Crude Enzyme Solution Research Articles

PARTIAL PURIFICATION AND CHARACTERIZATION OF AN EXTRACELLULAR METALLOPEPTIDASE PRODUCED BY Bacillus amyloliquefaciens FE-K1

The aim of this study was to purify and characterize the peptidase of Bacillus amyloliquefaciens (Fukumoto) (strain FE-K1) isolated from ropey bread. Peptidases were purified from crude enzyme solution by affinity chromatography with an efficiency of 25 % and a purification coefficient of 1.53. The optimum pH of partially purified peptidase (PPPase) solution was determined as 7.5 and the peptidases retained approximately 90 % of their initial activity in the pH range 7.0-8.5 following incubation at 37°C for 2 h. The optimum temperature for the PPPase was 60°C. The approximate molecular weight of the PPPase was determined as 36 kDa. Inactivation of the PPPase in the presence of O-FEN and EDTA showed them to be metallopeptidases and 5 mM of K+1 and 5 mM of Mn+2 ions increased the enzyme activity by 4 % and 6.15 %, respectively. The presence of Hg+2, Fe+3 and SDS (0.1-1.0 % w/v) caused inactivation whereas the enzyme retained most of its activity in the presence of 0.1-1.0 % (v/v) Triton X-100, Tween 20 and Tween 80 and 1-20 % (v/v) xylene, ethanol, acetone and acetonitrile. Characterization of the PPPase revealed the enzyme as a neutral serine metallopeptidase compatible with some organic solvents and surfactants.

Establishment of Biosynthetic Pathways To Generate Castasterone as the Biologically Active Brassinosteroid in Brachypodium distachyon.

Gas chromatography-mass spectrometry (GC-MS) analysis revealed that castasterone and its biosynthetic precursors are found in Brachypodium distachyon. In vitro conversion experiments with crude enzyme solutions prepared from B. distachyon demonstrated the presence of the following biosynthetic sequences: campesterol → campesta-4-en-3-one → campesta-3-one → campestanol → 6-deoxocathasterone → 6-deoxoteasterone → teasterone ↔ 3-dehydroteasterone ↔ typhasterol → castasterone. campesterol → 22-hydroxycampesterol → 22-hydroxy-campesta-4-en-3-one → 22-hydroxy-campesta-3-one → 6-deoxo-3-dehydroteasterone → 3-dehydroteasterone. 6-deoxoteasterone ↔ 6-deoxo-3-dehydroteasterone ↔ 6-deoxotyphasterol → 6-deoxocastasterone → castasterone. This shows that there are campestanol-dependent and campestanol-independent pathway in B. distachyon that synthesize 24-methylated brassinosteroids (BRs). Biochemical analysis of BRs biosynthetic enzymes confirmed that BdDET2, BdCYP90B1, BdCYP90A1, BdCYP90D2, and BdCYP85A1 are orthologous to BR 5α-reductase, BR C-22 hydroxylase, BR C-3 oxidase, BR C-23 hydroxylase, and BR C-6 oxidase, respectively. Brassinolide was not identified in B. distachyon. Additionally, B. distachyon crude enzyme solutions could not catalyze the conversion of castasterone to brassinolide, and the gene encoding an ortholog of CYP85A2 (a brassinolide synthase) was not found in B. distachyon. These results strongly suggest that the end product for brassinosteroid biosynthesis which controls the growth and development of B. distachyon is not brassinolide but rather castasterone.

Optimization of fermentation condition for cellulase enzyme production from Bacillus sp.

Abstract Cellulase is an important enzyme in present-day of industrial biotechnology. The current study is concerned with the production and partial characterization of cellulase enzyme from Bacillus sp. The effect of various fermentation conditions for cellulase production through shake-flask culture was investigated. Maximum enzyme production was obtained after 24 hours of incubation in fermentation medium with pH 3.5 at 35°C under having agitation at 150 rpm while inoculums volume 1% was applied. Enzyme production was 1.91 times higher after optimizing the production condition as compared to the basal media. Cellulase characterization revealed that optimum activity was at pH 5.5 and 50°C for 50 minutes. About 68% of the activity retained after heating the crude enzyme solution at 50°C for 30 minutes. This nature makes cellulase a suitable candidate for current mainstream biomass conversions for sustainable agriculture and industrial processes.

Development and Characterization of an Enzyme Membrane Reactor for Fructo-Oligosaccharide Production.

Fructo-oligosaccharides (FOS) are linear fructans comprising 2–5 fructose units linked to a terminal glucose residue. They are widely used as food and feed additives due to their sweetness, low calorific value, and prebiotic properties. Here we describe the synthesis of FOS catalyzed by a cell-free crude enzyme solution containing recombinant fructosyltransferase (1-FFT) produced in the yeast Kluyveromyces lactis. During the enzyme catalysis, glucose accumulates as a by-product and eventually inhibits FOS production. We therefore used an enzyme membrane reactor (EMR) to achieve the continuous removal of glucose and the simultaneous replenishment of sucrose. We observed a loss of flux during the reaction with the characteristics of complete pore blocking, probably caused by a combination of proteins (enzyme molecules) and polysaccharides (FOS). Such complex fouling mechanisms must be overcome to achieve the efficient production of FOS using EMR systems.

English

The leather industry is one among the most vital sectors for economic development contributing to high earnings. However, tanning of hides and skins is constrained by primitive technology and the use of hazardous chemicals that contribute to environmental pollution. This study was conducted to evaluate the efficacy of alkaline protease on dehairing animal hides and skins under different conditions. Alkaline protease was extracted from Aspergillus oryzae MG429773 cultured by solid state fermentation with a medium containing rice husks supplemented with 1% mineral solution incubated at 30 to 35°C for 7 days at pHs ranging from 7.5 to 9.5. The filtrate was used as a crude enzyme solution and also partially purified for a dehairing bioassay. Four pieces of cattle hide and goat skin (10 cm × 10 cm) were soaked or sprayed with enzyme solutions for a maximum of 5 days. Percentages of depilation of the hides and skins were recorded at 6 and 12 h intervals. Enzyme produced by A. oryzae showed the highest dehairing activity from 15 to 20% concentration of partially purified enzyme whereby 100% of goat and cattle hairs were removed within 24 and 48 h, respectively. For 5 and 10% enzyme concentration, 93.75 and 68.75% of hide and skin depilated in 72 h, whereas for the control 0.0 to 6.5% depilation was achieved in 72 h in hide and skins, respectively. Application of the A. oryzae protease by spray method was superior over a dipping method that released hair in effluent and caused skin decay after 5 days of exposure. This study indicates that enzyme extracted from cheaply available resources is efficient for dehairing by a spray method could be a potential technology for application by local tanners to improve the leather industry in Tanzania. Key words: Alkaline protease, enzymatic dehairing, hides and skins, leather industry, fungal biotechnology.

Influence of different parameters on reverse micelle extraction combined with acetone precipitation to purify sn-1,3 extracellular lipase from Aspergillus niger GZUF36

There are few reports on the feasibility of combined reverse micelle extraction and acetone precipitation to obtain electrophoretic pure enzymes. We aimed to purify a sn-1,3 extracellular lipase from a novel Aspergillus niger GZUF36 through this combination in this work. This lipase preliminarily purified by controlling the volume ratio (1:2.5) of crude enzyme solution and acetone. Then, we studied effects of different parameters on reverse micelle extraction. The suitable surfactant, pH, salt and cosolvent and extraction time for forward extraction were 125mM cetyl trimethylammonium bromide (CTAB), 9.0, 0.075M NaCl, 10% n-hexanol and 30min, respectively. Under these conditions, the forward extraction rate reached 90.3% ± 3.2%. The suitable salt, pH, extraction time and short chain alcohol for backward extraction were consecutively 1.5M KCl, 6.5, 60min and 10% ethanol. Adding 10% ethanol shows a significant advantage of improvement the extraction rate. Under these optimal conditions, the total extraction rate and purification factor of lipase reached 76.8% and 10.14, respectively. SDS-PAGE showed that molecular weight of the pure protein was 42.7kDa and TLC exhibited sn-1,3 selectivity of this lipase. LC-MS/MS analysis revealed that the lipase had 297 amino acid residues and was likely to glycosylate. Through the study of different parameters, it demonstrated that the new and simple combination of reverse micelle extraction using CTAB as surfactant and n-hexanol as cosolvent for forward extraction and adding ethanol for backward extraction and acetone precipitation is a promising method to get almost an electrophoretically pure sn-1,3 lipase.

Variation in metabolism and degradation of di-n-butyl phthalate (DBP) by high- and low-DBP accumulating cultivars of rice (Oryza sativa L.) and crude enzyme extracts

Crops can take up and accumulate di-n-butyl phthalate (DBP), an extensively used plasticizer with endocrine disrupting effect, which poses potential risk to human health. Our previous study found the genotype variation in accumulation of DBP by different cultivars of rice (Oryza sativa L.). Nevertheless, the effect of DBP metabolism in vivo on the accumulation variation among different plant cultivars remains unknown. In this study, metabolism variation of DBP by low (Fengyousimiao) and high (Peizataifeng) DBP-accumulating cultivars of rice and the key enzymes involving in DBP metabolism in rice plants were investigated using in vivo exposure of rice plants and in vitro exposure of root crude enzyme extracts. Both mono-n-butyl phthalate (MBP) and phthalic acid (PA) were detected as DBP metabolites in all rice tissues (i.e., roots, stems, leaves) and crude enzyme extracts with MBP predominance. DBP metabolism occurred simultaneously when DBP uptake with the highest metabolism in roots in vivo. Degradation of DBP in root crude enzyme extracts fitted well with the first order kinetics (R2 = 0.49–0.76, P < 0.05). The activity of carboxylesterase (CXE) in root crude enzyme extracts was significantly positively correlated with DBP degradation rates. CXE played an important role in DBP metabolism of rice plants, confirming by the fact that triphenyl phosphate of CXE inhibitor could inhibit DBP metabolism of in vivo and in vitro exposure. This result was further confirmed by in vitro degradation of DBP with the commercial pure CXE. The crude enzyme solution from roots of Fengyousimiao with higher CXE activity had significantly higher DBP degradation rates than that of Peizataifeng. However, Fengyousimiao with lower tolerance to DBP stress and higher inhibition by triphenyl phosphate displayed lower DBP metabolism ability in vivo than Peizataifeng.

Study on transformation and degradation of bisphenol A by Trametes versicolor laccase and simulation of molecular docking

As a typical class of environmental endocrine disruptors, bisphenol A poses a potential threat to the sustainable survival and reproduction of living beings and human beings. In this study, the interaction between Trametes versicolor laccase and bisphenol A (BPA) was studied by molecular docking simulation, and the catalytic degradation of BPA was verified by experiments. The conditions for the laccase production of T. versicolor were optimized by orthogonal design, and the degradation of BPA was studied using its crude enzyme solution. The optimum degradation conditions were obtained by response surface methodology (RSM). Ultimately, the transformation products after 3 and 6 h of reaction were detected by gas chromatography–mass spectrometry. Docking results demonstrated that the reaction between laccase and BPA was spontaneous, and the degradation rate in 24 h reached 88.76%. RSM results showed that the highest BPA degradation rate of 97.68% was reached after 1 h reaction at 44.6 °C, 5 mg/L initial BPA concentration, and pH 5.20. The intermediate products of BPA catalyzed by laccase included ethylbenzene, p-xylene, and cyclohexanone 1-methyl-4-isopropenyl-2-cyclohexenol. This finding reveals that BPA degradation by the crude laccase from T. versicolor starts from the C atoms between two benzene rings that connect BPA. Compared with expensive pure enzyme, the crude laccase solution prepared by T. versicolor showed greater efficiency in BPA degradation. This work provides theoretical references and experimental methods for the biological processing of harmful pollutants.

Effective Biodegradation of Aflatoxin B1 Using the Bacillus licheniformis (BL010) Strain.

Aflatoxin B1 (AFB1), a pollutant of agricultural products, has attracted considerable attention in recent years, due to its potential impact on health. In the present study, Bacillus licheniformis (BL010) was demonstrated to efficiently degrade AFB1, reducing over 89.1% of the toxin content within 120 h. A crude enzyme solution of BL010 exhibited the highest degradation level (97.3%) after three induction periods. However, uninduced BL010 bacteria was not capable of reducing AFB1. Furthermore, high performance liquid chromatography (HPLC) analysis showed that while a cell-free extract caused a significant decrease in AFB1 content (93.6%, p < 0.05), cell culture fluid treatment did not significantly degrade AFB1. The biotransformation products of AFB1 were detected and further identified by quadrupole time-of-flight liquid chromatography–mass spectrometry (LC-Q-TOF/MS); these corresponded to a molecular formula of C12H14O4. A sequence analysis of whole BL010 genes with a bioinformatics approach identified the secondary structures of two degrading enzymes (Chia010 and Lac010), providing an important basis for subsequent homology modeling and functional predictions.

Castasterone Can be Biosynthesized from 28-homodolichosterone in Arabidopsis thaliana

We recently demonstrated the biosynthesis of 24-ethylidene brassinosteroids in Arabidopsis thaliana. To determine the physiological role of biosynthesis of 24-ethylidene brassinosteroids, metabolism of 28-homodolichosterone as the end product of 24-ethylidene brassinosteroids biosynthesis was examined by a crude enzyme solution prepared from A. thaliana. In wild-type plants, dolichosterone and castasterone were identified as enzyme products on GC-MS analysis. In a mutant where DWARF1 was overexpressed (35S-DWF1), the conversion rate of 28-homodolichosterone to castasterone was significantly increased. These results indicate that conversion of 28-homodolichosterone to castasterone is mediated by dolichosterone in Arabidopsis. In the root growth assay, inhibitory activity was enhanced in the order of castasterone > dolichosterone > 28-homodolichosterone, demonstrating that conversion of 28-homodolichosterone to castasterone via dolichosterone is a biosynthetic reaction that increases BR activity in Arabidopsis. Compared to Arabidopsis grown under dark conditions, light-grown Arabidopsis showed up-regulated DWARF1 expression, resulting in an increased conversion rate of 28-homodolichosterone to castasterone, suggesting that light is an important regulatory factor for the biosynthetic connection of 24-ethylidene brassinosteroids and 24-methyl brassinosteroids in A. thaliana. Consequently, 24-ethylidene brassinosteroids biosynthesis to generate 28-homodolichosterone is a lightregulated alternative route for synthesis of the biologically-active BRs, castasterone and brassinolide in Arabidopsis plants.

Preparation of 1L-myo-Inositol 1-Phosphate as a Substrate of Phosphatidylinositol Phosphate Synthase.

Novel drugs possessing a mechanism of action specific to pathogenic mycobacteria, including Mycobacterium tuberculosis, are needed. In 2010, we discovered that the biosynthetic pathway of phosphatidylinositol, which is a membrane phospholipid, differs between humans and mycobacteria. The key enzyme responsible for this difference is phosphatidylinositol phosphate (PIP) synthase, which is present only in a few bacteria belonging to the phylum Actinobacteria. Discovering compounds that inhibit the activity of this enzyme will lead to the development of new drugs specific to pathogenic mycobacteria. Measuring PIP synthase activity requires the isotope-labeled substrate 1l-myo-inositol 1-phosphate (1l-Ino-1P). Because this substrate is not commercially available, we synthesized it from [14C] glucose 6-phosphate ([14C] Glc-6P), using a crude enzyme solution isolated from the methanoarchaeon 1l-Ino-1P synthase. The activity of 1l-Ino-1P synthase in the crude enzyme mixture was low, and quantitative analysis of the synthesized 1l-Ino-1P was inaccurate due to impurities present in the crude enzyme mixture. In the present study, we describe a method for synthesizing 1l-Ino-1P using a solution containing recombinant 1l-Ino-1P synthase derived from the hyperthermophilic archaeon Aeropyrum pernix. In addition, we elucidate the conditions leading to the almost complete conversion of Glc-6P into 1l-Ino-1P using this enzyme. Quantitation of the synthesized 1l -Ino-1P was performed by colorimetry and gas liquid chromatography. Further, we confirmed that isotope-labeled 1l-Ino-1P, which is difficult to quantitate by gas liquid chromatography, can be accurately quantified by colorimetry. We also confirmed that 1d-inositol 1-phosphate cannot be a substrate for PIP synthase.

The Preparation of Crystalline &amp;lt;i&amp;gt;O&amp;lt;/i&amp;gt;-D-Mannopyranosyl-(1&amp;#x2192;4)-O-D-Glucopyranosyl-(1&amp;#x2192;4)-D-Mannopyranose (MGM) from Konjac Glucomannan Using &amp;lt;i&amp;gt;Xylogone sphaerospora&amp;lt;/i&amp;gt; &amp;#x3B2;-mannanase System and &amp;lt;i&amp;gt;Candida guilliermondii&amp;lt;/i&amp;gt; Fermentation

O-D-mannopyranosyl-(1→4)-O-D-glucopyranosyl-(1→4)-D-Mannopyranose (MGM) was prepared via the enzymatic hydrolysis of konjac glucomannan and the subsequent elimination of monosaccharides from the resultant hydrolysate using yeast. The enzyme system hydrolyzed konjac glucomannan and produced monosaccharides and MGM without other oligomers at the 48 h reaction. Konjac 20 g was hydrolyzed at 60℃ and a pH 6.0 for 48 h with 200 mL crude enzyme solution from Xylogone sphaerospora. By eliminating monosaccharides from the hydrolysis products with yeast (Candida guilliermondii), 3.8 g of crystalline MGM was obtained, without the use of chromatographic techniques. After 48 h of yeast cultivation, the total sugar content fell from 5.2% to 3.7%, while the average degree of polymerization (D.P.) rose from 2.6 to 3.3.

Biodegradation of feather waste by keratinase produced from newly isolated Bacillus licheniformis ALW1.

Keratinase are proteolytic enzymes which have gained much attention to convert keratinous wastes that cause huge environmental pollution problems. Ten microbial isolates were screened for their keratinase production. The most potent isolate produce 25.2 U/ml under static condition and was primarily identified by partial 16s rRNA gene sequence as Bacillus licheniformis ALW1. Optimization studies for the fermentation conditions increased the keratinase biosynthesis to 72.2 U/ml (2.9-fold). The crude extracellular keratinase was optimally active at pH 8.0 and temperature 65 °C with 0.7% soluble keratin as substrate. The produced B. licheniformis ALW1 keratinase exhibited a good stability over pH range from 7 to 9 and over a temperature range 50–60 °C for almost 90 min. The crude enzyme solution was able to degrade native feather up to 63% in redox free system.

Improved biosynthesis of silver nanoparticles using keratinase from Stenotrophomonas maltophilia R13: reaction optimization, structural characterization, and biomedical activity.

In the present study, keratinase from Stenotrophomonas maltophilia R13 was used for the first time as a reducing agent for the eco-friendly synthesis of AgNPs. The keratinase produced by strain R13 was responsible for the reduction of silver ions and the subsequent formation of AgNPs. Maximum AgNP synthesis was achieved using 2mM AgNO3 at pH 9 and 40°C. Electron microscopy and dynamic light scattering analysis showed AgNPs were spherical and of average diameter ~ 8.4nm. X-ray diffraction revealed that AgNPs were crystalline. FTIR indicated AgNPs were stabilized by proteins present in the crude enzyme solution of strain R13. AgNPs exhibited a broad antimicrobial spectrum against several pathogenic microorganisms, and the antimicrobial mechanism appeared to involve structural deformation of cells resulting in membrane leakage and subsequent lysis. AgNPs also displayed 1,1-diphenyl-2-picrylhydrazyl (IC50 = 0.0112mg/ml), 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonate radical scavenging (IC50 = 0.0243mg/ml), and anti-collagenase (IC50 = 23.5mg/ml) activities.

Expression of Aspergillus niger N5-5 in E. coli and purification and identification of products.

Due to the feature of high hydrolysis, tannase is widely used in food, beverage, brewing and other fields. However, high cost in producing natural tannase makes it difficult to apply tannase to industry in a large-scale. Microbial expression systems can be used for preparing numerous amount of enzyme at low cost, so in this paper Aspergillus niger N5-5 was expressed using E. coli system. Specific primers were designed based on the Aspergillus niger N5-5 sequence N3 (GenBank, No.: KP677552), and tannase gene tan was promoted to carry 6 His tag and enzyme cutting site which contains NdeI/HindIII using PCR amplification. Then, tannase gene tan was connected to expression vector by NdeI/HindIII enzyme cutting. In this way, recombinant expression vector tan-pET43.1a was formed. Then, the expression vector pET43.1a by NdeI/HindIII enzyme cutting was transformed into E. coli BL21 (DE3) to induce expression of Aspergillus niger N5-5. When the induced fungi were disrupted by the ultrasonic wave, the crude enzyme was extracted and purified by using the IMAC, and then the activity of the crude enzyme and pure enzyme was determined. According to the results of determination of the tannase activity, the tannase activity of the crude enzyme was greatly improved after the crude enzyme was purified, and the specific activity of the pure enzyme was about 8 times of that of the crude enzyme. The results of SDS-PAGE of the pure enzyme showed that the molecular mass of the pure enzyme was about 65 kDa/64–65 kDa, which was consistent with the expected result (64.2 kDa), It can be concluded that the crude enzyme solution was purified successfully. The results of pure enzyme’s protein identification by Western Blotting showed that clear protein bands pro-3 were observed. Molecular mass of clear protein bands pro-3 was about 65 kDa, which was in line with the expected results (64.2 kDa). It can be seen that the aforementioned expression protein could be specifically combined with His tag. It proved expression protein to be a recombinant fusion protein with 6 His tag.

Determination of Aldehyde Dehydrogenase Activity by Fluorescence Spectrophotometer

A method for the determination of aldehyde dehydrogenase activity by fluorescence spectrophotometer was established. The precipitate of yeast was added by three volumes of phosphate buffers after centrifugation. Ultrasonic fragmentation of cells is under ice-cooling condition. The supernatant was collected by centrifugation as a crude enzyme solution of aldehyde dehydrogenase after sonication. The effects of pH and temperature on enzyme activity were investigated. Ammonium sulfate precipitation method and polyethylene glycol (PEG) precipitation method were alternative to purify the crude enzyme, and the effect of PEG precipitation was better. The purification factor of the enzyme protein was 2.32. NADH (nicotinamide adenine dinucleotide) solutions were scanned to determine the wavelength of fluorescence detection: excitation wavelength at 345 nm and emission wavelength of 460 nm. The fluorescence spectrophotometer detection limit was 0.2 mg/L, the linear range was 0.75-60 mg/L and the correlation coefficient was 0.995. The recoveries were 95.62% -99.21% with the relative standard deviations (RSD) of 0.60%-0.98% (n=6). This method was utilized to detect the activity of aldehyde dehydrogenase and the results were compared with the results of UV spectrophotometry. The results indicate that fluorescence spectrophotometer is a suitable method for the detection of aldehyde dehydrogenase enzyme activity.

Biodegradation of chlorothalonil by Enterobacter cloacae TUAH-1

The long-term excessive use of chlorothalonil (CTN) can lead to serious environmental pollution, which presents a cause for concern. Here, we investigated the degradation of CTN by using an effective strain, TUAH-1, which was identified as Enterobacter cloacae according to the morphological approach, 16S rDNA gene sequence and phylogenetic tree analysis. Treatment with TUAH-1 demonstrated that the maximum processing capability was 74 mg CTN per gram dry cells in 48 h under optimum conditions (30 °C–35 °C, pH 7.0). After treatment with 21.3 mg dry cells, the degradation efficiency was 97.4% for 20 mg l-1 CTN in the aqueous phase. Meanwhile, treatment with crude enzyme solution containing 2.0 mg ml-1 protein led to complete degradation of 20.0 mg l-1 CTN within 10 h, which is significantly faster than that of TUAH-1. The results also showed that CTN could not be detected after treatment with 5.68 g TUAH-1 per kg soil for 48 h when the original CTN content in the soil was 10 mg kg-1. After treatment with THAH-1 or its crude proteins, many degradation products were detected by HPLC-MS, suggesting two possible degradation pathways. The mechanism of CTN degradation by TUAH-1 was noted to be multienzymatic catalysis comprising glyceraldehyde-3-phosphate dehydrogenase and glutathione S-transferase.

The Absence of the Transcription Factor Yrr1p, Identified from Comparative Genome Profiling, Increased Vanillin Tolerance Due to Enhancements of ABC Transporters Expressing, rRNA Processing and Ribosome Biogenesis in Saccharomyces cerevisiae.

Enhancing the tolerance of Saccharomyces cerevisiae to inhibitors derived from lignocellulose is conducive to producing biofuel and chemicals using abundant lignocellulosic materials. Vanillin is a major type of phenolic inhibitor in lignocellulose hydrolysates for S. cerevisiae. In the present work, the factors beneficial to vanillin resistance in yeast were identified from the vanillin-resistant strain EMV-8, which was derived from strain NAN-27 by adaptive evolution. We found 450 SNPs and 44 genes with InDels in the vanillin-tolerant strain EMV-8 by comparing the genome sequences of EMV-8 and NAN-27. To investigate the effects of InDels, InDels were deleted in BY4741, respectively. We demonstrated that the deletion of YRR1 improved vanillin tolerance of strain. In the presence of 6 mM vanillin, deleting YRR1 increase the maximum specific growth rate and the vanillin consumption rate by 142 and 51%, respectively. The subsequent transcriptome analysis revealed that deleting YRR1 resulted in changed expression of over 200 genes in the presence of 5 mM vanillin. The most marked changes were the significant up-regulation of the dehydrogenase ADH7, several ATP-binding cassette (ABC) transporters, and dozens of genes involved in ribosome biogenesis and rRNA processing. Coincidently, the crude enzyme solution of BY4741(yrr1Δ) exhibited higher NADPH-dependent vanillin reduction activity than control. In addition, overexpressing the ABC transporter genes PDR5, YOR1, and SNQ2, as well as the RNA helicase gene DBP2, increased the vanillin tolerance of strain. Interestingly, unlike the marked changes we mentioned above, under vanillin-free conditions, there are only limited transcriptional differences between wildtype and yrr1Δ. This indicated that vanillin might act as an effector in Yrr1p-related regulatory processes. The new findings of the relationship between YRR1 and vanillin tolerance, as well as the contribution of rRNA processing and ribosome biogenesis to enhancing S. cerevisiae vanillin tolerance, provide novel targets for genetic engineering manipulation to improve microbes’ tolerance to lignocellulose hydrolysate.

Enhancement of soluble expression of codon-optimized Thermomicrobium roseum sarcosine oxidase in Escherichia coli via chaperone co-expression

The codon-optimized sarcosine oxidase from Thermomicrobium roseum (TrSOX) was successfully expressed in Escherichia coli and its soluble expression was significantly enhanced via the co-expression of chaperones. With the assistance of whole-genome analysis of T. roseum DSM 5159, the sox gene was predicated and its sequence was optimized based on the codon bias of E. coli. The TrSOX gene was successfully constructed in the pET28a plasmid. After induction with IPTG for 8h, SDS-PAGE analysis of crude enzyme solutions showed a significant 43kDa protein band, indicating SOX was successfully expressed in E. coli. However, the dark band corresponding to the intracellular insoluble fraction indicated that most of TrSOX enzyme existed in the inactive form in “inclusion bodies” owing to the “hot spots” of TrSOX. Furthermore, the co-expression of five different combinations of chaperones indicated that the soluble expression of TrSOX was greatly improved by the co-expression of molecular chaperones GroES-GroEL and DnaK-DnaJ-GrpE-GroES-GroEL. Additionally, the analysis of intramolecular forces indicated that the hydrophobic amino acids, hydrogen bonds, and ionic bonds were favorable for enhancing the interaction and stability of TrSOX secondary structure. This study provides a novel strategy for enhancing the soluble expression of TrSOX in E. coli.

Isolation and characterization of a novel Penicillium oxalicum strain Z1-3 with enhanced cellobiohydrolase production using cellulase-hydrolyzed sugarcane bagasse as carbon source

Biofuels from lignocellulosic biomass are acknowledged to be a promising alternative to fossil fuels. During biomass saccharification, nearly complete conversion is rarely achieved due to the recalcitrance of the residual biomass to enzymatic hydrolysis. Few studies have examined schemes for searching for novel fungal strains that could improve the conversion. In the current study, for the first time, we searched for efficient fungal producers of cellulases in a natural decay system, using a screening process containing cellulase-hydrolyzed sugarcane bagasse as carbon source. Three hundred and seventy-three fungal strains were isolated from 69 samples collected in Guangxi Zhuang Autonomous Region, China. Twelve of the 373 strains produced more than 0.5 filter paper units (FPU)/mL and were identified as Penicillium oxalicum strains. Among them, P. oxalicum Z1-3 yielded the highest cellulase production at 2.74FPU/ml, especially cellobiohydrolase, leading to about 0.5–2.0-fold higher synergistic enzyme activities including sugarcane bagasse residue cellulase, potassium hydroxide (KOH)-pretreated sugarcane bagasse cellulase, filter paper cellulase (FPase) and Avicelase, than the four isolates which yielded higher or similar FPase activities in comparison with the P. oxalicum reference strain HP7-1. Furthermore, a crude enzyme solution of Z1-3 was able to hydrolyze 92.5% of KOH-pretreated sugarcane bagasse to glucose within 96h. It was also shown that the cellobiohydrolase genes of Z1-3 and their promoter regions were the same as those of the reference strain HP7-1. Altogether, this scheme, which used recalcitrant biomass residues after cellulase hydrolysis, is feasible for searching for efficient cellulase producers. This work demonstrates the great potential of P. oxalicum Z1-3 in future biotechnological applications of lignocellulosic biomass collected from industry and agriculture.