Immobilization of catalase into chemically crosslinked chitosan beads

Bovine liver catalase was immobilized into chitosan beads prepared in crosslinking solution. Various characteristics of immobilized catalase such as the pH–activity curve, the temperature–activity curve, thermal stability, operational stability, and storage stability were evaluated. Among them the pH optimum and temperature optimum of free and immobilized catalase were found to be pH 7.0 and 35 °C. The K_m value of immobilized catalase (77.5 mM) was higher than that of free enzyme (35 mM). Immobilization decreased in V_max value from 32,000 to 122 μmol (min mg protein)⁻¹. It was observed that operational, thermal and storage stabilities of the enzyme were increased with immobilization.     

Immobilization of anaerobic sludge using chitosan crosslinked with lignosulfonate

A new method for immobilization of anaerobic sludge in chitosan is described. It is based on the reaction of basic NH₂ groups of chitosan with the acidic sulfonic-groups of lignosulfonate to form sulfonilamide linkages. The new procedure features simplicity, low-cost and mild immobilization conditions. Batch tests of acetate consumption along with a continuous reactor operation confirmed the effectiveness of the immobilization technique for maintenance of long-term stability of the polymer. Received 27 March 1997/ Accepted in revised form 01 October 1997     

纳米磁性壳聚糖微球固定化酵母醇脱氢酶的研究

建立了以纳米级磁性壳聚糖微球(magnetic chitosan microspheres , M-CS)为载体固定化酵母醇脱氢酶(yeast alcohol dehydrogenase,YADH)的方法,优化了YADH的固定化条件,考察了固定化酶的性质。结果表明,M-CS 呈规则的圆球形,粒径在30nm 左右,具有较好的磁响应性。酵母醇脱氢酶固定化适宜条件为:50 mg 磁性壳聚糖微球,加入20mL 0.25 mg/mL 酵母醇脱氢酶(蛋白质含量)磷酸盐缓冲液(0.05 mol/L ,pH 7.0) ,在4 ℃固定2h。M-CS 容易吸附酵母醇脱氢酶,但吸附的酶量受载体与酶的比例、溶液的离子浓度、溶液pH的影响明显,而温度对吸附的酶量的影响则相对较弱。相对于游离的酵母醇脱氢酶,固定化酶的最适温度略有升高,可明显改善其热稳定性、酸碱稳定性、操作稳定性和贮存稳定性。     

Matrices (re)loaded: Durability,viability, and fermentative capacity of yeast encapsulated in beads of different composition during long-term fed-batch culture

Encapsulated microbes have been used for decades to produce commodities ranging from methyl ketone to beer. Encapsulated cells undergo limited replication, which enables them to more efficiently convert substrate to product than planktonic cells and which contributes to their stress resistance. To determine how encapsulated yeast supports long-term, repeated fed-batch ethanologenic fermentation, and whether different matrices influence that process, fermentation and indicators of matrix durability and cell viability were monitored in high-dextrose, fed-batch culture over 7 weeks. At most timepoints, ethanol yield (g/g) in encapsulated cultures exceeded that in planktonic cultures. And frequently, ethanol yield differed among the four matrices tested: sodium alginate crosslinked with Ca²⁺ and chitosan, sodium alginate crosslinked with Ca²⁺, Protanal alginate crosslinked with Ca²⁺ and chitosan, Protanal alginate crosslinked with Ca²⁺, with the last of these consistently demonstrating the highest values. Young's modulus and viscosity were higher for matrices crosslinked with chitosan over the first week; thereafter values for both parameters declined and were indistinguishable among treatments. Encapsulated cells exhibited greater heat shock tolerance at 50°C than planktonic cells in either stationary or exponential phase, with similar thermotolerance observed across all four matrix types. Altogether, these data demonstrate the feasibility of re-using encapsulated yeast to convert dextrose to ethanol over at least 7 weeks.     

Immobilization of a protease on modified chitosan beads for the depolymerization of chitosan

Neutral protease was immobilized on chitosan (CS), carboxymethyl chitosan (CMCS), and N-succinyl chitosan (NSCS) hydrogel beads. And the biocatalysts obtained were used to prepare low molecular weight chitosan (LMWC) and chitooligomers. Weight-average molecular weight of LMWC produced by neutral protease immobilized on CS, CMCS and NSCS hydrogel beads were 3.4 kDa, 3.2 kDa and 1.9 kDa, respectively. The effects of immobilization support and substrate on enzymatic reaction were analyzed by measuring classical Michaelis-Menten kinetic parameters. The FT-IR, XRD and potentiometric determination results indicated decrease of molecular weight led to transformation of crystal structure, but the degree of N-deacetylation and chemical structures of residues were not changed compared to initial chitosan. The degree of polymerization of chitooligomers was mainly from 2 to 7. We observed a strong dependence of the immobilized enzyme properties on the chemical nature of the supports, which leads to different microenvironment of neutral protease and changes the hydrolyzing process.     

Microfiltration of recombinant yeast cells using a rotating disk dynamic filtration system

To develop a highly efficient cell harvest step under time constraint, a novel rotating disk dynamic filtration system was studied on the laboratory scale (0.147-ft.(2) nylon membrane) for concentrating recombinant yeast cells containing an intracellular product. The existing cross-flow microfiltration method yielded pseudo-steady state flux values below 25 LMH (L/m(2). h) even at low membrane loadings (10 L/ft.(2)). By creating high shear rates (up to 120,000(-1)) on the membrane surface using a rotating solid disk, this dynamic filter has demonstrated dramatically improved performance, presumably due to minimal cake buildup and reduced membrane fouling. Among the many factors investigated, disk rotating speed, which determines shear rates and flow patterns, was found to be the most important adjustable parameter. Our experimental results have shown that the flux increases with disk rotating speed, increases with transmembrane pressure at higher cell concentrations, and can be sustained at high levels under constant flux mode. At a certain membrane loading level, there was a critical speed below which it behaved similarly to a flat sheet system with equivalent shear. Average flux greater than 200 LMH has been demonstrated at 37-L/ft.(2) loading at maximum speed to complete sixfold concentration and 15-volume diafiltration for less than 100 min. An order of magnitude improvement over the crossflow microfiltration control was projected for large scale production. This superior performance, however, would be achieved at the expense of additional power input and heat dissipation, especially when cell concentration reaches above 80 g dry cell weight (DCW)/L. Although a positive linear relationship between power input and dynamic flux at a certain concentration factor has been established, high cell density associated with high viscosity impacted adversely on effective average shear rates and, eventually, severe membrane fouling, rather than cake formation, would limit the performance of this novel system. (c) 1995 John Wiley & Sons, Inc.     

History,lineage and phenotypic differentiation of sake yeast

ABSTRACT

Sake yeast was first isolated as a single yeast strain, Saccharomyces sake, during the Meiji era. Yeast strains suitable for sake fermentation were subsequently isolated from sake brewers and distributed as Kyokai yeast strains. Sake yeast strains that produce characteristic flavors have been bred in response to various market demands and individual preferences. Interestingly, both genetic and morphological studies have indicated that sake yeast used during the Meiji era differs from new sake yeasts derived from Kyokai Strain No. 7 lineage. Here, we discuss the history of sake yeast breeding, from the discovery of sake yeast to the present day, to highlight the achievements of great Japanese scientists and engineers.     

Production of ethanol from guava pulp by yeast strains

Guava pulp used for ethanol production by three yeast strains contained 10% (w/v) total sugars and was pH 4.1. Ethanol production at the optimum sugar concentration of 10%, at pH 4.1 and 30°C was 1.5%, 3.6% and 3.9% (w/v) by Saccharomyces cerevisiae MTCC 1972, Isolate-1 and Isolate-2, respectively, at 60 h fermentation. Higher sugar concentrations at 15 and 20% were inhibitory for ethanol production by all test cultures. The maximum production of ethanol at optimum natural sugar concentration (10%) of guava pulp, was 5.8% (w/v) at pH 5.0 by Isolate-2 over 36 h fermentation, which was only slightly more than the quantity of ethanol produced by Saccharomyces cerevisiae (5.0%) and Isolate-1 (5.3%) over 36 and 60h fermentation, respectively.     

Increasing yeast secretion of heterologous proteins by regulating expression rates and post-secretory loss

Heterologous protein secretion involves the coupled processes of protein synthesis, protein folding, and secretory trafficking. A more complete understanding of how these processes interrelate could help direct optimization of secretion systems. Here we provide a detailed study regarding the dynamics of heterologous protein secretion from yeast in terms of intracellular protein levels, secreted protein levels, and unfolded protein response (UPR). Three different protein expression induction temperatures (20, 30, and 37 degrees C) were investigated as a means to modulate expression rates and thus cellular responses. Inducing at 20 degrees C yielded the slowest initial secretion rate, but the highest absolute level of product. Correspondingly, the level and the rate of both intracellular protein accumulation and unfolded protein response (UPR) activation were also the lowest at 20 degrees C. In addition, secretion ceased after approximately 22 h at 30 and 37 degrees C, respectively, while it was continuous until nutrient depletion at 20 degrees C. Maxima in secretion levels were observed that were a result of the additive effects of secretion cessation and post-secretory protein loss. The post-secretory loss of protein did not appear to result from solution phase proteolysis or aggregation, but required the presence of yeast cells. Refeeding of both yeast nitrogen base and casamino acids successfully prevented the post-secretory loss of protein at both high (37 degrees C) and low (20 degrees C) temperatures, and further increased secretion levels 1.5-fold at 20 degrees C where the secretory pathway was still functioning. Taken together, these findings suggest that there exists an appropriate balance between protein synthesis, processing and secretion rates required for secretion optimization.     

Enhanced stabilization of mungbean thiol protease immobilized on glutaraldehyde-activated chitosan beads

A thiol protease purified from mungbean seedlings was immobilized on chitosan beads cross-linked with glutaraldehyde. The yield of the immobilized enzyme was maximum (～99%) at 1% concentration each of chitosan and glutaraldehyde. The immobilized enzyme showed reusability for 15 batch reactions. Immobilization shifted the optimum pH of the enzyme to a more acidic range and enhanced its stability both at acidic as well as alkaline pH values compared to the free enzyme. The stability of the enzyme to temperature and in aqueous non-conventional medium (ethanol and DMSO) was significantly improved by the immobilization process. The immobilized enzyme exhibited mass transfer limitation reflected by a higher apparent K_m value. This study produced an immobilized biocatalyst having improved characteristics and better operational stability than the soluble enzyme. The increase in stability in the presence of high concentrations of ethanol and DMSO may make it useful for catalyzing organic reactions such as trans-esterification and trans-amidation similar to other cysteine proteinases.     

壳聚糖球固定化酵母细胞的研究

以戊二醛为交联剂,将壳聚糖球交联引入醛基,然后将交联的壳聚糖球浸泡在酵母细胞悬浮液中,制备了固定化酵母细胞壳聚糖球。以苯乙酮酸为底物,催化合成了D-扁桃酸。最优固定化条件是戊二醛的质量分数w(GA)=1%,酵母细胞与交联壳聚糖球的质量比m(Y):m(CB)0=0.5,交联时间为6h,固定化时间为18h,底物浓度为10mmol/L,在此条件下反应最大转化率和产物光学纯度分别高达67.86%和98.05?。固定化酵母壳聚糖球具有良好的重复使用性和贮存稳定性。     

Immobilization of invertase via carbohydrate moiety on chitosan to enhance its thermal stability

A new technique using chitosan as support for covalent coupling of invertase via carbohydrate moiety improved the activity and thermal stability of immobilized invertase. The best preparation of immobilized invertase retained 91% of original specific activity (412 U mg^–1). The half-life at 60°C was increased from 2.3 h (free invertase) to 7.2 h (immobilized invertase). In contrast, the immobilization of invertase via protein moiety on chitosan or using Sepharose as support resulted in less thermostable preparations. Additionally, immobilization of invertase on both supports caused the optimal reaction pH to shift from 4.5 to 2.5 and the substrate (sucrose) concentration for maximum activity to increase from 0.5 M to 1.0 M.     

Immobilization and characterization of Saccharomyces cerevisiae in crosslinked,prepolymerized polyacrylamide-hydrazide

Summary Baker's yeast (Saccharomyces cerevisiae) was immobilized in gels made of prepolymerized, linear, water soluble polyacrylamide, partially substituted with acylhydrazide groups. Gelation was effected by the addition of controlled amounts of dialdehydes (e.g. glyoxal). The immobilized yeasts retained full glycolytic activity. Moreover, the entrapped cells were able to grow inside the chemically corsslinked gel during continuous alcohol production. Glyoxal was found to be the most favourable crosslinking agent for this system. the system employed allowed for the free exchange of substrate and products. The gel surrounding the entrapped cells had no effect on temperature stability profile. On the other hand, substantial enhancement in survival of cells in presence of high ethanol concentrations was recorded for the entrapped yeast. The capability of the immobilized yeast to carry out continuous conversion of glucose to ethanol was demonstrated.     

Acquisition of thermotolerant yeast Saccharomyces cerevisiae by breeding via stepwise adaptation

A thermotolerant Saccharomyces cerevisiae yeast strain, YK60‐1, was bred from a parental strain, MT8‐1, via stepwise adaptation. YK60‐1 grew at 40°C, a temperature at which MT8‐1 could not grow at all. YK60‐1 exhibited faster growth than MT8‐1 at 30°C. To investigate the mechanisms how MT8‐1 acquired thermotolerance, DNA microarray analysis was performed. The analysis revealed the induction of stress‐responsive genes such as those encoding heat shock proteins and trehalose biosynthetic enzymes in YK60‐1. Furthermore, nontargeting metabolome analysis showed that YK60‐1 accumulated more trehalose, a metabolite that contributes to stress tolerance in yeast, than MT8‐1. In conclusion, S. cerevisiae MT8‐1 acquired thermotolerance by induction of specific stress‐responsive genes and enhanced intracellular trehalose levels. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1116–1123, 2013     

Metabolic engineering of sesquiterpene metabolism in yeast

Terpenes are structurally diverse compounds that are of interest because of their biological activities and industrial value. These compounds consist of chirally rich hydrocarbon backbones derived from terpene synthases, which are subsequently decorated with hydroxyl substituents catalyzed by terpene hydroxylases. Availability of these compounds is, however, limited by intractable synthetic means and because they are produced in low amounts and as complex mixtures by natural sources. We engineered yeast for sesquiterpene accumulation by introducing genetic modifications that enable the yeast to accumulate high levels of the key intermediate farnesyl diphosphate (FPP). Co-expression of terpene synthase genes diverted the enlarged FPP pool to greater than 80 mg/L of sesquiterpene. Efficient coupling of terpene production with hydroxylation was also demonstrated by coordinate expression of terpene hydroxylase activity, yielding 50 mg/L each of hydrocarbon and hydroxylated products. These yeast now provide a convenient format for investigating catalytic coupling between terpene synthases and hydroxylases, as well as a platform for the industrial production of high value, single-entity and stereochemically unique terpenes.     

Genetic and fermentation properties of the K2 killer yeast, Saccharomyces cerevisiae T206

Saccharomyces cerevisiae T206 K⁺R⁺, a K₂ killer yeast, was differentiated from other NCYC killer strains of S. cerevisiae on the basis of CHEF-karyotyping and mycoviral RNA separations. Genomic DNA of strain T206 was resolved into 13 chromosome bands, ranging from approximately 0.2 to 2.2 Mb. The resident virus in strain T206 yielded L and M RNA species of approximately 5.1 kb and 2.0 kb, respectively. In micro-scale vinifications, strain T206 showed a lethal effect on a K^-R^- mesophilic wine yeast. Metabolite accumulation and toxin activity were measured over a narrow pH range of 3.2 to 3.5. Contrary to known fermentation trends, the challenged fermentations were neither stuck nor protracted although over 70% of the cell population was killed. Toxin-sensitive cells showed cytosolic efflux.     

cAMP-dependent phosphoprotein changes in the yeast Saccharomyces cerevisiae

Abstract cAMP-dependent phosphoprotein changes were determined using 1-dimensional SDS-gel electrophoresis in a cAMP-requiring yeast mutant ( Saccharomyces cerevisiae AM18). During cAMP starvation, the yeast cells accumulated 3 ³²P-labeled bands with M _r/ 72000, 54000, and 37000. The M _r/ 72000 protein was the most prominent phosphorylated protein. After the readdition of cAMP, these phosphoproteins lost their ³²P-label while phosphoproteins with M _r/ 76000, 65000, 56000 and 30000 were accumulated. Similar phosphoprotein changes were also detected in cdc35 at the nonpermissive temperature, but not in wildtype (A363A) or cdc7 strains of S. cerevisiae .     

Increased protein productivity from immobilized recombinant yeast

The Saccharomyces cerevisiae strain Mc16/p520 has an unstable plasmid, p520, which directs production of a wheat alpha-amylase. The effects of immobilizing this microorganism on the plasmid stability and the specific productivity of the secreted alpha-amylase were investigated. Small gelatin beads were used as the support in both fluidized and packed bed configurations, and the yeast cells were attached by covalent cross-linking with glutaraldehyde. These data were then compared to those for nonimmobilized, suspension cells.Plasmid stability was increased for the immobilized cells during continuous culture at dilution rates both above and below washout. Continuous suspension cultures were not stable and rapidly lost the plasmid. Immobilization caused an increase in specific and volumetric productivity during continuous culture, with a packed bed design resulting in the highest specific productivity.     

Immobilization of yeast alcohol dehydrogenase on magnetic nanoparticles for improving its stability

Yeast alcohol dehydrogenase (YADH) was immobilized covalently on Fe₃O₄ magnetic nanoparticles (10.6 nm) via carbodiimide activation. The immobilization process did not affect the size and structure of magnetic nanoparticles. The YADH-immobilized magnetic nanoparticles were superparamagnetic with a saturation magnetization of 61 emu g^–1, only slightly lower than that of the naked ones (63 emu g^–1). Compared to the free enzyme, the immobilized YADH retained 62% activity and showed a 10-fold increased stability and a 2.7-fold increased activity at pH 5. For the reduction of 2-butanone by immobilized YADH, the activation energies within 25–45 °C, the maximum specific activity, and the Michaelis constants for NADH and 2-butanone were 27 J mol^–1, 0.23 mol min^–1 mg^–1, 0.62 mM, and 0.43 M, respectively. These results indicated a structural change of YADH with a decrease in affinity for NADH and 2-butanone after immobilization compared to the free enzyme.     

Intracellular trehalose is neither necessary nor sufficient for desiccation tolerance in yeast

Trehalose is thought to be important for desiccation tolerance in a number of organisms, including Saccharomyces cerevisiae, but there is limited in vivo evidence to support this hypothesis. In wild-type yeast, the degree of desiccation tolerance has been shown previously to increase in cultures after diauxic shift and also in exponential-phase cultures after exposure to heat stress. Under both these conditions, increased survival of desiccation correlates with elevated intracellular trehalose concentrations. Our data confirm these findings, but we have tested the apparent importance of trehalose using mutant strains with a deleted trehalose-6-phosphate synthase gene (tps1Delta). Although tps1Delta strains do not produce trehalose, they are nevertheless capable of desiccation tolerance, and the degree of tolerance also increases after diauxic shift or heat stress, albeit slightly less than in the wild type. Conversely, when wild-type yeast is subjected to osmotic stress, mid-exponential-phase cultures produce high concentrations of intracellular trehalose but show little improvement in desiccation tolerance. These results show that there is no consistent relationship between intracellular trehalose levels and desiccation tolerance in S. cerevisiae. Trehalose seems to be neither necessary nor sufficient for, although in some strains might quantitatively improve, survival of desiccation, suggesting that other adaptations are more important.