Kinetics of an extracellular exo-inulinase production from a 5-flourocytosine resistant mutant of Geotrichum candidum using two-factorial design

In the present study, eight different strains of Geotrichum candidum were isolated and screened for an extracellular exo-inulinase production using chemically enriched sucrose–mineral media. The isolate (Zool-3i) with a better enzyme activity (1.38 IU/ml) was subjected to induced mutagenesis using methyl methane sulphonate (MMS) and a mutant with an enzyme activity of 32.06 IU/ml was obtained. Further exposure to ethyl methane sulphonate (EMS) and ultraviolet (UV) radiations yielded a mutant exhibiting an improved activity of 39.34 IU/ml. The potential mutant was cultured overnight and plated on 5fc–YPR agar medium and thus made resistant against 5-flourocytocine. Over 50-fold enhancement in enzyme production (71.85 IU/ml) was achieved when the process parameters including incubation period (48 h), sucrose concentration (5.0 g/L), pH (6.0), inoculum size (2.0%, 16 h old) and urea (0.2%) were identified using Plackett–Burman design. On the basis of kinetic variables, notably Q_p (0.723 U/g/h), Y_p/s (2.036 U/g) and q_p (0.091 U/g cells/h), the mutant MEU-5fc-6 was found to be a hyper producer of exo-inulinase (HS, LSD 0.045, p ? 0.05).     

Purification and properties of thermostable lipase from a thermophilic Bacillus stearothermophilus MC 7

Extracellular thermostable lipase produced by the thermophilic Bacillus stearothermophilus MC 7 was purified to 19.25-fold with 10.2% recovery. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was shown to be 62 500 Da. The purified enzyme expressed maximum activity at 75–80 °C and its half life was 30 min at 70 °C. The K_m and V_max were calculated to be, respectively, 0.33 mM and 188 μM min⁻¹ mg⁻¹ with p-nitrophenyl palmitate (pNPP) as a substrate. Enzyme activity was inhibited by divalent ions of heavy metals, thiol and serine inhibitors, whereas calcium ion stimulated its activity. The most advantageous method for immobilization was found to be ionic binding to DEAE Cellulose. The enzyme was able to hydrolyze both soluble and insoluble emulsified substrates and was classified as a lipase, expressing some esterase activity as well.     

Immobilization of laccase from Streptomyces psammoticus and its application in phenol removal using packed bed reactor

Laccase was produced from Streptomyces psammoticus under solid-state fermentation. The enzyme was partially purified by ammonium sulphate precipitation and was immobilized in alginate beads by entrapment method. Calcium alginate beads retained 42.5% laccase activity, while copper alginate beads proved a better support for laccase immobilization by retaining 61% of the activity. Phenol and colour removal from a phenol model solution was carried out using immobilized laccase. Batch experiments were performed using packed bed bioreactor, containing immobilized beads. Reusability of the immobilized matrix was studied for up to 8 successive runs, each run with duration of 6 h. The system removed 72% of the colour and 69.9% of total phenolics from the phenol model solution after the initial run. The immobilized system maintained 50% of its efficiency after eight successive runs. The degradation of phenolic compounds by immobilized laccase was evaluated and confirmed by Thin layer chromatography and nuclear magnetic resonance spectroscopy.     

Immobilization of esterase SulE in cross-linked gelatin/chitosan and its application in remediating soils polluted with tribenuron-methyl and metsulfuron-methyl

The esterase SulE is known to be an important and primary enzyme for the degradation of many sulfonylurea herbicides such as tribenuron-methyl, metsulfuron-methyl, and chlorimuron-ethyl. The disadvantages of applying free enzyme include poor stability and rapid deactivation in the natural environment, but these problems can be mitigated by using immobilized enzymes. In this study, cross-linked gelatin/chitosan (GLT/CTS) was selected as the enzyme immobilization carrier because its SulE activity recovery was higher than those of γ-polyglutamate/gelatin (γ-PGA/GLT) and γ-polyglutamate/chitosan (γ-PGA/CTS). The immobilization conditions were further optimized, and the effects of pH and temperature on GLT/CTS-SulE and free SulE activities to degrade tribenuron-methyl and metsulfuron-methyl were investigated. A microcosm experiment was conducted to assess the effectiveness of GLT/CTS-SulE in remediating soils polluted with tribenuron-methyl and metsulfuron-methyl. The results showed that, compared with free SulE, GLT/CTS-SulE displayed better pH and temperature adaptabilities and higher degradation efficiencies for both herbicides in soil, especially tribenuron-methyl. The results indicate that this is an excellent method to immobilize SulE for enhanced remediation of soils polluted with tribenuron-methyl or metsulfuron-methyl in situ.     

Immobilization of cellulase onto electrospun polyacrylonitrile (PAN) nanofibrous membranes and its application to the reducing sugar production from microalgae

This study demonstrates a method to prepare an immobilized cellulase by using an electrospun polyacrylonitrile (PAN) nanofibrous membrane as the support. To obtain an immobilized cellulase with high hydrolytic activity, the immobilization conditions including activation time, enzyme concentration, immobilization time, and temperature were optimized. Under those conditions, the immobilized cellulase possessed a protein loading of 30 mg/g-support and a specific activity of 3.2 U/mg-protein. After immobilization, the enzymatic stability of cellulase against pH and thermal stresses was improved. Fourier transform infrared spectroscopy (FTIR) measurements also revealed that the cellulase was covalently bonded to the supports. The immobilized cellulase was then used to hydrolyze cell wall of microalgae for the production of reducing sugars. Analyses using response surface methodology (RSM) show that the hydrolysis yield was affected by the reaction temperature, pH, and substrate/cellulase mass ratio, and a hydrolysis yield of 60.86% could be obtained at 47.85 °C, pH 5.82, and a substrate/cellulase mass ratio of 40 g-substrate/g-cellulase. This result suggests that the proposed scheme for the cellulase immobilization has great potential for the application to the reducing sugar production.     

漆酶在磁性壳聚糖微球上的固定及其酶学性质研究

以磁性壳聚糖微球为载体,戊二醛为交联剂,共价结合制备固定化漆酶。探讨了漆酶固定化的影响因素,并对固定化漆酶的性质进行了研究。确定漆酶固定化适宜条件为：50 mg磁性壳聚糖微球,加入10mL 0.8mg/mL 漆酶磷酸盐缓冲液（0.1mol/L,pH 7.0）,在4℃固定2h。固定化酶最适pH为3.0, 最适温度分别为10℃和55℃,均比游离酶降低5℃。在pH 3.0,温度37℃时,固定化酶对ABTS的表观米氏常数为171.1μmol/L。与游离酶相比,该固定化漆酶热稳定性明显提高,并具有良好的操作和存储稳定性。     

Immobilization of a lipoxygenase in silica gels for application in aqueous media

The encapsulation of soybean lipoxygenase-1 (LOX-1) in silica gels and its application in an aqueous medium, were studied. The main silica precursor was tetramethoxysilane (TMOS) but the introduction of hydrophobic SiCH₃ groups brought with methyltrimethoxysilane (MTMS) was evaluated. Other sol–gel synthesis parameters investigated comprised partial or complete drying by evaporation and CO₂ supercritical drying. The influence on LOX-1 activity of the various chemicals with which the enzyme was in contact during encapsulation (acetone, methanol, polyvinyl alcohol), as well as the temperature and pH, were examined. The activity of free and encapsulated LOX-1 was assayed on the oxygenation reaction of linoleic acid by dioxygen from air dissolved in aqueous medium, in a UV–vis spectrophotometer. With free LOX-1, the reaction advancement could be followed in continuous in the spectrophotometer. With the gels, in a first approach, the conversion was simply determined after 15 min reaction after filtration of the liquid, to discriminate between active and inactive gels. For the most interesting gels, the kinetics were then assessed by continuous recording in the UV spectrophotometer, after placing a small piece of gel (≈15 mg) directly in the cell. The best gels had an activity ≈30% of free LOX. The present studies, supplemented by characterization of the gels texture and structure, respectively by nitrogen adsorption and ²⁹Si MAS NMR, showed that drying a gel before use in aqueous media was detrimental to the activity. This effect is due to a contraction of the gel network which occurs when a dry aerogel sample is dipped in water after drying. Hence gels containing LOX-1 enzyme must not be dried but kept in water impregnated state, for optimum use.     

Inulinase overproduction by a mutant of the marine yeast Pichia guilliermondii using surface response methodology and inulin hydrolysis

In this study, in order to isolate inulinase overproducers from the marine yeast Pichia guilliermondii, its cells were treated by using UV light and LiCl. The mutant M-30 with enhanced inulinase production was obtained and was found to be stable after cultivation for 20 generations. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant M-30 in liquid fermentation. Inulin, yeast extract, NaCl, temperature, pH for maximum inulinase production by the mutant M-30 were found to be 20.0 g/l, 5.0 g/l, 20.0 g/l, 28 °C and 6.5, respectively. Under the optimized conditions, 127.7 U/ml of inulinase activity was reached in the liquid culture of the mutant M-30 whereas the predicted maximum inulinase activity of 129.8 U/ml was derived from RSM regression. Under the same conditions, its parent strain only produced 48.1 U/ml of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far. We also found that inulin could be actively converted into monosaccharides by the crude inulinase.     

Production of novel alkalitolerant and thermostable inulinase from marine actinomycete Nocardiopsis sp. DN-K15 and inulin hydrolysis by the enzyme

An actinomycete strain Nocardiopsis sp. DN-K15 showing high inulinolytic activity was isolated from marine sediment of Jiaozhou Bay in China. Under optimal conditions, Nocardiopsis sp. DN-K15 produced 25.1 U/ml of inulinase within 60 h of fermentation at shake flask level, which was 2.7-fold higher than the level in the basal medium. The optimal pH and temperature of the inulinase from strain DN-K15 were determined to be 60 °C and pH 8.0, respectively. The inulinase was highly active over a wide pH range (5.0–11.0) and retained more than 81 % of residual activity after incubation at 60 °C for 1 h, indicating its alkali-tolerant and thermostable nature. Thin layer chromatography analysis revealed that fructose was the main product of inulin hydrolysis, indicating its exoinulinase activity. The high yield of extracellular inulinase combined with its novel enzymatic property made Nocardiopsis sp. DN-K15 a potential candidate in biotechnological and industrial applications.     

Immobilization of diverse foreign proteins in viral polyhedra and potential application for protein microarrays

Cypoviruses are insect viruses that produce a cytoplasmic crystalline particle called the polyhedron in which progeny virions are occluded. The virion structural protein, VP3, is implicated in the occlusion of viral particles into polyhedra. In this study, we determined the amino acid sequence of VP3 required for occlusion of viral particles into polyhedra and proposed that this sequence could be used as an immobilization signal to direct the stable incorporation of foreign proteins into polyhedra. A large-scale survey revealed that the immobilization signal could, in fact, direct the incorporation of a variety of human proteins into polyhedra. Immune reactivity and protein-protein interactions were detected on the surface of polyhedra containing immobilized foreign proteins, and these particles were shown to be highly stabilized against dehydration. We showed that these particles could be arrayed onto a glass slide by standard spotting and laser manipulation methods. Thus, this approach is well suited for protein expression, purification, and the development of protein microarrays.     

Immobilization of epoxide hydrolase from Aspergillus niger onto DEAE-cellulose: enzymatic properties and application for the enantioselective resolution of a racemic epoxide

Recombinant epoxide hydrolase (EH) from Aspergillus niger can be a very promising tool for the resolution of various racemic epoxides by enantioselective hydrolysis. The enzyme was successfully immobilized by ionic adsorption onto DEAE-cellulose (99% yield, 70% of retention activity). The temperature for maximal activity (40 °C) and the activation energy (38.8 kJ/mol) were similar for both the immobilized and free EHs, whereas the optimal pH was about one unit less for the immobilized enzyme. Thermal stability was also affected by immobilization; the immobilized enzyme appeared to be slightly less stable than the free one. However, a gram-scale resolution of racemic para-chlorostyrene oxide (pCSO) was successfully carried out in a repeated batch reactor, operated for seven cycles. Furthermore, using a very high substrate concentration of 2 M (306 g/L), i.e. biphasic conditions, the resolution of 3 g of pCSO was also achieved in a repeated batch reactor using approximately 300 mg of immobilized EH, corresponding to less than 3 mg of the enzymatic powder.     

Determination and characterization of thermostable esterolytic activity from a novel thermophilic bacterium Anoxybacillus gonensis A4

A novel hot spring thermophile, Anoxybacillus gonensis A4 (A. gonensis A4) was investigated in terms of capability of tributyrin degradation and characterization of its thermostable esterase activity by the hydrolysis of p-nitrophenyl butyrate (PNPB). It was observed that A. gonensis A4 has an esterase with a molecular weight of 62 kDa. The extracellular crude preparation was characterized in terms of substrate specificity, pH and temperature optima and stability, kinetic parameters and inhibition/activation behaviour towards some chemicals and metal ions. Tributyrin agar assay showed that A. gonensis A4 secreted an esterase and V(max) and K(m) values of its activity were found to be 800 U/L and 176.5 microM, respectively in the presence of PNPB substrate. The optimum temperature and pH, for A. gonensis A4 esterase was 60-80 degrees C and 5.5, respectively. Although the enzyme activity was not significantly changed by incubating crude extract solution at 30-70 degrees C for 1 h, the enzyme activity was fully lost at 80 degrees C for same incubation period. The pH-stability profile showed that original crude esterase activity increased nearly 2-fold at pH 6.0. The effect of some chemicals on crude esterase activity indicated that A. gonensis A4 produce an esterase having serine residue in active site and -SH groups were essential for its activity.     

Immobilization of acetylcholinesterase in nanofibrous PVA/BSA membranes by electrospinning

Electrospinning, a simple and versatile method to fabricate nanofibrous supports, has attracted continuous attention in the field of enzyme immobilization. In this study, acetylcholinesterase (AChE) has been successfully immobilized in PVA nanofibers via electrospinning of a mixture of AChE, BSA as an enzyme stabilizing additive and PVA. The maximum activity recovery of immobilized AChE was about 40%. In comparison with free enzyme, the immobilized AChE showed improved stability while retaining a considerable amount of activity at lower pH values. Moreover, the immobilized AChE retained >34% of its initial activity when stored at 30°C for 100 days and retained 70% of its initial activity after ten consecutive reactor batch cycles.     

漆酶固定化及其在小型酶反应器中的应用

以尼龙网为载体,戊二醛为交联剂,固定化真菌漆酶。用固定化酶在自行设计的小反应器中降解低浓度造纸废水,试验结果如下：当废水COD的浓度控制在3000 mg/L左右,降解时间为26h,废水的COD去除率达到35%。     

Immobilization of Candida rugosa lipase on sporopollenin from Lycopodium clavatum

Sporopollenin is a natural polymer obtained from Lycopodium clavatum, which is highly stable with constant chemical structure and has high resistant capacity to chemical attack. In this study, immobilization of lipase from Candida rugosa (CRL) on sporopollenin by adsorption method is reported for the first time. Besides this, the enzyme adsorption capacity, activity and thermal stability of immobilized enzyme have also been investigated. It has been observed that under the optimum conditions (Spo-E_(0.3)), the specific activity of the immobilized lipase on the sporopollenin by adsorption was 16.3 U/mg protein, which is 0.46 times less than that of the free lipase (35.6 U/mg protein). The pH and temperature of immobilized enzyme were optimized, which were 6.0 and 40 °C respectively. Kinetic parameters V_max and K_m were also determined for the immobilized lipase. It was observed that there is an increase of the K_m value (7.54 mM) and a decrease of the V_max value (145.0 U/mg-protein) comparing with that of the free lipase.     

Immobilization of Lipase from Rhizopus Niveus: A Way to Enhance its Synthetic Activity in Organic Solvent

Lipase (EC 3.1.1.3) from Rhizopus niveus was immobilized by physical adsorption on various carriers, including different types of Celite, Spherosil and Duolite. After the enzyme immobilization, the recovered hydrolytic and synthetic activities on the different carriers were then determined. The results showed that the highest synthetic activity was obtained when Duolite XAD 761 was used as the carrier. However the recovered hydrolytic activity after the immobilization on this resin was relatively low although this carrier showed the best protein loading capacity. The highest recovered hydrolytic activity was observed when the lipase was immobilized on Celite Hyflo-Supercel using an immobilization buffer adjusted to pH 4. The comparison of the free and immobilized lipase specific activities suggest that the immobilization on Celite Hyflo-Supercel, Spherosil XOA 200 and silica has enhanced the lipase hydrolytic activity. On the other hand, the use of the lipase immobilized on Duolite XAD 761 as biocatalyst of synthetic reaction, compared to that of the free enzyme, allows the reaction initial velocity to be increased 12.2-fold. In addition, the synthetic activity of the lipase immobilized on Duolite XAD 761 was shown to be maximum at a water activity in the range of 0.32-0.52.     

Enhanced expression of the codon-optimized exo-inulinase gene from the yeast Meyerozyma guilliermondii in Saccharomyces sp. W0 and bioethanol production from inulin

In the present study, after the exo-inulinase gene INU1 from Meyerozyma guilliermondii was optimized according to the codon usage bias of Saccharomyces cerevisiae, both the optimized gene INU1Y and the native gene INU1 were ligated into the homologous integration expression vector pMIRSC11 and expressed in Saccharomyces sp. W0. It was determined that the inulinase activity of the recombinant yeast Y13 with the optimized gene INU1Y was 43.84 U/mL, which was obviously higher than that (31.39 U/mL) produced by the recombinant yeast EX3 with the native gene INU1. Moreover, it was indicated that the recombinant yeast Y13 could produce 126.30 mg/mL ethanol from 300.0 g/L inulin while the recombinant yeast EX3 and Saccharomyces sp. W0 produced 122.75 mg/mL and 114.15 mg/mL ethanol, respectively, under the same conditions. In addition, the ethanol productivity of the recombinant yeast Y13 was 2.25 mg/mL/h within 48 h of the fermentation, which was obviously higher than that of the recombinant yeast EX3 (1.97 mg/mL/h) and Saccharomyces sp. W0 (1.77 mg/mL/h) within the same period. The results demonstrated that the recombinant yeast Y13 had higher ethanol production and productivity than the recombinant yeast EX3 and Saccharomyces sp. W0. Therefore, it was concluded that the codon optimization of the exo-inulinase gene from M. guilliermondii effectively enhanced inulinase activity and improved ethanol production from inulin by Saccharomyces sp. W0 carrying the optimized inulinase gene.     

Immobilization of glucoamylase by adsorption on carbon supports and its application for heterogeneous hydrolysis of dextrin

Glucoamylase (GA) was immobilized by adsorption on carbon support: on Sibunit, on bulk catalytic filamentous carbon (bulk CFC) and on activated carbon (AC). This was used to prepare heterogeneous biocatalysts for the hydrolysis of starch dextrin. The effect of the texture characteristics and chemical properties of the support surface on the enhancement of the thermal stability of the immobilized enzyme was studied, and the rates of the biocatalyst's thermal inactivation at 65-80 degrees C were determined. The thermal stability of glucoamylase immobilized on different carbon supports was found to increase by 2-3 orders of magnitude in comparison with the soluble enzyme, and decrease in the following order: GA on Sibunit>GA on bulk CFC>GA on AC. The presence of the substrate (dextrin) was found to have a significant stabilizing effect. The thermal stability of the immobilized enzyme was found to increase linearly when the concentration of dextrin was increased from 10 wt/vol % to 50 wt/vol %. The total stabilization effect for glucoamylase immobilized on Sibunit in concentrated dextrin solutions was about 10(5) in comparison with the enzyme in a buffer solution. The developed biocatalyst, 'Glucoamylase on Sibunit' was found to have high operational stability during the continuous hydrolysis of 30-35 wt/vol % dextrin at 60 degrees C, its inactivation half-time (t1/2) exceeding 350 h. To improve the starch saccharification productivity, an immersed vortex reactor (IVR) was designed and tested in the heterogeneous process with the biocatalyst 'Glucoamylase on Sibunit'. The dextrin hydrolysis rate, as well as the process productivity in the vortex reactor, was found to increase by a factor of 1.2-1.5 in comparison with the packed-bed reactor.     

Production of a thermostable metal-tolerant laccase from Trametes versicolor and its application in dye decolorization

Production of laccase using a submerged culture of Trametes versicolor sdu-4 was optimized using a central composite design of the Response Surface Methodology. Optimized conditions gave a laccase yield of 4,213 U/L which was approximately three times of that in basal medium. The laccase was purified to homogeneity using a three-step process. The overall yield of the purification was 58%, with a purification fold of 11.4 and a specific activity of 1320.7 U/mg protein. The molecular mass of the laccase was 60 kDa. The optimum pH values of the enzyme were 2.2, 3.7, and 7 for the oxidations of ABTS, DMP, and syringaldazine, respectively. The enzyme had adaptability to a broad pH range and high temperature and wsa stable at pH 3.0 ∼ 10.0. The half-life of this laccase at 70°C was 2.2 h. Methyl red, 2-bromophenol, and 4-bromophenol were oxidized by the purified laccase in the absence of mediators. Purified laccase was effective in the decolorization of several dyes and was not inhibited by Cu²⁺, Mn²⁺, Zn²⁺, Na⁺, K⁺, Mg²⁺, Ba²⁺, and Ca²⁺ at 5 mM. These excellent characteristics made it a highly attractive candidate for industrial use.     

嗜热真菌Thermomyces lanuginosus热稳定几丁质酶基因的cDNA克隆及表达

根据Thermomyces lanuginosus热稳定几丁质酶Chit的N-端氨基酸序列和同源保守序列设计简并引物,通过RT-PCR及快速扩增cDNA末端(RACE)的方法,克隆了该几丁质酶的编码基因chit,全长cDNA为1500bp,包含一个由442个氨基酸组成的开放阅读框。该基因已在GenBank中注册,登录号为DQ092332。将成熟肽几丁质酶Chit阅读框与酵母表达载体pPIC9K连接,构建重组质粒pPIC9K/chit,转化毕赤酵母GS115,在甲醇的诱导下,成功地分泌出具生物活性的几丁质酶,诱导6d后酶活性达2.261U/mL,酶蛋白表达量为0.36mg/mL。该酶的最适反应温度和pH值分别为60℃和5.5,该酶在50℃以下稳定;65℃的半衰期为40min。