Expression of cellobiose dehydrogenase from Neurospora crassa in Pichia pastoris and its purification and characterization

A gene encoding cellobiose dehydrogenase (CDH) from Neurospora crassa strain FGSC 2489 has been cloned and expressed in the heterologous host Pichia pastoris, under the control of the AOX1 methanol inducible promoter. Recombinant CDH without the native signal sequence and fused with a His₆-tag (rNC-CDH1) was successfully expressed and secreted. rNC-CDH1 was produced at the level of 652 IU/L after 2 days of cultivation in the induction medium. The His₆-tagged rNC-CDH1 was purified through a one-step Ni–NTA affinity column under non-denaturing conditions. The purified rNC-CDH1 has a CDH activity of 7451 IU/L (0.89 mg protein/mL), with a specific CDH activity of 8.37 IU/mg. The purity of the enzyme was examined by SDS–PAGE, and a single band corresponding to a molecular weight of about 120 kDa was observed. Activity staining confirmed the CDH activity of the protein band. The purified rNC-CDH1 has maximum CDH activity at pH 4.5, and a rather broad temperature optimum of 25–70 °C. Kinetic analysis showed cellobiose and cellooligosaccharides are the best substrates for rNC-CDH1. The K_m value of the rNC-CDH1 for cellooligosaccharide increases with the elongation of glucosyl units. k_cat remains relatively constant when the chain length changes.     

Cellobiose dehydrogenase from the ligninolytic basidiomycete Phlebia lindtneri

Cellobiose dehydrogenase (CDH), an extracellular flavocytochrome produced by several wood-degrading fungi, was detected in the culture supernatant of the selective delignifier Phlebia lindtneri maintained on a cellulose-based liquid medium. Cellobiose dehydrogenase was purified to homogeneity by a rapid procedure, using ammonium sulfate precipitation, ion-exchange chromatography, and chromatofocusing. The enzyme was recovered with a 61.2 fold increased specific activity and a yield of 47.5%. As determined by SDS-PAGE, the molecular mass of the purified enzyme was found to be 104.5 kDa and its isoelectric point was 4.0. The carbohydrate content of the purified enzymes was 22%. In this work, the cellobiose dehydrogenase gene cdh1 and its corresponding cDNA from fungi Phlebia lidnteri were isolated, cloned, and characterized. The 2319 bp full-length cDNA of cdh1 encoded a mature CDH protein containing 755 amino acids, which was preceded by a signal peptide of 17 amino acids. The deduced protein sequence of cdh1 shared significant similarity with other known fungal cellobiose dehydrogenase.     

Aislamiento y evaluación de la actividad enzimática de hongos descomponedores de madera (Quindío,Colombia)

White rot fungi (Ascomycota and Basidiomycota) were collected on fallen trunks with different decay stages, in a subandean forest (La Montaña del Ocaso nature reserve), and it was evaluated their ligninolitic activity. They were cultured on malt extract agar. Then it was performed semiquantitative tests for laccase and cellobiose dehydrogenase (CDH) activity using ABTS and DCPIP as enzymatic inducers. Based on the results of these tests, the fungi with higher activities from trunks with different decay stages were selected: Cookeina sulcipes (for stage 1), a fungus from the family Corticiaceae (for stage 2), Xylaria polymorpha (for stage 3) and Earliella sp. (for stage 4). A fermentation was performed at 28 °C, during 11 days, in a rotatory shaker at 150 rpm. Biomass, glucose, proteins and enzyme activities measurements were performed daily. The fungi that were in the trunks with decay states from 1 to 3, showed higher laccase activity as the state of decay increased. A higher DCH activity was also associated with a higher. Also, there was a positive relationship between both enzymes' activities. Erliella was the fungus which presented the highest biomass production (1140,19 g/l), laccase activity (157 UL^?1) and CDH activity (43,50 UL^?1). This work is the first report of laccase and CDH activity for Cookeina sulcipes and Earliella sp. Moreover, it gives basis for the use of these native fungi in biotechnological applications and the acknowledgment of their function in the wood decay process in native forest.     

Optimization and immobilization of amylase obtained from halotolerant bacteria isolated from solar salterns

The objective of the present study was to isolate halotolerant bacteria from the sediment sample collected from Marakanam Solar Salterns, Tamil Nadu, India using NaCl supplemented media and screened for amylase production. Among the 22 isolates recovered, two strains that had immense potential were selected for amylase production and designated as P1 and P2. The phylogenetic analysis revealed that P1 and P2 have highest homology with Pontibacillus chungwhensis (99%) and Bacillus barbaricus (100%). Their amylase activity was optimized to obtain high yield under various temperature, pH and NaCl concentration. P1 and P2 strain showed respective, amylase activity maximum at 35 °C and 40 °C; pH 7.0 and 8.0; 1.5 M and 1.0 M NaCl concentration. Further under optimized conditions, the amylase activity of P1 strain (49.6 U mL^?1) was higher than P2 strain. Therefore, the amylase enzyme isolated from P. chungwhensis P1 was immobilized in sodium alginate beads. Compared to the free enzyme form (49.6 U mL^?1), the immobilized enzyme showed higher amylase activity as 90.3 U mL^?1. The enzyme was further purified partially and the molecular mass was determined as 40 kDa by SDS–PAGE. Thus, high activity of amylase even under increased NaCl concentration would render immense benefits in food processing industries.     

The effect of limited hydrolysis with Neutrase and ultrafiltration on the anti-adipogenic activity of soy protein

Limited hydrolysis of isolated soy protein (ISP) with Neutrase for 4 h to obtain the hydrolysate (NH4h) revealed the ability to suppress glycerol-3-phosphate dehydrogenase (GPDH) activity and relative lipid accumulation (RLA) in 3T3-L1 cells during differentiation. Lower GPDH activity or RLA indicates higher anti-adipogenic activity. Sequentially fractionating NH4h with 30–1 kDa (kilo-daltons) molecular weight cut-off (MWCO) membranes to obtain the 1 kDa concentrate resulted in further enhancing its anti-adipogenic activity in the cells. The GPDH activity significantly decreased from 280 to 100 U/mg protein (p < 0.05). When comparing the high-performance size-exclusion chromatography (HPSEC) profiles, the most active peptide for the anti-adipogenic activity was primarily composed of peptides with molecular weight between 1300 and 2200 Da. The in vitro effect of gastrointestinal protease on the anti-adipogenic activity of 1 kDa concentrate was also investigated. The results suggested that gastrointestinal proteases have very little effect on anti-adipogenic activity of the concentrate. According to the Western immunoblot analysis, 1 kDa concentrate inhibits adipogenesis by affecting the expression of peroxisome proliferators-activated receptor γ (PPARγ) and the CCAAT/enhancer binding protein α (C/EBPα) during 3T3-L1 cells differentiation.     

Production of β-fructofuranosidases by Aspergillus niveus using agroindustrial residues as carbon sources: Characterization of an intracellular enzyme accumulated in the presence of glucose

The production of β-fructofuranosidases by Aspergillus niveus, cultivated under submerged fermentation using agroindustrial residues, was investigated. The highest productivity of β-fructofuranosidases was obtained in Khanna medium supplemented with sugar cane bagasse as carbon source. Glucose enhanced the production of the intracellular enzyme, whereas that of the extracellular one was decreased. The intracellular β-fructofuranosidase was a trimeric protein of approximately 141 kDa (gel filtration) with 53.5% carbohydrate content, composed of 57 kDa monomers (SDS-PAGE). The optimum temperature and optimum pH were 60 °C and 4.5, respectively. The purified enzyme showed good thermal stability and exhibited a half-life of 53 min at 60 °C. β-Fructofuranosidase activity was slightly activated by Cu²⁺, Mn²⁺, Mg²⁺, and Na⁺ at 1 mM concentration. The enzyme hydrolyzed sucrose, raffinose, and inulin, with K_d values of 5.78 mM, 5.74 mM, and 1.74 mM, respectively.     

Enantioselective synthesis of (S)-phenylephrine by whole cells of recombinant Escherichia coli expressing the amino alcohol dehydrogenase gene from Rhodococcus erythropolis BCRC 10909

(R)-phenylephrine [(R)-PE] is an α₁-adrenergic receptor agonist that is widely used in over-the-counter drugs to treat the common cold. We found that Rhodococcus erythropolis BCRC 10909 can convert detectable level of 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) to (S)-PE by high performance liquid chromatography tandem mass spectrometry analysis. An amino alcohol dehydrogenase gene (RE_AADH) which possesses the ability to convert HPMAE to (S)-PE was then isolated from R. erythropolis BCRC 10909 and expressed in Escherichia coli NovaBlue. The purified RE_AADH, tagged with 6×His, had a molecular mass of approximately 30 kDa and exhibited a specific activity of 0.19 μU/mg to HPMAE in the presence of NADPH, indicating this enzyme could be categorized as NADP⁺-dependent short-chain dehydrogenase reductase. E. coli NovaBlue cell expressing the RE_AADH gene was able to convert HPMAE to (S)-PE with more than 99% enantiomeric excess (ee), 78% yield and a productivity of 3.9 mmol (S)-PE/L h in 12 h at 30 °C and pH 7. The (S)-PE, recovered from reaction mixture by precipitation at pH 11.3, could be converted to (R)-PE (ee > 99%) by Walden inversion reaction. This is the first reported biocatalytic process for the production of (S)-PE from HPMAE.     

Purification and characterization of aminopeptidase N from chicken intestine with potential application in debittering

An aminopeptidase with broad substrate specificity was purified to homogeneity (123.7-fold) with a yield of 3.43% from chicken (Gallus gallus) intestine using a combination of chromatographic separation strategies. The enzyme was identified as alanyl aminopeptidase or aminopeptidase N (APN) by Peptide Mass Fingerprinting. The molecular weight of the enzyme was estimated to be ∼180 kDa by SDS-PAGE and gel filtration chromatography. The enzyme was found to be a glycoprotein, having 40% sugar residue and a molecular mass of 108 kDa after deglycosylation. The enzymatic activity was optimal at 60 °C and pH 6.0. The enzyme preferentially hydrolyzed Leu-β-NA (K_m = 0.1 mM) followed by Ala, Phe, Tyr and Gly at N-terminal. The enzyme activity was completely inhibited by 1,10 phenanthroline (1 mM) and bestatin (1 mM) confirming it as a metalloprotease. Potential of this enzyme in combination with other endoproteases for the production of debittered protein hydrolysates has been discussed.     

Superoxide dismutases from larvae of the camel tick Hyalomma dromedarii

Three superoxide dismutases (EC 1.15.1.1) (TLSOD1, TLSOD2 and TLSOD3) were purified from larvae of the camel tick Hyalomma dromedarii by ammonium sulfate precipitation, ion exchange and gel filtration columns. SDS-PAGE revealed that the subunit molecular masses of the SODs are 40 ± 2 kDa, 67 ± 1.5 kDa and 45 ± 2.6 kDa for TLSOD1, TLSOD2 and TLSOD3, respectively. TLSOD1 and TLSOD2 are monomeric proteins, while TLSOD3 isoenzyme exhibits dimeric structure with native molecular mass of 90 kDa. The pI values are estimated at pH 8.0, pH 7.2 and pH 6.6 for the three SODs which displayed pH optima at 7.6, 8.0 and 7.8, respectively. CuCl₂ and ZnCl₂ increase the activity of TLSOD2 and TLSOD3, while MnCl₂ increases the activity of TLSOD1. KCN inhibits the activity of TLSOD2 and TLSOD3, while a remarkable resistance of TLSOD1 isoenzyme was detected. TLSOD1 is suggested to be a manganese containing isoenzyme while TLSOD2 and TLSOD3 are suggested to be copper/zinc-containing isoenzymes. These results indicate the presence of three different forms of SODs in the larval stage of camel tick. This finding will contribute to our understanding of the physiology of these ectoparasites and the development of non-traditional methods to control them.     

Metabolic engineering of Corynebacterium glutamicum for the production of itaconate

The capability of Corynebacterium glutamicum for glucose-based synthesis of itaconate was explored, which can serve as building block for production of polymers, chemicals, and fuels. C. glutamicum was highly tolerant to itaconate and did not metabolize it. Expression of the Aspergillus terreus CAD1 gene encoding cis-aconitate decarboxylase (CAD) in strain ATCC13032 led to the production of 1.4 mM itaconate in the stationary growth phase. Fusion of CAD with the Escherichia coli maltose-binding protein increased its activity and the itaconate titer more than two-fold. Nitrogen-limited growth conditions boosted CAD activity and itaconate titer about 10-fold to values of 1440 mU mg⁻¹ and 30 mM. Reduction of isocitrate dehydrogenase activity via exchange of the ATG start codon to GTG or TTG resulted in maximal itaconate titers of 60 mM (7.8 g l⁻¹), a molar yield of 0.4 mol mol⁻¹, and a volumetric productivity of 2.1 mmol l⁻¹ h⁻¹.     

Purification and characterization of a novel thermostable α-l-arabinofuranosidase (α-l-AFase) from Chaetomium sp.

The purification and characterization of an extracellular α-l-arabinofuranosidase (α-l-AFase) from Chaetomium sp. was investigated in this report. The α-l-AFase was purified to homogeneity with a purification fold of 1030. The purified α-l-AFase had a specific activity of 20.6 U mg^?1. The molecular mass of the enzyme was estimated to be 52.9 kDa and 51.6 kDa by SDS–PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.0 and 70 °C, respectively. The enzyme was stable over a broad pH range of 4.0–10.0 and also exhibited excellent thermostability, i.e., the residual activities reached 75% after treatment at 60 °C for 1 h. The enzyme showed strict substrate specificity for the α-l-arabinofuranosyl linkage. The K_m and V_max values for p-nitrophenyl (pNP)-α-l-arabinofuranoside were calculated to be 1.43 mM and 68.3 μmol min^?1 mg^?1 protein, respectively. Furthermore, the gene encoding α-l-AFase was cloned and sequenced and found to contain a catalytic domain belonging to the glycoside hydrolase (GH) family 43 α-l-AFase. The deduced amino acid sequence of the gene showed the highest identity (67%) to the putative α-l-AFase from Neurospora crassa. This is the first report on the purification, characterization and gene sequence of an α-l-AFase from Chaetomium sp.     

Conversion of squid pen by using Serratia sp. TKU020 fermentation for the production of enzymes,antioxidants, and N-acetyl chitooligosaccharides

A chitinase (CHT), a chitosanase (CHS) and a protease (PRO) were purified from the culture supernatant of Serratia sp. TKU020 with squid pen as the sole carbon/nitrogen source. The molecular masses of CHT, CHS and PRO determined by SDS-PAGE were approximately 65 kDa, 55 kDa and 55 kDa, respectively. CHT and CHS were inhibited by Mn²⁺, EDTA and PRO was inhibited by Mg²⁺, EDTA. The antioxidant activity of TKU020 culture supernatant was 78% (DPPH scavenging ability). N-Acetylglucosamine (GlcNAc) and N-acetyl chitobiose (GlcNAc)₂ were also produced from the culture supernatant by using TKU020 strain fermentation. The maximum production of GlcNAc and (GlcNAc)₂ was 1.3 mg/mL and 2.7 mg/mL, respectively, after 4 days of fermentation. With this method, we have shown that squid pen wastes can be utilized and it is effective in the production of enzymes, antioxidants, and N-acetyl chitooligosaccharides, facilitating its potential use in industrial applications and functional foods.     

Enhanced production of insecticidal prodigiosin from Serratia marcescens TKU011 in media containing squid pen

Using fishery-processing wastes of squid pen powder (SPP) as the sole carbon and nitrogen (C/N) source, Serratia marcescens TKU011 produced prodigiosin. The culture was incubated in 50 mL of medium in an Erlenmeyer flask (250 mL) containing 1.5% SPP at 30 °C for 1 day and then changed to 25 °C for 2 more days. The culture broth had high prodigiosin (0.978 mg/mL). S. marcescens TKU011 grown under illumination conditions in a shaking culture exhibited higher prodigiosin production than when grown under dark conditions contrary to previous reports. The culture supernatant reduced surface tension of water, and the surfactant activity increased when prodigiosin production increased. In this study, the fishery-processing waste, squid pen, was used to produce prodigiosin at greater quantities than reported in other studies, and we found that the prodigiosin had a novel property of insecticidal activity. This method has the potential for developing mass production of prodigiosin.     

Purification and characterization of an extracellular antifungal chitinase from Penicillium ochrochloron MTCC 517 and its application in protoplast formation

A highly chitinolytic strain Penicillium ochrochloron MTCC 517 was procured from MTCC, Chandigarh, India. Culture medium supplemented with 1% chitin was found to be suitable for maximum production of chitinase. Purification of extracellular chitinase was done from the culture medium by organic solvent precipitation and DEAE-cellulose column chromatography. The chitinase was purified 6.92-fold with 29.9% yield. Molecular mass of purified chitinase was found to be 64 kDa by SDS-PAGE. The chitinase showed optimum temperature 40 °C and pH 7.0. The enzyme activity was completely inhibited by Hg²⁺, Zn²⁺, K⁺ and NH₄⁺. The enzyme kinetic study of purified chitinase revealed the following characteristics, such as apparent K_m 1.3 mg ml^?1, V_max 5.523 × 10^?5 moles l^?1 min^?1 and K_cat 2.37 s^?1 and catalytic efficiency 1.82 s^?1 M^?1. The enzyme hydrolyzed colloidal chitin, glycol chitin, chitosan, glycol chitosan, N,N′-diacetylchitobiose, p-nitrophenyl N-acetyl-β-d-glucosaminide and 4-methylumbelliferyl N-acetyl-β-d-glucosaminide. The chitinase of P. ochrochloron MTCC 517 is an exoenzyme, which gives N-acetylglucosamine as the main hydrolyzate after hydrolysis of colloidal chitin. Protoplasts with high regeneration capacity were obtained from Aspergillus niger using chitinase from P. ochrochloron MTCC 517. Since it also showed antifungal activity, P. ochrochloron MTCC 517 seems to be a promising biocontrol agent.     

Cadmium distribution and its effects on molybdate-containing hydroxylases in Phragmites australis

The influence of cadmium (Cd) on physiological and biochemical parameters was studied to elucidate the mechanism of Cd resistance in Phragmites australis. Cadmium concentrations in roots, stems and leaves increased with exogenous Cd concentration, but Cd content in roots was much higher than in shoots. X-ray microanalysis was used to reveal compartments in which Cd accumulated in root cortex. Cadmium concentrations followed a gradient with the sequence: intercellular space > cell wall > vacuole > cytoplasm, indicating that most Cd was immobilized in the apoplast or sequestered into the vacuolar lumen. Sequential extraction of various Cd chelates revealed that more than half of extractable Cd was bound to proteins, whereas 26% was bound to organic acids. Cd-binding protein fractions were found in the roots after gel filtration chromatography, among which a polypeptide with an apparent molecular mass of 14 kDa bound Cd most avidly. One newly synthesized polypeptide of low molecular mass (1 kDa) appeared under Cd pollution, whereas a prominent fraction of 72 kDa disappeared. Four aldehyde oxidase (AO) isoenzyme activities increased significantly in roots under Cd pollution. Cd stress also enhanced xanthine dehydrogenase (XDH) activities in roots. Two AO polypeptides of different molecular sizes were detected in the roots by Western blot assay. The abundance of the 160 kDa subunit correlated with Cd stress, but the amount of the 90 kDa polypeptide did not change under Cd treatment. Enhanced abscisic acid (ABA) contents were observed in roots of P. australis exposed to Cd. The involvement of Cd distribution in plant tissues and subcellular compartments and of AO and XDH enzymatic activities in the acclimation mechanism of P. australis to Cd pollution is discussed herein.     

Association of TP53 codon 72 and CDH1 genetic polymorphisms with colorectal cancer risk in Bangladeshi population

Till now no pharmacogenetic study of TP53 codon 72 (Arg72Pro) and CDH1 rs16260 (-160C<A) genes has been reported on Bangladeshi population relating those with colorectal cancer. So the aim of the study is to determine whether there is an elevated risk of colorectal cancer development with TP53 codon 72 and CDH1 rs16260 genetic polymorphism in Bangladeshi population for the first time. To investigate the association of these two SNPs, we conducted a case-control study with 288 colorectal cancer patients and 295 healthy volunteers by using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. We found an increased risk of association between Arg/Pro heterozygosity (adjusted OR = 2.58, 95% CI = 1.77–3.77, p < 0.05) and Pro/Pro mutant homozygosity (adjusted OR = 2.92, 95% CI = 1.78–4.78, p < 0.05) along with the combined genotype (Arg/Pro + Pro/Pro) (adjusted OR = 2.70, 95% CI = 1.90–3.82, p < 0.05) and colorectal cancer predisposition. In case of CDH1 rs16260 polymorphism, C/A heterozygous and A/A mutant homozygous are significantly (p < 0.05) found to be associated with colorectal cancer risk with adjusted OR of 1.94 and 2.63, respectively. The combined genotype of C/A and A/A was also found to be strongly associated with colorectal cancer risk compared to C/C genotype (adjusted OR = 2.02, 95% CI = 1.42–2.87, p < 0.05). In conclusion, heterozygosity and mutant homozygosity as well as the combination of both TP53 Arg72Pro and CDH1 rs16260 polymorphisms are responsible to increase the risk of colorectal cancer development in Bangladeshi population.     

Structural characterization of thermostable,solvent tolerant,cytosafe tannase from Bacillus subtilis PAB2

Tannase production by Bacillus subtilis PAB2, was investigated under solid state fermentation using tamarind seed as sole carbon source and it was found as the highest titer (73.44 U/gds). The enzyme was purified to homogeneity, which showed the molecular mass around 52 kDa (K_m = 0.445 mM, V_max = 125.8 mM/mg/min and K_cat = 2.88 min^–1). The enzyme was found stable in a range of pH (3.0–8.0) and temperature (30–70 °C) with an optimal activity at pH 5.0, pI of 4.4 and at 40 °C temperature. It exhibited half-life (t_1/2) of 4.5 h at 60 °C. The enzyme comprised a typical secondary structure containing α-helix (9.3%), β-pleated sheet (33.6%) and β-turn (17.2%). The native conformation of the enzyme was alike a 44 nm spherical nanoparticle upon aggregation. Thermodynamic parameters of tannase revealed that it was stable at 40 °C and showed Q₁₀, ΔG_d and ΔS_d values of 2.08, 99.37 KJ/mol and 252.38 J mol⁻¹ K⁻¹, respectively. Organic solvents were stimulatory with regard to enzyme activity. Moreover, the altered enzyme activity was determined to be correlated with the changes in structural conformation in presence of inducer and inhibitor. Tannase was explored to have no cytotoxicity on Vero cell line as well as rat model study.     

Purification and characterization of a new carbonyl reductase from Leifsonia xyli HS0904 involved in stereoselective reduction of 3,5-bis(trifluoromethyl) acetophenone

Leifsonia xyli HS0904 can stereoselectively catalyze the bioreduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to its corresponding alcohol, which is a valuable chiral intermediate in the pharmaceuticals. In this study, a new carbonyl reductase derived from L. xyli HS0904 was purified and its biochemical properties were determined in detail. The carbonyl reductase was purified by 530-fold with a specific activity of 13.2 U mg⁻¹ and found to be a homodimer with a molecular mass of 49 kDa, in which the subunit molecular-weight was about 24 kDa. The purified enzyme exhibited a maximum enzyme activity at 34 °C and pH 7.2, and retained over 90% of its initial activity at 4 °C and pH 7.0 for 24 h. The addition of various additives, such as Ca²⁺, Mg²⁺, Mn²⁺, l-cysteine, l-glutathione, urea, PEG 1000 and PEG 4000, could enhance the enzyme activity. The maximal reaction rate (V_max) and apparent Michaelis–Menten constant (K_m) of the purified carbonyl reductase for BTAP and NADH were confirmed as 33.9 U mg⁻¹, 0.383 mM and 69.9 U mg⁻¹, 0.412 mM, respectively. Furthermore, this enzyme was found to have a broad spectrum of substrate specificity and can asymmetrically catalyze the reduction of a variety of ketones and keto esters.     

Biochemical properties of a β-mannanase and a β-xylanase produced by Ceriporiopsis subvermispora during biopulping conditions

One mannanase and one of the three xylanases produced by Ceriporiopsis subvermispora grown on Pinus taeda wood chips were characterized. A combination of ion exchange chromatography and SDS-PAGE data revealed the existence of a high-molecular-weight mannanase of 150 kDa that was active against galactoglucomannan and xylan. Its activity was optimal at pH 4.5. The K_m value with galactoglucomannan as substrate was 0.50 mg ml^?1. One xylanase with molecular mass of 79 kDa was also purified and characterized. Its activity was optimal at 60 °C and pH 8.0. Its K_m value with birchwood xylan as substrate was 1.65 mg ml^?1. Both the mannanase and the 79 kDa xylanase displayed relatively high activity on carboxymethyl cellulose. The sensitivity of the xylanase and mannanase to various salts was evaluated. None of the tested salts inhibited the xylanase, but Mn⁺², Fe⁺³, and Cu⁺² were strong inhibitors for the mannanase.     

Molecular cloning and high-level expression of a β-galactosidase gene from Paecilomyces aerugineus in Pichia pastoris

A β-galactosidase gene (designated PaGalA) was cloned for the first time from Paecilomyces aerugineus and expressed in Pichia pastoris under the control of the AOX1 promoter. The coding region of 3036 bp encoded a protein of 1011 amino acids with a deduced molecular mass of 108.7 kDa. The PaGalA without the signal peptide was cloned into a vector pPIC9K and was expressed successfully in P. pastoris as active extracellular β-galactosidase. The recombinant β-galactosidase (PaGalA) was secreted into the medium at an extremely high levels of 22 mg ml⁻¹ having an activity of 9500 U ml⁻¹ from high density fermentation culture, which is by far the highest yield obtained for a β-galactosidase. The purified enzyme with a high specific activity of 820 U mg⁻¹ had a molecular mass of 120 kDa on SDS-PAGE. PaGalA was optimally active at pH 4.5 and a temperature of 60 °C. The recombinant β-galactosidase was able to hydrolyze lactose efficiently at pH 5.0 and 50 °C. It also possessed transglycosylation activities at high concentrations of lactose. PaGalA exhibited better lactose hydrolysis efficiency in whey than two other widely used commercial lactases. The extremely high expression levels coupled with favorable biochemical properties make this enzyme highly suitable for commercial purposes in the hydrolysis of lactose in milk or whey.