Optimization of the fermentation process and antioxidant activity of mixed lactic acid bacteria for honeysuckle beverage.

The aim of the research was to obtain a high healthcare honeysuckle beverage with strong antioxidant activity. Honeysuckle (Lonicera japonica Thunb) was used as the raw material in this experiment. The effects of fermentation temperature, fermentation time, lactic acid bacteria inoculation amount, and sugar addition amount on the sensory quality of honeysuckle beverage were investigated by single factor test and orthogonal test, and the best process was obtained. The physicochemical indexes and antioxidant activity of honeysuckle beverages fermented with lactic acid bacteria were studied. The results showed that the fermentation temperature of the beverage was 37 °C, the fermentation time was 24 h, the inoculation amount of Lactiplantibacillus plantarum and Lactobacillus acidophilus mixed starter (1:1) was 3%, and 8% white granulated sugar was added. The highest sensory score was 87.30 ± 0.17, which was the optimal process. The honeysuckle liquid mixed inoculation with Lactiplantibacillus plantarum and Lactobacillus acidophilus was fermented for 24 h. The number of viable bacteria reached 9.84 ± 0.02 lg cfu/mL, the pH value was 3.10 ± 0.01, and the total polyphenol content was 7.53 ± 0.03 mg GAE/g. The number of lactic acid bacteria, pH, total polyphenol content, and free radical scavenging rate were significantly increased (p < 0.05) compared with the non-inoculated and single-inoculated lactic acid bacteria. To sum up, it was concluded that a better quality beverage could be obtained by fermenting a solution of honeysuckle with Lactiplantibacillus plantarum and Lactobacillus acidophilus mixed fermentation agent, providing a new approach and new ideas for the development of deep processing and fermented beverages using honeysuckle.

Antibacterial mechanism of inosine against Alicyclobacillus acidoterrestris.

Inosine could potentially become a novel antibacterial agent against Alicyclobacillus acidoterrestris as low doses of inosine can prevent its contamination. However, until now the antibacterial mechanism of inosine targeting A. acidoterrestris is still unknown. In this study, to unravel the mechanism of inosine against A. acidoterrestris puzzle, the effects of inosine on bacterial surface hydrophobicity, intracellular protein content, cell membrane damage extent, and permeability of the A. acidoterrestris were investigated. The results showed that inosine can effectively inhibit the growth and reproduction of A. acidoterrestris by destroying the integrity of cell membrane and increasing its permeability, causing the leakage of intracellular nutrients. Furthermore, the interaction networks of inosine target proteins were analyzed. The interaction networks further revealed that damage to bacterial cell membranes might be relevant to inosine's effect on bacterial DNA replication and cell energy metabolism through regulating nucleotide synthesis and metabolism and the activity of translation initiation factors. Finally, the antibacterial mechanism of inosine against A. acidoterrestris was proposed.

Inhibitory mechanism of quercetin on Alicyclobacillus acidoterrestris.

In this the antibacterial of quercetin against Alicyclobacillus acidoterrestris was evaluated by measuring the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). Subsequently, the effect of quercetin on A. acidoterrestris cell membrane was evaluated through scanning electron microscopy (SEM), surface hydrophobicity determination, diacetate fluorescein staining and propidium iodide (PI) staining. Additionally, the effects of quercetin on intracellular macromolecules and cell metabolism were explored by measuring the culture medium protein, bacterial protein and intracellular sodium and potassium adenosine triphosphate (ATP) enzyme activity. The results revealed that quercetin exhibited the MIC and MBC values of 100 ug/mL and 400 ug/mL, respectively, against A. acidoterrestris. The SEM results revealed that quercetin could induce irreversible damage to the cell membrane effectively. Moreover, quercetin could enhance the surface hydrophobicity of A. acidoterrestris. The results of flow cytometry and fluorescence microscopy analyses revealed that quercetin could promote cell damage by altering the cell membrane permeability of A. acidoterrestris, inducing the release of nucleic acid substances from the cells. Furthermore, the determination of protein content in the culture medium, bacterial protein content, and the Na(+)/K(+)-ATPase activity demonstrated that quercetin could reduce the intracellular protein content and impedes protein expression and ATPase synthesis effectively, leading to apoptosis.

Low-frequency alternating magnetic field thawing of frozen pork meat: Effects of intensity on quality properties and microstructure of meat and structure of myofibrillar proteins.

The purpose of the study was to evaluate the changes in quality properties and microstructure of pork meat as well as structural variation in myofibrillar proteins (MPs) after low-frequency alternating magnetic field thawing (LF-MFT) with different intensities (1-5 mT). LF-MFT at 3-5 mT shortened the thawing time. LF-MFT treatment significantly influenced the quality properties of meat and notably improved the structure of MPs (P < 0.05), compared to atmosphere thawing (AT). Especially, among the thawing treatments, LF-MFT-4 (LF-MFT at 4 mT) had the lowest values of thawing loss and drip loss, and the least changes in the color and myoglobin content. Regarding the results of rheological properties and micrographs, an optimal gel structure and a more compact muscle fiber arrangement formed during LF-MFT-4. Moreover, LF-MFT-4 was beneficial for improving the conformation of MPs. Therefore, LF-MFT-4 reduced the deterioration of porcine quality by protecting MPs structure, indicating a potential use in the meat thawing industry.

Study on the Interaction Mechanism of Methoxy Polyethylene Glycol Maleimide with Sweet Potato ß-Amylase.

In this study, sweet potato ß-amylase (SPA) was modified by methoxy polyethylene glycol maleimide (molecular weight 5000, Mal-mPEG5000) to obtain the Mal-mPEG5000-SPA modified ß-amylase and the interaction mechanism between SPA and Mal-mPEG5000 was investigated. the changes in the functional groups of different amide bands and modifications in the secondary structure of enzyme protein were analyzed using infrared spectroscopy and circular dichroism spectroscopy. The addition of Mal-mPEG5000 transformed the random curl in the SPA secondary structure into a helix structure, forming a folded structure. The Mal-mPEG5000 improved the thermal stability of SPA and protected the structure of the protein from breaking by the surrounding. The thermodynamic analysis further implied that the intermolecular forces between SPA and Mal-mPEG5000 were hydrophobic interactions and hydrogen bonds due to the positive values of ΔHθ and ΔSθ. Furthermore, the calorie titration data showed that the binding stoichiometry for the complexation of Mal-mPEG5000 to SPA was 1.26, and the binding constant was 1.256 × 107 mol/L. The binding reaction resulted from negative enthalpy, indicating that the interaction of SPA and Mal-mPEG5000 was induced by the van der Waals force and hydrogen bonding. The UV results showed the formation of non-luminescent material during the interaction, the Fluorescence results confirmed that the mechanism between SPA and Mal-mPEG5000 was static quenching. According to the fluorescence quenching measurement, the binding constant (KA) values were 4.65 × 104 L·mol-1 (298K), 5.56 × 104 L·mol-1 (308K), and 6.91 × 104 L·mol-1 (318K), respectively.

Understanding the influence of fluctuated low-temperature combined with high-humidity thawing on gelling properties of pork myofibrillar proteins.

The present study further investigated the effects of fluctuated low-temperature combined with high-humidity thawing (FLHT) on the gelling properties of pork myofibrillar proteins (MPs). Results showed that compared with refrigerator thawing (RT) and low-temperature combined with high-humidity thawing (LHT), FLHT effectively reduced the protein aggregation and maintained the relative stability by decreasing the variation in the turbidity and absolute ζ-potential value. The rheological results confirmed its improved elastic gel network. Meanwhile, FLHT samples exhibited markedly higher WHC with lower cooking loss (P < 0.05). The whiteness and strength of MPs gel were significantly higher in the FLHT group (P < 0.05). Moreover, there was no difference in textural properties between FLHT samples and fresh meat (FS) (P > 0.05), due to its homogeneous and compact microstructure. Therefore, FLHT plays an essential role in holding a superior gel quality and a compact structure, thereby evidencing its potential application in meat thawing.

Acid adaptive response of Alicyclobacillus acidoterrestris: A strategy to survive lethal heat and acid stresses.

Alicyclobacillus acidoterrestris causes the spoilage of pasteurized acidic fruit juice, seriouslydecreasing quality and posing a significant safety concern. We previously discovered that acid adaptation could induce stress adaptive responses of A. acidoterrestris, however, the underlying mechanisms of this induction have not been fully elucidated. In this work, the effects of acid adaptation (pH = 3.0, 1 h) on intracellular pH (pHi) and the morphophysiological properties of A. acidoterrestris under lethal heat and acid stresses were investigated, and gene expression profiles after acid adaptation were measured by transcriptomic analysis. The results showed that acid adaptation increased the pHi of A. acidoterrestris cells in response to lethal stresses, enhanced membrane integrity, decreased surface shrinkage and roughness, and altered the Fourier transform infrared spectra profiles. After acid adaptation of A. acidoterrestris, 517 differentially expressed genes (DEGs) were detected. Consistent with resistance phenotypes, DEGs included genes related to cell surface modification and pHi homeostasis. Specifically, the barrier function of cell membrane was strengthened during acid adaptation by increasing fatty acid (FA) chain length, promoting unsaturated FA biosynthesis, and maintaining balanced synthesis of zwitterionic and acidic phospholipids. To reduce excessive intracellular protons, cells upregulated glutamate decarboxylation, urease system, and branched-chain amino acid synthesis. Additionally, the nucleotide salvage pathway was activated, and homologous recombination, UvrD-mediated transcription-coupled, and ribonucleotide excision repair pathways were applied to repair DNA lesions. Sporulation metabolism was also induced. The findings of this study provide insight into the multiple layers of acid adaptive response strategies of A. acidoterrestris, with implications for the formulation of improved control measures in the fruit juice industry.

Integrated transcriptomic and proteomic analysis reveals the response mechanisms of Alicyclobacillus acidoterrestris to heat stress.

Alicyclobacillus acidoterrestris can survive pasteurization and is implicated in pasteurized fruit juice spoilage. However, the mechanisms underlying heat responses remain largely unknown. Herein, gene transcription changes of A. acidoterrestris under heat stress were detected by transcriptome, and an integrated analysis with proteomic and physiological data was conducted. A total of 911 differentially expressed genes (DEGs) was observed. The majority of DEGs and differentially expressed proteins (DEPs) were exclusively regulated at the mRNA and protein level, respectively, whereas only 59 genes were regulated at both levels and had the same change trends. Comparative analysis of the functions of the specifically or commonly regulated DEGs and DEPs revealed that the heat resistance of A. acidoterrestris was primarily based on modulating peptidoglycan and fatty acid composition to maintain cell envelope integrity. Low energy consumption strategies were established with attenuated glycolysis, decreased ribosome de novo synthesis, and activated ribosome hibernation. Terminal oxidases, cytochrome bd and aa3, in aerobic respiratory chain were upregulated. Meanwhile, the MarR family transcriptional regulator was upregulated, reactive oxygen species (ROS) was discovered, and the concentration of superoxide dismutase (SOD) increased, indicating that the accompanied oxidative stress was induced by high temperature. Additionally, DNA and protein damage repair systems were activated. This study provided a global perspective on the response mechanisms of A. acidoterrestris to heat stress, with implications for better detection and control of its contamination in fruit juice.

Transcriptome-Based Selection and Validation of Reference Genes for Gene Expression Analysis of Alicyclobacillus acidoterrestris Under Acid Stress.

Alicyclobacillus acidoterrestris is a major concern in fruit juice industry due to its spoilage potential of acidic fruit juice. Quantifying the expression levels of functional genes by real-time quantitative polymerase chain reaction (RT-qPCR) is necessary to elucidate the response mechanisms of A. acidoterrestris to acid stress. However, appropriate reference genes (RGs) for data normalization are required to obtain reliable RT-qPCR results. In this study, eight novel candidate RGs were screened based on transcriptome datasets of A. acidoterrestris under acid stress. The expression stability of eight new RGs and commonly used RG 16s rRNA was assessed using geNorm, NormFinder, and BestKeeper algorithms. Moreover, the comprehensive analysis using the RefFinder program and the validation using target gene ctsR showed that dnaG and dnaN were the optimal multiple RGs for normalization at pH 4.0; ytvI, dnaG, and 16s rRNA at pH 3.5; icd and dnaG at pH 3.0; and ytvI, dnaG, and spoVE at pH 2.5. This study revealed for the first time that A. acidoterrestris had different suitable RGs under different acid conditions, with implications for further deciphering the acid response mechanisms of this spoilage-causing bacterium.

Characterization of a novel antifungal protein produced by Paenibacillus polymyxa isolated from the wheat rhizosphere.

BACKGROUND: Fusarium head blight (FHB) is one of the disasters that seriously harm wheat and other small grain crops. It causes spoilage and mildew of the grain leading to a significant decline in the yield and quality of the grain. This research aimed to isolate antagonistic bacteria to purify antifungal proteins. A strain was isolated from the rhizosphere of healthy wheat in a wheat field affected by a severe FHB epidemic. This isolated strain was tentatively identified as Paenibacillus polymyxa 7F1, which displayed a strong inhibitory effect against several other pathogens. One novel antifungal protein was purified from the P. polymyxa 7F1 and successfully expressed. RESULTS: A crude culture of P. polymyxa 7F1 demonstrated antifungal activity that was stable at a temperature range of 60-90 °C and a pH range of 2.6-9.0. However, the antifungal activity of the P. polymyxa 7F1 was inhibited with proteinase K, trypsin, and neutral protease treatment. A 36 kDa protein with broad-spectrum antifungal activity was purified from the P. polymyxa 7F1. A glycosyl hydrolase domain was identified from this protein through liquid chromatography-mass spectrometry (LC-MS) analysis. A recombinant plasmid pET32a(+)/36kd for prokaryotic expression was constructed, and the renatured p36kd protein demonstrated similar antifungal activity to the 36 kDa protein purified from the P. polymyxa 7F1. CONCLUSION: A novel antifungal protein produced by P. polymyxa 7F1 was purified and expressed. The recombinant protein showed good antifungal activity as the novel purified protein. The novel antifungal protein provides an effective way to control the Fusarium head blight. © 2020 Society of Chemical Industry.

Influence of adding steam-exploded apple pomace on wheat flour characteristics and biscuit quality.

Apple pomace treated by steam explosion (SE-AP) was mixed with wheat flour, the wheat dough characteristics and biscuit quality are deserved to investigate. In this paper, the characteristics of wheat dough blended with SE-AP, including sedimentation values, pasting properties, and farinographic features were measured; the textural properties and sensory evaluation of the blended biscuits were analyzed. The results showed that the sedimentation values of wheat dough gradually decreased when SE-AP was less than 10%, which was almost no influence on the biscuit quality. The more SE-AP was added, the less values of peak viscosity, trough viscosity and final viscosity, which was disadvantage to the processing quality of wheat flour; however, the values of breakdown and setback increased with the addition of SE-AP, which improved the processing quality. Dough development time, stability time, and farinograph quality number decreased with the addition of SE-AP, which was unfavourable to the quality of wheat flour. When the addition of SE-AP was less than 10%, the hardness of biscuits decreased, springiness and resilience increased, and the chewability improved. According to the texture properties and organoleptic evaluation, the sensor score of the biscuits made from weak-gluten wheat with 10% (m/m) SE-AP added was the highest.

Analysis of differential expression proteins reveals the key pathway in response to heat stress in Alicyclobacillus acidoterrestris DSM 3922T.

For the purpose of investigating the heat resistance mechanism of Alicyclobacillus acidoterrestris, label-free quantification was used to reveal some cellular changes in A. acidoterrestris during heat stress. Totally, 545 differential expression proteins were respectively identified at heat stress of 65 °C for 5 min, of which 258 proteins were up-regulated and 287 proteins were down-regulated. These significantly changed proteins were mapped to 100 pathways and some of them were mostly related to protection or repair of macromolecules such as proteins and DNA, cell wall formation, which indicated that these proteins might play crucial roles in response to heat stress. The KEGG pathway analysis combined with protein functional analysis and further validation at mRNA level suggested that A. acidoterrestris sensed the temperature rise in environment through alterations in the secondary structure of DNA and RNA molecules. The biosynthesis of antibiotics pathway and the ribosomes might be involved in signal transduction in heat stress and further trigger a large number of proteins playing a critical role in the regulation of heat stress in A. acidoterrestris. The study firstly demonstrated the global physiological response to heat stress and the results provided a better understanding of thermal adaption mechanism of A. acidoterrestris.

Label free quantitative analysis of Alicyclobacillus acidoterrestris spore germination subjected to low ambient pH.

Inhibition of spore germination or sterilization after induction of spore germination would effectively control low pH food spoilage caused by Alicyclobacillus acidoterrestris spores. However, the characteristics and mechanisms of A. acidoterrestris spore germination in low ambient pH remains poorly understood. In this study, the germination rate of A. acidoterrestris spores at different ambient pH conditions was determined, and subsequently the proteomic profiles of A. acidoterrestris in spore germination were analysed by label-free quantification, in which the specific metabolic pathways involved were identified and key functional proteins were screened and validated using RT-qPCR (real time quantitative PCR). The suitable ambient pH value for the spore germination of A. acidoterrestris ranged from 3.0 to 5.0 with the optimum pH of 4.0. According to the LC-ESI-MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry) analysis, 98 proteins of geminated spores of A. acidoterrestris incubated for 2 h at pH 3.0 were changed significantly in comparison to non-germinated spores, the expression of 20 proteins were up-regulated and that of 78 proteins down-regulated respectively. Those differential expressed proteins were mainly involved in cell wall hydrolysis, cell morphological changes, protein synthesis and folding, perception of external stimuli and signal transduction etc., and we observed that germination receptor D (GerD), cell wall hydrolase, transpeptidase, peptidase S1 and two-component regulatory system phoR were significantly up-regulated, but hydrolase NlpC/P60, peptidoglycan glycosyltransferase, spore coat proteins CotX, CotJB and the Lrp/AsnC (leucine-responsive regulatory protein/asparagine synthase C products) protein were significantly down-regulated in the experiment, which implied the important roles of identified proteins during the spore germination. Furthermore, the pathway analysis showed the possible involvement of differentially expressed proteins in the ß-lactam resistance, ribosome, biosynthesis of secondary metabolites, pyruvate metabolism, two-component system and other metabolic pathways, which indicated that synthesis and hydrolysis of cell wall, intracellular substance synthesis, energy generation and signal transduction were likely associated with the initiation of spore germination and restoration of vegetative growth. In conclusion, the quantitative proteomic landscape of A. acidoterrestris spores could provide the theoretic and experimental evidences for the hazard control of A. acidoterrestris spores in the thermal pasteurization process of acidic beverages industry.

Chemical Modification of Sweet Potato ß-amylase by Mal-mPEG to Improve Its Enzymatic Characteristics.

The sweet potato ß-amylase (SPA) was modified by 6 types of methoxy polyethylene glycol to enhance its specific activity and thermal stability. The aims of the study were to select the optimum modifier, optimize the modification parameters, and further investigate the characterization of the modified SPA. The results showed that methoxy polyethylene glycol maleimide (molecular weight 5000, Mal-mPEG5000) was the optimum modifier of SPA; Under the optimal modification conditions, the specific activity of Mal-mPEG5000-SPA was 24.06% higher than that of the untreated SPA. Mal-mPEG5000-SPA was monomeric with a molecular weight of about 67 kDa by SDS-PAGE. The characteristics of Mal-mPEG5000-SPA were significantly improved. The Km value, Vmax and Ea in Mal-mPEG5000-SPA for sweet potato starch showed that Mal-mPEG5000-SPA had greater affinity for sweet potato starch and higher speed of hydrolysis than SPA. There was no significant difference of the metal ions' effect on Mal-mPEG5000-SPA and SPA.

Site-saturation mutagenesis library construction and screening for specific broad-spectrum single-domain antibodies against multiple Cry1 toxins.

Potential ecological environmental and food safety risks of various Cry toxins of Bacillus thuringiensis (Bt) in transgenic food have received gradually increasing attention, which urged to establish an efficient and broad-spectrum detection technology for Cry toxins. Based on the single-domain antibody (sdAb) A8 against Bt Cry1Ab toxin screened from the humanized domain antibody library, the key amino acids of sdAb (A8) binding five kinds of Cry1 toxins were predicted using homology modeling and molecular docking technology, and the results showed that 105th asparagine, 106th arginine, 107th valine, and 114th arginine, respectively, located in heavy-chain complementarity-determining region 3 were common key amino acid sites. Subsequently, site-saturation cooperative mutagenesis of the four key sites was performed using overlap extension PCR, and multiple site-saturation mutagenesis sdAb library with the capacity of 1.2 × 105 colony-forming units (CFU) was successfully constructed. With alternating five Cry1 toxins as coating antigen, two generic sdAbs (2-C1, 2-C9) were screened out from the mutagenesis library, which could detect six kinds of Cry1 toxins at least. Through ELISA analysis, the binding activity of 2-C9 was significantly enhanced, and its OD values versus Cry1Aa, Cry1Ab, Cry1B, Cry1C, and Cry1E increased to 1.34, 1.53, 1.82, 2.39, and 2.7 times, respectively, compared with maternal antibody A8. The IC50 values of 2-C9 against Cry1Aa, Cry1Ab, Cry1B, and Cry1C were lower than that of A8, which showed that the affinity of 2-C9 against Cry1 toxins was enhanced. The results were beneficial to developing high-throughput and high-sensitive immune-detecting technology for Cry toxins.

Heterogeneous expression of DnaK gene from Alicyclobacillus acidoterrestris improves the resistance of Escherichia coli against heat and acid stress.

Alicyclobacillus acidoterrestris, an acidophilic and thermophilic bacteria, is an important microbial resource for stress resistance genes screening. In this study, DnaK gene from A. acidoterrestris was subcloned to construct the recombinant plasmid pET28a-DnaK. The successful construction of the plasmid was verified by double-enzyme digestion and sequencing analysis. The recombinant plasmid was transformed into Escherichia coli BL21 and isopropy-ß-D-thiogalactoside (IPTG) was used to induce recombinant E. coli to express DnaK gene. A 70 kD fusion protein was identified by SDS-PAGE, which suggested that DnaK gene from A. acidoterrestris was successfully expressed. The recombinant and wild BL21 were treated with high temperatures of 54, 56 and 58 °C at pH values of 5.0-7.0 to compare the effects of heterogeneous expression of the DnaK gene from A. acidoterrestris on the stress resistance. The experimental results showed that survival rate of recombinant BL21-DnaK has been improved considerably under heat and acid stresses in contrast with the wild BL21, and D-values of recombinant BL21 were 14.7-72% higher than that of wild BL21, which demonstrated that heterogeneous expression of DnaK gene from A. acidoterrestris could significantly enhance the resistance of host bacteria E. coli against heat and acid stresses.

Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay.

3-Phenoxybenzoic acid (3-PBA) is a general metabolite of synthetic pyrethroids. It could be used as a generic biomarker for multiple pyrethroids exposure for human or pyrethroid residues in the environment. In this study, monoclonal antibodies (mAbs) against 3-PBA were developed by using PBA-bovine serum albumin (BSA) as an immunogen. In the competitive enzyme-linked immunosorbent assay (ELISA) format, the I50 and I10 values of purified mAbs were 0.63 and 0.13 µg/ml, respectively, with a dynamic range between 0.19 and 2.04 µg/ml. Then, the colloidal gold (CG)-based lateral flow immunoassay was established based on the mAbs. The working concentration of coating antigen and CG-labeled antibodies and the blocking effects were investigated to get optimal assay performance. The cutoff value for the assay was 1 µg/ml 3-PBA, and the detection time was within 10 min. A total of 40 river water samples were spiked with 3-PBA at different levels and determined by the lateral flow immunoassay without any sample pretreatments. The negative false rate was 2.5%, and no positive false results were observed at these levels. This lateral flow immunoassay has the potential to be an on-site screening method for monitoring 3-PBA or pyrethroid residues in environmental samples.

Expression of DnaJ gene in Alicyclobacillus acidoterrestris under stress conditions by quantitative real-time PCR.

UNLABELLED: This article describes the cloning, sequence analysis and expression of the DnaJ gene from Alicyclobacillus acidoterrestris. The genome walking technique was used to clone the full-length sequence of DnaJ and quantitative real-time PCR was used to analyze DnaJ expression under stress conditions. AadnaJ (GenBank accession nr: HQ893544) containing an open reading frame of 1137 bp encoding 378 amino acid residues was cloned from A. acidoterrestris DSM 3922(T). The nucleotide sequence of AadnaJ shows 77% homology with the DnaJ of A. acidocaldarius LAA1. The DnaJ expression level was upgraded rapidly under heat or acid stress. Its mRNA expression level reached a peak value at 25 min after the onset of heat stress (70 °C) and at 1 h after the onset of acid stress (pH = 1). Acid stress at pH 1 for 25 and 60 min led to the DnaJ expression levels 2.1 times and 35.7 times above that of the control, respectively. In response to cold stress at 0 °C, the DnaJ expression level decreased drastically to 0.04 times that of the control level after 1 h. The expression patterns of DnaJ in response to the stress conditions shown here explained the heat and acidity endurance of A. acidoterrestris. PRACTICAL APPLICATION: This study directly addresses the role of the DnaJ gene in temperature and acid endurance in A. acidoterrestris. This provides a basis for the development of genetic and molecular techniques that may minimize the adverse effects of A. acidoterrestris in fruit juice production. This study also sheds light on the design of heat- and acid-tolerant recombinases and the understanding of the molecular mechanisms underlying heat and acid resistance in A. acidoterrestris.

Cloning and expression of the endo-1,3(4)-ß-glucanase gene from Paecilomyces sp. FLH30 and characterization of the recombinant enzyme.

The cDNA encoding ß-1,3(4)-glucanase, named PsBg16A, from Paecilomyces sp. FLH30 was cloned, sequenced, and over expressed in Pichia pastoris, with a yield of about 61,754 U mL⁻¹ in a 5-L fermentor. PsBg16A has an open reading frame of 951 bp encoding 316 amino acids, and the deduced amino acid sequence of PsBg16A revealed that it belongs to glycoside hydrolase family 16. The purified recombinant PsBg16A had a pH optimum at 7.0 and a temperature optimum at 70 °C, and randomly hydrolyzed barley ß-glucan, lichenin, and laminarin, suggesting that it is a typical endo-1,3(4)-ß-glucanase (EC 3.2.1.6) with broad substrate specificity for ß-glucans.