Effect of post-induction nutrient feeding strategies on the production of bioadhesive protein in Escherichia coli

The effect of post-induction nutrient feeding strategies on the production of bioadhesive protein using an IPTG inducible expression system in Escherichia coli was investigated. Cells were cultured in an exponential fed-batch mode to the OD600 of ca. 100 (48 gDCW/L) prior to induction. Six different post-induction nutrient feeding strategies (pH-stat, exponential, constant and linear change in feeding rate with three different slopes) were then applied, and bioadhesive protein production was examined. It was found that post-induction cell growth was independent of nutrient feeding rate. However, bioadhesive protein production was significantly affected by post-induction feeding strategies. Linearly changing post-induction feeding rate with a suitable slope allowed production of bioadhesive protein up to 5.3 g/L, which was higher than that obtained by the other post-induction feeding strategies.     

Bioprocess development to produce a hyperthermostable S-methyl-5′-thioadenosine phosphorylase in Escherichia coli

Nucleoside phosphorylases are important biocatalysts for the chemo-enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S-methyl-5′-thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide-spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24-well plates using a fed-batch medium. Subsequently, HCD cultivations were performed using E. coli BL21-Gold cells, by employing a glucose-limited fed-batch strategy. Comparing different growth rates in stirred-tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500-mL scale, final cell dry weights of 87.1–90.1 g L⁻¹ were observed together with an overproduction of ApMTAP in a 1.9%–3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg⁻¹ (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli.     

The methylotrophic yeast Pichia pastoris as a host for the expression and production of thermostable xylanase from the bacterium Rhodothermus marinus

A thermostable glycoside hydrolase family-10 xylanase originating from Rhodothermus marinus was cloned and expressed in the methylotrophic yeast Pichia pastoris (SMD1168H). The DNA sequence from Rmxyn10A encoding the xylanase catalytic module was PCR-amplified and cloned in frame with the Saccharomyces cerevisiae alpha-factor secretion signal under the control of the alcohol oxidase (AOX1) promotor. Optimisation of enzyme production in batch fermentors, with methanol as a sole carbon source, enabled secretion yields up to 3gl(-1) xylanase with a maximum activity of 3130Ul(-1) to be achieved. N-terminal sequence analysis of the heterologous xylanase indicated that the secretion signal was correctly processed in P. pastoris and the molecular weight of 37kDa was in agreement with the theoretically calculated molecular mass. Introduction of a heat-pretreatment step was however necessary in order to fold the heterologous xylanase to an active state, and at the conditions used this step yielded a 200-fold increase in xylanase activity. Thermostability of the produced xylanase was monitored by differential-scanning calorimetry, and the transition temperature (T(m)) was 78 degrees C. R. marinus xylanase is the first reported thermostable gram-negative bacterial xylanase efficiently secreted by P. pastoris.     

Xylanase production by Trichoderma reesei Rut C-30 grown on L-arabinose-rich plant hydrolysates

The suitability of L-arabinose-rich plant hydrolysates as carbon sources and inducers of xylanase production in Trichoderma reesei Rut C-30 was tested. Significantly higher xylanase activities were obtained in cultures on oat husk and sugar beet pulp hydrolysates than on lactose. In batch culture with oat husk hydrolysate and lactose, the xylanase activity was about 9 times higher ( approximately 510 IU/ml) than in lactose ( approximately 60 IU/ml). Even higher xylanase activity ( approximately 630 IU/ml) was obtained when the batch cultivations were done on sugar beet pulp hydrolysate and lactose. In a fed-batch culture using oat husk hydrolysate-lactose the xylanase activity was as high as 1350 IU/ml in 4 days. The cellulase production clearly decreased when T. reesei was cultured on both hydrolysates compared to the cultivation on lactose. Moreover, the relative amounts of the xylanases I-III were similar regardless the used carbon source.     

Characterization of major hydrolytic enzymes secreted by Pythium myriotylum, causative agent for soft rot disease

Pythium myriotylum, an oomycetous necrotroph is the causal agent of soft rot disease affecting several crops. Successful colonization by necrotrophs depends on their secretion of a diverse array of plant cell wall degrading enzymes (CWDEs). The induction dynamics of CWDEs secreted by P. myriotylum was analysed as little information is available for this pathogen. Activities of CWDEs that included pectinase, cellulase, xylanase and protease were detected using radial diffusion assay and differential staining. In Czapek Dox minimal medium supplemented with respective substrates as carbon source, the increase in CWDE activities was observed till 8 days of incubation after which a gradual decline in enzymatic activities was observed. With sucrose as sole carbon source, all the enzymes studied showed increase in activity with fungal growth while with cell wall material derived from ginger rhizome as sole carbon source, an initial spurt in cellulase, xylanase and pectinase activities was observed 3 days post incubation while protease activity increased from three days of incubation and reached maximum at 13 days of incubation. To further evaluate the role of CWDEs in pathogenicity, UV-induced mutants (pmN14uv1) were generated wherein significant reduction in cellulase, pectinase and protease activities were observed while that of xylanase remained unchanged compared to wild type isolate (RGCBN14). Bioassays indicated changes in infection potential of pmN14uv1 thereby suggesting the crucial role played by P. myriotylum CWDEs in initiating the rotting process. Hence appropriate strategies that target the production/activity of these secretory hydrolytic enzymes will help in reducing disease incidence/pathogen virulence.     

Morphological and Molecular Characterization of Pomegranate Fruit Rot Pathogen, <Emphasis Type="Italic">Chaetomella raphigera</Emphasis>, and its Virulence Factors

A new fungal pathogen was isolated from rotten pomegranates collected from the orchards of different parts of Maharashtra. The pathogen was morphologically identified as Chaetomella raphigera followed by sequencing of ITS and D1/D2 hypervariable region of LSU (28S) of rRNA gene. The pathogen produced pectinase, cellulase, xylanase and protease in liquid medium at a concentration of 71, 13.8, 54.3 and 7 U/ml respectively. Enzyme activity was also determined during pathogenesis in the tissues artificially infected by C. raphigera. Xylanase activity was maximum (25.1 U/g) followed by pectinase (19.2 U/g) and cellulase (1.5 U/g), whereas, protease activity was unnoticed. There was significant correlation (P < 0.05) between disease rating scale and pectinase, xylanase and cellulase activity in infected tissues. This indicates the simultaneous production of hydrolytic enzymes that aids in necrosis of fruit tissues. The elevated levels of these enzymes in infected tissues as compared with control suggest their possible role in pathogenesis. Thus, pectinase, cellulase and xylanase produced by C. raphigera acts as major virulence factors in the development of fruit rot in pomegranates. This is a first report of fungal fruit rot caused by C. raphigera in pomegranate.     

The effect of protease on stability of cellulase and xylanase fromCellulomonas flavigena

Summary Crude preparations of extracellular cellulase and xylanase fromCellulomonas flavigena at 4°C show a rapid loss of activity. With the protease inhibitors aprotinin and -2 macroglobulin this loss of activity could be dramatically reduced. Cellulase and xylanase extracted from a protease negative mutant were also more stable. When the cellulase and xylanase was purified by DEAE sepharose from wild type strains, the protease activity could be separated, such preparations of cellulase and xylanase were extremely stable.     

Expression of xylanase enzymes from thermophilic microorganisms in fungal hosts

Bulk production of xylanases from thermophilic microorganisms is a prerequisite for their use in industrial processes. As effective secretors of gene products, fungal expression systems provide a promising, industrially relevant alternative to bacteria for heterologous enzyme production. We are currently developing the yeast Kluyveromyces lactis and the filamentous fungus Trichoderma reesei for the extracellular production of thermophilic enzymes for the pulp and paper industry. The K. lactis system has been tested with two thermophilic xylanases and secretes gram amounts of largely pure xylanase A from Dictyoglomus thermophilum in chemostat culture. The T. reesei expression system involves the use of the cellobiohydrolase I (CBHI) promoter and gene fusions for the secretion of heterologous thermostable xylanases of both bacterial and fungal origin. We have reconstructed the AT-rich xynB gene of Dictyoglomus thermophilum according to Trichoderma codon preferences and demonstrated a dramatic increase in expression. A heterologous fungal gene, Humicola grisea xyn2, could be expressed without codon modification. Initial amounts of the XYN2 protein were of a gram per liter range in shake-flask cultivations, and the gene product was correctly processed by the heterologous host. Comparison of the expression of three thermophilic heterologous microbial xylanases in T. reesei demonstrates the need for addressing each case individually.     

Cell wall degrading enzymes in Orobanche aegyptiaca and its host Brassica campestris

Activities of the cell-wall degrading enzymes cellulase (EC 3. 2. 1. 4). poly-galacturonase (EC 3. 2. 1. 15) and xylanase (EC 3. 2. 1. 8), and also that of protease (EC 3. 4. 21. 14) have been determined in the parasite Orobanche aegyiptiaca Pers. and in the roots of infected and control host plants of Brassica campestris L. var. rapa Hartm. Shoots, roots and root-freed tubers of Orobanche showed pronounced activities of these enzymes. When expressed in terms of specific activity, protease, cellulase and polygalacturonase activities were higher and xylanase lower in all parts of Orobanche than in the roots of infected or control host plants. Orobanche roots had higher activities of cellulase, polygalacturonase and protease than tuber or shoot. While xylanase activity was higher in the shoot than in roots or tuber. Infection by Orobanche significantly enhanced the cellulase activity of host roots. Cellulase and polygalacturonase in the Orobanche roots appear as the main enzymes involved in establishing haustorial connection with the host root. Protease might be involved in degrading proteins and lipoproteins of the cell walls and membranes of the host tissues.     

Thermostable, alkalophilic and cellulase free xylanase production by Thermoactinomyces thalophilus subgroup C

Thermoactinomyces thalophilus produced cellulase free extracellular endo-1,4-beta-xylanase (EC 3.2.1.8) at 50 degrees C and pH 8.5. Maximum xylanase production was achieved in fermentation medium using birchwood xylan as substrate after 96 h of growth at 50 degrees C. Other agricultural substrates such as wheat bran, wheat straw, sugarcane bagasse and cornstover produced less xylanase. The crude enzyme preparation from mutant T. thalophilus P2 grown under optimised fermentation conditions showed no cellulase contamination and maximum xylanase activity of 42 U/ml at 65%deg;C and pH 8.5-9.0. This enzyme with initial xylanase activity of 42 U/ml was found thermostable up to 65 degrees C and retaining 50% of its activity after its incubation for 125 min at 65 degrees C.     

Production of xylanase by Thermoascus aurantiacus from sugar cane bagasse in an aerated growth fermentor

In recent years, the use of xylanases has been adopted by many processing industries, such as pulp and paper, food and textile factories. This study demonstrates that Thermoascus aurantiacus ATCC 204492 is able to produce a high level of thermostable xylanase when sugar cane bagasse is used as a substrate. Fermentations were performed in a glass-column reactor with forced aeration. A xylanase activity of 1597 U/g was attained after 10 days of solid-state fermentation. The effects of different airflow rates (0, 3.0, 6.0 l/(h g) bagasse) and initial mass of bagasse (8, 12.5, 17 g) on the production of xylanase were investigated using a statistical experimental design. The airflow rates had a significant effect on enzyme activity, whereas initial mass of bagasse had no significant effect on enzyme activity. 6 l/(h g) airflow rate and 8 g substrate resulted in the highest yields of xylanase (1597 U/g).     

草酸青霉纤维素酶基因和木聚糖酶基因的调控基因POX05145的鉴定

草酸青霉能产生完整的纤维素酶和木聚糖酶酶系,其纤维素酶基因的表达主要受转录因子的调控。前期工作中,通过对草酸青霉菌株HP7-1在不同碳源培养基培养条件下转录组的比较分析,获得了调控纤维素酶和木聚糖酶产量的候选调控基因集。本研究以草酸青霉ΔPoxKu70为出发菌株,通过同源重组法,构建并获得了其中一个候选调控基因POX05145的缺失突变株ΔPOX05145。在微结晶纤维素Avicel诱导培养条件下,与出发菌株ΔPoxKu70相比,ΔPOX05145的纤维素酶产量和木聚糖酶产量发生了显著改变。其中,在诱导第2天时,ΔPOX05145对硝基苯-β-D-纤维二糖苷酶产量和木聚糖酶产量分别上升43.4%和164.7%,对硝基苯-β-D-半乳糖吡喃葡萄糖苷酶产量下降92.8%,但是,滤纸酶产量和羧甲基纤维素酶产量没有显著变化。然而,在诱导第4天时,所有纤维素酶产量和木聚糖酶产量上升100.4%~294.0%。实时荧光定量PCR检测表明POX05145在不同的时间不同程度的调控主要的纤维素酶基因和木聚糖酶基因的表达。序列分析表明POX05145含有一个GAL4类锌指结构的DNA结合功能域和一个保守的真菌特有的转录因子结构域(Fungal_TF_MHR)。     

Interrelationship of Xylanase Induction and Cellulase Induction of Trichoderma longibrachiatum

Xylose oligomers rapidly induced xylanase activity of Trichoderma longibrachiatum, whereas induction was delayed in the presence of glucose. Cellobiose, cellopentaose, and xylobiose did not induce detectable levels of cellulase activity. However, mixtures of xylobiose with cellobiose or cellopentaose rapidly induced cellulase activity. In addition, mixtures of xylobiose with cellopentaose or cellobiose induced xylanase activity more effectively than xylobiose alone. Both xylanase and cellulase activity were detected after a lag period in the presence of lactose.     

Detection and recovery of hydrolytic enzymes from spent compost of four mushroom species

To evaluate the potential of using the enzymes from spent mushroom compost (SMC) as an industrial enzyme, the production of alpha-amylase, cellulase, beta-glucosidase, laccase, and xylanase was determined from the SMC of four edible mushroom species (Pleurotus ostreatus, Lentinula edodes, Flammulina velutipes and Hericium erinaceum). Among the tested SMC, the SMC of L. edodes showed the highest enzyme activity in alpha-amylase (229 nkat/g), cellulase (759 nkat/g) and beta-glucosidase (767 nkat/g) in 0.5% Triton X-100, and that of P. ostreatus showed the highest activity in laccase (1452 nkat/g) in phosphate-buffered 0.2% Triton X-100. The highest xylanase activity (119 nkat/g) was found in the SMC of F. velutipes.     

Ethylene biosynthesis-inducing protein from cellulysin is an endoxylanase

The proteinaceous ethylene biosynthesis-inducing factor (EIF) that was purified from Cellulysin was also shown to contain a xylanase activity. In all nondenaturing protein separation methods employed (Sephacryl S-200 chromatography, and preparative isoelectric focusing and agarose electrophoresis), xylanase activity copurified with the ethylene biosynthesis-inducing activity. Treatment with heat (60°C) or proteases in 8 molar urea inhibited both ethylene-inducing and xylanase activities. Antibodies raised against purified EIF, which contains three polypeptides of 18, 14, and 10 kilodaltons, immunoprecipitated both ethylene biosynthesis-inducing and xylanase activities. The purified EIF contained no detectable cellulase, polygalacturonase, or protease activity. Other hydrolytic activities as estimated by using p-nitrophenyl derivatives of several sugars as substrates also were not detected. Different commercially available hydrolytic enzyme preparations were tested for both ethylene biosynthesis-inducing and xylanase activities. All enzymes tested contained xylanase activity, but only a few induced ethylene biosynthesis. Western blots of proteins separated by SDS-PAGE, using antibodies prepared against the non-denatured purified EIF, revealed two major bands of about 18 and 14 kilodaltons in EIF. These antibodies seem to be specific for these proteins from Trichoderma viride, because there was little cross-reactivity with the other proteins in Cellulysin and other commercial enzyme preparations. Based on these data, we suggest that EIF contains a specific xylanase activity which is involved in inducing ethylene biosynthesis.     

High-level xylanase production by alkaliphilic Bacillus pumilus ASH under solid-state fermentation

Bacillus pumilus ASH produced a high level of an extracellular and thermostable xylanase enzyme when grown using solid-state fermentation (SSF). Among a few easily available lignocellulosics tested, wheat bran was found to be the best substrate (5,300 U/g of dry bacterial bran). Maximum xylanase production was achieved in 72 h (5,824 U/g). Higher xylanase activity was obtained when wheat bran was moistened with deionized water (6,378 U/g) at a substrate-to-moisture ratio of 1:2.5 (w/v). The optimum temperature for xylanase production was found to be 37°C. The inoculum level of 15% was found to be the most suitable for maximum xylanase production (7,087 U/g). Addition of peptone stimulated enzyme production followed by yeast extract and mustard oil cake, whereas glucose, xylose and malt extract greatly repressed the enzyme activity. Repression by glucose was concentration-dependent, repressing more than 60% of the maximum xylanase production at a concentration of 10% (w/v). Cultivation in large enamel trays yielded a xylanase titre that was slightly lower to that in flasks. The enzyme activity was slightly lower in SSF than in SmF but the ability of the organism to produce such a high level of xylanase at room temperature and with deionized water without addition of any mineral salts in SSF, could lead to substantial reduction in the overall cost of enzyme production. This is the first report on production of such a high level of xylanase under SSF conditions by bacteria.     

Growth and enzyme production by Polyporus hirsutus in presence of ammonium sulphate

Summary Growth of Polyporus hirsutus on rice straw rapidly increases its susceptibility to cellulase and xylanase. Addition of ammonium sulphate to the straw (0.1 g/g) enhances cellulase and xylanase production but does not affect laccase production by the fungus although it appears to inhibit its growth.     

The effects of bioprocess parameters on extracellular proteases in a recombinant <Emphasis Type="Italic">Aspergillus niger</Emphasis> B1-D

Although host proteases are often considered to have a negative impact upon heterologous protein production by filamentous fungi, relatively little is known about the pattern of their appearance in recombinant fungal bioprocesses. In the present study, we investigated extracellular proteases from a filamentous fungus, Aspergillus niger B1-D, genetically modified to secrete hen egg white lysozyme (HEWL). Our findings indicate that extracellular protease activity is only detected after the carbon source is completely utilised in batch cultures. The proteases are predominantly acid proteases and have optimal temperature for activity at around 45°C. Their activity could be partially inhibited by protease inhibitors, indicating the existence of at least four kinds of proteases in these culture fluids, aspartic-, serine-, cysteine-, and metallo-proteases. Oxygen enrichment does not have any noticeable effects on extracellular protease activity except that the onset of protease activity appears earlier in oxygen enrichment runs. Oxygen enrichment stimulates HEWL production substantially, and we propose that it is related to fungal morphology. Thermal stress imposed by raising process temperature (from 25 to 30 and 35°C) in early exponential phase, led to appearance of protease activity in the medium following the heat shock. Continued cultivation at high temperatures significantly reduced HEWL production, which was associated with increased activity of the extracellular proteases in these cultures.     

Attenuated secretion of the thermostable xylanase xynB from Pichia pastoris using synthesized sequences optimized from the preferred codon usage in yeast

Xylanase has been used extensively in the industrial and agricultural fields. However, the low-yield production of xylanase from native species cannot meet the increasing demand of the market. Therefore, improving the heterologous expression of xylanase through basic gene optimization may help to overcome the shortage. In this study, we synthesized a high-GC-content native sequence of the thermostable xylanase gene xynB from Streptomyces olivaceoviridis A1 and, also designed a slightly AT-biased sequence with codons completely optimized to be favorable to Pichia pastoris. The comparison of the sequences' expression efficiencies in P. pastoris X33 was determined through the detection of single-copy-number integrants, which were quantified using qPCR. Surprisingly, the high GC content did not appear to be detrimental to the heterologous expression of xynB in yeast, whereas the optimized sequence, with its extremely skewed codon usage, exhibited more abundant accumulation of synthesized recombinant proteins in the yeast cell, but an approximately 30% reduction of the secretion level, deduced from the enzymatic activity assay. In this study, we developed a more accurate method for comparing the expression levels of individual yeast transformants. Moreover, our results provide a practical example for further investigation of what constitutes a rational design strategy for a heterologously expressed and secreted protein.     

Increased heterologous protein production in Aspergillus niger fermentation through extracellular proteases inhibition by pelleted growth

The dependence of filamentous fungal protease secretion on morphology was investigated by employing the recombinant Aspergillus niger strain AB4.1[pgpdAGLAGFP] which contains a gene for the glucoamylase-GFP (green fluorescence protein) fusion protein. Different inoculum levels were used to obtain different sizes of pellet or free mycelia. The extracellular protease activity of the cultures varied with the pellet size and decreased dramatically when the morphology was changed from free mycelia to pellets. The culture with an optimal pellet size of 1.6 mm was obtained from an inoculum of 4 x 10(6) spores/mL. It resulted in a specific protease activity of 158 units/L, only one-third of that in free mycelial growth, and a maximum specific GFP yield of 0.98 mg/g (cell mass) compared to 0. 29 mg/g for free mycelial growth with an inoculum of 10(7) spores/mL. The results indicate that this bioprocessing strategy can be effectively used to inhibit protease activity in filamentous fungal fermentation and thereby to enhance heterologous protein production.