibiting carrageenase activity. The isolated strain was identified by morphology observation and 16S rDNA sequencing. κ-carrageenase produced by Pseudoalteromonas sp. ASY5 was purified and characterized by DNS method. RESULTS: A bacterial strain ASY5 with high carrageenase activity was isolated from mangrove soil humus, and was identified as Pseudoalteromonas sp. The molecular mass of the purifiedenzyme was estimated to be 30 kDa. The optimal temperature and pH of the enzyme were 60°C and 7.5, respectively. The enzyme was stabileat 50°C, and more stable between pH 7.0 and 9.0. The enzyme could convert κ-carrageenan. The Km and Vmax values of the enzyme for κ-carrageenan was 2.28 mg /mL and 147.06 µmol/(min · mg), respectively. The enzyme was significantly stimulated by Na+, K+, Ca2+, Mg2+ and Al3+. The enzyme was inhibited strongly by Ag+, Zn2+, Cd2+ and SDS. CONCLUSION: κ-carrageenase produced by Pseudoalteromonas sp. ASY5 was stable at high temperature and alkaline pH, with potential application in carrageenan oligosaccharides production.

Cloning and characterization of a new manganese superoxide dismutase from deep-sea thermophile Geobacillus sp. EPT3.

A new gene encoding a superoxide dismutase (SOD) was identified from a thermophile Geobacillus sp. EPT3 isolated from a deep-sea hydrothermal field in east Pacific. The open reading frame of this gene encoded 437 amino acid residues. It was cloned, overexpressed in Escherichia coli (DE3), and the recombinant protein was purified to homogeneity. Geobacillus sp. EPT3 SOD was of the manganese-containing SOD type, as judged by the insensitivity of the recombinant enzyme to both KCN and H2O2, and the activity analysis of Fe or Mn reconstituted SODs by polyacrylamide gel electrophoresis. The recombinant SOD was determined to be a homodimer with monomeric molecular mass of 59.0 kDa. In comparison with other Mn-SODs, the manganese-binding sites are conserved in the sequence (His260, His308, Asp392, His396). The recombinant enzyme had high thermostability at 50 °C. It retained 57 % residual activity after incubation at 90 °C for 1 h, which indicated that this SOD was thermostable. The enzyme also showed striking stability over a wide range of pH 5.0-11.0. At tested conditions, the recombinant SOD from Geobacillus sp. EPT3 showed a relatively good tolerance to some inhibitors, detergents, and denaturants, such as ß-mercaptoethanol, dithiothreitol, phenylmethylsulfonyl fluoride, Chaps, Triton X-100, urea, and guanidine hydrochloride.

[Isolation, identification and characterization of an agarase-producing marine bacterial strain Stenotrophomonas sp. NTa].

OBJECTIVE: To identify and characterize a marine bacterial strain producing agarase. METHODS: The agarase-producing bacterium was isolated from coastal sediments in Xiamen using agar as the sole carbon source. The strain was identified by the analyses of 16S rRNA gene sequence, phenotype and biochemical reactions. Agarase activity was determined by dinitrosalicylic acid method, and the category of agarase was assayed using chromogenic substrate. At last, the characteristics of agarase were determined. RESULTS: The results of the 16S rRNA phylogenetic, phenotypic and biochemical analyses showed that: the agar-degrading bacterium NTa belonged to the genus Stenotrophomonas sp.. The strain could produce extracellular agarases, including alpha-agarase and beta-agarase. The optimum temperature and pH of strain NTa agarase were 40 degrees C and 7.0, respectively. The enzymatic activity was stable below 30 degrees C. It also showed stability over a pH range between 7.0 and. 0. Ca2+ could activate agarase activity, and Na+, K+ and Mg2+ had no significant influence. However, Ag', Ba2 , Fe2' , Mn2', Cu2', Zn2+ and Fe3' inhibited the enzyme activity. The enzymatic activity of stain NTa agarase was inhibited by EDTA. The agarase had good resistance to some inhibitors, detergents and denaturant. CONCLUSION: Stenotrophomonas sp. NTa is a new type of agarase-producing strain, which can produce both alpha-agarase and beta-agarase and has potential applications in the production of agaro-oligosaccharide.

[Identification and characterization of Aspergillus aculeatus JMUdb058 for naringinase production].

OBJECTIVE: A new naringinase-producing strain, JMUdb058 was identified and characterized. METHODS: The strain was identified by morphological observation and 28S rDNA homogeneous analysis. Naringinase was identified by monitoring the hydrolysis of naringin to prunin and naringenin using a reversed phase High Performance Liquid Chromatography (HPLC). The regulation of naringinase expression was studied by measuring naringinase activity of 11 different carbon sources and 7 nitrogen sources in shaking cultivation. The naringinase-producing capacity was investigated in both solid-state fermentation and submerged fermentation. RESULTS: The macro-morphology and micro-morphology of JMUdb058 corresponded to the characteristics of Aspergillus section Nigri Gams, and the 28S rDNA sequences showed homogeneity at 100% to Aspergillus aculeatus. Crude enzymes prepared by both submerged fermentation and solid-state fermentation could hydrolyze naringin to prunin and naringenin. In addition, the enzyme could remove naringin from citrus juice effectively. Carbon resources, including hesperidin, naringin, rutin and rhamnose, and organic nitrogen resources, i. e., tryptone, soybean meal, yeast extract and corn syrup were shown to express the naringinase. The strain had an outstanding ability to yield naringinase in the solid-state fermentation, which showed an alpha-L-rhamnosidase activity of 5903 U/gds by HPLC, and the naringinase of 1939U/gds by HPLC and 72232 U/gds by Davis method. CONCLUSION: It is the first time to report a stain of Aspergillus aculeatus can produce naringinase, carbon source containing rhamnose groups are able to induce the enzyme expression. The stain JMUdb058 is a new microorganism source for high yield of naringinase, in particularly by the solid-state fermentation.

[Characterization of Cryptococcus sp. Jmudeb008 and regulation of naringinase activity by glucose].

OBJECTIVE: We identified a new isolated naringinase-producing yeast strain named as Jmudeb008, and analyzed its naringinase-producing ability cultured with different composition and concentration of carbon sources. METHODS: The strain was identified based on conventional phenotypic methods and sequences of the D1/D2 region of 26S rDNA and 5.8S-ITS. Media with different composition and concentration of carbon sources were used in shaking culture of Jmudeb008. The activity of naringinase was evaluated by analyzing the concentration of naringin, naringenin and glucose during 48 h culture. RESULTS: The Jmudeb008's sequences of the D1/D2 region of 26S rDNA and 5.8S-ITS were 99% identical with Cryptococcus laurentii. Further glucose fermentation test, urease test, DBB (diazotization based blue) test and nitrate reduction test were coincided with results of DNA sequencing. Therefore, Jmudeb008 was identified as Cryptococcus laurentii. When Jmudeb008 was cultured in the medium with naringin as the only carbon source, it could synthesize naringinase. However, when glucose was available, the synthesis of naringinase was repressed. CONCLUSION: The new isolated naringinase-producing yeast strain JmudebO08 was identified as Cryptococcus laurentii. The glucose in medium repressed naringinase expression.

Characterization and immobilization of arylsulfatase on modified magnetic nanoparticles for desulfation of agar.

Carboxyl functioned magnetic nanoparticles (CMNPs) were prepared by a simple co-precipitation method and characterized by Fourier transform infrared spedtroscopy and scanning electron microscope. The prepared CMNPs were used for covalent immobilization of the arylsulfatase which could be applied in desulfation of agar. The optimal immobilizaion conditions were obtained as follows: glutaraldehyde concentration 1.0% (v/v), cross-linking time 3h, immobilization time 3h, immobilization temperature 5°C and enzyme dose 0.62U. Increase in properties of the arylsulfatase such as optimum temperature and pH was observed after immobilization. Immobilization led to increased tolerance of enzyme to some metal ions, inhibitors and detergents. The Km and kcat of the immobilized enzyme for hydrolysis of p-NPS at pH 7.5 and at 50°C were determined to be 0.89mmol/L and 256.91s-1, respectively. The relative desulfuration rates of immobilized arylsulfatase maintained 61.7% of its initial desulfuration rates after seven cycles. After the reaction of agar with immobilized arylsulfatase for 90min at 50°C, 46% of the sulfate in the agar was removed. These results showed that the immobilization of arylsulfatase onto CMNPs is an efficient and simple way for preparation of stable arylsulfatase and have a great potential for application in enzymatic desulfation of agar.

Characterization of an extracellular biofunctional alginate lyase from marine Microbulbifer sp. ALW1 and antioxidant activity of enzymatic hydrolysates.

A novel alginate-degrading marine bacterium Microbulbifer sp. ALW1 was isolated from rotten brown alga. An extracellular alginate lyase was purified to electrophoretic homogeneity and had a molecular mass of about 26.0 kDa determined by SDS-PAGE and size exclusion chromatography. This enzyme showed activities towards both polyguluronate and polymannuronate indicating its bifunctionality while with preference for the former substrate. Using sodium alginate as a substrate, strain ALW1 alginate lyase was optimally active at 45 °C and pH 7.0. It was stable at 25 °C, 30 °C, 35 °C and 40 °C, but not stable at 50 °C. This alginate lyase showed good stability over a broad pH range (5.0-9.0). The enzyme activity was increased to 5.1 times by adding NaCl to a final concentration of 0.5M. Strain ALW1 alginate lyase produced disaccharide (majority) and trisaccharide from alginate indicating that this enzyme could be a good tool for preparation of alginate oligosaccharides with low degree of polymerization (DP). The alginate oligosaccharides displayed the scavenging abilities towards radicals (DPPH, ABTS(+) and hydroxyl) and the reducing power. Therefore, the hydrolysates exhibited the antioxidant activity and had potential as a natural antioxidant.

Preparation and characterization of tannase immobilized onto carboxyl-functionalized superparamagnetic ferroferric oxide nanoparticles.

Tannase from Aspergillus tubingensis was immobilized onto carboxyl-functionalized Fe3O4 nanoparticles (CMNPs), and conditions affecting tannase immobilization were investigated. Successful binding between CMNPs and tannase was confirmed by Fourier transform infrared spectroscopy and thermogravimetric analysis. Vibrating sample magnetometry and X-ray diffraction showed that the CMNPs and immobilized tannase exhibit distinct magnetic responses and superparamagnetic properties. Free and immobilized tannase exhibited identical optimal temperatures of 50°C and differing pH optima at 6 and 7, respectively. The thermal, pH, and storage stabilities of the immobilized tannase were superior to those of free tannase. After six cycles of catalytic hydrolysis of propyl gallate, the immobilized tannase maintained over 60% of its initial activity. The Michaelis constant (Km) of the immobilized enzyme indicated its higher affinity for substrate binding than the free enzyme.

Investigation of sunlight-induced deterioration of aroma of pummelo (Citrus maxima) essential oil.

Deterioration of aromas of pummelo essential oil (EO) induced by sunlight was compared to those induced by heat and oxygen exposure using the techniques of sensory evaluation and GC-MS analysis. The sunlight-exposed EO was found to possess an oily off-flavor odor, which was significantly different from its counterparts induced by oxygen and heat. The strong oily note of the sunlight-exposed EO was attributed to the existence of linalool oxides and limonene oxides, as well as the lack of neral and geranial, for which UV sunlight was revealed to be the critical contributor causing the chemical reactions for the aroma changes. The results demonstrated that UV sunlight could significantly affect the aroma of the pummelo EO, providing valuable information that will benefit the production and storage of EO-based aromatic products.

Purification and characterization of a naringinase from Aspergillus aculeatus JMUdb058.

A naringinase from Aspergillus aculeatus JMUdb058 was purified, identified, and characterized. This naringinase had a molecular mass (MW) of 348 kDa and contained four subunits with MWs of 100, 95, 84, and 69 kDa. Mass spectrometric analysis revealed that the three larger subunits were ß-D-glucosidases and that the smallest subunit was an α-L-rhamnosidase. The naringinase and its α-L-rhamnosidase and ß-D-glucosidase subunits all had optimal activities at approximately pH 4 and 50 °C, and they were stable between pH 3 and 6 and below 50 °C. This naringinase was able to hydrolyze naringin, aesculin, and some other glycosides. The enzyme complex had a K(m) value of 0.11 mM and a k(cat)/K(m) ratio of 14,034 s(-1) mM(-1) for total naringinase. Its α-L-rhamnosidase and ß-D-glucosidase subunits had K(m) values of 0.23 and 0.53 mM, respectively, and k(cat)/K(m) ratios of 14,146 and 7733 s(-1) mM(-1), respectively. These results provide in-depth insight into the structure of the naringinase complex and the hydrolyses of naringin and other glycosides.

Characterization of a new and thermostable esterase from a metagenomic library.

A new gene encoding an esterase (designated as EstEP16) was identified from a metagenomic library prepared from a sediment sample collected from a deep-sea hydrothermal field in east Pacific. The open reading frame of this gene encoded 249 amino acid residues. It was cloned, overexpressed in Escherichia coli, and the recombinant protein was purified to homogeneity. The monomeric EstEP16 presented a molecular mass of 51.7 kDa. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding highest specific activity with p-nitrophenyl acetate. When p-nitrophenyl butyrate was used as a substrate, recombinant EstEP16 exhibited highest activity at pH 8.0 and 60°C. The recombinant enzyme retained about 80% residual activity after incubation at 90°C for 6 h, which indicated that EstEP16 was thermostable. Homology modeling of EstEP16 was developed with the monoacylglycerol lipase from Bacillus sp. H-257 as a template. The structure showed an α/ß-hydrolase fold and indicated the presence of a typical catalytic triad. The activity of EstEP16 was inhibited by addition of phenylmethylsulfonyl fluoride, indicating that it contains serine residue, which plays a key role in the catalytic mechanism.

Development and evaluation of an HPLC method for accurate determinations of enzyme activities of naringinase complex.

An HPLC method that can separate naringin, prunin, and naringenin was used to help accurately measure the activities of naringinase and its subunits (α-L-rhamnosidase and ß-D-glucosidase). The activities of the naringinase and ß-d-glucosidase were determined through an indirect calculation of the naringenin concentration to avoid interference from its poor solubility. The measured enzymatic activities of the naringinase complex, α-L-rhamnosidase, and ß-D-glucosidase were the as same as their theoretical activities when the substrates' (i.e., naringin or prunin) concentrations were 200 µg/mL, and the enzyme concentrations were within the range of 0.06-0.43, 0.067-0.53, and 0.15-1.13 U/mL, respectively. The ß-D-glucosidase had a much higher Vmax than either naringinase or α-L-rhamnosidase, implying the hydrolysis of naringin to prunin was the limiting step of the enzyme reaction. The reliability of the method was finally validated through the repeatability test, indicating its feasibility for the determinations of the naringinase complex.

Characterization and preparation of Aspergillus niger naringinase for debittering citrus juice.

UNLABELLED: Naringinase from Aspergillus niger was prepared and characterized to evaluate its effectiveness in debittering citrus juice. The enzyme was purified to homogeneity by sulfate fractionation and chromatographies on Q-Sepharose, Sephacryl S-200, and S-100 HR columns, and estimated by gel filtration chromatography (GFC) to have a molecular weight (MW) of 131 kDa, of which its subunit was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be around 65.5 kDa. The enzyme showed active and stable pH ranges both within 4.5 to 5.0. Its optimal temperature was in the range of 45 to 55 °C. Freeze drying provided an estimated enzymatic recovery of 95.9%, greater than spray drying with the recovery at 55.6%. The freeze-drying powder could retain its enzymatic activity stably at 4 °C for 6 mo. Also, the enzyme in 0.220 U/mL citrus juice could sufficiently remove the naringin for the bitterness. Oral acute toxicity study revealed the maximum tolerated dose (MTD) of the naringinase powder was >10 g/kg in mice. The contents of arsenic (As), lead (Pb), mercury (Hg), the aerobic plate count, and coliform number in the enzyme powder all met the criteria for food use. These characteristics suggest that the naringinase from A. niger is efficient and suitable for debittering the citrus juice, and the process consisting of fermentation, salt precipitation, ion exchange, ultrafiltration, and freeze drying is a promising means to prepare the naringinase for food industry, setting up a strong base to enzymatically debitter citrus juice. PRACTICAL APPLICATION: This study focused on characterization, preparation, and validation of naringinase from A. niger, which provided useful information on how to prepare, store, and use the naringinase. In addition, this naringinase met the safety standards for food use and showed strong ability to remove the bitter taste from citrus juice, which provided useful information for interested readers, and the food industry.

Efficient immobilization of agarase using carboxyl-functionalized magnetic nanoparticles as support

Background: A simple and efficient strategy for agarase immobilization was developed with carboxyl-functionalized magnetic nanoparticles (CMNPs) as support. The CMNPs and immobilized agarase (agarase-CMNPs) were characterized by transmission electron microscopy, dynamic light scattering, vibrating sample magnetometry, scanning electron microscopy, X-ray diffraction, thermogravimetric analysis, and zeta-potential analysis. The hydrolyzed products were separated and detected by ESI-TOF-MS. Results: The agarase-CMNPs exhibited a regular spherical shape with a mean diameter of 12 nm, whereas their average size in the aqueous solution was 43.7 nm as measured by dynamic light scattering. These results indicated that agarase-CMNPs had water swelling properties. Saturation magnetizations were 44 and 29 emu/g for the carriers and agarase-CMNPs, respectively. Thus, the particles had superparamagnetic characteristics, and agarase was successfully immobilized onto the supports. Agaro-oligosaccharides were prepared with agar as substrate using agarase-CMNPs as biocatalyst. The catalytic activity of agarase-CMNPs was unchanged after six reuses. The ESI-TOF mass spectrogram showed that the major products hydrolyzed by agarase-CMNPs after six recycle uses were neoagarotetraose, neoagarohexaose, and neoagarooctaose. Meanwhile, the end-products after 90 min of enzymatic treatment by agarase-CMNPs were neoagarobiose and neoagarotetraose. Conclusions: The enhanced agarase properties upon immobilization suggested that CMNPs can be effective carriers for agarase immobilization. Agarase-CMNPs can be remarkably used in developing systems for repeated batch production of agar-derived oligosaccharides.

[Repeated batch and fed-batch process for astaxanthin production by Phaffia rhodozyma].

A comparative study of batch and repeated batch process was carried out for astaxanthin fermentation of Phaffia rhodozyma to develop a more economical method for astaxanthin industrial production. In shaking flask fermentation, the change of biomass and astaxanthin production was studied to compare the five-day cycle with four-day cycle of repeated batch culture of P. rhodozyma. Astaxanthin production increased at first and then decreased subsequently in seven cycles, yet the yield of astaxanthin in the next six cycles remains higher than that of the first cycle. Comparing the average production of astaxanthin in the seven cycles, four-day cycle performed even better than five-day cycle. Subsequently, a repeated fed-batch process was used in a 5-1 bioreactor. The experimental data showed that biomass and astaxanthin production of the second batch could reach the level of the first batch, no matter that the carbon source was glucose or hydrolysis sugar of starch. This result showed that this strain had good stability, and thus repeated batch and fed-batch process could be applied in astaxanthin fermentation for economical purpose.

[Characterization and evaluation of an astaxanthin over-producing Phaffia rhodozyma].

We evaluated an astaxanthin overproducing Phaffia rhodozyma JMU-MVP14, and developed astaxanthin high-yielding fermentation process. We analyzed several fermentation parameters, i.e., biomass, astaxanthin and total carotenoids content to compare the characteristics of P rhodozyma JMU-MVP14 and the original strain through flask fermentation experiments. We conducted batch and fed-batch fermentation experiments in 7 L fermentor to investigate the effects of pH controlling models and feeding medium compositions on the production of astaxanthin. We further evaluated the capability and practical value of P rhodozyma JMU-MVP14 by fed-batch cultivation in the 1 m3 fermentor. Flask fermentation experiments revealed that P. rhodozyma JMU-MVP14 produced high yield of astaxanthin and carotenoids with specific productivity of astaxanthin and specific productivity of total carotenoids of 6.01 mg/g and 10.38 mg/g. Results of batch culture experiments in the 7 L fermentor showed that controlling the pH by ammonia auto-feeding was better than discontinuously adjusting pH value at 6.0 with regard to the high productivities of biomasses and astaxanthin. This P. rhodozyma strain synthesized astaxanthin partially linked to the growth with the Ks and pmax of 0.20 h ' and 21.73 g/L, respectively. Results of batch-fed fermentations in 7 L fermentor indicated that the complex feeding medium consisted of 50% glucose, 0.5% yeast extract and 0.3% corn steep syrup had lower astaxanthin productivity than the simple feeding medium containing only 50% glucose, which produced biomass, volumetric productivity of astaxanthin, volumetric productivity of total carotenoids, specific productivity of astaxanthin and total carotenoids at 32.81 g/L, 155.99 mg/L, 4.94 mg/g, 399.99 mg/L and 12.19 mg/g, respectively. As fed-batch cultured in 1 m3 fermentor, P rhodozyma JMU-MVP14 yielded 85.11 g/L of biomass, 279.96 mg/L of volumetric productivity of astaxanthin, 618.01 mg/L of volumetric productivity of total carotenoids, 3.29 mg/g of specific productivity of astaxanthin and 7.26 mg/g of specific productivity of total carotenoids. Additionally, P rhodozyma JMU-MVP14 cell contained 21.54% of protein, 41.34% of carbohydrate and 34.31% of lipid. These comprehensive results suggest that P. rhodozyma JMU-MVPl14 has great practical prosperity related to its strong ability to produce astaxanthin and good value byproducts.

Preparation and characterization of κ-carrageenase immobilized onto magnetic iron oxide nanoparticles

Background Carboxyl-functionalized magnetic nanoparticles were synthesized via chemical co-precipitation method and modified with oleic acid which was oxidized by potassium permanganate, and κ-carrageenase from Pseudoalteromonas sp. ASY5 was subsequently immobilized onto them. The immobilization conditions were further optimized, and the characterizations of the immobilized κ-carrageenase were investigated. Results The κ-carrageenase was immobilized onto magnetic iron oxide nanoparticles, and the bonding was verified by Fourier transform infrared spectroscopy. The optimal conditions for κ-carrageenase immobilization were 2.5% (w/v) glutaraldehyde, 13.9 U κ-carrageenase for 20 mg of magnetic nanoparticles, a 2-h cross-linking time, and a 2-h immobilization time at 25°C. Under these conditions, the activity of the immobilized enzyme and the enzyme recovery rate were 326.0 U · g- 1 carriers and 46.9%, respectively. The properties of the immobilized κ-carrageenase were compared with those of the free enzyme. The optimum temperatures of the free and immobilized κ-carrageenase were 60 and 55°C, respectively, and the optimum pH of κ-carrageenase did not change before and after immobilization (pH 7.5). After immobilization, κ-carrageenase exhibited lower thermal stability and improved pH stability, as well as better storage stability. The immobilized κ-carrageenase maintained 43.5% of the original activity after being used 4 times. The kinetic constant value (Km) of κ-carrageenase indicates that the immobilized enzyme had a lower binding affinity for the substrate. Conclusions Under optimal conditions, the activity of the immobilized enzyme and enzyme recovery rate were 326.0 U · g- 1·κ-carrageenase-CMNPs and 46.9%, respectively. The thermal, pH, and storage stabilities of κ-carrageenase-CMNPs were relatively higher than those of free κ-carrageenase.