Secretory expression of human growth hormone inSaccharomyces cerevisiae using three different leader sequences

A recombinant human growth hormone (hGH) was expressed as a secretory product in the yeastSaccharomyces cerevisiae. Three different leader sequences derived from the mating factor α1 (MFα1), inulinase and invertase were used to direct the secretion of hGH into the extracellular medium. Among three leader sequences tested, the inulinase leader sequence was found to be the most efficient in the secretory expression of hGH. In contrast, no hGH was detected in the extracellular medium with the invertase leader sequence. After 48 h shake-flask culture, the yields of hGH secreted into the medium by the invertase, MFα1, inulinase and invertase leader sequences were approximately 0, 0.3 and 0.9 mg/L, respectively. The secretion efficiencies were also found to be 0, 3.8 and 13% for the invertase, MFα1 and inulinase leader sequences, respectively.     

Studies on a beta-fructosidase (inulinase) produced bySaccharomyces fragilis

Summary A study was made of a β-fructosidase, which is produced extracellularly and intracellularly bySaccharomyces fragilis. The enzyme catalyzes the hydrolysis of inulin, bacterial levans, sucrose, and the fructose portion of raffinose, by splitting off terminal fructosyl units. It attacks β-2,1 as well as β-2,6 linkages. The enzyme content of inulin-grown cells is sufficient to allow fermentation of inulin at the same rate as glucose. The ratio of hydrolysis rates with sucrose and inulin was about 25 for the β-fructosidase ofS. fragilis and about 14,000 for invertase.S. fragilis does not contain significant amounts of invertase and it ferments inulin, sucrose and raffinose with the aid of a related, but different enzyme, inulinase. Conditions of growth were established which favor inulinase synthesis. Highest yields were obtained with inulin as the carbon source, and somewhat lower yields with raffinose. Glucose, fructose and sucrose were poor inducers of inulinase. The pH of the medium during growth on inulin had to be in the range where inulinase could act, otherwise growth was tardy and poor. In an inulin containing medium aeration favored enzyme production as a result of stimulation of growth. The inulinase content of the cells in a unit volume was generally greater than that in the culture medium. The intracellular inulinase could be solubilized quantitatively by autolysis. The intra-and extracellular inulinases were concentrated and purified to the same extent. Comparison of the two preparations with respect to substrate specificity, rate of inactivation by heat, pH optima with sucrose (4.2) and with inulin (5.0), and elution patterns from a column of diethylaminoethyl cellulose, indicated that the intra-and extracellular enzymes were identical.     

The β-fructofuranosidase activities of a strain of Clostridium acetobutylicum grown on inulin

Summary The -fructofuranosidase activities of a strain of Clostridium acetobutylicum, selected for its capacity to grow on inulinic substrates, were investigated. When grown on inulin, this strain produced extracellular and intracellular -fructofuranosidases, both of which hydrolysed inulin (inulinase activity) and sucrose (invertase activity). Inulinase activity was higher than invertase activity in the extracellular preparation, the opposite being observed for the cellular preparation. The effects of pH and temperature, substrate specificity and the kinetic constants for inulin and sucrose were studied on both preparations, as well as induction by inulin and repression by glucose and fructose of inulinase and invertase activities. The overall results were consistent with the existence of a least one inulinase, (EC 3.2.1.7), mainly but not entirely released in the extracellular medium, and an invertase (3.2.1.26) localized within the cell.Time course hydrolysis experiments of dalhia inulin and Jerusalem artichoke inulofructans by extracellular inulinase showed that this preparation had a remarkably high specificity for hydrolysis of long chain inulofructans.     

Mutual influence of invertase and acid phosphatase of the yeastSaccharomyces cerevisiae on their secretion into the cultivation medium

The hypothesis that various extracellular enzymes produced by the yeastSaccharomyces cerevisiae exert a mutual influence on their secretion into the cultivation medium was tested experimentally. The statistically processed results indicate that extracellular invertase affects the secretion of acid phosphatase, and acid phosphatase affects the secretion of invertase. In addition, the secretion of each of these enzymes was shown to be subject to autoregulation.     

Discrimination of Mammalian GPI-Anchored Proteins by Hydropathy and Amino Acid Propensities

A 1.7-kb DNA fragment cloned from Zymomonas mobilis genomic DNA complemented the inability to grow on sucrose of a Sue ^? mutant of Z. mobilis that was deficient in the production of both extracellular levansucrase and invertase. Analysis of the nucleotide sequence of the fragment found two open reading frames (ORFs), both of which did not correspond to the structural gene for the levansucrase or the invertase. By subcloning each ORF into two different Suc ^? mutants of Z. mobilis, it has been found that the first ORF (gene zliE) activates the production of the extracellular levansucrase and invertase, and the second ORF (gene zliS) stimulates the secretion of the two enzymes. Gene zliS might contribute to the secretion of proteins having no signal peptide. The expression of zliE and zliS seemed to be under the control of the same promoter.     

Invertase and phosphatase of yeast in a phosphate-limited continuous culture

Summary Deficiency of inorganic phosphate caused the hyper production of invertase and the derepression of acid phosphatase in a continuous culture ofSaccharomyces carlsbergensis. The specific invertase activity was 40,000 enzyme units per g dry cell weight at a dilution rate lower than 0.05 h^–1 with a synthetic glucose medium of which the molecular ratio of KH₂PO₄ to glucose was less than 0.006. This activity is eight fold higher than in a batch growth and 1.5 fold as much as the highest enzyme activity observed so far in a glucose-limited continuous culture.For the hyper production of invertase, it is necessary to culture the yeast continuously by keeping the Nyholm's conservative inorganic phosphate concentration at less than 0.2 m mole per g dry weight cell. The derepression of acid phosphatase brought about by phosphate deficiency, was similar in both batch and continuous cultures.Nomenclature D dilution rate of continuous culture (h^–1) - E_i invertase concentration in culture (enzyme unit l^–1) - E_p acid phosphatase concentration in culture (enzyme unit l^–1) - P inorganic phosphate concentration in culture (mM) - S glucose concentration in culture (mM) - X cell concentration in culture (g dry weight cell l^–1) Greek Letter specific rate of growth (h^–1) Suffix f feed - 0 initial value     

Optimized invertase expression and secretion cassette for improving Yarrowia lipolytica growth on sucrose for industrial applications

Yarrowia lipolytica requires the expression of a heterologous invertase to grow on a sucrose-based substrate. This work reports the construction of an optimized invertase expression cassette composed of Saccharomyces cerevisiae Suc2p secretion signal sequence followed by the SUC2 sequence and under the control of the strong Y. lipolytica pTEF promoter. This new construction allows a fast and optimal cleavage of sucrose into glucose and fructose and allows cells to reach the maximum growth rate. Contrary to pre-existing constructions, the expression of SUC2 is not sensitive to medium composition in this context. The strain JMY2593, expressing this new cassette with an optimized secretion signal sequence and a strong promoter, produces 4,519 U/l of extracellular invertase in bioreactor experiments compared to 597 U/l in a strain expressing the former invertase construction. The expression of this cassette strongly improved production of invertase and is suitable for simultaneously high production level of citric acid from sucrose-based media.     

Biosynthesis of Inulinases by Bacillus Bacteria

Biosynthesis of extracellular inulinase by bacteria Bacillus polymyxa 29,B. polymyxa 722, and B. subtilis 68 was studied. The optimal parameters for the producer growth were as follows: pH 7.0, 33–35°C, growth duration 72 h. The presence of reduced mineral nitrogen or organic nitrogen was necessary for the enzyme biosynthesis. The inulinase biosynthesis was sharply activated in the presence of carbohydrates. B. polymyxa 722 and B. polymyxa 29 displayed the maximum activities on a starch-containing culture medium; the maximum activity of B. subtilis 68 was in the presence of sucrose. Inulin did not induce inulinase biosynthesis by the strains studied. The time course of bacteria growth and enzyme biosynthesis was studied.     

Alkaline inulinase production by a newly isolated bacterium Marinimicrobium sp. LS–A18 and inulin hydrolysis by the enzyme

To date, all of microbial inulinases reported showed optimal activity at pH values ranging from 3.5 to 7.0. A bacterial strain, Marinimicrobium sp. LS-A18, showing high extracellular inulinolytic activity was isolated from a marine solar saltern of the Yellow Sea in China. Maximum enzyme activity was obtained at 55°C and pH 9.0, respectively. The inulinase activity was induced by inulin, but not by the other carbon sources employed. Under the optimal medium and culture condition, the highest inulinase activity, 14.6 U/ml, was obtained after 96 h of incubation at shake flask level. The optimal medium for inulinase production was MHI medium containing 4% inulin, 1% peptone and 5% NaCl, while the optimal culture condition for inulinase production were pH 7.5, temperature 37°C, agitation speed 210 rpm, medium volume 40 ml in 250 ml shake flask, and incubation time 96 h. A large amount of monosaccharides was released after inulin hydrolysis by the inulinase from strain LS-A18. This is the first report on alkaline inulinase production from microorganism.     

Secretion of invertase from Schwanniomyces occidentalis

Invertase synthesis in Schwanniomyces occidentalis is regulated by catabolite repression and is derepressed by raffinose and low concentrations of glucose. Efficiency of a carbon source in derepression of invertase is dependent upon the type of culture medium: either raffinose in a rich medium or a low concentration of glucose in a yeast minimal medium. The kinetics of derepression can be modulated by changing the carbon source. When cells are grown in a rich medium with 0.5% raffinose as the sole carbon source, Schwanniomyces occidentalis secretes 80 times more invertase than Saccharomyces cerevisiae grown in the same conditions. About 50% of the total amount of invertase produced by Schwanniomyces occidentalis is secreted in the extracellular medium in contrast to Saccharomyces cerevisiae where only 6 to 15% of the protein is secreted in the medium.     

不同信号肽对酿酒酵母蔗糖转化酶Suc2分泌表达水平的影响

目前，绝大多数酿酒酵母（Saccharomyces cerevisiae）菌株利用菊糖生产乙醇的能力有限，而蔗糖转化酶Suc2是酿酒酵母水解菊糖的关键酶，其分泌水平直接影响酿酒酵母转化菊糖为乙醇的性能。为提高酿酒酵母中蔗糖转化酶Suc2的分泌表达水平，利用生物信息学的分析方法选择出11种不同的分泌信号肽，包括酿酒酵母内源性、其他菌株来源以及已报道序列优化改造的信号肽，将它们融合至Suc2并构建了相应的酿酒酵母BY4741重组菌。其中，酿酒酵母内源分泌信号肽AGA2能使蔗糖转化酶Suc2更有效的分泌，含有信号肽AGA2的重组菌BY-AG的蔗糖酶酶活和菊糖酶酶活相对于含有天然信号肽的原始菌BY-S分别提高42%和26%，其利用菊糖产乙醇能力较原始菌提高了32%，乙醇产量达到78.11 g/L。在使用毕赤酵母（Pichia pastoris）分泌信号肽MSB2时，蔗糖转化酶Suc2的分泌水平也有提高，含有信号肽MSB2的重组菌BY-MS较原始菌BY-S的蔗糖酶酶活和菊糖酶酶活分别提高了80%和74%，同时，利用菊糖产乙醇能力也提高了56%，产量达到86.31 g/L。最后，对重组菌BY-MS摇瓶发酵过程中的生物量、蔗糖酶酶活、残糖总量和乙醇产量进行了监测，结果表明，重组菌BY-MS的发酵性能较原始菌BY-S有显著提高。本研究为提高蔗糖转化酶Suc2的分泌水平、构建高效菊糖基乙醇生产菌株提供参考。     

Effects of Octadecanoylsucrose Derivatives on the Production of Inulinase by Apergillus niger SL-09

Summary The effect of the addition of octadecanoylsucrose esters to the growth medium on the production of inulinase by Aspergillus niger SL-09 was studied in batch culture using shake flasks. The activities of inulinase in vitro and in vivo formed by Aspergillus niger SL-09 was enhanced dramatically by the addition of sucrose ester S-770 to the medium, and it was confirmed that sucrose ester acted as a very efficient inducer for inulinase production. As a result, with the addition of 6 g sucrose ester l⁻¹ at the beginning of the culture, the enzyme activities were enhanced near 7-fold higher than that obtained in the basal medium.     

Mutual effect of invertase and acid phosphatase from the yeast Saccharomyces cerevisiae on their secretion into culture media

The hypothesis that various extracellular enzymes produced by the yeast Saccharomyces cerevisiae exert a mutual influence on their secretion into the culture medium was tested experimentally. The statistically processed results indicate that extracellular invertase affects the secretion of acid phosphatase, and acid phosphatase affects the secretion of invertase. In addition, the secretion of each of these enzymes was shown to be subject to autoregulation.     

Production and localization of beta-fructosidase in asynchronous and synchronous chemostat cultures of yeasts

In synchronized continuous cultures of Saccharomyces cerevisiae CBS 8066, the production of the extracellular invertase (EC 3.2.1.26) showed a cyclic behavior that coincided with the budding cycle. The invertase activity increased during bud development and ceased at bud maturation and cell scission. The cyclic changes in invertase production resulted in cyclic changes in amounts of invertase localized in the cell wall. However, the amount of enzyme invertase present in the culture liquid remained constant throughout the budding cycle. Also, in asynchronous continuous cultures of S. cerevisiae, the production and localization of invertase showed significant fluctuation. The overall invertase production in an asynchronous culture was two to three times higher than in synchronous cultures. This could be due to more-severe invertase-repressive conditions in a synchronous chemostat culture. Both the intracellular glucose-6-phosphate concentration and residual glucose concentration were significantly higher in synchronous chemostat cultures than in asynchronous chemostat cultures. In the asynchronous and synchronous continuous cultures of S. cerevisiae, about 40% of the invertase was released into the culture liquid; it has generally been believed that S. cerevisiae releases only about 5% of its invertase. In contrast to invertase production and localization in the chemostat cultures of S. cerevisiae, no significant changes in inulinase (EC 3.2.1.7) production and localization were observed in chemostat cultures of Kluyveromyces maxianus CBS 6556. In cultures of K. marxianus about 50% of the inulinase was present in the culture liquid.     

Production and localization of beta-fructosidase in asynchronous and synchronous chemostat cultures of yeasts.

In synchronized continuous cultures of Saccharomyces cerevisiae CBS 8066, the production of the extracellular invertase (EC 3.2.1.26) showed a cyclic behavior that coincided with the budding cycle. The invertase activity increased during bud development and ceased at bud maturation and cell scission. The cyclic changes in invertase production resulted in cyclic changes in amounts of invertase localized in the cell wall. However, the amount of enzyme invertase present in the culture liquid remained constant throughout the budding cycle. Also, in asynchronous continuous cultures of S. cerevisiae, the production and localization of invertase showed significant fluctuation. The overall invertase production in an asynchronous culture was two to three times higher than in synchronous cultures. This could be due to more-severe invertase-repressive conditions in a synchronous chemostat culture. Both the intracellular glucose-6-phosphate concentration and residual glucose concentration were significantly higher in synchronous chemostat cultures than in asynchronous chemostat cultures. In the asynchronous and synchronous continuous cultures of S. cerevisiae, about 40% of the invertase was released into the culture liquid; it has generally been believed that S. cerevisiae releases only about 5% of its invertase. In contrast to invertase production and localization in the chemostat cultures of S. cerevisiae, no significant changes in inulinase (EC 3.2.1.7) production and localization were observed in chemostat cultures of Kluyveromyces maxianus CBS 6556. In cultures of K. marxianus about 50% of the inulinase was present in the culture liquid.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

Cloning and characterization of the inulinase gene from a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The extracellular inulinase structural gene was isolated from the genomic DNA of the marine yeast Pichia guilliermondii strain 1 by PCR. The gene had an open reading frame of 1,542 bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 514 amino acid residues of a protein with a putative signal peptide of 18 amino acids and the calculated molecular mass of 58.04 kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNXPNGL) and (RDPKVF). It also had ten conserved putative N-glycosylation sites. The inulinase from P. guilliermondii strain 1 was found to be closely related to that from Kluyveromyces marxianus. The inulinase gene without the signal sequence was subcloned into pPICZαA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum activity of 58.7 ± 0.12 U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. A large amount of monosaccharides, disaccharides and oligosaccharides were detected after the hydrolysis of inulin with the crude recombinant inulinase.     

Increase in extracellular inulinase production for a new Rhizoctonia ssp. strain by using buckwheat (Fagopyrum esculentum) flour as a single carbon source

Aims: A newly isolated strain of Rhizoctonia ssp. was used for the production of extracellular inulinase. Previously, the qualitative effects of some carbon and nitrogen sources from fermentative media and the physicochemical parameters for growth were established by Plackett–Burman analysis, and the main parameters that affect extracellular inulinase yield were identified. In this study, the quantitative effect of the carbon to nitrogen ratio in the fermentative medium and the growth temperature were studied and optimized using central composite design and response surface methodology. Methods and results: On the basis of optimization, the maximum extracellular inulinase activity was achieved when 2·5–6·5% buckwheat flour was used as a single carbon source and 4·6–5·0% yeast extract was used as nitrogen source, by submerged cultivation, after 48 h at an incubation temperature between 15 and 27·5°C. Conclusions: Under the fermentative conditions established in this study, a maximum extracellular inulinase yield of 1·8 UI ml^?1 was achieved. Rhizoctonia ssp. strain can be used for extracellular inulinase production. Also, buckwheat flour proved to be an inexpensive and abundant substrate suitable for obtaining inulinase. Significance and impact of the study: Inulinases are versatile tools for biotechnology as they can be used for a wide range of applications, including production of bioethanol, fructose syrup and inulo‐oligosaccharides, lactic acid, citric acid and butanediol.     

Acid and alkaline invertases in roots and nodules of Lupinus angustifolius infected with Rhizobium lupini

Summary Both acid and alkaline invertase activity were found in tips and cortical tissue of Lupinus angustifolius L. roots infected with Rhizobium lupini NZP 2257. Only the alkaline invertase was detected in the nodule cytoplasm. Weak invertase activity found in the bacteroids was probably a contamination from plant invertase. The alkaline invertase activity in the nodule cytoplasm was 250 times that detected in the bacteroids and 8 times that detected in cortical tissue. No intracellular or extracellular invertase was detected in R. lupini cultured in liquid medium containing sucrose.