Murray Moo-Young

Disruption of microbial cells for intracellular products

Abstract Disintegration of microbial cells is a necessary first step for the production of intracellular enzymes and organelles. With increasing use of intracellular microbial material in industry and medicine, the cell disruption unit operation is gaining in importance. This review examines the state of the art of the large-scale cell disruption technology and disruption methods of potential commercial value.

Ester synthesis in lipase-catalyzed reactions

This review discusses the myriad of reaction systems employed in ester production catalyzed by lipases. Enzyme behavior in reaction systems is a consequence of certain structural patterns typical of lipases. Water has a profound effect on the lipase behavior either directly by affecting the hydration of the enzyme or indirectly by changing the nature of the reaction media and/or enzyme support materials. The significance of water is further emphasized with the understanding of its role in various reaction media and support materials. In addition to water, the media and support material are seldom inert and often interact with other factors affecting the reaction. Several methods of water removal and their effectiveness in lipase-catalyzed reactions are discussed. There is a recent surge of interest in modifying lipase systems with surfactants as studies demonstrate the potential of the enhanced performance of surfactant-modified lipases.

Fermentation optimization for the production of poly(β-hydroxybutyric acid) microbial thermoplastic

Abstract Batch culture of Alcaligenes latus, American Type Culture Collection 29713, was investigated for producing the intracellular bioplastic poly(β-hydroxybutyric acid) (PHB). A central, composite experimental design was used to optimize the composition of the culture medium for maximizing the productivity of PHB. Investigated were the effects of temperature, the initial culture pH, the ionic strength of the medium, the concentration of trace elements, the type of nitrogen source, and the carbon-to-nitrogen ratio. The optimal temperature for growth and PHB synthesis appeared to be 33°C; however, over the 25–37°C range, the effect of temperature was negligible. An initial pH value of 6.5 gave the best results; pH values that differed even slightly from the optimum reduced the culture performance. Typical culture characteristics were: 0.075/h maximum specific growth rate, 0.38 g/l h maximum specific sucrose consumption rate, and 0.15 g/l h maximum specific PHB production rate. PHB was lost because of hydrolysis in the stationary phase, suggesting critical importance of timing the harvest. Under the best conditions, PHB constituted up to 63% of dry cell mass after 93 h of culture. The average biomass yield coefficient on sucrose was about 0.4 kg/kg. Of the four nitrogen sources—ammonium chloride, ammonium sulfate, ammonium nitrate, and urea—used, only the first two supported the culture satisfactorily. The biomass and PHB showed clear yield maxima at 1.5 g/l ammonium chloride (C:N ratio = 21.5) and 1.4 g/l ammonium sulfate (C:N ratio = 28.3). The yields were higher with ammonium sulfate and were relatively more sensitive to changes in its concentration. Ionic strength had a strong negative effect on PHB productivity. The highest PHB yield occurred at 4 g/l phosphate buffer concentration. Iron appeared to have the potential to enhance the proportion of PHB in the cells.

Recent advances in bioprocessing application of membrane chromatography

Compared to traditional chromatography using resins in packed-bed columns, membrane chromatography is a relatively new and immature bioseparation technology based on the integration of membrane filtration and liquid chromatography into a single-stage operation. Over the past decades, advances in membrane chemistry have yielded novel membrane devices with high binding capacities and improved mass transfer properties, significantly increasing the bioprocessing efficiency for purification of biomolecules. Due to the disposable nature, low buffer consumption, and reduced equipment costs, membrane chromatography can significantly reduce downstream bioprocessing costs. In this review, we discuss technological merits and disadvantages associated with membrane chromatography as well as recent bioseparation applications with a particular attention on purification of large biomolecules.

Enzymatic Degradation of Cell Wall and Related Plant Polysaccharides

Polysaccharides such as starch, cellulose and other glucans, pectins, xylans, mannans, and fructans are present as major structural and storage materials in plants. These constituents may be degraded and modified by endogenous enzymes during plant growth and development. In plant pathogenesis by microorganisms, extracellular enzymes secreted by infected strains play a major role in plant tissue degradation and invasion of the host. Many of these polysaccharide-degrading enzymes are also produced by microorganisms widely used in industrial enzyme production. Most commerical enzyme preparations contain an array of secondary activities in addition to the one or two principal components which have standardized activities. In the processing of unpurified carbohydrate materials such as cereals, fruits, and tubers, these secondary enzyme activities offer major potential for improving process efficiency. Use of more defined combinations of industrial polysaccharases should allow final control of existing enzyme processes and should also lead to the development of novel enzymatic applications.

Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli.

ABSTRACT To date, most genetic engineering approaches coupling the type II Streptococcus pyogenes clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to lambda Red recombineering have involved minor single nucleotide mutations. Here we show that procedures for carrying out more complex chromosomal gene replacements in Escherichia coli can be substantially enhanced through implementation of CRISPR/Cas9 genome editing. We developed a three-plasmid approach that allows not only highly efficient recombination of short single-stranded oligonucleotides but also replacement of multigene chromosomal stretches of DNA with large PCR products. By systematically challenging the proposed system with respect to the magnitude of chromosomal deletion and size of DNA insertion, we demonstrated DNA deletions of up to 19.4 kb, encompassing 19 nonessential chromosomal genes, and insertion of up to 3 kb of heterologous DNA with recombination efficiencies permitting mutant detection by colony PCR screening. Since CRISPR/Cas9-coupled recombineering does not rely on the use of chromosome-encoded antibiotic resistance, or flippase recombination for antibiotic marker recycling, our approach is simpler, less labor-intensive, and allows efficient production of gene replacement mutants that are both markerless and “scar”-less.

ESTIMATION OF OXYGEN PENETRATION DEPTH IN IMMOBILIZED CELLS

SummaryA simple method is proposed for calculating oxygen pentration depth in immobilized cells by assuming zero order kinetics in the presence of several external oxygen transport resistances. Calculations indicate that typical penetration depths of oxygen for immobilized microbial cells are in the range of 50–200 μ and those for immobilized or encapsulated animal and plant tissue culture are about 500–1000 μ. Based on calculations, oxygen transport in microencapsulation and microcarriers for tissue cultures are not transport-limited, but a slight limitation is expected for those in a hollow fiber reactor.

Disulfide bond formation and its impact on the biological activity and stability of recombinant therapeutic proteins produced by Escherichia coli expression system

Therapeutic proteins require correct disulfide bond formation for biological activity and stability. This makes their manufacturing and storage inherently challenging since disulfide bonds can be aberrantly formed and/or undergo significant structural changes. In this paper the mechanisms of disulfide bond formation and scrambling are reviewed, with a focus on their impact on the biological activity and storage stability of recombinant proteins. After assessing the research progress in detecting disulfide bond scrambling, strategies for preventing this phenomenon are proposed.

Culture of Saccharomyces cerevisiae on hydrolyzed waste cassava starch for production of baking-quality yeast

Abstract A fermentation medium based on waste cassava starch hydrolysate and a four-phase feeding strategy for a fed-batch culture of Bakers yeast Saccharomyces cerevisiae are presented. Cassava starch isolated from the wastewater produced in processing of cassava mash into gari was liquefied with a thermostable 1.4-α- d -glucanohydrolase (EC 3.2.1.1) in the presence of 100 ppm Ca 2+ at 80°C and pH 6.1–6.3 for one h. The liquefied material was saccharified with 1.4-α- d -glucan glucohydrolase (EC 3.2.1.3) at 55°C and pH 5.5 for two h. Over 98% of the starch was hydrolyzed; about 80.7% of the hydrolysate was glucose. The fermentation feeding profile which was based on a desired specific growth rare range of 0.18–0.23 h −1 , a biomass yield coefficient of 0.5 g g −1 , and a feed substrate concentration of 200 g l −1 was implemented manually using the cassava hydrolysate feed in test experiments and glucose feed in control experiments. The fermentation off-gas was analyzed on-line by mass spectrometry for the calculation of the oxygen uptake rate, the carbon dioxide evolution rate, and the respiratory quotient. Off-line determinations of biomass, ethanol, and glucose were done, respectively, by dry weight, gas chromatography, and spectrophotometry. Cell mass concentrations of 50–58 g l −1 were achieved in all experiments within 28 h of which the last 15 h were in the fed-batch mode. The average biomass yields for the cassava and glucose media were identical at 0.49 g g −1 . No significant differences were observed between the leavening activities of the products of the test, the control media, and a commerical preparation of instant active dry yeast. Waste cassava starch hydrolysate was established as a suitable low cost replacement for glucose in the production of baking-quality yeast.

The scale-up of plant cell culture: Engineering considerations

The enormous versatility of plants has continued to provide the impetus for the development of plant tissue culture as a commercial production strategy for secondary metabolites. Unfortunately problems with slow growth rates and low products yields, which are generally non-growth associated and intracellular, have made plant cell culture-based processes, with a few exceptions, economically unrealistic. Recent developments in reactor design and control, elicitor technology, molecular biology, and consumer demand for natural products, are fuelling a renaissance in plant cell culture as a production strategy. In this review we address the engineering consequences of the unique characteristics of plant cells on the scale-up of plant cell culture.