Two extracellular endoxylanases from alkaliphilic Bacillus firmus differ in their synthesis

To investigate the synthesis of two extracellular endoxylanases, xylan-binding and unbound xylanases from an alkaliphilic Bacillus firmus, washed cells were incubated in alkaline mineral salt media containing various carbon sources. The 23 kDa xylan-binding endoxylanase (XBE), which hydrolyses insoluble xylan, was produced before the 45 kDa, unbound endoxylanase. All the carbon sources tested at 5 mg ml^–1, including glucose, induced production of XBE but the unbound xylanase was totally repressed by glucose. The production of XBE increased when glucose concentration increased but was not synthesized until the glucose in the medium was less than 1 mg ml^–1.     

High-level expression of an endoxylanase gene from Bacillus sp. in Bacillus subtilis DB104 for the production of xylobiose from xylan

To produce xylobiose from xylan, high-level expression of an endoxylanase gene from Bacillus sp. was carried out in Bacillus subtilis DB104. A 1.62-kb SmaI DNA fragment, coding for an endoxylanase of Bacillus sp., was ligated into the Escherichia coli/B. subtilis shuttle vector pJH27Δ88, producing pJHKJ4, which was subsequently transformed into B. subtilis DB104. A maximum endoxylanase activity of 105 U/ml was obtained from the supernatant of B. subtilis DB104 harboring pJHKJ4. The endoxylanase was purified to homogeneity by ion-exchange chromatography and the production profile of xylooligosaccharides from xylan by the endoxylanase was examined by HPLC with a carbohydrate analysis column. Xylobiose was the major product from xylan at 40 °C and its proportion in the xylan hydrolyzates increased with the reaction time; at 12 h, over 60% of the reaction products was xylobiose. These results suggest that xylobiose, which has a stimulatory effect on the selective growth of the intestinal bacterium Bifidobacterium, can be mass-produced effectively by the endoxylanase of Bacillus sp. cloned in B. subtilis. Received: 2 January 1998 / Received revision: 4 March 1998 / Accepted: 4 March 1998     

Use of lignocellulose biomass for endoxylanase production by Streptomyces termitum

Actinobacteria isolates from Brazilian Cerrado soil were evaluated for their ability to produce enzymes of the cellulolytic and xylanolytic complex using lignocellulose residual biomass. Preliminary semiquantitative tests, made in Petri plates containing carboxymethylcellulose and beechwood xylan, indicated 11 potential species producing enzymes, all belonging to the genus Streptomyces. The species were subsequently grown in pure substrates in submerged fermentation and analyzed for the production of enzymes endoglucanase, β-glucosidase, endoxylanase, and β-xylosidase. The best results were obtained for endoxylanase enzyme production with Streptomyces termitum(UFLA CES 93). The strain was grown on lignocellulose biomass (bagasse, straw sugarcane, and cocoa pod husk) that was used in natura or acid pretreated. The medium containing sugarcane bagasse in natura favored the production of the endoxylanase that was subsequently optimized through an experimental model. The highest enzyme production 0.387?U?mL^?1, (25.8 times higher), compared to the lowest value obtained in one of the trials, was observed when combining 2.75% sugar cane bagasse and 1.0?g?L^?1 of yeast extract to the alkaline medium (pH 9.7). This is the first study using S. termitum as a producer of endoxylanase.     

Molecular expression of xylanase gene in Cryptococcus albidus

In the yeast Cryptococcus albidus, the utilization of xylan as compared to xylose requires at least an inducible endoxylanase enzyme, secreted in the culture medium. The endoxylanase induction was monitored by immunoprecipitation of in vivo and in vitro synthesized products. The mature endoxylanase is a highly glycosylated enzyme with an apparent molecular weight of 48000. Upon chemical deglycosylation with trifluoromethanesulfonic acid, the molecular weight was reduced to 40000. Addition of tunicamycin to the culture medium resulted in the synthesis of a modified polypeptide having a molecular weight of 40000. Poly(A)-containing RNA isolated from the yeast was translated in the rabbit reticulocyte protein-synthesizing system. The appearance of a translatable xylanase mRNA was observed in xylan-grown cells but not in xylose-grown cells. The polypeptide identified as xylanase had a molecular weight of 44000. This suggests that the xylanase is synthesized as a precursor, containing a peptide signal sequence of 35 residues.     

The fermentation of xylose —an analysis of the expression of Bacillus and Actinoplanes xylose isomerase genes in yeast

Summary The genes encoding xylose isomerase from Bacillus subtilis and Actinoplanes missouriensis have been isolated by complementation of a xylose isomerase defective Escherichia coli mutant. The xylose isomerase gene from A. missouriensis could be expressed in E. coli under the control of its own promoter, whereas the cloned Bacillus gene was expressed in E. coli only after the spontaneous integration of the E. coli IS5 element. After fusion of the Bacillus gene to the yeast PDC1 promoter, transformants of Saccharomyces cerevisiae contained the xylose isomerase protein. Approx. 5% of the total cellular protein of transformants consisted of xylose isomerase that was found to be at least partly insoluble. Neither the insoluble protein nor Triton X-114 solubilized isomerase was catalytically active. To investigate whether the xylose isomerase of A. missouriensis can be expressed in S. cerevisiae the coding region was fused to the yeast GAL1 promoter. Analysis of total RNA from yeast transformants containing this construction showed a xylose isomerase specific mRNA.Dedicated to Professor Karl Esser on the occasion of his 60th birthday     

Expression of a catalytic domain of a Neocallimastix frontalis endoxylanase gene (xyn3) in Kluyveromyces lactis and Penicillium roqueforti

A cDNA fragment encoding the A catalytic domain of the Neocallimastix frontalis endoxylanase XYN3 was amplified and cloned by the polymerase chain reaction technique. The xyn3A DNA fragment was inserted between the Saccharomyces cerevisiae phosphoglycerate kinase gene promoter and terminator sequences on a multicopy episomal plasmid for Kluyveromyces lactis. The XYN3A domain was successfully expressed in K. lactis and functional endoxylanase was secreted by the yeast cells with the K. lactis killer toxin secretion signal. The XYN3A domain was also expressed in a strain of Penicillium roqueforti as a fusion protein (ShBLE::XYN3A) of the phleomycin-resistance gene product and the endoxylanase. Active endoxylanase was efficiently secreted from the fungal cells with the Trichoderma viride cellobiohydrolase (CBH1) secretion signal and processed by a related KEX2 endoprotease of the secretion pathway. Several differently glycosylated forms of the recombinant enzymes were secreted by the yeast and the filamentous fungus. Received: 10 November 1998 / Received revision: 8 March 1999 / Accepted: 14 March 1999     

Development of strains of the thermotolerant yeast Hansenula polymorpha capable of alcoholic fermentation of starch and xylan

The thermotolerant yeast Hansenula polymorpha ferments glucose and xylose to ethanol at high temperatures. However, H. polymorpha cannot utilize starchy materials or xylans. Heterologous amylolytic and xylanolytic enzymes have to be expressed in this yeast to provide for utilization and growth on starch and xylan. Genes SWA2 and GAM1 from the yeast Schwanniomyces occidentalis, encoding α-amylase and glucoamylase, respectively, were expressed in H. polymorpha. The expression was achieved by integration of the SWA2 and GAM1 genes under the strong constitutive promoter of the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase gene (HpGAP) into H. polymorpha genome. Resulting transformants acquired the ability to grow on a minimal medium containing soluble starch as a sole carbon source. Ethanol production at high-temperature fermentation from starch by the recombinant strains was up to 10 g/L. The XYN2 gene encoding endoxylanase of the fungus Trichoderma reseei was expressed in H. polymorpha. Co-expression of xlnD gene coding for β-xylosidase of the fungus Aspergillus niger and the XYN2 gene in H. polymorpha was achieved by integration of these genes under control of the HpGAP promoter. Resulting transformants were capable of growth and alcoholic fermentation on a minimal medium supplemented with birchwood xylan as a sole carbon source at 48 °C.     

Production of extracellular protease from halotolerant bacterium, Bacillus aquimaris strain VITP4 isolated from Kumta coast

A newly isolated halotolerant Bacillus sp. VITP4 was investigated for the production of extracellular protease. 16S rRNA gene analysis identified it as Bacillus aquimaris. Enzyme secretion corresponded with growth (G_t, 38 min) in the basal Zobell medium, reaching a maximum during stationary phase (630 U/ml, 48 h). Protease production was investigated in different salt concentrations (0–4 M). While growth was optimum in the basal medium, higher levels of protease activity were observed in 0.5 M salt medium (728 U/ml, 48 h) and 1 M salt medium (796 U/ml, 78 h) with 21% and 32% increase in production, respectively. Salt concentrations above 2.5 M did not support bacterial growth. The optimum pH and temperature for production were pH 7.5 and 37 °C, respectively. A combination of peptone and yeast extract yielded optimum protease secretion. Inorganic nitrogen sources proved to be less favourable. Production was reduced in the presence of readily available carbon sources owing to catabolic repression. Effect of various salts (1–6%) indicated favourable bacterial growth in these conditions for producing proteolytic molecules with increased activity. The study assumes significance in the ability of the halotolerant bacterium to survive in a wide range of salinity and yield optimum levels of extracellular protease.     

鲈鱼生长激素在甲醇酵母中的胞内表达

甲醇酵母pichia pastoris是一种理想的真核蛋白高水平表达系统.将鲈鱼(Lateolabrax japonicus)生长激素基因克隆到酵母整合型质粒载体pHIL-D2,经转化his4缺陷型酵母GS115,用PCR方法筛选阳性转化子,并用斑点印迹法筛选多拷贝转化子,经甲醇诱导表达,SDS-PAGE和蛋白质印迹杂交结果证实了表达产物为重组的鲈鱼生长激素.     

A Novel Culture Method for High Level Production of Heterologous Protein in Saccharomyces cerevisiae

A high level production system for heterologous protein by cold culture of yeast transformants at 15°C was developed. The yeast transformants, carrying a plasmid containing cDNA for Aspergillus oryzae α-amylase (Taka-amylase A) or human lysozyme synthetic DNA, were cultivated in a selective medium for 1 or 2 days until full growth at 30°C. The yeast cells were harvested by centrifugation from the culture fluid and then were transferred to YPD medium. These inoculated broths were incubated for 2 days at 15°C and then for another 2 days at 30°C. By the cold culture method described above, higher amounts of Taka-amylase A (28.6 mg/liter) and human lysozyme (6.1 mg/liter) were produced by the yeast transformants compared to those by conventional methods.

Heterologous protein productions using YEp, YCp, and YIp types of yeast expression vectors with ADH1 or GAPDH promoter by the cold culture method showed effective productivity of about 2-fold compared to those by the conventional method of culture at 30°C. The high level production of heterologous protein by this method was not specific to the S. cerevisiae strains examined.     

Extracellular production of human immunoglobulin G Fc region (hIgG-Fc) by Escherichia coli

Summary We have constructed the recombinant plasmid for the extracellular production of human immunoglobulin G Fc region (hIgG-Fc) in Escherichia coli. The excretion vector pEXFC10 contained the weakly activated kil gene of plasmid pMB9 and the DNA fragment encoding a fused protein, in which the codons for the alkalophilic Bacillus sp. No. 170 penicillinase signal peptide and the hIgG-Fc were fused through the one additional amino acid Ser, which was identical with the N-terminus of alkalophilic Bacillus mature penicillinase. By cultivating E. coli carrying pEXFC10, about 40% of hIgG-Fc was excreted into the culture medium. The N-terminal amino acid sequence of the extracellular hIgG-Fc indicated that processing occurred correctly between Ala and Ser. From the sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) in the nonreducing condition, it was suggested that most of the extracellular hIgG-Fc proteins took the dimeric form via disulfide bonds.     

Efficient secretory production of alkaline phosphatase by high cell density culture of recombinant Escherichia coli using the Bacillus sp. endoxylanase signal sequence

New secretion vectors containing the Bacillus sp. endoxylanase signal sequence were constructed for the secretory production of recombinant proteins in Escherichia coli. The E. coli alkaline phosphatase structural gene fused to the endoxylanase signal sequence was expressed from the trc promoter in various E. coli strains by induction with IPTG. Among those tested, E. coli HB101 showed the highest efficiency of secretion (up to 25.3% of total proteins). When cells were induced with 1 mM IPTG, most of the secreted alkaline phosphatase formed inclusion bodies in the periplasm. However, alkaline phosphatase could be produced as a soluble form without reduction of expression level by inducing with less (0.01 mM) IPTG, and greater than 90% of alkaline phosphatase could be recovered from the periplasm by the simple osmotic shock method. Fed-batch cultures were carried out to examine the possibility of secretory protein production at high cell density. Up to 5.2 g/l soluble alkaline phosphatase could be produced in the periplasm by the pH-stat fed-batch cultivation of E. coli HB101 harboring pTrcS1PhoA. These results demonstrate the possibility of efficient secretory production of recombinant proteins in E. coli by high cell density cultivation. Received: 8 September 1999 / Received revision: 3 January 2000 / Accepted 4 January 2000     

Screening and identification of extracellular lipase-producing thermophilic bacteria from a Malaysian hot spring

Seven lipase-producing thermophilic bacteria (ST 1, ST 4, ST 6, ST 7, ST 8, ST 9 and ST 10) were isolated from the Setapak hot spring in Malaysia. The crude extracellular lipases recovered by ultrafiltration of cell-free culture supernatant were reacted in an olive oil mixture and their lipolytic activities were compared. Identification of the bacteria was carried out using the Biolog system and biochemical tests. Strain ST 7 that exhibited the highest lipolytic activity of 4.58 U/ml was identified as belonging to the Bacillus genus. Strain ST 6 with an activity of 3.51 U/ml, was identified as Ralstonia paucula. The lipolytic activities of strains ST 1, ST 4, ST 8, ST 9 and ST 10 were 2.39, 1.84, 2.38, 1.80 and 2.62 U/ml respectively. Strains ST 1, ST 4, and ST 10 were identified as Ralstonia paucula while strains ST 8 and ST 9 were Bacillus spp. Strains ST 7 and ST 9 were tentatively identified as Bacillus thermoglucosidasius, Bacillus stearothermophilus or Bacillus coagulans, whereas strain ST 8 was tentatively identified as Bacillus subtilis.     

Some factors affecting lipase production by yeasts and filamentous fungi

Summary The highest activity of extracellular lipases was obtained after 24 h of cultivation of R. miehei NH in the medium with 0.5% beef tallow and medium working volume of 100 ml (2302U/ug of protein). For yeast after 36h of cultivation for Y.lipolytica 2, in 50 ml medium with 0.5% beef tallow (1796 U/ug of protein). Lipases activity increased by lowering the working volume of medium and decreasing the glucose content.     

Phytase production by high cell density culture of recombinant Bacillus subtilis

Bacillus subtilis BD170, harboring a plasmid pGT44[phyC] carrying the phytase gene (phyC) and a phosphate-depletion inducible pst-promoter, was grown in a 2 l bioreactor. Using a controlled feeding of glucose, high cell densities of 32 and 56 g dry cell weight l^–1 were achieved with peptone and yeast extract, respectively, as the complex nitrogen sources in a semi-defined growth medium. The fed-batch protocol was applied to production of recombinant phytase and a high extracellular phytase activity (48 U ml^–1) was reached with peptone. Although the yeast extract feeding resulted in a higher cell density, it was unsuitable as a medium component for phytase expression due to its relatively high phosphate content.     

Purification and characterization of cutinase from Bacillus sp. KY0701 isolated from plastic wastes

A total of approximately 400 bacterial strains were isolated from 73 plastic wastes collected from 14 different regions. Nineteen isolates that form clear zones both on tributyrin and poly ε-caprolactone (PCL) agar, were identified based on 16S rRNA gene sequences. Among these, Bacillus sp. KY0701 that caused the highest weight loss of PCL films in minimal salt medium, was selected for cutinase production. The highest enzyme activity (15 U/mL) was obtained after 4 days of incubation at 50°C, pH 7.0 and 200?rpm in a liquid medium containing 1.5% (w/v) apple cutin and 0.1% (w/v) yeast extract. The purified enzyme was stable at high temperatures (50–70°C) and over a wide pH range (5.5–9.0). The relative activity of cutinase was at least 75% in the percent of various organic solvents. The apparent K_m and V_max values of the cutinase for p-nitrophenyl butyrate were 0.72?mM and 336.8?µmol p-nitrophenol/h/g, respectively. In addition, it showed high stability and compatibility with commercial detergents. These features of cutinase obtained from Bacillus sp. KY0701 make it a promising candidate for application in the detergent and chemical industries. In our best knowledge, this is the first report for cutinase production and characterization produced by a Bacillus strain.     

Studies on a mixed culture of Candida parapsilosis and different bacilli on alkane

Summary Eight strains of Bacillus were able to grow on alkane in a mixed culture with Candida parapsilosis. The growth of Bacillus was dependent on that of the yeast. Every variation of culture parameters influenced directly the growth of the yeast and then that of Bacillus. Myristic acid, produced by Candida parapsilosis, was presumably the principal carbon source for the growth of Bacillus in a mixed culture.Dedicated to Prof. Dellweg to his 60th birthday     

Production of surfactant and detergent-stable,halophilic, and alkalitolerant alpha-amylase by a moderately halophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. Strain <Emphasis Type="Italic">TSCVKK</Emphasis>

A moderately halophilic alkalitolerant Bacillus sp. Strain TSCVKK, with an ability to produce extracellular halophilic, alkalitolerant, surfactant, and detergent-stable alpha-amylase was isolated from soil samples obtained from a salt-manufacturing industry in Chennai. The culture conditions for higher amylase production were optimized with respect to NaCl, substrate, pH, and temperature. Maximum amylase production of 592 mU/ml was achieved in the medium at 48 h with 10% NaCl, 1% dextrin, 0.4% yeast extract, 0.2% tryptone, and 0.2% CaCl₂ at pH 8.0 at 30 °C. The enzyme activity in the culture supernatant was highest with 10% NaCl at pH 7.5 and 55 °C. The amylase that was partially purified by acetone precipitation was highly stable in various surfactants and detergents. Glucose, maltose, and maltooligosaccharides were the main end products of starch hydrolysis indicating that it is an alpha-amylase.     

Expression of a Synthetic Gene Encoding the Anticoagulant-Antimetastatic Protein Ghilanten by the Methylotropic YeastPichia pastoris

Ghilantens are a family of cysteine-rich inhibitors of the clotting enzyme, factor Xa, that are produced in the salivary glands of the South American leechHaementeria ghilianii.In this study, a gene, designed from the amino acid sequence of a specific ghilanten isoform, was assembled from eight double-stranded oligonucleotide fragments. A yeast expression plasmid, pPIC9HG-2, was constructed by making an in-frame fusion of the ghilanten-coding sequences with the region encoding the pre-pro α-mating factor signal sequence for secretion. The expression of ghilanten in pPIC9HG-2 was under the control of the methanol-inducible, alcohol oxidase (AOX1) promoter.Pichia pastorisyeast strains KM 71 and SMD 1168 were transformed with linearized pPIC9HG-2 to target integration of the plasmid to the chromosomal 5′-AOX1locus via homologous recombination. Both strains yielded His⁺transformants that secreted a potent anticoagulant activity into the medium. Product yield was improved by using buffered media (pH 6.0) supplemented with either casamino acids or a mixture of yeast extract and peptone. The protease-deficient strain, SMD 1168, secreted about a twofold higher level ofr-ghilanten than KM 71. Significant clonal variation in the expression ofr-ghilanten was found among the His⁺transformants. A high producing clone was selected for production at the 2-liter shake flask and 10-liter bioreactor scales.r-Ghilanten was recovered from the fermentation broths in a single step by heparin Sepharose affinity chromatography. Protein sequence analysis of the amino terminus showed that the correct processing to yield mature ghilanten varied with the fermentation conditions.