Regulation of the β-hydroxyacyl ACP dehydratase gene of <Emphasis Type="Italic">Picea mariana</Emphasis> by alternative splicing

The gene for β-hydroxyacyl ACP dehydratase, a de novo fatty acid biosynthetic enzyme, was cloned from Picea mariana (black spruce) and consists of five exons and four introns. The first intron of the β-hydroxyacyl ACP dehydratase mRNA is alternatively spliced. Retention of intron 1 in splice variants results in truncation of the β-hydroxyacyl ACP dehydratase ORF at a premature termination codon. In addition, splicing of intron 1 was found to be associated with cold temperature. mRNAs retaining intron 1 increase with seed imbibition at 22°C but not 4°C, whereas, splicing of intron 1 increases in winter weeks with temperatures below freezing. These results provide evidence that alternative splicing may also contribute to regulation of lipid biosynthesis in Picea mariana.     

Physiological studies on xylose induction and glucose repression of xylanolytic enzymes in Aspergillus sydowii MG49

Abstract Synthesis of extracellular xylanase and intracellular β-xylosidase in Aspergillus sydowii is induced in the presence of both d- and l-xylose in addition to xylobiose and β-d-methyl xyloside. Glucose exhibits a transient catabolite repression which can be partially overcome by external addition of 100 μM dibutyryl 3',5'-cAMP but not by that of cAMP itself. In the presence of xylose or other inducers this cyclic nucleotide stimulates the rate of xylanolytic enzyme synthesis by 85% and 129% for xylanase and β-xylosidase, respectively.     

Production, characterization and properties of polysaccharide depolymerizing enzymes from a strain ofCurvularia inaequalis

Xylanase, β-glucosidase, β-xylosidase, endoglucanase and polygalacturonase production fromCurvularia inaequalis was carried out by means of solid-state and submerged fermentation using different carbon sources. β-Glucosidase. β-xylosidase, polygalacturonase and xylanase produced by the microorganisms were characterized. β-Glucosidase presented optimum activity at pH 5.5 whereas xylanase, poly-galacturonase and β-xylosidase activities were optimal at pH 5.0. Maximal activity of β-glucosidase was determined at 60°C, β-xylosidase at 70°C, and polygalacturonase and xylanase at 55°C. These enzymes were stable at acidic to neutral pH and at 40–45 °C. The crude enzyme solution was studied for the hydrolysis of agricultural residues.     

Thermolabile xylanase of the Antarctic yeast Cryptococcus adeliae: production and properties

Xylanase production by the Antarctic psychrophilic yeast Cryptococcus adeliae was increased 4.3 fold by optimizing the culture medium composition using statistical designs. The optimized medium containing 24.2 g l⁻¹ xylan and 10.2 g l⁻¹ yeast extract and having an initial pH of 7.5 yielded xylanase activity at 400 nkat (nanokatal) ml⁻¹ after 168-h shake culture at 4°C. In addition, very little endoglucanase, β-mannanase, β-xylosidase, β-glucosidase, α-l-arabinofuranosidase, and no filter paper cellulase activities were detected. Among 12 carbon sources tested, maximum xylanase activity was induced by xylan, followed by lignocelluloses such as steamed wheat straw and alkali-treated bagasse. The level of enzyme activity produced on other carbon sources appeared to be constitutive. Among the complex organic nitrogen sources tested, the xylanase activity was most enhanced by yeast extract, followed by soymeal, Pharmamedia (cotton seed protein), and Alburex (potato protein). A batch culture at 10°C in a 5-l fermenter (3.5-1 working volume) using the optimized medium gave 385 nkat at 111 h of cultivation. The crude xylanase showed optimal activity at pH 5.0–5.5 and good stability at pH 4–9 (21 h at 4°C). Although the enzyme was maximally active at 45°–50°C, it appeared very thermolabile, showing a half-life of 78 min at 35°C. At 40°–50°C, it lost 71%–95% activity within 5 min. This is the first report on the production as well as on the properties of thermolabile xylanase produced by an Antarctic yeast. Received: December 10, 1999 / Accepted: March 23, 2000     

The regulation of xylanolytic enzyme formation by Butyrivibrio fibrisolvens NCFB 2249

Butyrivibrio fibrisolvens NCFB 2249 formed xylan-degrading enzymes on a wide range of carbohydrate growth substrates. The specific activities of α-L-arabinofuranosidase and β-D-xylosidase were increased (up 20-fold) after growth on xylan or xylose-containing saccharides. Xylose was not an effective substrate for xylanase production although its formation was induced on xylobiose and higher DP xylose-containing saccharides. Acetyl esterase activity was also highest after growth on xylan. The synthesis of xylanase and β-xylosidase was repressed by glucose and hemicellulosic pentoses and although α-L-arabinofuranosidase formation was also subject to catabolite regulation, xylose did not repress its synthesis.     

Induction and preliminary characterization of intracellular β-glucosidases from a cellulolytic Streptomyces strain

Abstract The cellulolytic actinomycete Streptomyces sp. QM-B814 posasses an intracellular β-glucosidase system which is induced by cellobiose and carboxymethylcellulose. Maximal β-glucosidase activity was attained 8–10 h after inducer addition to exponential phase growing cultures. The induction is depressed in the presence of glucose. The system is composed of two electrophoretically different β-glucosidase forms showing relative molecular masses of about 60 and 35 kDa, and p I values in the range 4.2–4.5. Both β-glucosidases are synthesized de novo. The enzymes share substrate preference and are both inhibited by δ-gluconolactone and p -chloromercuribenzoate. The induction pattern and glucose inhibition are similar for both enzymes.     

Characterization of a bifunctional xylanase/endoglucanase from yak rumen microorganisms

A new gene, RuCelA, encoding a bifunctional xylanase/endoglucanase, was cloned from a metagenomic library of yak rumen microorganisms. RuCelA showed activity against xylan and carboxymethylcellulose (CMC), suggesting bifunctional xylanase/endoglucanase activity. The optimal conditions for xylanase and endoglucanase activities were 65°C, pH 7.0 and 50°C, pH 5.0, respectively. In addition, the presence of Co⁺ and Co²⁺ can greatly improve RuCelA's endoglucanase activity, while inhibits its xylanase activity. Further examination of substrate preference showed a higher activity against barley glucan and lichenin than against xylan and CMC. Using xylan and barley glucan as substrates, RuCelA displayed obvious synergistic effects with β-1,4-xylosidase and β-1,4-glucosidase. Generation of soluble oligosaccharides from lignocellulose is the key step in bioethanol production, and it is greatly notable that RuCelA can produce xylo-oligosaccharides and cello-oligosaccharides in the continuous saccharification of pretreated rice straw, which can be further degraded into fermentable sugars. Therefore, the bifunctional RuCelA distinguishes itself as an ideal candidate for industrial applications.     

β-Methyl-xyloside: positive effect on xylanase induction in Cellulomonas flavigena

Synthesis of extracellular xylanase in Cellulomonas flavigena is induced in the presence of xylan and sugarcane bagasse as substrates. The essential factors for efficient production of xylanase are the appropriate medium composition and an inducing substrate. The increase in xylanase production levels in C. flavigena were tested with a number of carbon sources and different culture conditions. Xylose, arabinose, glycerol and glucose did not induce xylanase production in this microorganism. β-Methyl-xyloside (β-mx), a structural analog of xylobiose, also did not induce xylanase when used as the sole carbon source, but when xylan or sugar cane bagasse was supplemented with β-mx, extracellular xylanase production increased by 25 or 46%, respectively. The response of C. flavigena to xylan plus β-mx was accompanied by a significant accumulation of reducing sugar, an effect not observed with the combination sugarcane bagasse plus β-mx as substrate. To our knowledge, this is the first report on the effect of β-mx on the induction of xylanase in C. flavigena.     

The production of hemicellulases by Thermomyces lanuginosus strain SSBP: influence of agitation and dissolved oxygen tension

Shake-flask cultivation of T. lanuginosus strain SSBP on coarse corn cobs yielded β-xylanase levels of 56,500 nkat/ml at 50 °C, whereas other hemicellulases (β-xylosidase, β-glucosidase, and α-l-arabinofuranosidase) were produced at levels less than 7 nkat/ml. Cultivation on d-xylose yielded much lower levels of xylanase (350 nkat/ml), although other hemicellulase levels were similar to those produced on corn cobs. The influence of agitation rate and dissolved oxygen tension (DOT) on hemicellulase production was studied further in a bioreactor. On xylose, xylanase activities of 4,330 nkat/ml and 4,900 nkat/ml were obtained at stirrer speeds up to 1,400 rpm to control DOT. At a constant stirrer speed of 400 rpm, xylanase activities of 10,930 nkat/ml and 15,630 nkat/ml were obtained when cultivated on xylose and beechwood xylan respectively, despite DOT levels below 5% for the duration of fermentation. The results indicate that there is an interaction between agitation rate and DOT, impacting on xylanase and accessory enzyme production. Higher agitation rates favoured the production of xylosidase, arabinofuranosidase and glucosidase by T. lanuginosus strain SSBP, whereas the lower agitation rates favoured xylanase production. Rheological difficulties precluded cultivation on corn cobs in the bioreactor. Volumetric xylanase productivities of 1,060,000 nkat/l · h and 589,000 nkat/l · h obtained on beechwood xylan and xylose indicate that T. lanuginosus strain SSBP is a hyper-xylanase producer with considerable industrial potential. Received: 5 May 1999 / Received revision: 31 May 2000 / Accepted: 3 June 2000     

De novo synthesis,constitutive expression of <Emphasis Type="Italic">Aspergillus sulphureus</Emphasis> β-xylanase gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and partial enzymic characterization

The endo-β-1, 4-xylanase gene xynA from Aspergillus sulphureus, encoded a lack-of-signal peptide protein of 184 amino acids, was de novo synthesized by splicing overlap extension polymerase chain reaction according to Pichia pastoris protein’s codon bias. The synthetic DNA, composed of 572 nucleotides, was ligated into the downstream sequence of an α-mating factor in a constitutive expression vector pGAPzαA and electrotransformed into the P. pastoris X-33 strain. The transformed yeast screened by Zeocin was able to constitutively secrete the xylanase in yeast–peptone–dextrose liquid medium. The heterogenous DNA was stabilized in the strain by 20-times passage culture. The recombinant enzyme was expressed with a yield of 120 units/mL under the flask culture at 28°C for 3 days. The enzyme showed optimal activity at 50°C and pH 2.4–3.4. Residual activity of the raw recombinant xylanase was not less than 70% when fermentation broth was directly heated at 80°C for 30 min. However, the dialyzed xylanase supernatant completely lost the catalytic activity after being heated at 60°C for 30 min. The recombinant xylanase showed no obvious activity alteration by being pretreated with Na₂HPO₄-citric acid buffer of pH 2.4 for 2 h. The xylanase also showed resistance to certain metal ions (Na⁺, Mg²⁺, Ca²⁺, K⁺, Ba²⁺, Zn²⁺, Fe²⁺, and Mn²⁺) and EDTA. These biochemical characteristics suggest that the recombinant xylanase has a prospective application in feed industry as an additive.     

Optimization of xylanase production using inexpensive agro-residues by alkalophilic Bacillus subtilis ASH in solid-state fermentation

Alkalophilic Bacillus subtilis ASH produced high levels of xylanase using easily available inexpensive agricultural waste residues such as wheat bran, wheat straw, rice husk, sawdust, gram bran, groundnut and maize bran in solid-state fermentation (SSF). Among these, wheat bran was found to be best substrate. Xylanase production was highest after 72 h of incubation at 37 °C and at a substrate to moisture ratio of 1:2 (w/v). The inoculum level of 15% resulted in maximum production of xylanase. The enzyme production was stimulated by the addition of nutrients such as yeast extract, peptone and beef extract. In contrast, addition of glucose and xylose repressed the production of xylanase. The extent of repression by glucose (10%, w/v) was 81% and it was concentration-dependent. Supplementation of the medium with 4% xylose caused 59% repression. Under optimized conditions, xylanase production in SSF (8,964 U of xylanase/g dry wheat bran) was about twofold greater than in submerged fermentation. Thus, B. subtilis produced a very high level of xylanase in SSF using inexpensive agro-residues, a level which is much higher than that reported by any other bacterial isolate. Furthermore, the enzyme was produced at room temperature and with tap water without the addition of any mineral salt in SSF, leading to a marked decrease in the cost of xylanase production, which enhances its industrial potential.     

Rice straw fermentation by Schizophyllum commune ARC-11 to produce high level of xylanase for its application in pre-bleaching

Rice straw is valuable resource that has been used as substrate for cost effective production of xylanase under solid-state fermentation by a newly isolated white rot fungi, S. commune ARC-11. Out of eleven carbon sources tested, rice straw was found most effective for the induction of xylanase that produced 4288.3?IU/gds of xylanase by S. commune ARC-11. Maximum xylanase production (6721.9?IU/gds) was observed on 8^th day of incubation at temperature (30?°C), initial pH (7.0) and initial moisture content (70.0%). The supplementation of ammonium sulphate (0.08% N, as available nitrogen) enhanced the xylanase production up to 8591.4?IU/gds. The xylanase production by S. commune ARC-11 was further improved by the addition of 0.10%, (w/v) of Tween-20 as surfactant. The maximum xylanase activities were found at pH 5.0 and temperature 55?°C with a longer stability (180?min) at temperature 45, 50 and 55?°C. This xylanase preparation was also evaluated for the pre-bleaching of ethanol-soda pulp from Eulaliopsis binata. An enzyme dosage of 10?IU/g of xylanase resulted maximum decrease in kappa number (14.51%) with a maximum improvement 2.9% in ISO brightness compared to control.     

Simplified feeding strategies for the fed-batch cultivation of Kluyveromyces lactis GG799 for enhanced recombinant xylanase production

A xylanase gene (xyn2) from Trichoderma reesei ATCC 58350 was previously cloned and expressed in Kluyveromyces lactis GG799. The production of the recombinant xylanase was conducted in a developed medium with an optimised batch and with fed-batches that were processed with glucose. The glucose served as a carbon source for cell growth and as an inducer for xylanase production. In a 1-L batch system, a glucose concentration of 20 g L^?1 and 80 % dissolved oxygen were found to provide the best conditions for the tested ranges. A xylanase activity of 75.53 U mL^?1 was obtained. However, in the batch mode, glucose depletions reduced the synthesis of recombinant xylanase by K. lactis GG799. To maximise the production of xylanase, further optimisation was performed using exponential feeding. We investigated the effects of various nitrogen sources combined with the carbon to nitrogen (C/N) molar ratio on the production of xylanase. Of the various nitrogen sources, yeast extract was found to be the most useful for recombinant xylanase production. The highest xylanase production (110.13 U mL^?1) was measured at a C/N ratio of 50.08. These conditions led to a 45.8 % increase in xylanase activity compared with the batch cultures. Interestingly, the further addition of 500 g L^?1 glucose led to a 6.2-fold increase (465.07 U mL^?1) in recombinant xylanase activity. These findings, together with those of the exponential feeding strategy, indicate that the composition of the C/N molar ratio has a substantial impact on recombinant protein production in K. lactis.