Purification and characterization of cutinase from Bacillus sp. KY0701 isolated from plastic wastes

A total of approximately 400 bacterial strains were isolated from 73 plastic wastes collected from 14 different regions. Nineteen isolates that form clear zones both on tributyrin and poly ε-caprolactone (PCL) agar, were identified based on 16S rRNA gene sequences. Among these, Bacillus sp. KY0701 that caused the highest weight loss of PCL films in minimal salt medium, was selected for cutinase production. The highest enzyme activity (15 U/mL) was obtained after 4 days of incubation at 50°C, pH 7.0 and 200?rpm in a liquid medium containing 1.5% (w/v) apple cutin and 0.1% (w/v) yeast extract. The purified enzyme was stable at high temperatures (50–70°C) and over a wide pH range (5.5–9.0). The relative activity of cutinase was at least 75% in the percent of various organic solvents. The apparent K_m and V_max values of the cutinase for p-nitrophenyl butyrate were 0.72?mM and 336.8?µmol p-nitrophenol/h/g, respectively. In addition, it showed high stability and compatibility with commercial detergents. These features of cutinase obtained from Bacillus sp. KY0701 make it a promising candidate for application in the detergent and chemical industries. In our best knowledge, this is the first report for cutinase production and characterization produced by a Bacillus strain.     

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。     

Effect of immobilization support, water activity, and enzyme ionization state on cutinase activity and enantioselectivity in organic media

We studied the reaction between vinyl butyrate and 2-phenyl-1-propanol in acetonitrile catalyzed by Fusarium solani pisi cutinase immobilized on zeolites NaA and NaY and on Accurel PA-6. The choice of 2-phenyl-1-propanol was based on modeling studies that suggested moderate cutinase enantioselectivity towards this substrate. With all the supports, initial rates of transesterification were higher at a water activity (a(w)) of 0.2 than at a(w) = 0.7, and the reverse was true for initial rates of hydrolysis. By providing acid-base control in the medium through the use of solid-state buffers that control the parameter pH-pNa, which we monitored using an organo-soluble chromoionophoric indicator, we were able, in some cases, to completely eliminate dissolved butyric acid. However, none of the buffers used were able to improve the rates of transesterification relative to the blanks (no added buffer) when the enzyme was immobilized at an optimum pH of 8.5. When the enzyme was immobilized at pH 5 and exhibited only marginal activity, however, even a relatively acidic buffer with a pK(a) of 4.3 was able to restore catalytic activity to about 20% of that displayed for a pH of immobilization of 8.5, at otherwise identical conditions. As a(w) was increased from 0.2 to 0.7, rates of transesterification first increased slightly and then decreased. Rates of hydrolysis showed a steady increase in that a(w) range, and so did total initial reaction rates. The presence or absence of the buffers did not impact on the competition between transesterification and hydrolysis, regardless of whether the butyric acid formed remained as such in the reaction medium or was eliminated from the microenvironment of the enzyme through conversion into an insoluble salt. Cutinase enantioselectivity towards 2-phenyl-1-propanol was indeed low and was not affected by differences in immobilization support, enzyme protonation state, or a(w).     

Arthrobacter sp. lipase immobilization for improvement in stability and enantioselectivity

Arthrobacter sp. lipase (ABL, MTCC no. 5125) is being recognized as an efficient enzyme for the resolution of drugs and their intermediates. The immobilization of ABL on various matrices for its enantioselectivity, stability, and reusability has been studied. Immobilization by covalent bonding on sepharose and silica afforded a maximum of 380 and 40 IU/g activity, respectively, whereas sol–gel entrapment provided a maximum of 150 IU/g activity in dry powder. The immobilized enzyme displayed excellent stability in the pH range of 4–10 and even at higher temperature, i.e., 50–60°C, compared to free enzyme, which is unstable under extreme conditions. The resolution of racemic auxiliaries like 1-phenyl ethanol and an intermediate of antidepressant drug fluoxetine, i.e., ethyl 3-hydroxy-3-phenylpropanoate alkyl acylates, provided exclusively R-(+) products (∼99% ee, E=646 and 473), compared to cell free extract/whole cells which gave a product with ∼96% ee (E=106 and 150). The repeated use (ten times) of covalently immobilized and entrapped ABL resulted in no loss in activity, thus demonstrating its prospects for commercial applications.     

Bioremediation of crude oil polluted seawater by a hydrocarbon-degrading bacterial strain immobilized on chitin and chitosan flakes

In this laboratory-scale study, we examined the potential of chitin and chitosan flakes obtained from shrimp wastes as carrier material for a hydrocarbon-degrading bacterial strain. Flakes decontamination, immobilization conditions and the survival of the immobilized bacterial strain under different storage temperatures were evaluated. The potential of immobilized hydrocarbon-degrading bacterial strain for crude oil polluted seawater bioremediation was tested in seawater microcosms. In terms of removal percentage of crude oil after 15 days, the microcosms treated with the immobilized inoculants proved to be the most successful. The inoculants formulated with chitin and chitosan as carrier materials improved the survival and the activity of the immobilized strain. It is important to emphasize that the inoculants formulated with chitin showed the best performance during storage and seawater bioremediation.     

Application of chitosan beads immobilized Rhodococcus sp. NCIM 2891 cholesterol oxidase for cholestenone production

The cell-bound cholesterol oxidase from the Rhodococcus sp. NCIM 2891 was purified three fold by diethylaminoethyl–sepharose chromatography. The estimated molecular mass (SDS-PAGE) and K_m of the enzyme were ∼55.0 kDa and 151 μM, respectively. The purified cholesterol oxidase was immobilized on chitosan beads by glutaraldehyde cross-linking reaction and immobilization was confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy and energy dispersive X-ray analysis. The optimum temperature (45 °C, 5 min) for activity of the enzyme was increased by 5 °C after immobilization. Both the free and immobilized cholesterol oxidases were found to be stable in many organic solvents except for acetone. Fe²⁺ and Pb²⁺ at 0.1 mM of each acted as inhibitors, while Ag⁺, Ca²⁺, Ni²⁺ and Zn²⁺ activated the enzyme at similar concentration. The biotransformation of cholesterol (3.75 mM) with the cholesterol oxidase immobilized beads (3.50 U) leads to ∼88% millimolar yield of cholestenone in a reaction time of 9 h at 25 °C. The immobilized enzyme retains ∼67% activity even after 12 successive batches of operation. The biotransformation method thus, shows a great promise for the production of pharmaceutically important cholestenone.     

Sulfonation of papain-treated chitosan and its mechanism for anticoagulant activity

The novel low-molecular-weight chitosan polysulfate (MW 5120-26,200 Da) was prepared using the depolymerization of chitosan with papain (EC. 3.4.22.2). The sulfonation of depolymerized products was performed using chlorosulfonic acid in N,N-dimethylformamide under semi-heterogeneous conditions. The structures of the products were characterized by FTIR, ¹³C NMR, and ¹H NMR (1D, 2D NMR) spectroscopy. The present study sheds light on the mechanism of anticoagulant activity of chitosan polysulfate. Anticoagulant activity was investigated by an activated partial thromboplastin assay, a thrombin time assay, a prothrombin time assay, and thrombelastography. Surface plasmon resonance also provided valuable data for understanding the relationship between the molecular binding of sulfated chitosan to two important blood clotting regulators, antithrombin III and heparin cofactor II. These results show that the principal mechanism by which this chitosan polysulfate exhibits anticoagulant activity is mediated through heparin cofactor II and is dependent on polysaccharide molecular weight.     

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.     

Preparation and characterization of chitosan encapsulated Sargassum sp. biosorbent for nickel ions sorption

A new biosorbent - Sargassum sp. encapsulated with epichlorohydrin (ECH) cross-linked chitosan (CS) was investigated for nickel ions removal. The prepared biosorbent with Sargassum sp. to cross-linked chitosan of 3 (weight ratio) had the highest sorption capacity. The biosorption kinetics can be well fitted by the diffusion-controlled model. The organic leaching of CS was 77-88% less than that of algae at different pH. The biosorption capacity of nickel on CS was much higher than that of cross-linked chitosan (CLC) bead and lower than that of raw algae due to encapsulation. In addition, the reusability of CS was further evaluated and confirmed through five adsorption-desorption cycles. Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) analysis demonstrated that the nickel ions sequestration mechanism included ion exchange and nickel complexation with the carboxyl, amino, alcoholic and ether groups in CS.     

Optimization of the masking molecule for active-site-protected immobilization of Taq DNA polymerase and its application

The method of oriented and activity-preserved immobilization of biologically active proteins based on concepts of active-site masking and kinetic control was further developed in this study. Minimal requirements for the masking DNA molecule were found to be a 5′overhang of 5–7 nucleotides and a double-stranded region of 11–13 bp to retain approximately 70% of the enzyme activity. The amplification range of protected immobilized (PIM) Taq DNA polymerase was over 1.2 kb. These data suggest that PIM Taq DNA polymerase can be used for various commercial applications.     

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%。     

Optimal immobilization of β-galactosidase from Pea (PsBGAL) onto Sephadex and chitosan beads using response surface methodology and its applications

Response surface methodology (RSM) and centre composite design (CCD) were used to optimize immobilization of β-galactosidase (BGAL) from Pisum sativum onto two matrices: Sephadex G-75 and chitosan beads. The immobilization efficiency of 75.66% and 75.19% were achieved with Sephadex G-75 and chitosan, respectively. There was broad divergence in physico-chemical properties of Sephadex-PsBGAL and chitosan-PsBGAL. Chitosan-PsBGAL was better suited for industrial application based on its broad pH and temperature optima, higher temperature stability, reusability etc. Sephadex-PsBGAL and chitosan-PsBGAL showed much variation in their catalytic properties with respect to soluble enzyme. About 50% loss in activity of Sephadex-PsBGAL and chitosan-PsBGAL were observed after 12 and 46 days at 4 °C, respectively. Chitosan-PsBGAL showed higher rate of lactose hydrolysis present in milk and whey at room temperature and 4 °C than Sephadex-PsBGAL. In both cases, lactose of milk whey was hydrolyzed at higher rate than that of milk.     

Improving the activity and stability of Yarrowia lipolytica lipase Lip2 by immobilization on polyethyleneimine-coated polyurethane foam

In this study, polyurethane foam (PUF) was used for immobilization of Yarrowia lipolytica lipase Lip2 via polyethyleneimine (PEI) coating and glutaraldehyde (GA) coupling. The activity of immobilized lipases was found to depend upon the size of the PEI polymers and the way of GA treatment, with best results obtained for covalent-bind enzyme on glutaraldehyde activated PEI-PUF (MW 70,000 Da), which was 1.7 time greater activity compared to the same enzyme immobilized without PEI and GA. Kinetic analysis shows the hydrolytic activity of both free and immobilized lipases on triolein substrate can be described by Michaelis–Menten model. The K_m for the immobilized and free lipases on PEI-coated PUF was 58.9 and 9.73 mM, respectively. The V_max values of free and immobilized enzymes on PEI-coated PUF were calculated as 102 and 48.6 U/mg enzyme, respectively. Thermal stability for the immobilization preparations was enhanced compared with that for free preparations. At 50 °C, the free enzyme lost most of its initial activity after a 30 min of heat treatment, while the immobilized enzymes showed significant resistance to thermal inactivation (retaining about 70% of its initial activity). Finally, the immobilized lipase was used for the production of lauryl laurate in hexane medium. Lipase immobilization on the PEI support exhibited a significantly improved operational stability in esterification system. After re-use in 30 successive batches, a high ester yield (88%) was maintained. These results indicate that PEI, a polymeric bed, could not only bridge support and immobilized enzymes but also create a favorable micro-environment for lipase. This study provides a simple, efficient protocol for the immobilization of Y. lipolytica lipase Lip2 using PUF as a cheap and effective material.     

Enhanced production of an alkaline pectinase from Streptomyces sp. RCK-SC by whole-cell immobilization and solid-state cultivation

An alkalophilic Streptomyces sp. RCK-SC, which produced a thermostable alkaline pectinase, was isolated from soil samples. Pectinase production at 45 °C in shaking conditions (200 rev min⁻¹) was optimal (76,000 IU l⁻¹) when a combination of glucose (0.25% w/v) and citrus pectin (0.25% w/v) was added along with urea (0.25% w/v) in the basal medium devoid of yeast extract and peptone. All the tested amino acids and vitamins greatly induced pectinase production and increased the specific productivity of pectinase up to 550%. In an immobilized cell system containing polyurethane foam (PUF), the pectinase production was enhanced by 32% (101,000 IU l⁻¹) compared to shake flask cultures. In solid-state cultivation (SSC) conditions, using wheat bran as solid substrate, pectinase yield of 4857 IU g⁻¹ dry substrate was obtained at substrate-to-moisture ratio of 1:5 after 72 h of incubation. The partially purified pectinase was optimally active at 60 °C and retained 80% of its activity at 50 °C after 2 h of incubation. The half life of pectinase was 3 h at 70 °C. Pectinase was stable at alkaline pH ranging from 6.0 to 9.0 for more than 8 h at room temperature retaining more than 50% of its activity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Synthesis of functionalized polyethylenimine-grafted mesoporous silica spheres and the effect of side arms on lipase immobilization and application

In the present study, silicate mesoporous materials (MCM-41), MCM-41-grafted polyethylenimine (MCM-41@PEI), and succinated PEI containing amine, amide, and acid groups were successfully synthesized and characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray diffraction, scanning electron microscopy, transmission electron microscopy, Brunauer–Emmett–Teller analysis, and X-ray photoelectron spectroscopy. Thermomyces lanuginosa lipase (TLL) was then immobilized onto MCM-41 and polymer-grafted MCM-41 by physical adsorption. Besides, for enzyme immobilization via covalent bonding, glutaraldehyde (GLU), and hexamethylene diisocyanate (HMDI) were used as the bridges for binding the enzyme to supports. The best result was obtained with the immobilized lipase on MCM-41@PEI-GLU. In the study of the enzyme reusability, it was shown that about 83% of the initial activity could be retained after 12 cycles of uses. The immobilized lipase on the selected support was also applied for the synthesis of ethyl valerate. Following 24 h incubation in n-hexane and solvent free media, the esterification percentages were 79% and 67%, respectively.     

Biodegradation of dipropyl phthalate and toxicity of its degradation products: a comparison of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">pisi</Emphasis> cutinase and <Emphasis Type="Italic">Candida cylindracea</Emphasis> esterase

The efficiency of two lypolytic enzymes (fungal cutinase, yeast esterase) in the degradation of dipropyl phthalate (DPrP) was investigated. The DPrP-degradation rate of fungal cutinase was surprisingly high, i.e., almost 70% of the initial DPrP (500 mg/l) was decomposed within 2.5 h and nearly 50% of the degraded DPrP disappeared within the initial 15 min. With the yeast esterase, despite the same concentration, more than 90% of the DPrP remained even after 3 days of treatment. During the enzymatic degradation of DPrP, several DPrP-derived compounds were detected and time-course changes in composition were also monitored. The final chemical composition after 3 days was significantly dependent on the enzyme used. During degradation with fungal cutinase, most DPrP was converted into 1,3-isobenzofurandione (IBF) by diester hydrolysis. However, in the degradation by yeast esterase, propyl methyl phthalate (PrMP) was produced in abundance in addition to IBF. The toxic effects of the final degradation products were investigated using various recombinant bioluminescent bacteria. As a result, the degradation products (including PrMP) from yeast esterase severely caused oxidative stress and damage to protein synthesis in bacterial cells, while in the fungal cutinase processes, DPrP was significantly degraded to non-toxic IBF after the extended period (3 days).     

Formulation of organic and inorganic hydrogel matrices for immobilization of β‐glucosidase in microfluidic platform

The aim of this study was to formulate silica and alginate hydrogels for immobilization of β‐glucosidase. For this purpose, enzyme kinetics in hydrogels were determined, activity of immobilized enzymes was compared with that of free enzyme, and structures of silica and alginate hydrogels were characterized in terms of surface area and pore size. The addition of polyethylene oxide improved the mechanical strength of the silica gels and 68% of the initial activity of the enzyme was preserved after immobilizing into tetraethyl orthosilicate–polyethylene oxide matrix where the relative activity in alginate beads was 87%. The immobilized β‐glucosidase was loaded into glass–silicon–glass microreactors and catalysis of 4‐nitrophenyl β‐d ‐glucopyranoside was carried out at various retention times (5, 10, and 15 min) to compare the performance of silica and alginate hydrogels as immobilization matrices. The results indicated that alginate hydrogels exhibited slightly better properties than silica, which can be utilized for biocatalysis in microfluidic platforms.     

Identification of novel denitrifying bacteria Stenotrophomonas sp. ZZ15 and Oceanimonas sp. YC13 and application for removal of nitrate from industrial wastewater

Two novel denitrifying bacteria were successfully isolated from industrial wastewater and soil samples. Using morphological, biochemical/biophysical and 16S rRNA gene analyses, these two bacteria were identified as Stenotrophomonas sp. ZZ15 and Oceanimonas sp. YC13, respectively. Both of these two bacteria showed efficient NO₃ ⁻-N removing abilities under a semi-anaerobic condition without obvious accumulation of NO₂ ⁻-N, N₂O-N and NH₄ ⁺-N. NO₃ ⁻-N removal from paper mill wastewater was also successful by treatments with either a denitrifier or an immobilization method. Therefore, this study provides valuable denitrifying bacteria in biotreatment of industrial wastewater and other environmental pollution caused by NO₃ ⁻/NO₂ ⁻.     

T2-2菌株对多菌灵的降解特性及生物修复试验

摘要：【目的】为获得降解多菌灵的微生物菌株,并用其制备生物修复剂,修复被污染的土壤。【方法】从耐药性木霉菌株诱变选育过程中,得到一株能降解多菌灵的变异菌株T2-2。该菌株在多菌灵浓度100 mg/L无机盐培养基中, 于25℃、200 r/min振荡培养取样,用HPLC-MS检测代谢产物;以玉米秸秆粉为原料经固体发酵制成T2-2生物修复剂;采用土壤人工接种,在T2-2菌剂接种量为107cfu/g干土、多菌灵含量为0.1 mg/g干土时进行灭菌土和自然土壤的修复试验;另外,还做了T2-2菌剂对黄瓜枯萎病的活体防效试验。【结果】处理2 d的培养液,HPLC-MS检测出代谢产物为：2-氨基苯并咪唑,苯并咪唑和2-氨基苯腈,处理5 d的培养液经检测未发现多菌灵和代谢产物;土壤修复试验中,灭菌土壤中的多菌灵接种6 d被完全降解,而自然土壤中的多菌灵被完全降解缩短到4 d。说明秸秆粉作为共代谢底物,促进了T2-2和土著微生物的共代谢降解作用;另外,T2-2菌剂对黄瓜枯萎病的活体防治效果达到81.7%,优于化学农药。【结论】木霉T2-2菌株即可降解土壤中的多菌灵,又可防治植物病害。     

Identification of Paenibacillus sp. 2S-6 and application of its xylanase on biobleaching

A Paenibacillus sp. strain 2S-6 was isolated from the black liquor of the first brownstock washing stage of kraft pulping process and identified by its 16S rDNA sequence. This bacterial strain utilized a variety of saccharides and polysaccharides as carbon source, but neither lignin nor lipids. Crude xylanase from Paenibacillus sp. 2S-6 was produced in a 5 L laboratory fermenter at 37 °C, pH 7. After 24 h, up to 10.5 IU xylanase per mg of protein in the crude extract of fermentation broth was obtained. After two-stage ultrafiltration, the optimal activity of partially purified xylanase reached 60.51 IU/mg at 50 °C, pH 6. A major band indicating molecular weight of 33 kDa was shown on SDS-PAGE for the partially purified xylanase. After 4 h at 60 °C, 48.99% and 31.25% residual xylanase activities were demonstrated at pH 7 and 9, respectively. Efficacy of its xylanase on the bleaching agent saving was demonstrated by using 5 IU xylanase per gram oven-dried pulp prior to bleaching, referred as biobleaching. Identical levels of brightness and higher levels of viscosity were obtained for the xylanase pretreated eucalypt kraft pulps followed by a 20% reduction of the bleaching agent dosage in the first step of a commercial C₇₀/D₃₀-E_o-D bleaching sequence.