Codon optimization of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> β-1,3-1,4-glucanase gene and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

β-1,3-1,4-glucanase (EC3.2.1.73) as an important industrial enzyme has been widely used in the brewing and animal feed additive industry. To improve expression efficiency of recombinant β-1,3-1,4-glucanase from Bacillus licheniformis EGW039(CGMCC 0635) in methylotrophic yeast Pichia pastoris GS115, the DNA sequence encoding β-1,3-1,4-glucanase was designed and synthesized based on the codon bias of P. pastoris, the codons encoding 96 amino acids were optimized, in which a total of 102 nucleotides were changed, the G+C ratio was simultaneously increased from 43.6 to 45.5%. At shaking flask level, β-1,3-1,4-glucanase activity is 67.9 and 52.3 U ml⁻¹ with barley β-glucan and lichenan as substrate, respectively. At laboratory fermentor level, the secreted protein concentration is approximately 250 mg l⁻¹. The β-1,3-1,4-glucanase activity is 333.7 and 256.7 U ml⁻¹ with barley β-glucan and lichenan as substrate, respectively; however, no activity of this enzyme on cellulose is observed. Compared to the nonoptimized control, expression level of the optimized β-1,3-1,4-glucanase based on preferred codons in P. pastoris shown a 10-fold higher level. The codon-optimized enzyme was approximately 53.8% of the total secreted protein. The optimal acidity and temperature of this recombinant enzyme were pH 6.0 and 45°C, respectively.     

Heterologous expression of an endo-β-1,4-glucanase gene from the anaerobic fungus <Emphasis Type="Italic">Orpinomyces</Emphasis> PC-2 in <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

The codon modified neutral endo-β-1,4-glucanase gene celEn, originating from the anaerobic fungus Orpinomyces sp. strain PC-2, was inserted between the strong promoter Pcel7A and the terminator Tcel7A from Trichoderma reesei. The resulting expression cassette was ligated to the pCAMBIA1300 Agrobacterium binary vector to construct pCB-hE that also contains a hygromycin B resistance marker. pCB-hE was introduced into T. reesei ZU-02 through an Agrobacterium tumefaciens–mediated transformation procedure that has been modified with an improved transformation efficiency of 12,500 transformants per 10⁷ conidia. Stable integration of the celEn gene into the chromosomal DNA of T. reesei ZU-02 was confirmed by PCR. After 48 h fermentation in shaking flasks, the endo-β-1,4-glucanase activities increased to 55–70 IU ml⁻¹ in transgenic strains, which were about 6–7 times higher than that of the original ZU-02 strain (9.5 IU ml⁻¹). When the avicel was added in fermentation medium, the endo-β-1,4-glucanase activity in the transgenic strains could be further increased to 193.6 IU ml⁻¹ after 84 h fermentation. Transgenic T. reesei strains with high neutral endo-β-1,4-glucanase activity will be particularly suitable for certain applications in textile industry. The improved procedures for overproduction and secretion of heterologous proteins in transgenic T. reesei can also be used to generate similar recombinant proteins for research or industrial purposes.     

High-level expression of a specific β-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris

In this study, a novel β-1,3-1,4-glucanase gene (designated as PtLic16A) from Paecilomyces thermophila was cloned and sequenced. PtLic16A has an open reading frame of 945 bp, encoding 314 amino acids. The deduced amino acid sequence shares the highest identity (61%) with the putative endo-1,3(4)-β-glucanase from Neosartorya fischeri NRRL 181. PtLic16A was cloned into a vector pPIC9K and was expressed successfully in Pichia pastoris as active extracellular β-1,3-1,4-glucanase. The recombinant β-1,3-1,4-glucanase (PtLic16A) was secreted predominantly into the medium which comprised up to 85% of the total extracellular proteins and reached a protein concentration of 9.1 g l⁻¹ with an activity of 55,300 U ml⁻¹ in 5-l fermentor culture. The enzyme was then purified using two steps, ion exchange chromatography, and gel filtration chromatography. The purified enzyme had a molecular mass of 38.5 kDa on SDS–PAGE. It was optimally active at pH 7.0 and a temperature of 70°C. Furthermore, the enzyme exhibited strict specificity for β-1,3-1,4-d-glucans. This is the first report on the cloning and expression of a β-1,3-1,4-glucanase gene from Paecilomyces sp.     

Ginsenoside Rd production from the major ginsenoside Rb<Subscript>1</Subscript> by <Emphasis Type="Italic">β</Emphasis>-glucosidase from <Emphasis Type="Italic">Thermus caldophilus</Emphasis>

Under optimum conditions (pH 5, 75°C, and 0.2 U purified enzyme ml⁻¹), 4 mg ginsenoside Rd was produced from 5 mg reagent-grade ginsenoside Rb₁ in 5 ml after 30 min by β-glucosidase from Thermus caldophilus GK24. Using a ginseng root extract containing 1 mg ginsenoside Rb₁ ml⁻¹ and 3.2 mg additional ginsenosides ml⁻¹, 1.23 mg ginsenoside Rd ml⁻¹ was produced after 18 h; the concentrations of ginsenosides Rb₁, Rb₂, and Rc used for ginsenoside Rd production were 0.77, 0.17, and 0.19 mg ml⁻¹, respectively.     

Screening for conditions of enhanced production of a recombinant β-glucanase secreted into the medium by <Emphasis Type="Italic">Escherichia coli</Emphasis>

The extracellular production of a hybrid bacterial β-glucanase using Escherichia coli was studied by using combinations of promoters of varying strength for both a β-glucanase as the target protein and the Kil protein as the releasing factor. Four strains with different combinations of promoter strengths were cultivated in shake-flasks on four different media to assess the cross-influence of promoter and medium in a general manner. Promoters were taken from natural as well as synthetic sequences known to exhibit either weak or strong promoter strength. By far the highest extracellular glucanase activity (>200 U ml⁻¹) was achieved when a strain harbouring the kil gene under control of a strong synthetic stationary-phase promoter and the glucanase gene under control of a strong synthetic constitutive promoter was cultivated on a complex medium mainly composed of casein peptone, yeast extract, and glycerol.     

Galactooligosaccharide production by a thermostable β-galactosidase from <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis>

A recombinant β-galactosidase from Sulfolobus solfataricus produced galactooligosaccharides (GOS) from lactose by transgalactosylation. The enzyme activity for GOS production was maximal at pH 6.0 and 85°C. The half-lives of the recombinant β-galactosidase at 70, 75, 80, 85, and 90°C were 700, 111, 72, 43, and 2.4 h, respectively, and its deactivation energy was 213 kJ mol⁻¹. The optimal amount of enzyme for effective GOS production was 3.6 U of enzyme ml⁻¹. GOS production increased with increasing lactose concentration, whereas the yield of GOS from lactose was almost constant. The rates of hydrolysis and transgalactosylation reactions increased with increasing temperature but the final concentration of GOS was maximal at 80°C. Under the conditions of pH 6.0, 80°C, 600 g lactose l⁻¹, and 3.6 U enzyme ml⁻¹, 315 g GOS l⁻¹ were obtained for 56 h with a yield of 52.5% (w/w). The β-galactosidase from S. solfataricus produced GOS with the highest concentration and yield among thermostable β-galactosidases reported to date.     

Production of the <Emphasis Type="Italic">Aspergillus aculeatus</Emphasis> endo-1,4-β-mannanase in <Emphasis Type="Italic">A. niger</Emphasis>

The β-mannanase gene (man1) from Aspergillus aculeatus MRC11624 (Izuka) was patented for application in the coffee industry. For production of the enzyme, the gene was originally cloned and expressed in Saccharomyces cerevisiae. However the level of production was found to be economically unfeasible. Here we report a 13-fold increase in enzyme production through the successful expression of β-mannanase of Aspergillus aculeatus MRC11624 in Aspergillus niger under control of the A. niger glyceraldehyde-3-phosphate dehydrogenase promoter (gpd _P) and the A. awamori glucoamylase terminator (glaA_T). The effect of medium composition on mannanase production was evaluated, and it was found that the glucose concentration and the organic nitrogen source had an effect on both the volumetric enzyme activity and the specific enzyme activity. The highest mannanase activity levels of 16,596 nkat ml⁻¹ and 574 nkat mg⁻¹ dcw were obtained for A. niger D15[man1] when cultivated in a process-viable medium containing corn steep liquor as the organic nitrogen source and high glucose concentrations.     

Enzymatic biotransformation of ginsenoside Rb1 to 20(<Emphasis Type="Italic">S</Emphasis>)-Rg3 by recombinant β-glucosidase from <Emphasis Type="Italic">Microbacterium esteraromaticum</Emphasis>

Microbacterium esteraromaticum was isolated from ginseng field. The β-glucosidase gene (bgp1) from M. esteraromaticum was cloned and expressed in Escherichia coli BL21 (DE3). The bgp1 gene consists of 2,496 bp encoding 831 amino acids which have homology to the glycosyl hydrolase family 3 protein domain. The recombinant β-glucosidase enzyme (Bgp1) was purified and characterized. The molecular mass of purified Bgp1 was 87.5 kDa, as determined by SDS-PAGE. Using 0.1 mg ml⁻¹ enzyme in 20 mM sodium phosphate buffer at 37°C and pH 7.0, 1.0 mg ml⁻¹ ginsenoside Rb1 was transformed into 0.444 mg ml⁻¹ ginsenoside Rg3 within 6 h. The Bgp1 sequentially hydrolyzed the outer and inner glucose attached to the C-20 position of ginsenosides Rb1. Bgp1 hydrolyzed the ginsenoside Rb1 along the following pathway: Rb1 → Rd → 20(S)-Rg3. This is the first report of the biotransformation of ginsenoside Rb1 to ginsenoside 20(S)-Rg3 using the recombinant β-glucosidase.     

Overexpression and characterization of cyprosin B in transformed suspension cells of <Emphasis Type="Italic">Cynara cardunculus</Emphasis>

Cynara cardunculus suspension cells were transformed by particle bombardment to overexpress the cypro11 gene coding for cyprosin B. Green fluorescent protein, used as a visual reporter through mgfp4-ER gene, facilitates the screening of transformed cells at the initial stages when antibiotics cause generalized cell death. mgfp4-ER lacks a cryptic intron and has an endoplasmic reticulum target sequence, these traits conferring an adequate use as screenable marker for transformed cells. Selected transformed cells, grown in a bioreactor, produced 3.8 g dcw l⁻¹ of biomass, 80 mg l⁻¹ of total protein and 2,060 U ml⁻¹ of enzymatic activity. Specific activity of cyprosin B, purified by anionic-exchange chromatography, was 215 U mg⁻¹ with a purification degree of 8.3-fold. The cyprosin B activity is optimal at 42°C for pH 5.1 and is inhibited by pepstatin A. The results encourage the overexpression of cypro11 gene in transformed C. cardunculus cells leading to high yields of cyprosin B production in bioreactor, which can be considered adequate for industrial production.     

A novel highly acidic β-mannanase from the acidophilic fungus Bispora sp. MEY-1: gene cloning and overexpression in Pichia pastoris

Using degenerate polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR, a 1,347-bp full-length complementary DNA fragment encompassing the gene man5A, which encodes a 429-amino acid β-mannanase with a calculated mass of 46.8 kDa, was cloned from acidophilic Bispora sp. MEY-1. The deduced amino acid sequence (catalytic domain) displayed highest identity (54.1%) with the Emericella nidulans endo-β-1,4-d-mannanase, a member of the glycoside hydrolase family 5. Recombinant MAN5A was overexpressed in Pichia pastoris, and its activity in the culture medium reached 500 U ml⁻¹. The enzyme was acidophilic, with highest activity at pH 1.0–1.5, lower than any known mannanases, and optimal temperature for activity was 65°C. MAN5A had good pH adaptability, excellent thermal and pH stability, and high resistance to both pepsin and trypsin. The specific activity, K _m, and V _max for locust bean gum substrate was 3,373 U mg⁻¹, 1.56 mg ml⁻¹, and 6,587.6 μmol min⁻¹ mg⁻¹, respectively. The enzymatic activity was not significantly affected by ions such as Ca²⁺, Cr³⁺, Co²⁺, Zn²⁺, Na⁺, K⁺, and Mg²⁺ and enhanced by Ni²⁺, Fe³⁺, Mn²⁺ and Ag⁺. These favorable properties make MAN5A a potential candidate for use in various industrial applications.     

Strain improvement of <Emphasis Type="Italic">Trichoderma reesei</Emphasis> Rut C-30 for increased cellulase production

The strain of Trichoderma reesei Rut C-30 was subjected to mutation after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NG) for 6 h followed by UV irradiation for 15 min. Successive mutants showed enhanced cellulase production, clear hydrolysis zone and rapid growth on Avicel-containing plate. Particularly, the mutant NU-6 showed approximately two-fold increases in activity of both FPA and CMCase in shake flask culture when grown on basal medium containing peptone (1%) and wheat bran (1%). The enzyme production was further optimized using eight different media. When a mixture of lactose and yeast cream was used as cellulase inducer, the mutant NU-6 yielded the highest enzyme and cell production with a FPase activity of 6.2 U ml⁻¹, a CMCase activity of 54.2 U ml⁻¹, a β-glucosidase activity of 0.39 U ml⁻¹, and a fungal biomass of 12.6 mg ml⁻¹. It deserved noting that the mutant NU-6 also secreted large amounts of xylanases (291.3 U ml⁻¹). These results suggested that NU-6 should be an attractive producer for both cellulose and xylanase production.     

Cellulase production from agricultural residues by recombinant fusant strain of a fungal endophyte of the marine sponge Latrunculia corticata for production of ethanol

Several fungal endophytes of the Egyptian marine sponge Latrunculia corticata were isolated, including strains Trichoderma sp. Merv6, Penicillium sp. Merv2 and Aspergillus sp. Merv70. These fungi exhibited high cellulase activity using different lignocellulosic substrates in solid state fermentations (SSF). By applying mutagenesis and intergeneric protoplast fusion, we have obtained a recombinant strain (Tahrir-25) that overproduced cellulases (exo-β-1,4-glucanase, endo-β-1,4-glucanase and β-1,4-glucosidase) that facilitated complete cellulolysis of agricultural residues. The process parameters for cellulase production by strain Tahrir-25 were optimized in SSF. The highest cellulase recovery from fermentation slurries was achieved with 0.2% Tween 80 as leaching agent. Enzyme production was optimized under the following conditions: initial moisture content of 60% (v/w), inoculum size of 10⁶ spores ml⁻¹, average substrate particle size of 1.0 mm, mixture of sugarcane bagasse and corncob (2:1) as the carbon source supplemented with carboxymethyl cellulose (CMC) and corn steep solids, fermentation time of 7 days, medium pH of 5.5 at 30°C. These optimized conditions yielded 450, 191, and 225 units/gram dry substrate (U gds⁻¹) of carboxylmethyl cellulase, filter-paperase (FPase), and β-glucosidase, respectively. Subsequent fermentation by the yeast, Saccharomyces cerevisiae NRC2, using lignocellulose hydrolysates obtained from the optimized cellulase process produced the highest amount of ethanol (58 g l⁻¹). This study has revealed the potential of exploiting marine fungi for cost-effective production of cellulases for second generation bioethanol processes.     

Improved guggulsterone production from sugars, precursors, and morphactin in cell cultures of Commiphora wightii grown in shake flasks and a bioreactor

Cell cultures of Commiphora wightii (Arnott.) Bhandari were grown in shake flasks and a bioreactor and an increase in guggulsterone accumulation up to 18 μg l⁻¹ was recorded in cells grown in the production medium containing a combination of sucrose:glucose (4% total), precursors (phenylalanine, pyruvic acid, xylose, and sodium acetate), morphactin, and 2iP. A yield of 10 g l⁻¹ biomass and ∼200 μg l⁻¹ guggulsterone was recorded in a 3-l flask and in a 2-l stirred tank bioreactor compared with 6.6 g biomass and 67 μg l⁻¹ guggulsterone in 250-ml flasks. Increased vessel size was correlated with increased biomass and guggulsterone accumulation. 2iP alone was not effective for biomass and guggulsterone accumulation in cell cultures of C. wightii.     

Taxane Production in Suspension Culture of <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">Media</Emphasis> var. <Emphasis Type="Italic">Hicksii</Emphasis> Carried Out in Flasks and Bioreactor

Paclitaxel and 10-deacetylbaccatin III (10-DAB III) were produced in suspension cultures of Taxus × media var. Hicksii grown in shake-flasks and in a 7-l bioreactor reaching, in the bioreactor, 4.4 mg l⁻¹ (on day 14) and 37.5 mg l⁻¹ (on day 11). In shake-flasks the highest total content of paclitaxel and 10-DAB III was 7.3 mg l⁻¹ (on day 4) and 8.8 mg l⁻¹ (on day 18). Phenylalanine, at 0.05 mM, increased paclitaxel accumulation in cells cultivated in bioreactor and flasks 30-fold and 9-fold (from 0.02 mg l⁻¹ to 0.6 mg l⁻¹ and to 0.2 mg l⁻¹, respectively). The 10-DAB III content in cells from flasks was increased from 0.4 mg l⁻¹ to 1.6 mg l⁻¹.     

Studies on inositol-mediated expression of MAL gene encoding maltase and phospholipid biosynthesis in Schizosaccharomyces pombe

In this study, the effects of inositol addition on expression of the MAL gene encoding maltase and phosphatidylinositol (PI) biosynthesis in Schizosaccharomyces pombe (a naturally inositol-requiring strain) were examined. We found that specific maltase activity was at its maximum when the concentration of added inositol reached 6 μg ml⁻¹ in a synthetic medium containing 2.0% (w/v) glucose. When the concentration of added inositol was 1 μg ml⁻¹ in the medium, repression of MAL gene expression occurred at glucose concentration higher than 0.2% (w/v). However, when S. pombe was cultured in the synthetic medium containing 6 μg ml⁻¹, repression of maltase gene expression occurred only at initial glucose concentration above 1.0% (w/v). More mRNA encoding maltase was detected in the cells grown in the medium with 6 μg ml⁻¹ inositol than in those grown in the same medium with 1 μg ml⁻¹ inositol. These results demonstrate that higher inositol concentrations in the synthetic medium could derepress MAL gene expression in S. pombe. PI content of the yeast cells grown in the synthetic medium with 6 μg ml⁻¹ of inositol was higher than that of the yeast cells grown in the same medium with 1 μg ml⁻¹ of inositol. This means that PI may be involved in the derepression of MAL gene expression in S. pombe.     

Purification,characterization, and potential applications of formate oxidase from <Emphasis Type="Italic">Debaryomyces vanrijiae</Emphasis> MH201

Formate oxidase was found in cell-free extracts of Debaryomyces vanrijiae MH201, a soil isolate. After purification by column chromatography, the preparation showed a protein band corresponding to a molecular mass (MM) of 64 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The MM, estimated by a gel filtration, was 99 kDa. The preparation showed two and three bands on isoelectric focusing under denaturing and native conditions, respectively. These results suggest that the preparation contained three isoforms, each of which might be composed of αα, αβ, and ββ subunits with apparently similar MM. The preparation acted on formate with K _m and V _max values of 11.7 mM and 262 μmol min⁻¹ mg⁻¹, respectively, at pH 4.5 and 25°C, but showed no evidence of activity on the other compounds tested. The optimum pH and temperature were pH 4.0 and 35°C, respectively. The preparation showed activities of 85% of the initial activity after storage at pH 6.0 and 4°C for 8 weeks. When 10 mM formaldehyde was reacted with 2.0 U ml⁻¹ of the enzyme preparation at pH 5.5 and room temperature in the presence of 2.0 U ml⁻¹ of a microbial aldehyde oxidase and 100 U ml⁻¹ of catalase for 180 min, neither of formate nor formaldehyde was detected, suggesting that the reaction involved the quantitative conversion of formaldehyde to carbon dioxide.     

Endocrinological investigation into the reproductive cycles of two sympatric skate species, Malacoraja senta and Amblyraja radiata, in the western Gulf of Maine

The smooth skate, Malacoraja senta, and thorny skate, Amblyraja radiata, are two commercially exploited batoids found within the Gulf of Maine. During the past five years, we conducted a large study to accurately describe important biological life history parameters previously lacking for these species. As part of that project, the current study reports our findings on the hormonal profiles associated with the reproductive cycles of M. senta and A. radiata. Blood samples were obtained from mature M. senta and A. radiata of both sexes from all months of the year, and plasma testosterone (T), estradiol (E₂) and progesterone (P₄) concentrations were determined using radioimmunoassay (RIA). In female M. senta and A. radiata, monthly T concentrations ranged from 4,522 pg ml⁻¹ to 1,373 pg ml⁻¹ and 31,940 pg ml⁻¹ to 14,428 pg ml⁻¹, E₂ concentrations from 831 pg ml⁻¹ to 60 pg ml⁻¹ and 8,515 pg ml⁻¹ to 2,902 pg ml⁻¹, and P₄ concentrations from 3,027 pg ml⁻¹ to 20 pg ml⁻¹ and 3,264 pg ml⁻¹ to 331 pg ml⁻¹, respectively. No statistical differences were detected between any months for any hormone. Estradiol concentrations were not correlated with ovary weight, shell gland weight, or diameter of the largest follicles in either species. Monthly T concentrations in male M. senta and A. radiata ranged from 23,146 to 12,660 pg ml⁻¹ and from 57,500pg ml⁻¹ to 24,737 pg ml⁻¹, while E₂ concentrations ranged from 7.5 pg ml⁻¹ to undetectable and 103 to 30 pg ml⁻¹, respectively. No statistical differences were observed between months for either steroid. Testosterone concentrations were weakly correlated with testes weight and percent of stage VI spermatocysts in A. radiata, however, no correlation was detected between T and stage VI spermatocysts in M. senta. Collectively, these data support the previous conclusion that M. senta and A. radiata of both sexes are capable of reproducing year round in the western Gulf of Maine.     

Cloning of β-1,3-1,4-glucanase gene from Bacillus licheniformis EGW039 (CGMCC 0635) and its expression in Escherichia coli BL21 (DE3)

β-1,3-1,4-Glucanase has been applied in the brewing and animal feed additive industry. It can effectively improve digestibility of barley-based diets and reduce enteritis. It also reduces viscosity during mashing for high-quality brewers malt. The aim of this work is to clone β-1,3-1,4-glucanase-encoding gene and express it heterogeneously. The gene was amplified by polymerase chain reaction using Bacillus licheniformis genomic DNA as the template and ligated into the expression vector pET28a. The recombinant vector was transformed into Escherichia coli. The estimated molecular weight of the recombinant enzyme with a six-His tag at the N terminus was about 28 kDa, and its activities in cell lysate supernatant were 1,286 and 986 U ml⁻¹ for 1% (w/v) barley β-glucan and 1% (w/v) lichenan, respectively. Accordingly, the specific activities were 2,479 and 1,906 U mg⁻¹ for these two substrates. The expression level of recombinant β-1,3-1,4-glucanase was about 60.9% of the total protein and about 12.5% of the total soluble protein in crude cell lysate supernatant. Acidity and temperature optimal for this recombinant enzyme was pH 5.6 and 40°C, respectively.