Byong H. Lee

Recombinant protein production in a variety of Nicotiana hosts: a comparative analysis

Although many different crop species have been used to produce a wide range of vaccines, antibodies, biopharmaceuticals and industrial enzymes, tobacco has the most established history for the production of recombinant proteins. To further improve the heterologous protein yield of tobacco platforms, transient and stable expression of four recombinant proteins (i.e. human erythropoietin and interleukin-10, an antibody against Pseudomonas aeruginosa, and a hyperthermostable α-amylase) was evaluated in numerous species and cultivars of Nicotiana. Whereas the transient level of recombinant protein accumulation varied significantly amongst the different Nicotiana plant hosts, the variety of Nicotiana had little practical impact on the recombinant protein concentration in stable transgenic plants. In addition, this study examined the growth rate, amount of leaf biomass, total soluble protein levels and the alkaloid content of the various Nicotiana varieties to establish the best plant platform for commercial production of recombinant proteins. Of the 52 Nicotiana varieties evaluated, Nicotiana tabacum (cv. I 64) produced the highest transient concentrations of recombinant proteins, in addition to producing a large amount of biomass and a relatively low quantity of alkaloids, probably making it the most effective plant host for recombinant protein production.

Transformation Systems of non-Saccharomyces Yeasts

ABSTRACT:  This review describes the transformation systems including vectors, replicons, genetic markers, transformation methods, vector stability, and copy numbers of 13 genera and 31 species of non-Saccharomyces yeasts. Schizosaccharomyces pombe was the first non-Saccharomyces yeast studied for transformation and genetics. The replicons of non-Saccharomyces yeast vectors are from native plasmids, chromosomal DNA, and mitochondrial DNA of Saccharomyces cerevisiae, non-Saccharomyces yeasts, protozoan, plant, and animal. Vectors such as YAC, YCp, YEp, YIp, and YRp were developed for non-Saccharomyces yeasts. Forty-two types of genes from bacteria, yeasts, fungi, and plant were used as genetic markers that could be classified into biosynthetic, dominant, and colored groups to construct non-Saccharomyces yeasts vectors. The LEU2 gene and G418 resistance gene are the two most popular markers used in the yeast transformation. All known transformation methods such as spheroplast-mediating method, alkaline ion treatment method, electroporation, trans-kingdom conjugation, and biolistics have been developed successfully for non-Saccharomyces yeasts, among which the first three are most widely used. The highest copy number detected from non-Saccharomyces yeasts is 60 copies in Kluyveromyces lactis. No general rule is known to illustrate the transformation efficiency, vector stability, and copy number, although factors such as vector composition, host strain, transformation method, and selective pressure might influence them.

Growth of Lactobacillus paracasei ssp. paracasei on tofu whey

The liquid by-product of the soybean product tofu, tofu whey (TW), was used as a growth medium for the production of Lactobacillus paracasei ssp. paracasei LG3 cultures. The TW used in this study contained stachyose, raffinose, sucrose, fructose and glucose, but the strain used could only utilize the three latter. The lactobacilli population obtained in MRS broth was three times higher than that in TW alone, and supplementation of TW was thus examined. Of 19 mixtures of yeast extracts (YE), peptones and potato extracts examined, the best nitrogen sources were YE and tryptone. The addition of YE, salts (phosphates, citrates, Mg and Mn), glucose as well as Tween to TW tripled the populations to 2.9 x 10(9) cfu/ml, which was as high as that obtained in MRS broth. Growth of L. paracasei LG3 in cow rehydrated skim milk was inferior to that in TW.

Cloning and expression of β-galactosidase gene from Bifidobacterium infantis into Escherichia coli

Bifidobacterium infantis HL96 produces three β-galactosidases (β-gal I, II and III). A genomic bank of B. infantis was constructed in E. coli by using pBR322 as a cloning vector. Two E. coli transformants, BIG1 and BIG4, possessing β-galactosidase activity, were selected from X-gal plates. They contained two different recombinant plasmids with insert DNA fragments of approx. 4.6 and 4.4 kb, respectively. The restriction maps of pBIG1 and pBIG4 were constructed. β-Galactosidases from crude cell-free extracts of B. infantis and of two E. coli recombinants were analyzed by native PAGE and characterized by activity staining. pBIG1 and pBIG4 were shown to carry the genes for β-gal I and β-gal III, respectively. Optimal pH and temperature for hydrolytic activity of the native enzyme were 7.5 and 40°C, while those for recombinant BIG1 and BIG4 were 7.5, 50°C and 8.0, 40°C, respectively.

Microencapsulation of a recombinant aminopeptidase (PepN) from Lactobacillus rhamnosus S93 in chitosan-coated alginate beads

A recombinant aminopeptidase (90 kDa) of Lactobacillus rhamnosus S93 produced by E. coli was encapsulated in alginate or chitosan-treated alginate beads prepared by an extrusion method. This study investigated the effects of alginate, CaCl2, chitosan concentrations, hardening time, pH and alginate/enzyme ratios on the encapsulation efficiency (EE) and the enzyme release (ER). Chitosan in the gelling solution significantly increased the EE from 30.2% (control) to 88.6% (coated). This polycationic polymer retarded the ER from beads during their dissolution in release buffer. An increase in alginate and chitosan concentrations led to greater EE and lesser ER from the beads. The greatest EE was observed in a pH 5.4 solution (chitosan-CaCl2) during bead formation. Increasing the CaCl2 concentration over 0.1 M neither affected the EE nor the ER. Increasing hardening time beyond 10 min led to a decrease in EE and the alginate:enzyme ratio (3 : 1) was optimal to prevent the ER.

Thermostability of Probiotics and Their α-Galactosidases and the Potential for Bean Products

Soybeans and other pulses contain oligosaccharides which may cause intestinal disturbances such as flatulence. This study was undertaken to investigate α-galactosidase-producing probiotics added to frozen foods which can survive warming treatments used in thawing and consumption of the pulses. The maximum α-galactosidase activity (1.26 U/mg protein) was found in Bifidobacterium breve S46. Lactobacillus casei had the highest α-galactosidase thermostability among the various strains, with D values of 35, 29, and 9.3 minutes at 50°C, 55°C, and 60°C, respectively. The enzyme activity was less affected than viable cells by heating. However, the D values of two bacterial enzymes were lower than those of three commercial α-galactosidase-containing products. Freshly grown cells and their enzymes were more stable than the rehydrated cultures and their enzymes. Practical Application. Enzymes and cultures can be added to foods in order to enhance the digestibility of carbohydrates in the gastrointestinal tract. However since many foods are warmed, it is important that the thermostability of the enzymes be assessed. This paper provides data on the stability of α-galactosidase, which could potentially be added to food matrices containing stachyose or raffinose, such as beans.

Effect of free or encapsulated recombinant aminopeptidase of Lactobacillus rhamnosus S93 on acceleration of Cheddar cheese ripening.

The use of recombinant aminopeptidase (PepN) from Lactobacillus rhamnosus S93 in free or encapsulated form was investigated to shorten the duration of Cheddar cheese ripening. Proteolysis was determined by measuring the soluble nitrogen as phosphotungstic acid (PTA-N) derivatives and free amino acids (FAA) over a 6-month period. The experimental cheeses received higher scores for sensory properties than the control cheese. The amounts of PTA-N and total FAA in the cheese with the encapsulated enzyme after 2 months of ripening were close to those of the control cheese after 6 months, suggesting the acceleration in proteolysis by about 4 months.

Immobilization of Cells and Enzymes for Fermented Dairy or Meat Products

Historically, we can find fermented products in almost all cultural backgrounds around the world. Notably, there are many different milk or meat-based foods and this chapter will focus on them (Kosikowski 1982; Wood 1998). Cheese, yoghurt, sour cream, kefir, or cultured butter are probably the most common fermented dairy products, but many regional varieties exist (Farnworth 2004). Fermented meats are typically found as dry sausages (Luke 1998). Yeasts are mostly involved in the manufacture of bread and alcoholic beverages, which are basically cereal- or fruit-based products. In fermented meat and milk, the main microorganisms used are the lactic acid bacteria (LAB). Yeast and molds are rather involved in ripening. Therefore, the LAB will constitute the main focus of this chapter.

Oligosaccharides extracted from cell walls of Kluyveromyces marxianus grown on whey

After whey fermentation by Kluyveromyces marxianus var. marxianus (30°C, pH 4.5, 24 h) and autolysis of the cells (50°C, pH 6.5, 12 h), the subsequent extracts were centrifuged (10,000 × g, 4°C, 15 min), and the cell walls were separated from the autolysates. Cell walls were then treated with: (i) 0.75M NaOH (75°C, 20 h) ; or (ii) lytic enzymes, 0.0025–5.0% (w/v), in 5 mM phosphate buffer (pH 6.5–7.0) (40°C, 24 h). The lytic enzymes were denaturated (80°C, 15 min), and the alkali solutions were neutralized with 0.5M acetic acid, before centrifugation. The supernatants were concentrated by a Speed-Vac concentrator, and analyzed by HPLC, equipped with a TSK-Amide 80 column (1.0 ml/ min of water/acetonitrile, 35/65 ratio, 60°C, 40 min). Tetrasaccharides were detected. Gels were formed when cell walls were treated with NaOH.