黑曲霉β-甘露聚糖酶ManA耐热突变体的筛选

为了改造黑曲霉（Aspergillus niger）来源的β-甘露聚糖酶ManA,获得耐热性高的突变体。利用易错聚合酶链式反应（PCR）技术对构建的表达质粒pGAPZαA-manA进行随机突变,将突变文库转化至毕赤酵母（Pichia pastoris）GS115,表达筛选耐热性高的突变体,并进一步对突变位点进行定点突变,筛选其他耐热性高的突变体。结果表明,ManA的H283R与H283K突变体相对于野生型在耐热性方面有显著提升,75 ℃加热30 min其相对酶活由26.66%分别提升至76.95%和83.57%;在pH 3.0～9.0的条件下,50 ℃保存24 h,H283K突变体与野生型的相对酶活均在90%以上,H283R突变体在pH 3.0时的相对酶活下降到了84.72%,两个突变体的最适温度、pH及比酶活与野生型没有明显差异。说明β-甘露聚糖酶ManA的283位组氨酸（H283）对其耐热性较为关键,将该位点突变为精氨酸（R）和赖氨酸（K）时,ManA的耐热性得到显著提升。     

Single-site mutation of C363G or N463T strengthens thermostability improvement of IG181–182 deleted acidic α-amylase from deep-sea thermophile Geobacillus sp.

The acidic α-amylase Gs4j-AmyA from deep-sea thermophile Geobacillus sp. is more acid-resistant than the commercial sources as it displays more than 50% of the optimum at a range of pH 4.5–7. This may allow the removal of the step of pH adjustment in starch processing and save costs. Unfortunately, this amylase is not very stable at 90–95°C. Therefore, to develop a new commercial acidic α-amylase targeted to the food industry, it is necessary to further improve the thermostability of the α-amylase Gs4j-AmyA. In this study, 11 different α-amylase mutants were obtained by site-mutagenesis. Among them, the mutant IG181–182* (IG181–182 deletion) showed significant improvement in thermostability, whose half-life at 70°C was 63.4 times longer than the wild type. Interestingly, single-site mutants C363G and N463T showed no enhancement on thermostability, while the half-lives of the combination mutants IG181–182*/C363G and IG181–182*/N463T at 70°C were further extended by 16.8 and 38.7%, respectively. Unfortunately, the catalytic constants (k_cat) of the mutants C363G, N463T, IG181–182*/C363G and IG181–182*/N463T declined by 59, 49, 37 and 16%, respectively. The optimum temperature (65–70°C) and pH (5.5–5.6) of the mutants was unchanged. The thermostability improvement by IG181–182 deletion could be strengthened by synchronous C363G or N463T mutation.     

GABA shunt mediates thermotolerance in Saccharomyces cerevisiae by reducing reactive oxygen production

The GABA shunt pathway involves three enzymes, glutamate decarboxylase (GAD), GABA aminotransferase (GAT) and succinate semialdehyde dehydrogenase (SSADH). These enzymes act in concert to convert glutamate (α‐ketoglutarate) to succinate. Deletion mutations in each of these genes in Saccharomyces cerevisiae resulted in growth defects at 45°C. Double and triple mutation constructs were compared for thermotolerance with the wild‐type and single mutant strains. Although wild‐type and all mutant strains were highly susceptible to brief heat stress at 50°C, a non‐lethal 30 min at 40°C temperature pretreatment induced tolerance of the wild‐type and all of the mutants to 50°C. The mutant strains collectively exhibited similar susceptibility at 45°C to the induced 50°C treatments. Intracellular reactive oxygen intermediate (ROI) accumulation was measured in wild‐type and each of the mutant strains. ROI accumulation in each of the mutants and in various stress conditions was correlated to heat susceptibility of the mutant strains. The addition of ROI scavenger N‐tert‐butyl‐α‐phenylnitrone (PBN) enhanced survival of the mutants and strongly inhibited the accumulation of ROI, but did not have significant effect on the wild‐type. Measurement of intracellular GABA, glutamate and α‐ketoglutarate during lethal heat exposure at 45°C showed higher levels of accumulation of GABA and α‐ketoglutarate in the uga1 and uga2 mutants, while glutamate accumulated at higher level in the gad1 mutant. These results suggest that the GABA shunt pathway plays a crucial role in protecting yeast cells from heat damage by restricting ROI production involving the flux of carbon from α‐ketoglutarate to succinate during heat stress. Copyright © 2013 John Wiley & Sons, Ltd.     

Role of ComFA in controlling the DNA uptake rate during transformation of competent Bacillus subtilis

The roles of ComFA and ComEC in DNA uptake by competent Bacillus subtilis were analyzed by transformation with DNA in protoplast lysates (LP transformation). Deletion mutants of comFA and comEC and putative Walker A mutants (K152N, K152Q, K152E) of comFA were constructed by fusion polymerase chain reaction. Transformants of comEC mutant with purified DNA and DNA in protoplast lysate were not obtained, which shows a lack of transformation ability and backwards recombination of the mutant. Transformants of the comFA mutant were obtained by LP transformation (1.8 × 10(4) transformants/μg DNA). Low relative efficiency of transformation (RET) of comFA compared to wild type (4.3 × 10(-4)) showed an important role for comFA in DNA uptake. Walker A mutants showed 1.8-19 × 10(-4) RET, suggesting a dependence on ATPase activity for transformation. Co-transformation between short linkages was only detected in comFA mutants. The results demonstrated that ComFA controlled the DNA uptake rate. The interpretation was further supported by analyzing the plasmid used in LP transformation of the comFA mutant. The RET of comFA compared to the wild type was 2.7 × 10(-2), 60-fold higher than that with chromosomal DNA (4.3 × 10(-4)). Following addition of DNA into comFA culture, transformants were obtained after 15 min, with the number of transformants increasing over time. The kinetics strongly suggested that in comFA mutants, formation of another DNA uptake complex without ComFA would be a lengthy process.     

Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae

A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β‐Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9‐fold higher than that of the wild‐type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild‐type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild‐type strain. The relative value (mnn2 deletion mutant/wild‐type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β‐glucosidase activity using p‐nitrophenyl‐β‐glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high‐molecular‐weight substrates to the active sites of the displayed enzymes. Copyright © 2014 John Wiley & Sons, Ltd.     

Mutation of Acetobacter pasteurianus by UV irradiation under acidic stress for high‐acidity vinegar fermentation

To obtain a mutant with higher vinegar production, a wild‐type industrial strain Acetobacter pasteurianus CICIM B7003 was pretreated in 50 g L^?1 acetic acid for 1 h and cultured for 12 h without adding any acetic acid; then, the enriched cells were mutated by UV under acidic stress (60 g L^?1 acetic acid). Using this method, A. pasteurianus CICIM B7003‐02, a mutant exhibiting best performance, was obtained. Notably, after repeated experiments, it was confirmed that B7003‐02 accumulated 103.81 ± 1.17 g L^?1 acetic acid within 160‐h batch cultivation in Erlenmeyer flasks, which was 49.2% higher than that of the wild type. Repeated batch fermentations with A. pasteurianus B7003‐02 were carried out for several runs in a Frings 8‐L acetator, and high‐acidity vinegars (90 ± 0.39 g L^?1) were produced. This work reveals the potential value for improvement in industrial vinegar production.     

Reduction of Methanol in Brewed Wine by the Use of Atmospheric and Room‐Temperature Plasma Method and the Combination Optimization of Malt with Different Adjuncts

Methanol, often generated in brewed wine, is highly toxic for human health. To decrease the methanol content of the brewed wine, atmospheric and room‐temperature plasma (ARTP) was used as a new mutagenesis tool to generate a mutant of Saccharomy cescerevisiae with lower methanol content. Headspace gas chromatography was used to determine the identity and concentration of methanol with butyl acetate as internal standard in brewed wine. With 47.4% higher and 26.3% positive mutation rates were obtained, the ARTP jet exhibited a strong effect on mutation breeding of S. cerevisiae. The mutant S. cerevisiae S12 exhibited the lowest methanol content, which was decreased by 72.54% compared with that of the wild‐type strain. Subsequently, the mutant S. cerevisiae S12 was used to ferment different combinations of malt and adjuncts for lower methanol content and higher alcoholic content. It was shown that the culture 6#, which was 60% malt, 20% wheat, and 20% corn, was the best combinations of malt and adjuncts, with the lowest methanol content (104.8 mg/L), and a relatively higher alcoholic content (15.3%, v/v). The optimal malt–adjunct culture 6#, treated with the glucoamylase dose of 0.04 U/mg of grain released the highest reducing sugars (201.6 mg/mL). It was indicated that the variation in reducing sugars among the combinations of malt and different adjuncts could be due to the dose of exogenous enzymes.     

Characterization of Phaffia rhodozyma 3A 4–8 Generated by Low-dose γ-irradiation

ABSTRACT: Astaxanthin content, superoxide dismutase activity, catalase activity, and transmission electron microscopy (TEM) of astaxanthin-hyperproducing mutant 3A 4–8, previously isolated through repeated rounds of γ-irradiation below 10 kGy and visual screening, was examined and compared with wild strain 67–385 and parent strain 2A2N to characterize its mutant. Astaxanthin content of Phaffia rhodozyma was determined using high-performance liquid chromatography. After 10 d culture, 3A 4–8 produced 2.5 mg/g yeast, 78% higher astaxanthin content than parent strain. Mutant exhibited lower superoxide dismutase and higher catalase activities than parent strain. TEM study showed mutant had smaller-sized mitochondria than parent strain. These results indicate γ-irradiation is an effective means of mutagenesis for production of carotenoidhyperproducing mutants.     

Strain Improvement Through UV and Chemical Mutagenesis for Enhanced Citric Acid Production in Molasses-Based Solid State Fermentation

Aspergillus niger was subjected to UV radiation and chemical mutagenesis to develop its hyper-producing mutants for enhanced citric acid production. Ethyl methane sulfonate (EMS) and Ethidium bromide (EB) were used for chemical mutagenesis of Aspergillus niger. UV, and chemically treated mutants of Aspergillus niger were identified by using 2-deoxy, D-glucose as selective marker. The selected mutants were cultured in solid state fermentation (SSF) of sugarcane molasses medium (10%) using corn cobs, banana stalk, sugarcane bagasse, wheat straw, and wheat bran as carrier substrates. After pH adjustment and sterilization, the triplicate flasks were inoculated with 5 mLof homogenous spore suspensions of selected mutants of A. niger and the flasks were subjected to SSF under still culture conditions. The mutant EB-3 (treated with 1 mg/mL ethidium bromide for 120 min) giving highest citric acid yield (64.2 mg/mL) in 72 h was selected as hyper-producing mutant. Citric acid production process using EB-3 mutant was then optimized to enhance citric acid production by the mutant in SSF. Aspergillus niger EB-3 mutant could produce 67.72 mg/mL citric acid in 72 h using banana stalks as support material under optimum conditions of pH (pH 6), incubation temperature (35°C) and inoculum size (5 mL) in SSF.     

Production of cordycepin by a repeated batch culture of a Cordyceps militaris mutant obtained by proton beam irradiation

Cordycepin (3′-deoxyadenosine) is one of the most versatile metabolites of Cordyceps militaris due to its broad spectrum of biological activity. In our previous study, the C. militaris mutant G81-3, which produces higher levels of cordycepin, was obtained by high-energy proton beam irradiation. In this study, the effects of adenosine on cordycepin production in a surface liquid culture of the mutant and the wild type strains were investigated. For the mutant strain, the optimum dose of adenosine yielded a 30% increase in cordycepin production; the maximum levels of production with adenosine and without adenosine were 8.6 g/l and 6.7 g/l, respectively. In contrast, the increase due to adenosine supplementation for the wild type strain was only 15% (3.1 g/l with adenosine and 2.7 g/l without adenosine). Furthermore, a repeated batch culture, an efficient production method, was carried out to eliminate the relatively long lag phase of the mutant culture. Over four cycles, both the mutant and the wild type strain maintained a production level of more than 85% of that of the initial cycle. As a result, the disadvantage of the mutant was successfully overcome, resulting in a productivity (0.48 g/(l d)) higher than that of the batch culture (0.29 g/(l d)). The productivity for cordycepin obtained in this study is the highest reported value to date, and this method could be applied to large-scale production of cordycepin at industrial levels.     

Genome‐wide screen of Saccharomyces cerevisiae for killer toxin HM‐1 resistance

We screened a set of Saccharomyces cerevisiae deletion mutants for resistance to killer toxin HM‐1, which kills susceptible yeasts through inhibiting 1,3‐beta‐glucan synthase. By using HM‐1 plate assay, we found that eight gene‐deletion mutants had higher HM‐1‐resistance compared with the wild‐type. Among these eight genes, five—ALG3, CAX4, MNS1, OST6 and YBL083C—were associated with N‐glycan formation and maturation. The ALG3 gene has been shown before to be highly resistant to HM‐1. The YBL083C gene may be a dubious open reading frame that overlaps partially the ALG3 gene. The deletion mutant of the MNS1 gene that encodes 1,2‐alpha‐mannosidase showed with a 13‐fold higher HM‐1 resistance compared with the wild‐type. By HM‐1 binding assay, the yeast plasma membrane fraction of alg3 and mns1 cells had less binding ability compared with wild‐type cells. These results indicate that the presence of the terminal 1,3‐alpha‐linked mannose residue of the B‐chain of the N‐glycan structure is essential for interaction with HM‐1. A deletion mutant of aquaglyceroporin Fps1p also showed increased HM‐1 resistance. A deletion mutant of osmoregulatory mitogen‐activated protein kinase Hog1p was more sensitive to HM‐1, suggesting that high‐osmolarity glycerol pathways plays an important role in the compensatory response to HM‐1 action. Copyright © 2010 John Wiley & Sons, Ltd.     

Use of chemical mutagenesis for the isolation of food grade beta-galactosidase overproducing mutants of bifidobacteria, lactobacilli and Streptococcus thermophilus

A classical chemical mutagenesis protocol was evaluated for increasing beta-galactosidase production by probiotic bacteria to improve their potential to treat symptoms of lactose malabsorption in humans. Two Bifidobacterium species (B. breve and B. longum) and one strain each of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus were tested by a single exposure to two chemical mutagens, ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). To screen for beta-galactosidase (beta-gal) overproducing mutants, optimized EMS and MNNG mutant pots for each strain were plated on BHI agar containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal). Colonies that exhibited a blue color were selected for quantitative beta-gal activities using the o-nitrophenyl-beta-galactoside (ONPG) assay. Seventy-five mutants were obtained out of more than 2 million colonies screened and showed increased beta-galactosidase activities compared with the wild-type strains. EMS gave a higher frequency of beta-gal overproducing mutants than MNNG for three of the four strains, S. thermophilus, B. breve, and B. longum, whereas the frequency of L. delbrueckii ssp. bulgaricus beta-gal mutants was similar with both mutagens. The highest beta-gal increases, when induced during growth in lactose, for mutants of each culture were 137% for L. delbrueckii ssp. bulgaricus; 104% for S. thermophilus; 70% for B. breve; and 222% for B. longum mutants. This food-grade classical approach has the ability to moderately increase beta-gal concentrations in probiotic cultures to improve their potential for treating the symptoms of lactose malabsorption in humans.     

USE OF A MODIFIED UREA GEL ISOELECTRIC FOCUSING METHOD FOR SPECIES IDENTIFICATION OF RAW OR BOILED WHITE,PINK, AND ROCK SHRIMP1

Isoelectric focusing (IEF) polyacrylamide gel containing an 80% pH 4–6.5 and 20% pH 3–10 ampholyte mixture greatly improved protein banding pattern for species identification of water extracts of raw pink, white and rock shrimp compared with the system using only the pH 3–10 range ampholyte. Identification of a specific species in mixture samples was achieved by the detection of water-extractable shrimp specific protein bands present in the gel. Sodium dodecyl sulfate (SDS) was a better protein extractant than water for cooked shrimp. Both water and SDS extracts of cooked shrimp showed specific protein banding patterns and improved resolution for species identification.     

Mutations in the N‐terminal region of the Schizosaccharomyces pombe glutathione transporter pgt1+ allows functional expression in Saccharomyces cerevisiae

Pgt1p encodes a glutathione transporter in Schizosaccharomyces pombe, orthologous to the Saccharomyces cerevisiae glutathione transporter, Hgt1p. Despite high similarity to Hgt1p, Pgt1p failed to display functionality during heterologous expression in S. cerevisiae. In the present study we employed a genetic strategy to investigate the reason behind the non‐functionality of pgt1⁺ in S. cerevisiae. Functional mutants were isolated after in vitro mutagenesis. Several mutants were obtained and four mutants analysed. Among these, three yielded different point mutations in the N‐terminal region (301–350 bp) of the transporter before the first transmembrane domain, while one mutant contained a deletion of 42 nucleotides within the same region. The mutant pgt1⁺ proteins not only expressed and localized correctly, but displayed high‐affinity glutathione transport capabilities in S. cerevisae. Comparison of wild‐type pgt1⁺ with the functional mutants revealed that a loss in protein expression was responsible for lack of functionality of wild‐type pgt1⁺ in S. cerevisiae. The mRNA levels in wild‐type and mutants were comparable, suggesting that the block was in translation. The formation of a strong stem–loop structure appeared to be responsible for inefficient translation in pgt1⁺ and disruption of these structures in the mutants was probably permitting translation. This was confirmed by making silent mutations in this region of wild‐type pgt1⁺, which led to their functionality in S. cerevisiae. This genetic strategy to relieve functional blocks in expression should greatly facilitate the study of these and other transporters from more intractable genetic organisms in a heterologous expression system. Copyright © 2012 John Wiley & Sons, Ltd.     

Site-directed mutagenesis study of the antibody 2D7 which catalyzes a reaction for insertion of Cu2+ into mesoporphyrin

Monoclonal antibody 2D7 generated against a transition-state analog N-methyl mesoporphyrin catalyzes a reaction for insertion of a cupric ion into mesoporphyrin. To investigate amino acid residues responsible for the catalytic activity, site-directed mutagenesis of the amino acid residues in the third complementarity determining region of the heavy chain (CDRH3) was performed on the antigen-binding fragment (Fab) of the antibody. Recombinant Fab mutants, in which Arg95 is replaced with Ala (R95A), Asp96 with Asn (D96N) and Met97 with Gly (M97G), were examined in terms of the catalytic efficiency of the reaction (k/K(S)) and the dissociation constant for N-methyl mesoporphyrin binding (K(d)) and these values were compared with those of the wild type. The k/K(S) values of the R95A and D96N mutants were 0.96% and 1.0% of that of the wild type, respectively, whereas the M97G mutant had no detectable catalytic activity. The K(d) values of the R95A and D96N mutants were 165 and 69 times that of the wild type, respectively, while that of the M97G mutant was similar to that of the wild type. The relationship between the k/K(S) and 1/K(d) values in the wild type and the R95A and D96N mutants suggests that Arg95 and Asp96 are responsible for stabilizing the transition-state in the catalytic reaction. The results of the M97G mutant allow us to propose that Met97 plays an important role in the catalytic activity probably due to a subtle and specific conformation of the antibody.     

Comparative proteomic analysis of a Candida albicans DSE1 mutant under filamentous and non‐filamentous conditions

Candida albicans is a common opportunistic pathogen that causes a variety of diseases in immunocompromised hosts. In a pathogen, cell wall proteins are important virulence factors. We previously characterized Dse1 as a cell wall protein necessary for virulence and resistance to cell surface‐disrupting agents, such as Calcofluor white, chitin deposition, proper adhesion and biofilm formation. In the absence of decomplexation, our objectives were to investigate differential proteomic expression of a DSE1 mutant strain compared to the wild‐type strain. The strains were grown under filamentous and non‐filamentous conditions. The extracted cell proteome was subjected to tryptic digest, followed by generation of peptide profiles using MALDI–TOF MS. Generated peptide profiles were analysed and unique peaks for each strain and growth condition mined against a Candida database, allowing protein identification. The DSE1 mutant was shown to lack the chitin biosynthesis protein Chs5, explaining the previously observed decrease in chitin biosynthesis. The wild‐type strain expressed Pra1, involved in pH response and zinc acquisition, Atg15, a lipase involved in virulence, and Sod1, required for oxidative stress tolerance, in addition to proteins involved in protein biosynthesis, explaining the increase in total protein content observed compared to the mutants strain. The mutant, on the other hand, expressed glucoamylase 1, a cell wall glycoprotein involved in carbohydrate metabolism cell wall degradation and biofilm formation. As such, MALDI–TOF MS is a reliable technique in identifying mutant‐specific protein expression in C. albicans. Copyright © 2014 John Wiley & Sons, Ltd.     

Asp238→Asn Creates a Novel Consensus N‐Glycosylation Site in Aspergillus awamori Glucoamylase

A single mutation, Asp238→Asn (D238N), of Aspergillus awamori glucoamylase (GA) was identified that increases extracellular production of the enzyme in Saccharomyces cerevisiae at 37 °C. The mutant was isolated as a suppressor of Gly396→Ser (G396S), a previously isolated temperature‐sensitive mutation that decreases the thermostability and extracellular production of GA expressed in S. cerevisiae. Culture supernatants of the double mutant G396S/D238N contained much more GA than supernatants of G396S at 33.5 and 37 °C but not at 30 °C. Additionally, culture supernatants of the D238N contained 1.5 to 2‐fold more GA than supernatants of wild‐type when grown at 37 °C but not at 30 or 33.5 °C. The D238N mutation creates a consensus N‐glycosylation site in GA. Mass spectrometry showed that the molecular weight of D238N was 2319 Da greater than that of the wild‐type GA and that of D238N/G396S was 3094 Da greater than that of G396S, suggesting the presence of an additional N‐linked glycan at residue 238. No difference in thermostability or activity was observed between the G396S and G396S/D238N mutants or between wild‐type and D238N GAs, and D238N did not affect intracellular GA levels at 30 or 37 °C.