Directed evolution of an aminoalcohol dehydrogenase for efficient production of double chiral aminoalcohols

The aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154, which can be used as a catalyst for the stereoselective reduction of (S)-1-phenyl-1-keto-2-methylaminopropane to d-pseudoephedrine (dPE), is inhibited by the accumulation of dPE in the reaction mixture, limiting the yield of dPE. To improve this weak point of the enzyme, random mutations were introduced into aadh, and a mutant enzyme library was constructed. The mutant library was screened with a color detectable high-throughput screening method to obtain the evolved enzymes showing the activity in the presence of a high concentration of dPE. Two mutant enzymes showed higher tolerability to dPE than the wild type enzyme. Each of these enzymes had a single amino acid substitution in a different position (G73S and S214R), and a third mutant enzyme carrying both of these amino acid substitutions was constructed. Escherichia coli transformant cells, which express mutant AADHs, showed activity in the presence of 100mg/ml dPE. A kinetic parameter analysis of the wild type and mutant enzymes was carried out. As compared with the wild type enzyme, the mutant enzymes carrying the S214R amino acid substitution or both the S214R and G73S substitutions showed higher k(cat) values, and the mutant enzymes carrying the G73S amino acid substitution or both the G73S and S214R substitutions showed higher K(m) values. These results suggest that the Ser214 residue plays an important role in enzyme activity, and that the Gly73 residue participates in enzyme-substrate binding.     

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.     

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.     

Replacement of His12 or His119 of bovine pancreatic ribonuclease A with acidic amino acid residues for the modification of activity and stability

In an attempt to produce a bovine pancreatic ribonuclease A (RNase A) with increased activity and stability, the catalytic pair of His12 and His119 was substituted with aspartic acid or glutamic acid, and aspartic acid, respectively, to evaluate the role of the two histidine residues in the activity and stability. Kinetic analysis revealed that k(cat)/K(m) values were significantly reduced for all mutant enzymes due to a decreased k(cat) rather than an increased K(m): the k(cat) values for both CpA and C>p of H12D and H12E decreased to about 1/1000; the k(cat) values of H119D decreased by 1/3300 for CpA and 1/80 for C>p. Thus, neither Asp nor Glu is able to act solely as an efficient catalytic residue of RNase A. Alkylation with iodoacetic acid (IAA) revealed that mutant enzymes had reduced reaction rates and that no modification was evident at Glu12 and Asp12 of H12E and H12D, respectively. This indicates that the low catalytic activity of mutant enzymes could be due to low basicity of Asp12 and Glu12. While the T(m) of H119D was almost the same as that of the wild-type enzyme, the T(m) of both H12D and H12E markedly decreased. It became apparent that His12 located at the bottom of the active site cleft contributes significantly to the structural stability of RNase A.     

Improvements in the cell-free production of functional antibodies using cell extract from protease-deficient Escherichia coli mutant

Expression of a functional antibody fragment (Fab) using an Escherichia coli cell-free expression system has been reported previously [Jiang et al., FEBS Lett., 514, 290-294 (2002)]. The low yield of the synthesized antibody, however, limits the usefulness of the cell-free expression system, partly due to the degradation of product by endogenous proteases from the E. coli extract. To determine which proteases are responsible for the degradation, we compared the expression of a 6D9 Fab fragment under conditions whereby several protease inhibitors were added into the cell-free system. The addition of serine protease inhibitor increased the amount of the Fab fragment, indicating that serine proteases caused the antibody degradation. Therefore, several serine protease-deficient mutants of E. coli BW25113 were constructed by targeted homologous recombination. The use of extract from a double protease-deficient mutant (DeltadegP-ompT) significantly increased the amount and antigen-binding activities of an anti-HSA scFv and a 6D9 Fab fragment. These results suggest that the DegP- and OmpT-deleted mutant is a useful source of S30 extract for the production or screening of antibodies using the cell-free expression system.     

Development and validation of an HPLC-based screening method to acquire polyhydroxyalkanoate synthase mutants with altered substrate specificity

A rapid and convenient method for the compositional analysis of polyhydroxyalkanoate (PHA) was developed using high-performance liquid chromatography (HPLC) and alkaline sample pretreatment in a 96-well plate format. The reliability of this system was confirmed by the fact that a mutant with a D171G mutation of Aeromonas caviae PHA synthase (PhaC(Ac)), which gained higher reactivity toward 3-hydroxyhexanoate (3HHx), was selected from the D171X mutant library. Together with D171G mutant, several single mutants showing high reactivity toward 3HHx were isolated by the HPLC assay. These new mutants and double mutants combined with an N149S mutation were used to synthesize P(3-hydroxybutyrate-co-3HHx) in Ralstonia eutropha PHB(-)4 from soybean oil as carbon source, achieving higher levels of 3HHx fraction than the wild-type enzyme. Based on these results, the high-throughput screening system will serve as a powerful tool for exploring new and beneficial mutations responsible for regulating copolymer composition of PHA.     

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.     

新型天冬氨酸激酶突变株构建及酶学性质表征

通过定点随机突变技术,提高天冬氨酸代谢途径中首个关键限速酶天冬氨酸激酶(aspartate kinase,AK)的催化活力,减弱或解除代谢产物对其协同反馈抑制,并通过Discovery Studio等软件对其空间结构进行分析,借助分子动力学模拟对其机制进行分析.首先选择ATP附近关键残基位点,在前期T379N/A380...     

普通烟草长叶柄突变性状的遗传分析

叶柄是烟草叶片的重要组成部分,烟草长叶柄性状遗传分析可用于研究烟草叶柄和叶片的发育。利用甲基磺酸乙酯（EMS）诱变普通烟草烤烟品种中烟100和红花大金元各获得一个（极）长叶柄突变体（M48和M50）,将两个突变体分别与各自的野生型中烟100和红花大金元及普通烟草烤烟品种革新三号杂交获得F₁,F₁自交获得F₂群体,F₁与其相应的野生型亲本回交获得BC₁F₁群体。对各类型材料进行表型调查和统计分析结果显示:F₁均表现为（极）长叶柄突变表型,在F₂群体和BC₁F₁群体中,经x2检验,（极）长叶柄突变表型与野生型表型的理论分离比均符合3:1和1:1,表明（极）长叶柄突变性状由染色体上一对显性单基因控制。上述结果为进一步定位和克隆（极）长叶柄突变基因,揭示其在烟草叶柄和叶片发育中的作用奠定基础。      

蛋白质工程改造磷脂酶D提高磷脂酰丝氨酸产量

为了进一步提高磷脂酰丝氨酸(PS)产量,将来源于Streptomyces katrae的野生型磷脂酶D(SkPLD)于E.coli BL21(DE3)中进行异源表达,获得菌株E.coli BL21(DE3)/pET28a-SkPLD,以磷脂酰胆碱(PC)和L-serine为底物,发现限制PS产量进一步提高的限制因素是转磷脂酰反应转化率低(<35%)而水解反应转化率过高(>30%)。在对SKPLD结构进行详尽分析的基础上,通过蛋白质工程改造策略扩大底物催化通道,降低位阻效应;同时提高His462与L-serine的亲和力,降低水解反应。结果表明,获得4个有益单突变体(Y405A、Q407G、D370P、K478P),对其进行组合突变,获得四突变体SkPLDY405A/Q407G/D370P/K478P,转化率、PS产量、k_cat/K_m分别为55.6%、42.3 g·L-1、1.53(mmol/L)-1s-1,比野生型分别提高了59.8%、59.6%、93.7%。PS产量进一步提高的原因在于四突变体催化腔体积扩大到718.6 ?3,且His462 N原子与L-serine O原子的催化距离(d1)缩短0.6 ?,与H₂O O原子的距离(d2)增加0.8 ?。在3 L规模转化体系中,PS产量为50.4 g·L-1、转化率达到66.3%,时空产率为12.6 g/L/h。本研究利用蛋白质工程改造获得了转磷脂酰活性提高的突变体,为提高磷脂酶D的工业生产奠定了坚实的基础。     

Conversion of a typical catalase from Bacillus sp. TE124 to a catalase-peroxidase by directed evolution

We have converted a typical catalase from Bacillus sp. TE124 to a catalase-peroxidase using DNA shuffling and error-prone PCR. A triple mutant, R47H/R356C/D374N, that showed significantly reduced catalase activity and increased peroxidase activity was identified by screening mutant libraries. When single mutant--R47H, R356C and D374N--were generated by site-directed mutagenesis, conserved Arg-47, located on the distal side of the prosthetic heme group in the superfamily of typical catalases, was found to be responsible for the conversion of catalase to catalase-peroxidase. To further clarify the role of Arg-47, arginine was replaced with different amino acids--alanine, lysine, aspartic acid, glutamic acid, glutamine, phenylalanine, tryptophan and tyrosine--and the mutant enzymes were assayed. All of the arginine mutants had increased peroxidase activity coupled with reduced catalase activity. Among these mutants, R47W exhibited the highest peroxidase activity, while R47E and R47Q not only had increased peroxidase activity but also retained relatively high catalase activity. These results suggest that tryptophan plays a key role in the catalytic mechanism of the peroxidase reaction and that glutamic acid and glutamine facilitate both catalatic and peroxidatic reactions.     

LITERATURE ABSTRACTS

GENERAL PRINCIPLES: Preparation and Functional Properties of Rapeseed Protein Products. E.H. Mansour, J. Perédi and E. Dworschák GENERAL PRINCIPLES: Determination of the Molecular Mass of Polygalacturonic Acid. Gy. Mady, B. Lakatos, J.J. Kever and N.V.D. Yakonova GENERAL PRINCIPLES: Crystallization Kinetics of Emulsified Triglycerides. S. Özilgen, C. Simoneau, J.B. German, M.J. McCarthy and D.S. Reid GENERAL PRINCIPLES: The Role of Phytase and Lignin in Decorticated Dry Bean (Phaseolus vulgaris) Hardening During Storage. M.M. Mafuleka, D.N. Ott, G.L. Hosfield and M.A. Uebersax GENERAL PRINCIPLES: Effect of Flour Quality Characteristics on Puff Pastry Baking Performance. R.L. Hay BASIC PRINCIPLES: Effects of pH On Gelaion and Emulsification of a β-Lactoglobulin Concentrate. P. Reikin, T. Liu and B. Launay METHODOLOGY AND INSTRUMENTATION: Analysis of the Helical Screw Rheometer for Fluid Food. M.S. Tamura, J.M. Henderson, R.L. Powell and C.F. Shoemaker METHODOLOGY AND INSTRUMENTATION: Stress Relaxation Test Conditions for Meat Products to Measure Viscoelasticity. G.S. Mittal, M. Zhang and S. Barbut METHODOLOGY AND INSTRUMENTATION: Impact Damage on Rough Rice. A.D. Sharma, O.R. Kunze and N.N. Sarker METHODOLOGY AND INSTRUMENTATION: Frequency Factor—Activation Energy Compensation Relations for Viscosity of Fruit Juices. M. Ozilgen and L. Bayindirli METHODOLOGY AND INSTRUMENTATION: Dynamic Measurement of Starch Granule Swelling During Gelatinization. G.R. Ziegler, D.B. Thompson and J. Casasnovas METHODOLOGY AND INSTRUMENTATION: Development of Two Instrumental Methods for Corn Masa Texture Evaluation. B. Ramirez-Wong, V.E. Sweat, P.I. Torres and L.W. Rooney INSTRUMENTAL MEASUREMENTS: Rheology of Bengal Gram (Cicer arientinum) Flour Suspensions. S. Bhattacharya, K.K. Bhat and K.G. Raghuveer INSTRUMENTAL MEASUREMENTS: Residence Time Distribution of Food Suspensions Containing Large Particles When Flowing in Tubular Systems. K. Palmieri, D. Cacace, G. Dipollina, G. Dall'Anglio and P. Masi SENSORY ASSESSMENT: Understanding Mouthfeel Attributes: A Multidimensional Scaling Aproach. M. Bertino and H.T. Lawless SENSORY ASSESSMENT: Effect of Homogenization and Fat Content on Oral Perception of Low and High Viscosity Model Creams. N.J. Richardson, D.A. Booth and N.L. Stanley SENSORY ASSESSMENT: Discrimination of Texture and Appearance in Cheddar Cheese Using Consumer Free-Choice Profiling. F.R. Jack, J.R. Piggott and A. Paterson SENSORY ASSESSMENT: Influence of Oat Gum, Guar Gum and Carboxymethyl Cellulose on the Perception of Sweetness and Flavour. Y. Mälkki, R.-L. Heiniö and K. Autio FACTORS AFFECTING TEXTURE: Physical, Chemical, and Dry-Milling Properties of Corn of Varying Density and Breakage Susceptibility. A.J. Peplinski, M.R. Paulsen and A Bouzaher FACTORS AFFECTING TEXTURE: Frozen Dought: Rheological Changes and Yeast Viability. K. Autio and E. Sinda FACTORS AFFECTING TEXTURE: Modified Atmosphere Storage of Peaches and Nectarines to Reduce Storage Disorders. S. Lurie FACTORS AFFECTING TEXTURE: Peach Firmness During Storage as Affected by Three Irrigation Schedules. X. Zhang, G.H. Brusewitz, S.M. Huslig and M.W. Smith FACTORS AFFECTING TEXTURE: Effect of Hot Processing on the Properties of Restructured Beef with Alginate/Calcium Binders. M.A. Al-Joher and A.D. Clarke FACTORS AFFECTING TEXTURE: Cohesion and Hardness of Extrusion-Cooked Mechanically- and Hand-Deboned Poultry Meat with Soy Protein Isolate and Kappa-Carrageenan. A.S. Ba-Jaber, J.N. Sofos, G.R. Schmidt and J.A. Maga FACTORS AFFECTING TEXTURE: Effect of Squid Liver Extract on Proteins and Texture of Cooked Squid Mantle. I. Kolodziejska, J. Pacana and Z.E. Sikorski FACTORS AFFECTING TEXTURE: Bacterial Cellulose. IV. Application to Processed Foods. A. Okiyama, M. Motoki and S. Yamanaka FACTORS AFFECTING TEXTURE: Physical and Chemical Evaluation of Peanut Butter During Storage. N.M. El-SHimi FACTORS AFFECTING TEXTURE: Effect of Emulsifiers on the Properties of Pasta Products. E. Kovács, É Vámos-Kardos, M. Kiss-Laszlavik and E. Pallagi FACTORS AFFECTING TEXTURE: Biochemical, Rheological, Cooking Quality and Acceptability of Defatted Soy-Supplemented Whole Durum Meal Noodles. S.A. Taha FACTORS AFFECTING TEXTURE: Physical Properties of Wheat Flour Extrudates Produced with Shortening and Starch Plus Several Baking Ingredients. G.H. Ryu and C.E. Walker FACTORS AFFECTING TEXTURE: Analytical Model for Plastic Impact of Fruit on Thin Plate. S. Gan-Mor and A. Mizrach FACTORS AFFECTING TEXTURE: Gel-Strength in Restructured Potato Products Affects Oil Uptake During Deep-Fat Frying. E.J. Pinthus, P. Weinberg and I.S. Saguy FACTORS AFFECTING TEXTURE: Texturization of Sweetened Mango Pulp: Optimization Using Response Surface Methodology. C. Mouquet, J-C Dumas and S. Guilbert FACTORS AFFECTING TEXTURE: Effect of Dextrans of Differing Molecular Weights on the Rheology of Wheat Flour Doughs and the Quality Characteristics of Pan and Arabic Breads. A.S. Ross, G.J. McMaster, J.D. Tomlinson and N.W.H. Cheetham FACTORS AFFECTING TEXTURE: Changes in Rheological Properties of Hydroxypropyl Potato Starch Pastes during Freeze–Thaw Treatments. III. Effects of Cooking Conditions and Concentration of the Starch Paste. H.R. Kim, P. Muhrbeck and A.-C. Eliasson FACTORS AFFECTING TEXTURE: Functional Properties of the Starchy Flour Extracted from Cassava on Fermentation with a Mixed Culture Inoculum. S.N. Moorthy, M. George and G. Padmaja FACTORS AFFECTING TEXTURE: Effect of Sorghum Flour Addition on the Characteristics of Wheat Flour Tortillas. P.I. Torres, B. Ramirez-Wong, S.O. Serna-Saldivar and L.W. Rooney FACTORS AFFECTING TEXTURE: Effects of Cooking Water Composition on Stickiness and Cooking Loss of Spaghetti. L.J. Malcolmson and R.R. Matsuo FACTORS AFFECTING TEXTURE: Effects of Emulsifiers and Hydrocolloids on Whole Wheat Bread Quality: A Response Surface Methodology Study. E. Mettler and W. Siebel     

INFLUENCE OF G187W/K188F/D189Y MUTATION IN THE SUBSTRATE BINDING POCKET OF TRYPSIN ON β-CASEIN PROCESSING

Undertaking to modulate the catalytic properties of trypsin, highly conserved G187, K188 and D189 were replaced with aromatic amino acid residues in order to perturb the electrostatics and to amplify hydrophobic interactions of the substrate binding site. The kinetic parameters of the wild-type trypsin and G187W/K188F/D189Y mutant were determined with synthetic tetrapeptide substrates and β-casein at different pHs. Compared with trypsin, the mutant G187W/K188F/D189Y exhibits 1.3-fold increase in K_m values for the substrates containing arginine and lysine. This mutant shows a 30- to 40-fold decrease of its kcat and its second-order rate constant k_cm/k_m decreases = 40- and 55-fold for substrates containing arginine and lysine, respectively. The kinetic analysis reveals that the mutant conserves the native trypsin capacity to hydrolyze peptide bonds containing arginyl and lysyl residues. Surprisingly, as demonstrated only by proteolysis of a natural substrate (β-casein), the mutant cleaves also peptide bonds involving asparagine and glutamine.     

近平滑假丝酵母ATCC7330羰基还原酶CpCR突变体酶的稳定性研究

通过蛋白质理性设计和定点突变技术,在近平滑假丝酵母ATCC 7330羰基还原酶wtCpCR的基础上,构建5个突变体酶A98N、S307N、G262N、S216N和S258N,考察了有机溶剂、温度、剪切力、氧气、辅酶NADPH浓度对突变体酶的稳定性影响.研究表明,A98 N在磷酸盐(potassium phosphate...     

Enhancement of thermostability of kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047 by random mutagenesis

Random mutation by error-prone PCR was introduced into kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047. One thermostable mutant enzyme, D513N, was isolated. The D513N mutant enzyme showed an optimum temperature of 67.5-70 degrees C (the wild type, 65 degrees C), and thermostability up to 67.5 degrees C (the wild type, up to 60 degrees C). The half-lives of D513N were estimated to be 135 h at 60 degrees C, 110 min at 70 degrees C and 6 min at 75 degrees C, respectively. They were about 1.6-fold, 7-fold and 6-fold longer than those of the wild-type enzyme, respectively.     

Growth stimulation of a proteinase positive Lactococcus lactis strain by a proteinase negative Lactococcus lactis strain

Lactococcus lactis AMP15/pAMP31(D471R) is a proteinase negative, lactose negative strain with a modified oligopeptide transport system, and potential as a debittering agent due to its efficient utilization of hydrophobic peptides. Five wild L. lactis strains of dairy origin, which produced cheeses of high flavour quality, were cocultured with L. lactis AMP15/pAMP31(D471R) in an attempt to select adequate combinations of strains for use as defined cheese starters with potential debittering ability. Four of these strains, L. lactis B6, K16, M21 and P21, inhibited growth of L. lactis AMP15/pAMP31(D471R) at a level of 10(6) to 10(7) cfu mL(-1) after 24 h of incubation, even though production of bacteriocin-like compounds could only be proven for L. lactis M21. When L. lactis AMP15/pAMP31(D471R) was cocultured with the fifth strain, L. lactis N22, its growth was significantly (P<0.001) inhibited whereas growth of L. lactis N22 was significantly stimulated. The nature of the interaction was studied and it was established that L. lactis N22 is auxotrophic for folate, a compound produced and excreted by L. lactis AMP15/pAMP31(D471R).     

LITERATURE ABSTRACTS

FACTORS AFFECTING TEXTURE: Breadmaking Quality of Ten Greek Breadwheats-Baking and Storage Tests on Bread Made by Long Fermentation and Activated (Chemical) Dough Development Processes, and the Effects of Bug-damaged Wheat. N. P. Matsoukas and W. R. Morrison GENERAL PRINCIPLES: Viscoelastic Properties of Whey Protein Gels: Mechanical Model and Effects of Protein Concentration on Creep. K. Katsuta and D. Rector and J. E. Kinsella GENERAL PRINCIPLES: Development of a Test to Predict Gelation Properties of Raw Turkey Muscle Proteins. E. A. Foegeding GENERAL PRINCIPLES: Rheology of the Breadmaking Process. A. H. Bloksma GENERAL PRINCIPLES: Influence of Temperature on the Viscous Behavior of Some Concentrated Dispersions. D. C. Lee and S. N. Bhatacharya INSTRUMENTATION AND METHODOLOGY: Detection of Firmness in Peaches by Impact Force Response. F. I. Meredith, R. G. Leffler and C. E. Lyon INSTRUMENTATION AND METHODOLOGY: Determination of Rheological Behavior of Tomato Concentrates Using Back Extrusion. (Res. Note) M.S.B. Alviar and D. S. Reid INSTRUMENTAL MEASUREMENTS: Characterization of a Pectin from Sunflower Heads Residues. M. L. Alarcão e Silva SENSORY ASSESSMENT: Temporal Aspects of Hedonic Responses. D. E. Taylor and R. M. Pangborn FACTORS AFFECTING TEXTURE: The Effect of Denaturation of β-Lactoglobulin on Renneting–A Quantitaative Study. D. G. Dalgliesh FACTORS AFFECTING TEXTURE: Control of Muscle Softening and Protease-Parasite Interactions in Arrow-tooth Atheresthes stomias. (Res. Note) D. H. Greene and J. K. Babbit FACTORS AFFECTING TEXTURE: Effects of Connective Tissue on Algin Restructured Beef. S. A. Ensor, J. N. Sofos and G. R. Schmidt FACTORS AFFECTING TEXTURE: Restructuring of Mechanically Deboned Chicken and Nonmeat Binders in a Twin-screw Extruder. V. B. Alvarez, D. M. Smith, R. G. Morgan and A. M. Booren FACTORS AFFECTING TEXTURE: Relationships Between Physicochemical Properties of Nonfish Protein and Textural Properties of Protein-incorporated Surimi Gel. K. H. Chung and C. M. Lee FACTORS AFFECTING TEXTURE: Strength of Gels Prepared from Washed and Unwashed Minces of Hoki (Macrunonus novaezelandiae) Stored in Ice. G. A. MacDonald, J. Lelievre and N.D.C. Wilson FACTORS AFFECTING TEXTURE: Textural Degradation of Cooked Fish Meat Gel (Kamaboko) by the Addition of an Edible Mushroom, Judas' Ear [Auricularia auriculajudae (Fr. ) Quel]. Y. Makinodan and M. Hujita FACTORS AFFECTING TEXTURE: Influence of Pasteurization Time and Temperature on the Rheology and Sensory Properties of a Type of Gazpacho. L. Jimenez and A. Lopez FACTORS AFFECTING TEXTURE: Investigation of the Relationship Between Wheat Lipids and Baking Properties. E. M. Kárpáti, F. Békés, R. Lásztity, F. Örsi and I. Smied and A. Mosonyi FACTORS AFFECTING TEXTURE: Prediction of Mechanical Dough Development, Water Absorption and Baking Performance from Farinograph Parameters. T. D. Atkins and N. G. Larsen     

基于定向进化技术提高地衣芽孢杆菌L-天冬酰胺酶活性

为提高地衣芽孢杆菌L-天冬酰胺酶活性,通过定向进化技术对其进行分子改造。经过两轮易错聚合酶链式反应和一轮DNA shuffling,从19 100多个突变株中筛选到突变体S10、S16和S21,其酶比活力较野生型分别提高了106%、74%和43%,且突变酶K_(cat)/K_m都有所增大。其中,突变体S10氨基酸序列发生3个突变,K43E、N67S和I269L。三维模拟结果显示,第43位氨基酸突变为谷氨酸、第67位氨基酸突变为丝氨酸,可能提高了底物亲和力和催化效率,从而提高酶活性。圆二色谱分析表明,相比野生酶,突变酶的α-螺旋数减少、无规则卷曲有所增加,表明其刚性略有降低,柔性有所增加。利用定向进化策略能够有效地提高地衣芽孢杆菌L-天冬酰胺酶活性。