Determination of the extracellular and cell-associated hydrolase profiles of Pseudomonas fluorescens sp. using the Analytab API ZYM system

The extracellular and cell-associated hydrolase profiles of a number of Pseudomonas fluorescens strains were examined with the Analytab API ZYM system. Esterase/lipase was the only strong extracellular enzyme activity detected (mean 3.33): weak esterase, lipase, and leucine aminopeptidase activities were found with some strains (mean activities of 1.08, 1.53, and 1.40, respectively). Very strong leucine aminopeptidase activity (4.5) was associated with the cells. Cell-associated trypsin, esterase/lipase, acid phosphatase, and phosphoamidase were also found. Neither extracellular nor cell-associated hydrolase profiles changed significantly when cells were grown in skim milk or mineral salts medium at either 5 or 20 degrees C. Similarly, added calcium did not seem required for synthesis of any of the enzymes. The extracellular enzyme profiles differed considerably from those of the cell-associated enzymes for all strains tested. An extracellular proteinase-deficient mutant of strain 32A (RM14) failed to produce significant quantities of extracellular esterase/lipase activity. Production of cell-associated enzymes was unaffected by the mutation. These results suggest that the Analytab API ZYM system may be useful in identifying psychrotrophs isolated from milk.     

定向进化植物乳杆菌和痤疮丙酸杆菌高产共轭亚油酸

为获得高产共轭亚油酸（conjugated linoleic acid，CLA）的菌株，对亚油酸异构酶系植物乳杆菌的肌球蛋白交叉反应抗原（myosin-cross-reactive antigen，MCRA）基因mcra和痤疮丙酸杆菌的亚油酸异构酶基因lai-p进行克隆，克隆成功后连接到骨架质粒pCold-SUMO上构建两种含mcra基因和lai-p基因的大肠杆菌重组菌株，利用异丙基-β-D-硫代半乳糖苷低温诱导阳性重组菌蛋白表达，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳验证得到目的蛋白表达条带，说明两种基因表达成功。采用易错聚合酶链式反应定向进化mcra基因和lai-p基因，构建重组菌突变体库，利用流式细胞术分选获得吸光度提高和CLA产量提高的重组菌株，其中pCold-ycmcra-gfp重组菌243 株，吸光度最高的为155号菌1.050 6±0.000 4，提高了5.02 倍；pCold-yclai-p-gfp重组菌156 株，产量最高的为39号菌（11.62±0.003 6）μg/mL，提高了2.99 倍。本研究为后期深入了解突变菌株CLA产量提高的原因，并获得两种亚油酸异构酶的产酶机制奠定了一定基础。     

利用易错PCR构建酱油酿造用T酵母spt15突变库

为得到酱油酿造优良性状的微生物,对酱油酿造用T酵母进行研究。首先利用易错聚合酶链式反应（polymerase chain reaction,PCR）技术构建spt15（可编码TATA结合蛋白）的突变基因库,然后重组于表达载体YEplac195中,并导入尿嘧啶营养缺陷型菌株W303和WM2中筛选。结果表明：在不同盐质量分数的固体培养基中经筛选获得4?个最佳突变菌株,其具有耐盐优势,在酱油发酵中可产生较高的氨基酸态氮。经PCR产物测序可知,重组质粒中目的基因的序列有碱基的增添、缺失和替换。     

易错PCR技术改造扩展青霉脂肪酶

利用易错PCR技术,建立脂肪酶基因随机突变文库,将随机突变脂肪酶基因转化毕赤酵母GS115,初步筛选了4000株突变菌株,对300株较优突变株进行酶的活力、耐热性、耐酸性的摇管复筛,进一步摇瓶复筛后获得优良突变株ep3,所产突变脂肪酶(ep3-GS)的适宜pH为9.4,适宜作用温度为35℃,与野生重组脂肪酶(PEL-GS)一致,该温度下酶的比活为3440 U/mg,比野生型脂肪酶提高17%。     

密码子优化及透明颤菌血红蛋白共表达提高耐热脂肪酶在毕赤酵母的表达

根据毕赤酵母密码子的偏好性,通过在线软件对Thermomyces lanuginosus脂肪酶基因（tll）和透明颤菌血红蛋白基因（vhb）进行密码子优化。优化后的脂肪酶基因（tll-opt）和透明颤菌血红蛋白基因（vhb-opt）中碱基GC含量分别由原来的46%提高到49%和42%提高到51%,碱基A、T、C、G均匀分布,减少了AT和GC富集区,利于tll-opt和vhb-opt基因在毕赤酵母X33中的表达。将tll和tll-opt连接到表达载体pPICZαA并转入毕赤酵母X33中。摇瓶培养条件下,含有tll和tll-opt重组工程菌的最大酶活力分别为7 U/mL和16 U/mL。50 L发酵罐培养条件下,含有tll和tll-opt重组工程菌的最大酶活力分别为201 U/mL和430 U/mL。为了进一步提高含tll-opt重组工程菌的表达酶活性,将vhb-opt转入该重组工程菌得到工程菌VHb＋（含有tll-opt和vhb-opt）。重组工程菌VHb＋在摇瓶培养条件下最大酶活力为23 U/mL,分别是优化后基因和原始基因最大表达酶活力的1.39 倍和3.28 倍。重组工程菌VHb＋在50 L发酵罐瓶培养条件下最大酶活力为610 U/mL,分别是优化后基因和原始基因最大表达酶活力的1.41 倍和3.03 倍。     

Isolation and characterization of novel lipases from a metagenomic library of the microbial community in the pitcher fluid of the carnivorous plant Nepenthes hybrida

Members of the genus Nepenthes are carnivorous plants that use the pitfall method of insect capture as a supplementary nutritional source. We extracted metagenomic DNA from the microbial community found in the pitcher fluid of Nepenthes and constructed a plasmid-based metagenomic library. An activity-based screening method enabled the isolation of two lipase genes, lip1 and lip2. Both Lip1 and Lip2 belong to a novel family or subfamily of lipases and show lipase activities in acidic conditions, such as those found in pitcher fluid. This study was conducted under the assumption that the secreted Lip1 and Lip2 were capable of enzymatic activity in the acidic pitcher fluid.     

圆弧青霉BD26碱性脂肪酶基因的克隆及其在大肠杆菌中的表达

采用反转录聚合酶链式反应(RT-PCR),以圆弧青霉BD26总RNA为模板,扩增出774 bp cDNA片段,将该基因片段克隆到表达载体pET-28a(+)上并转化Escherichia coil BL21(DE3),得到重组菌株BL21/ Lip PD。经IPTG诱导,菌体在固体琼脂显色平板上形成明显透明圈。SDS-PAGE电泳显示该脂肪酶分子质量约为27 ku。表达条件优化结果表明,当工程菌培养至OD_(600)为0.5时,加入IPTG至终浓度为1.0 mmol/L,在32℃诱导培养1 h,比酶活达29.30 U/mg。     

一株产耐高温碱性脂肪酶菌株的筛选及产酶条件优化

用Tween平板法从采集自河北省的122份土样中筛选到一株脂肪酶活性较高的菌株Lipa1318。综合其形态学和生理生化特征以及16S r DNA的序列比对结果,确定该菌株为粘质沙雷氏菌（Serratia marcescens）。其所产胞外脂肪酶的最适反应温度为60℃,最适p H为8.0,是一种耐热的碱性脂肪酶。摇瓶发酵实验证明该菌株的最适产酶条件为:葡萄糖1.0%,牛肉膏1.0%,Triton X-100 1.5%,橄榄油0.50%,Ca Cl20.50mmol/L,发酵液初始p H为8.0,30℃培养96h,通过发酵条件的优化使酶活提高了3.6倍。      

微生物脂肪酶资源挖掘研究进展

脂肪酶广泛分布于动物、植物和微生物中,工业化脂肪酶主要来源于微生物。脂肪酶制剂的进一步工业化 应用受限于生产成本和脂肪酶的温度和pH值耐受性、活性、专一性和溶剂耐受性等酶学性质,虽然微生物脂肪酶 基因和蛋白相关资源信息已经非常丰富,但适合食品、药品和能源等工业应用的脂肪酶制剂品种依然较少,研究者 仍然在为新型和理想的脂肪酶制剂资源的挖掘而努力。本文重点综述了微生物脂肪酶资源挖掘的主要研究方法,主 要包括通过环境筛选和宏基因组筛选挖掘新的具有优良特性的脂肪酶;脂肪酶微生物生产菌株的改良、脂肪酶基因 在重组工程菌株中的重组表达和优化、蛋白质工程方法对脂肪酶蛋白进行改造、脂肪酶固定化和催化工艺的改良 等。     

Effect of Bacillus subtilis spo0A mutation on cell wall lytic enzymes and extracellular proteases, and prevention of cell lysis

The Bacillus subtilis spo0A mutant is an adequate host for extracellular protein production (e.g., alpha-amylase). However the mutant was prone to cell lysis. SDS-PAGE and zymography of cell wall lytic proteins indicated that the spo0A mutant contained high amounts of two major autolysins (LytC [CwlB] and LytD [CwlG]) and two minor cell wall lytic enzymes (LytE [CwlF] and LytF [CwlE]). On the other hand, the expression of eight extracellular protease genes was very poor or absent in the spo0A mutant. An eight-extracellular-protease-deficient mutant (Dpr8 strain) was constructed and the strain also exhibited cell lysis. The autolysins from the spo0A mutant were degraded by the supernatant of the wild type but not degraded by that of the Dpr8 mutant. These results suggest that the extensive cell lysis of the spo0A mutant was partially caused by the stability of autolysins via the decrease of the extracellular proteases. The introduction of a major autolysin and/or SigD mutations into the spo0A mutant was effective for preventing cell lysis.     

Identification of a triacylglycerol lipase gene family in Candida deformans: molecular cloning and functional expression

The yeast Candida deformans CBS 2071 produces an extracellular lipase which was shown to catalyse the production of various esters by the esterification of free fatty acids, even in the presence of a large molar excess of water. To clone the gene encoding this extracellular lipase, Saccharomyces cerevisiae was transformed with C. deformans genomic libraries and screened for lipolytic activity on a medium containing rapeseed oil emulsion and rhodamine B. Three members of a lipase gene family (CdLIP1, CdLIP2 and CdLIP3) were cloned and characterized. Each deduced lipase sequence has a Gly-His-Ser-Leu-Gly-(Gly/Ala)-Ala conserved motif, eight cysteine residues and encodes an N-terminal signal sequence. MALDI-TOF mass spectrometry analysis of a proteolytic digest of the lipase produced was used to obtain experimental evidence that the CdLIP1 gene encoded the extracellular lipase. Recombinant expression studies confirmed that the cloned genes encoded functional lipases. The three lipases are very similar to lipases from the related species Yarrowia lipolytica. Significant homologies were also found with several yeast and fungal lipases. As C. deformans CBS 2071 was previously considered to be synonymous with Y. lipolytica, the strains were compared for the extent of nucleotide divergence in the variable regions (D1/D2) at the 5'-end of the large-subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region has diverged sufficiently to suggest that C. deformans is a separate species. The nucleotide sequences of the CdLIP1, CdLIP2 and CdLIP3 genes will appear in the EMBL nucleotide sequence database under Accession Nos AJ428393, AJ428394 and AJ428395, respectively.     

Detection of bacteria using foreign DNA: the development of a bacteriophage reagent for Salmonella

A phage-based reagent was developed for the detection of Salmonella in food samples. The parental phage was Felix 01, which lyses practically all Salmonella. Using data obtained about the molecular biology of the phage, a recombinant phage that carried the bacterial genes specifying luciferase was produced. The method involved the isolation of amber nonsense mutations and subsequent crosses to render doubly mutant phage with a very low reversion rate on strains lacking an amber suppressor. A plasmid was constructed that contained a segment of Felix 01 DNA with two adjacent genes, one dispensable and the other essential, and their flanking sequences. Recombinant DNA technology was used to remove the two genes and the luxA and luxB genes for luciferase, and a gene specifying a tRNA that recognizes amber codons (supF=tyrT) was put in their stead. This region could be transferred into the genome of the phage by homologous recombination. The recombinant phage cannot grow because it lacks an essential gene. However, it can grow in a host that synthesizes the missing protein. This technique allows the construction of "locked" recombinant phages that carry foreign DNA but which cannot propagate themselves in nature.     

Purification and partial characterization of psychrotrophic Serratia marcescens lipase

Serratia marcescens isolated from raw milk was found to produce extracellular lipase. The growth of this organism could contribute to flavor defects in milk and dairy products. Serratia marcescens was streaked onto spirit blue agar medium, and lipolytic activity was detected after 6 h at 30 degrees C and after 12 h at 6 degrees C. The extracellular crude lipase was collected after inoculation of the organism into nutrient broth and then into skim milk. The crude lipase was purified to homogeneity by ion-exchange chromatography and gel filtration. The purified lipase had a final recovered activity of 45.42%. Its molecular mass was estimated by SDS-PAGE assay to be 52 kDa. The purified lipase was characterized; the optimum pH was likely between 8 and 9 and showed about 70% of its activity at pH 6.6. The enzyme was very stable at pH 8 and lost about 30% of its activity after holding for 24 h at 4 degrees C in buffer of pH 6.6. The optimum temperature was observed at 37 degrees C and exhibited high activity at 5 degrees C. The thermal inactivation of S. marcescens lipase was more obvious at 80 degrees C; it retained about 15% of its original activity at 80 degrees C and was completely inactivated after heating at 90 degrees C for 5 min. Under optimum conditions, activity of the enzyme was maximum after 6 min. The Michaelis-Menten constant was 1.35 mM on tributyrin. The enzyme was inhibited by a concentration more than 6.25mM. Purified lipase was not as heat-stable as other lipases from psychrotrophs, but it retained high activity at 5 degrees C. At pH 6.6, the pH of milk, purified lipase showed some activity and stability. Also, the organism demonstrated lipolytic activity at 6 degrees C after 12 h. Therefore, S. marcescens and its lipase were considered to cause flavor impairment during cold storage of milk and dairy products.     

The effects of environmental conditions on the lipolytic activity of strains of Penicillium roqueforti

The lipolytic activity of 30 strains of Penicillium roqueforti was investigated by agar diffusion tests on tributyrin (esterase activity) and olive oil agar (lipase activity), by titration of the free fatty acids (FFA) produced and by gas chromatographic analysis of the individual FFA released after growth at 25 or 10 degrees C in butterfat emulsions containing 0, 2 or 7% NaCl. All strains investigated by the agar diffusion tests possessed esterase activity and 23 strains were also able to hydrolyse olive oil, but differences in esterase activity were seen. The agar diffusion tests and the titration of FFA showed that the amount of FFA released by a strain of P. roqueforti is determined by both esterase and lipase activity. A large release of FFA was only seen for strains with the ability to hydrolyse both short- and long-chained fatty acids, while strains with esterase activity produced smaller amounts of FFA. Between 7 and 14 days of incubation a steep increase in the release of FFA was observed both by the titration and by GC analysis, and then a decline from 14 to 21 days, probably caused by conversion of FFA to methyl ketones. Identical FFA profiles were found for two strains with different lipolytic activity. Long-chained fatty acids dominated the profile, while the short-chained fatty acids only were detected in small amounts and mainly in the end of incubation. Both strains were stimulated by NaCl in the emulsions.     

耐受性枯草芽孢杆菌的脉冲强光诱变筛选及产酶活力分析

采用脉冲强光对枯草芽孢杆菌进行诱变处理,通过耐受性测试,在抗性菌株中筛选出优良耐受菌株,并与原始菌株对比分析产酶活力及遗传稳定性,以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳比较分析菌体蛋白质表达的差异。结果表明,在脉冲电压2?450?V,照射距离5?cm,脉冲40?次条件下,筛选出8?株抗性菌株;经初筛和复筛得到兼具高温耐受、强酸耐受和高浓度胆盐耐受性变异菌株B3和B7,产α-淀粉酶、蛋白酶、脂肪酶活力分别比原始菌株提高了67%和77%（P＜0.01）、56%和71%（P＜0.01）、34%和42%（P＜0.05）,变异菌株产酶稳定性较高（P＞0.05）,可遗传变异;诱变前后菌体蛋白表达出现差异;结果证明脉冲强光用于枯草芽孢杆菌的诱变具有可行性。     

Role of repetitive nine-residue sequence motifs in secretion, enzymatic activity, and protein conformation of a family I.3 lipase

A family I.3 lipase from Pseudomonas sp. MIS38 (PML) contains 12 repeats of a nine-residue sequence motif in the C-terminal region. To elucidate the role of these repetitive sequences, mutant proteins PML5, PML4, PML1, and PML0, in which 7, 8, 11, and all 12 of the repetitive sequences are deleted, and PMLdelta19, in which 19 C-terminal residues are truncated, were constructed. Escherichia coli DH5 cells carrying the Serratia marcescens Lip system permitted the secretion of the wild-type and all of the mutant proteins except for PMLdelta19, although they were partially accumulated in the cells in an insoluble form as well. Both the secretion level and cellular content of the proteins decreased in the order PML > PML5 > PML4 > PML1 > PML0, indicating that repetitive sequences are not required for secretion of PML but are important for its stability in the cells. All the mutant proteins were purified in a refolded form and their biochemical properties were characterized. CD spectra, the Ca2+ contents, and susceptibility to chymotryptic digestion strongly suggested that the five repetitive sequences remaining in PML5 are sufficient to form a beta-roll structure, whereas the four in PML4 are not. PML5 and PMLdelta19 showed both lipase and esterase activities, whereas PML4, PML1, and PML0 were inactive. These results suggest that the enzymatic activity of PML is not seriously affected by a deletion or truncation at the C-terminal region as long as a succession of repetitive sequences can build a beta-roll structure.     

Molecular typing of industrial strains of Pseudomonas spp. isolated from milk and genetical and biochemical characterization of an extracellular protease produced by one of them

P. fluorescens is responsible for the highest depredation of milk because of its capacity to synthesize extracellular lipase and protease which hydrolyze milk fat and proteins. Several P. fluorescens synthesize an extracellular caseinolytic metalloprotease, called AprX. It is important to rapidly detect the presence of a contamination of raw milk by a strain, especially a P. fluorescens strain, having a high potential of depredation. If standard plate count procedures are often employed, they are time consuming and do not permit to rapidly evaluate the potential of depredation. An alternative method consists to search the aprX gene, but such a method remains of low sensitivity and does not allow evaluating the real potential of depredation of the contaminant. After a milk depredation event, three strains of Pseudomonas spp. (F, 2312 and 2313) have been isolated from a dairy plant. Using molecular and phenotypic approaches, these strains were identified as P. fluorescens strains. Their respective extracellular caseinolytic potential was characterized as well as that of several collection strains of P. fluorescens. It appeared that these strains secreted one protease of about 45 kDa, that their extracellular caseinolytic potential was highly variable for one strain to another and that the one of strain F was the highest. The protease secreted by the strain F was purified and its N-terminal sequence established. It shared 100% identity with the domain 14-34 of extracellular alkaline endoprotease sequences which are called AprX for some of them. Its gene was sequenced as well as that of two collection strains of P. fluorescens having a significant lower extracellular caseinolytic potential. The genomic environment of the aprX gene as well as its expression during the strain growth was investigated. It appears that the difference of extracellular caseinolytic potential which has been observed between the three strains does not mainly result from the AprX sequence/structure but it might rather result from the aprX level of expression.     

Enhanced production of prodigiosin-like pigment from Serratia marcescens SMdeltaR by medium improvement and oil-supplementation strategies

Serratia marcescens SMdeltaR, an SpnR-defective isogenic mutant of S. marcescens SS-1, was used to produce a prodigiosin-like pigment (PLP). Luria-Bertani (LB) broth, frequently used for prodigiosin biosynthesis with S. marcescens strains, was modified by increasing the concentrations of tryptone and yeast extract while completely removing NaCl from the medium. The resulting modified LB (MLB) medium achieved an almost 3.0-fold increase in PLP yield (152 mg l(-1)) when compared with the original LB broth. The addition of vegetable oils (2-6% [v/v]) to the fermentation broth markedly enhanced PLP production. PLP yields of 525, 579, and 790 mg l(-1) were obtained when the MLB medium was supplemented with 4% soybean oil, 4% olive oil and 6% sunflower oil, respectively. PLP production was found to be positively correlated with extracellular surface emulsification activity, suggesting a link between the PLP production and the presence of biosurfactant. This work shows that the optimal medium for PLP yield was sunflower oil (6%)-supplemented MLB medium, which resulted in an approximately 14-fold higher PLP yield than that in LB broth. Mass spectrometry and NMR analysis indicated that the PLP product is a prodigiosin derivative, called undecylprodigiosin.