Optimization of Enzymatically Prepared Hexyl Butyrate by Lipozyme IM-77

Hexyl butyrate, a green note flavour compound, is widely used in the food industry. The ability of immobilised lipase (Lipozyme IM-77) from Rhizomucor miehei to catalyse the transesterification of hexanol and tributyrin was investigated in this study. Response surface methodology (RSM) and five-level-five-factor central composite rotatable design (CCRD) were employed to evaluate the effects of synthesis parameters, such as reaction time (2 to 10 h), temperature (25 to 65 °C), enzyme amount (10 to 50 %), substrate amount (in mol) ratio of tributyrin to hexanol (1:1 to 3:1), and added water content (0 to 20 %), on percentage amount (in mol) conversion of hexyl butyrate by transesterification. Reaction time and enzyme amount were the most important variables and substrate amount (in mol) ratio had less effect on the percentage of amount (in mol) conversion. Based on canonical analysis, the optimum synthesis conditions were: reaction time 8.3 h, temperature 50 °C, enzyme amount 42.7 %, substrate amount (in mol) ratio 1.8:1, and added water 12.6 %. The predicted value was 96.2 % and actual experimental value 95.3 % of the amount (in mol) conversion

Engineering of Escherichia coli protein expression process development

It almost 30% protein drugs are expression by Escherichia coli, because of rapid growth and high production yield. We have developed E.coli base system for recombinant protein expression, scFv, Fab and vaccine. In this study we introduce example about process development for nutrient components selection. Shaker flasks were used for different nitrogen and carbon components screening by DoE. Seven media formulations for E. coli fermentation were used in this study. By changing nitrogen and carbon source ratio, product titer of target protein could be optimized, at least 1.4 folds increased. The best result from shaker flask was used in 250 mL parallel fermenter and pH, dissolved oxygen, feeding/induction strategy were evaluated. The processes from seed culture to harvest only require 64 hours. The optimized time was reduced to 32 hours. The result showed that both target protein expression and cell density value were comparable, but the total process time was significantly reduced by half Please click Additional Files below to see the full abstract

Therapeutic protein expression platform of microbial system

A number of expression systems have been developed for the production of pharmaceutical products. Pichia pastoris and Escherichia coli expression system operate in our lab and express antibody fragment (scFv), cytokine, protein base adjuvant and vaccine and process enzyme. The expression platform are consisted of three part, first is strain generation , the second is fermentation process development in 250 ml fermentor and the last is process scale-up to 5 litter fermentor. Please click Additional Files below to see the full abstract

The microbial antibodies secretion expression platform with scale down fermentors

Therapeutic antibodies have become one of the most effective therapeutics for human diseases such as cancer, inflammation and viral infection. The production of antibody-based drugs using microbial expression systems is more cost effective with ease of gene manipulation compared to mammalian expression systems. In our team, antibody fragments (ex: BsAb, scFv and Fab) were produced from methylotrophic yeast Pichia pastoris secretion expression system with the AOX1 as driven promoter or E. coli secretion expression system. To achieve high production yield for both system, we investigated fermentation parameter such as base medium, induction medium, induction condition, feeding strategy and pH. For the 250 ml fermentor Pichia system, the nitrogen have been add into glycerol fed medium and/or methanol induction medium and also compared base-medium, buffered glycerol-complex medium (BMGY) and basal salt medium (BS). The highest scFv production was yielded from the basal salt medium as base medium, glycerol fed medium plus nitrogen and multiple carbon source methanol induction medium. This process can yielded over 500 mg/L scFv. After scale-up from 250 ml fermentor to 5L fermentor, the methanol fed-back control system also applied on the 5 L fermentor, can achieve 1.7 g/L scFv in 5 days. The E. coli expression process has passed through screening for high production yield clones in 2 ml deep-well then confirmed by using 250 ml flask scale. Feeding medium, DO, pH etc, parameters were investigated by parallel 250 ml-fermenter. The parameters from 250 ml fermentor were validated by using 5 L fermenter. Under this scale-up procedure, the antibody Fab was 100 folds production yield, production deep well stage at 1 mg/L, production from 250 ml fermentor stage is 50-100 mg/L and production 5 L fermentor stage is over 35-90 mg/L. Although different antibodies will result in different production yield, building a reliable platform to predict production yield from antibody cell clones under deep well and shake flask stage serves a good scale-down model for future scale-up prediction

The evolutionary history of histone H3 suggests a deep eukaryotic root of chromatin modifying mechanisms

<p>Abstract</p> <p>Background</p> <p>The phenotype of an organism is an outcome of both its genotype, encoding the primary sequence of proteins, and the developmental orchestration of gene expression. The substrate of gene expression in eukaryotes is the chromatin, whose fundamental units are nucleosomes composed of DNA wrapped around each two of the core histone types H2A, H2B, H3 and H4. Key regulatory steps involved in the determination of chromatin conformations are posttranslational modifications (PTM) at histone tails as well as the assembly of histone variants into nucleosomal arrays. Although the mechanistic background is fragmentary understood, it appears that the chromatin signature of metazoan cell types is inheritable over generations. Even less understood is the conservation of epigenetic mechanisms among eukaryotes and their origins.</p> <p>Results</p> <p>In the light of recent progress in understanding the tree of eukaryotic life we discovered the origin of histone H3 by phylogenetic analyses of variants from all supergroups, which allowed the reconstruction of ancestral states. We found that H3 variants evolved frequently but independently within related species of almost all eukaryotic supergroups. Interestingly, we found all core histone types encoded in the genome of a basal dinoflagellate and H3 variants in two other species, although is was reported that dinoflagellate chromatin is not organized into nucleosomes.</p> <p>Most probably one or more animal/nuclearid H3.3-like variants gave rise to H3 variants of all opisthokonts (animals, choanozoa, fungi, nuclearids, Amoebozoa). H3.2 and H3.1 as well as H3.1t are derivatives of H3.3, whereas H3.2 evolved already in early branching animals, such as <it>Trichoplax</it>. H3.1 and H3.1t are probably restricted to mammals.</p> <p>We deduced a model for protoH3 of the last eukaryotic common ancestor (LECA) confirming a remarkable degree of sequence conservation in comparison to canonical human H3.1. We found evidence that multiple PTMs are conserved even in putatively early branching eukaryotic taxa (Euglenozoa/Excavata).</p> <p>Conclusions</p> <p>At least a basal repertoire of chromatin modifying mechanisms appears to share old common ancestry and may thus be inherent to all eukaryotes. We speculate that epigenetic principles responsive to environmental triggers may have had influenced phenotypic variation and concomitantly may potentially have had impact on eukaryotic diversification.</p

Gram Level scFv expression platform of Pichia pastoris

The methylotrophic yeast Pichia pastoris secretion expression system has been developed for the antibody fragments (scFv) production platform. The platform includes three technology platforms, the first one is strain generation, the second is fermentation process development in 250 ml fermentor and the last is process scale up to 5 L. A recombinant scFv went through clone generation, include signal peptide tool box, normally yield 2.5 mg/L titer in deep well. Through the fermentation process development of induction medium composition and feeding strategy by Eppendorf Dasgip parallel 250 ml mini fermentor. During induction step, feeding 100% methanol as induction medium can only produce less than 50 mg/L scFv while feeding methanol-sorbitol mixture can significant increase the production yield to 306 mg/L in five days, about 6-folds increase in productivity. With the supply of additional nitrogen source during glycerol feeding step or at induction step, higher scFv production with 510 mg/L can be achieved. Thus, following the medium composition optimization, the production titer was improved 10 folds in 250 ml mini-fermentor stage. Moreover, when we switched the induction medium feeding strategy from DO-stat to the stepwise feeding, the titer increased form 510 mg/L to ~1000 mg/L and yielded another 2- folds improvement. During medium composition and feeding strategy optimization at 250 ml mini fermentor scale, the production titer could increase 20 folds. Overall, the production titer increased 400 folds from cell line generation to 250 ml fermentation parameter optimization. Furthermore, the process parameter can be scale-up to 5 L fernentor achieving \u3e 1 g/L. Recent progress to include BIP in the expression vector gave at least 2 fold improvement in scFv titer in shake flask, the new clone will be optimized in our established 250 ml and 5 L fermentation platform. Please click Additional Files below to see the full abstract

Gram level scFv expression platform of Phichi pastoris

The methylotrophic yeast Pichia pastoris secretion expression system has been developed for the antibody fragments (scFv) production platform. The platform includes three technology platforms, the first one is strain generation, the second is fermentation process development in 250 ml fermentor and the last is process scale up to 5 L. A recombinant scFv went through clone generation, include signal peptide tool box, normally yield 2.5 mg/L titer in deep well. Through the fermentation process development of induction medium composition and feeding strategy by Eppendorf Dasgip parallel 250 ml mini fermentor. During induction step, feeding 100% methanol as induction medium can only produce less than 50 mg/L scFv while feeding methanol-sorbitol mixture can significant increase the production yield to 306 mg/L in five days, about 6-folds increase in productivity. With the supply of additional nitrogen source during glycerol feeding step or at induction step, higher scFv production with 510 mg/L can be achieved. Thus, following the medium composition optimization, the production titer was improved 10 folds in 250 ml mini-fermentor stage. Moreover, when we switched the induction medium feeding strategy from DO-stat to the stepwise feeding, the titer increased form 510 mg/L to ~1000 mg/L and yielded another 2- folds improvement. During medium composition and feeding strategy optimization at 250 ml mini fermentor scale, the production titer could increase 20 folds. Overall, the production titer increased 400 folds from cell line generation to 250 ml fermentation parameter optimization. Furthermore, the process parameter can be scale-up to 5 L fernentor achieving \u3e 1 g/L. Recent progress to include BIP in the expression vector gave at least 2 fold improvement in scFv titer in shake flask, the new clone will be optimized in our established 250 ml and 5 L fermentation platform Please click Additional Files below to see the full abstract

On the Momentum Dependence of the Flavor Structure of the Nucleon Sea

Difference between the $\bar u$ and $\bar d$ sea quark distributions in the proton was first observed in the violation of the Gottfried sum rule in deep-inelastic scattering (DIS) experiments. The parton momentum fraction $x$ dependence of this difference has been measured over the region $0.02 < x < 0.35$ from Drell-Yan and semi-inclusive DIS experiments. The Drell-Yan data suggested a possible sign-change for $\bar d(x)-\bar u(x)$ near $x \sim 0.3$, which has not yet been explained by existing theoretical models. We present an independent evidence for the $\bar d(x)-\bar u(x)$ sign-change at $x \sim 0.3$ from an analysis of the DIS data. We further discuss the $x$-dependence of $\bar d(x)-\bar u(x)$ in the context of meson cloud model and the lattice QCD formulation.Comment: 5 pages, 5 figures, final versio