Adsorptive Immobilization of a Pseudomonas Strain on Solid Carriers for Augmented Decolourization in a Chemostat Bioreactor

Pseudomonas GM3, a highly efficient strain in cleavage of azo bonds of synthetic dyes under anoxic conditions, was immobilized via adsorption on two types of carriers, porous glass beads and solid PVA particles. The cells were cultivated in a nutrient medium, adsorbed on sterile carriers, stabilized as biofilms in repeated batch cultures, and introduced into a chemostat activated sludge reactor for augmented decolourization. The microbial cells were quickly adsorbed and fixed on the PVA surface, compared to a slow and linear immobilization on the glass surface. The porous structure of glass beads provided shelter for the embedded cells, giving a high biomass loading or thick biofilm (13.3 mg VS ml^?1 carrier) in comparison with PVA particles (4.8 mg VS ml^?1 carrier), but the mass transfer of substrate in the biofilm became a significant limiting factor in the thicker biofilms (effectiveness factor η = 0.31). The microbial decolourization rate per volume of carriers was 0.15 and 0.17 mg dye ml^?1 of glass beads and PVA particles, respectively. In augmented decomposition of a recalcitrant azo dye (60 mg l^?1), the immobilized Pseudomonas cells in porous glass beads gave a stable decolourization efficiency (80 - 81%), but cells fixed on solid PVA particles showed an initial high colour removal of 90% which then declined to a stable removal efficiency of 81%. In both cases, the colour removal efficiency of the chemostat bioreactor was increased from < 10% by an activated sludge to ～80% by the augmented system.     

Electrospun polyvinyl alcohol/bovine serum albumin biocomposite membranes for horseradish peroxidase immobilization

Electrospinning, a simple and versatile method to fabricate nanofibrous supports, has attracted attention in the field of enzyme immobilization. Biocomposite nanofibers were fabricated from mixed PVA/BSA solution and the effects of glutaraldehyde treatment, initial BSA concentration and PVA concentration on protein loading were investigated. Glutaraldehyde cross-linking significantly decreased protein release from nanofibers and BSA loading reached as high as 27.3% (w/w). In comparison with the HRP immobilized into the nascent nanofibrous membrane, a significant increase was observed in the activity retention of the enzyme immobilized into the PVA/BSA biocomposite nanofibers. The immobilized HRP was able to tolerate much higher concentrations of hydrogen peroxide than the free enzyme and thus the immobilized enzyme did not demonstrate substrate inhibition. The immobilized HRP retained ⿼50% of the free enzyme activity at 6.4 mM hydrogen peroxide and no significant variation was observed in the KM value of the enzyme for hydrogen peroxide after immobilization. In addition, reusability tests showed that the residual activity of the immobilized HRP were 73% after 11 reuse cycles. Together, these results demonstrate efficient immobilization of HRP into electrospun PVA/BSA biocomposite nanofibers and provide a promising immobilization strategy for biotechnological applications.     

固定化微生物在生物膜式生化需氧量传感器中的应用现状

生化需氧量(Biochemical oxygen demand,BOD)微生物传感器是一种快速检测水样中有机污染物含量的设备,固定化微生物是其核心部件之一,对其稳定性、响应时间、使用寿命及实际应用范围等性能有着重要影响。生物膜式BOD传感器较其他类型的BOD微生物传感器具有结构简单、灵敏度高、响应时间短等优点,受到广泛的研究和应用。本文主要针对固定化微生物在生物膜式BOD传感器中的应用情况,概述较典型的微生物固定化方式的原理、特点及应用;总结几类应用较多或具有较好前景的载体材料,并讨论载体特性与传感器性能之间的关系;综述微生物在该领域的应用现状;简要介绍生物膜式BOD传感器的实际应用及商业化现状,比较其与另外几种BOD微生物传感器的优缺点;分析生物膜式BOD传感器中固定化微生物现存的一些问题及其发展趋势。     

添加剂对大孔吸附树脂固定化脂肪酶的影响

利用大孔吸附树脂DA-201为载体对海洋脂肪酶固定化,并探寻添加剂对固定化过程的影响。分别以NH_4Cl、甘露糖和甘氨酸为添加剂,采用单因素和正交实验相结合的方法优化条件。结果显示,以NH_4Cl为添加剂的最优条件:柠檬酸-柠檬酸钠缓冲液pH 6. 0,固定化温度30℃,载体投放量0. 5g,NH_4Cl浓度25mmol/L,固定化时间3. 0h,酶活力达到115. 27U/g;比不含有添加剂的固定化酶固定化效率提高47. 42%。以甘露糖为添加剂最优条件:磷酸二氢钾-氢氧化钠缓冲液pH7. 0,固定化温度35℃,载体投放量0. 5g,甘露糖浓度10mmol/L,固定化时间4. 5h;酶活力达到122. 75U/g,比不含有添加剂的固定化酶固定化效率提高6. 50%。以甘氨酸为添加剂的最优条件:磷酸二氢钾-氢氧化钠缓冲液pH7. 0,固定化温度20℃,载体投放量0. 5g,甘氨酸浓度为25mmol/L,固定化时间7. 5h;酶活力达到141. 69U/g,比不含有添加剂的固定化酶固定化效率提高26. 12%。采用不同添加剂对大孔吸附树脂DA-201的吸附固定化过程有较大影响,可以极大地提高吸附效率;同时发现缓冲液类型、pH、温度、添加剂浓度和固定化时间等对DA-201树脂吸附脂肪酶有很大影响,对后续吸附固定化工业酶研究有较好的参考价值。     

Hydrolysis of soluble starch using Bacillus licheniformis alpha-amylase immobilized on superporous CELBEADS

In the present work, indigenously prepared rigid superporous (pore size of approximately 3 microm) cross-linked cellulose matrix (CELBEADS) has been used as a support for the immobilization of Bacillus licheniformis alpha-amylase (BLA). Optimum pH and temperature, and Michaelis-Menten constants were determined for both free and immobilized BLA. Immobilized BLA was observed to produce a different saccharide profile than free BLA at any value of dextrose equivalent. It was observed that pH, temperature, and initial starch concentration has a significant effect on the saccharide profile of starch hydrolysate produced using immobilized BLA in the batch mode, whereas the ratio of concentration of enzyme units to initial starch concentration has no influence on the same. Hence immobilized BLA can be used as an additional tool for production of maltodextrins with different saccharide profiles. Immobilized BLA has better thermostability than free BLA. Immobilized BLA was found to retain full activity even after eight batches of hydrolysis, each of 8h duration at 55 degrees C and 90 mg/mL initial starch concentration. A semiempirical model has been used for the prediction of saccharide composition of starch hydrolysate with respect to time.     

One-step immobilization and purification of genetic engineering CBD fusion EndoS on cellulose for antibodies Fc-glycan remodeling

The endoglycosidase (EndoS and its glycosynthase mutants D233A, D233Q) gene was fused with cellulose binding domain (CBD) using pET-35b vector and the fusion enzymes were successfully expressed in Escherichia coli. Then a simplified approach for one-step immobilization and purification of EndoS enzymes using cellulose as matrices were developed and excellent loading efficiency (81–90%) was achieved in optimal condition. The cellulose immobilized CBD-EndoS and the glycosynthase mutants presented high catalytic activity and were successfully applied in a two-step antibody Fc N-glycan remodeling, generating a therapeutic antibody with homogeneous glycoform in high efficiency. The cellulose immobilized CBD-EndoS and its mutants (D233A and D233Q) displayed excellent storage stability when stored at 4 degrees for one month. Reusability studies demonstrated that the cellulose immobilized CBD-EndoS and its mutants could be recycled for five times without obvious activity loss.     

Purification of Lac repressor protein using polymer displacement and immobilization of the protein

Lac repressor protein was purified from E. coli BMH8117 harboring plasmid pWB1000 and E. coli K₁₂BMH 71-18 strains. Displacement of the protein with poly(ethyleneimine) (PEI) from phosphocellulose cation exchange column was shown to be an effective elution strategy. It resulted in better recoveries and sharper elution profiles than traditional salt elution without effecting the purity of the protein. The elution is assumed to proceed via displacement of bound protein by PEI when the polymer binds to the ion exchanger. The minor impurities in the protein solution were finally removed by chromatography on immobilized metal affinity column. The repressor protein undergoes distinct conformational changes upon addition of specific inducer isopropyl--D-thiogalactoside (IPTG), which is evidenced by changes in ultraviolet absorption spectrum. The protein was immobilized covalently to the Sepharose matrix. The intact biological activity of the protein after immobilization was shown by binding of genomic DNA and lac operator plasmid DNA from E. coli to the immobilized lac repressor.     

Effect of chlorhexidine on molecular weight distribution of fructans produced by fructosyltransferase in solution and immobilized on surface

The effect of chlorhexidine (CHX), a potent antibacterial agent, was tested on the molecular weight distribution (MWD) of fructans synthesized by cell-free fructosyltransferase (FTF) in solution in comparison to FTF immobilized onto hydroxyapatite (HA). Size-exclusion chromatography (SEC) analysis has shown that cell-free FTF, both in solution and immobilized on HA, produces both low MW (1.9-2.2 kDa) and high MW (913-1047 kDa) fructans. CHX at a concentration of 0.02% altered the MWD of the fructans by reducing the polydispersity ratio and changing the MWD of the fructans synthesized both by immobilized FTF and by FTF in solution. These changes of the fructans in the presence of CHX adds a new prospective to the anticaries effect of CHX in addition to its antibacterial properties.     

Sorption isotherms and kinetics in the primary biodegradation of anionic surfactants by immobilized bacteria: I. Pseudomonas C12B

The surfactant-degrading biocatalyst Pseudomonas C12B was immobilized by covalent linking on silanized inorganic supports and by physical entrapment of cells within reticulated polyurethane foam. Both immobilized biocatalysts have been shown to be appropriate for the effective primary biodegradation of the anionic surfactants sodium dodecyl sulphate (SDS), dodecylbenzene sulphonic acid (DBS), dioctyl sulphosuccinate (DOSS) and dihexyl sulphosuccinate (DHSS). The overall surfactant removal from water by cells entrapped in reticulated polyurethane foam exhibits a biphasic process, a rapid sorption step of the surfactant onto the cell-loaded support and the intrinsic primary biodegradation slower step, both acting cooperatively. The optimization of variables for the adsorption and the biodegradation processes (flow rate, particle size, substrate concentration) have been studied. Sorption isotherms for the surfactants on reticulated polyurethane foam have been established as type II of the Brunauer, Deming, Deming and Teller (BDDT) classification. The kinetics of the primary biodegradation of SDS by cells covalent linked on sepiolite treated with 3-aminopropyl triethoxysilane (APTS) were found to be first-order. In this case, surfactant adsorption does not exist.