Bioinformatic characterization of the extracellular lipases from Kluyveromyces marxianus

Lipases are hydrolytic enzymes that break the ester bonds of triglycerides, generating free fatty acids and glycerol. Extracellular lipase activity has been reported for the nonconventional yeast Kluyveromyces marxianus, grown in olive oil as a substrate, and the presence of at least eight putative lipases has been detected in its genome. However, to date, there is no experimental evidence on the physiological role of the putative lipases nor their structural and catalytic properties. In this study, a bioinformatic analysis of the genes of the putative lipases from K. marxianus L-2029 was performed, particularly identifying and characterizing the extracellular expected enzymes, due to their biotechnological relevance. The amino acid sequence of 10 putative lipases, obtained by in silico translation, ranged between 389 and 773 amino acids. Two of the analysed putative proteins showed a signal peptide, 25 and 33 amino acids long for KmYJR107Wp and KmLIP3p, and a molecular weight of 44.53 and 58.23 kDa, respectively. The amino acid alignment of KmLIP3p and KmYJR107Wp with the crystallized lipases from a patatin and the YlLip2 lipase from Yarrowia lipolytica, respectively, revealed the presence of the hydrolase characteristic motifs. From the 3D models of putative extracellular K. marxianus L-2029 lipases, the conserved pentapeptide of each was determined, being GTSMG for KmLIP3p and GHSLG for KmYJR107Wp; besides, the genes of these two enzymes (LIP3 and YJR107W) are apparently regulated by oleate response elements. The phylogenetic analysis of all K. marxianus lipases revealed evolutionary affinities with lipases from abH15.03, abH23.01, and abH23.02 families.     

Application of multiple sensory evaluations to produce fermented beverages made from sole whey using Kluyveromyces marxianus

Various fermented whey-based beverages have been developed to reduce waste. However, approaches to increase the preference of whey-based beverages are required because of the low sensory acceptability of whey. Here, we identified the better starting material (whey type), between raw whey (RW) and demineralised whey (DMW), and determined the optimal initial concentration using multiple sensory evaluations to develop acceptable fermented beverages made from sole whey with pure cultured Kluyveromyces marxianus (i.e. without additional ingredients and processing methods). Acceptance tests showed that fermented beverages made from DMW were superior to RW as the starting material. The amounts of ethanol produced were 5.0%, 7.6% and 9.5% v/v from the different initial DMW concentrations of 10%, 15% and 20% w/v, respectively. We observed a significant positive correlation (r_s = 0.32, P < 0.05) between the assessment attributes, strength of taste as alcohol beverage and overall acceptability, indicating that higher concentrations of DMW yielded a more desirable product.     

The terminal protein of the linear DNA plasmid pGKL2 shares an N-terminal domain of the plasmid-encoded DNA polymerase

The 36K protein attached at the 5′ end of the linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis was first purified and characterized. The terminal protein was purified from cells (1 kg wet weight) by ammonium sulphate precipitation and two rounds of centrifugation to equilibrium in CsCl gradients. The pGKL2 was present only in the post-microsomal supernatant. Approximately 10 mg of the purified pGKL2 was recovered and digested with DNase I. The terminal protein (final ca. 0·8 mg) was homogeneous by electrophoresis and we determined the N-terminal amino acid sequence up to ten residues, showing that it existed in the cryptic N-terminal domain of pGKL2-ORF2 (DNA polymerase) sequence.     

超声波协同马克思克鲁维酵母Z17胞内粗酶处理降低乳清蛋白的致敏性

以超声预处理过的乳清蛋白为酶解底物,采用OPA法、ELISA分析等手段,探究马克思克鲁维酵母Z17粗酶水解乳清蛋白、降低乳清蛋白致敏性【以α-乳白蛋白(α-LA)和β-乳球蛋白(β-LG)为抗原性表征】的最优超声预处理-酶解条件。结果表明:乳清蛋白水解度受初始pH值和酶解温度的影响显著,α-LA、β-LG抗原性受初始pH值的影响显著,超声间歇时间和超声功率的交互作用对α-LA、β-LG抗原性影响显著。采用响应面法获得马克思克鲁维酵母Z17转化乳清蛋白的最优酶解条件是:超声间歇时间16 s,超声功率400 W,初始pH 6.16,酶解温度18.48℃,预测α-LA抗原性、β-LG抗原性的降低率达到最大值,分别为65.56%和57.96%。     

克鲁维酵母突变株UV-G-40-3菊粉酶性质的研究

研究了克鲁维酵母突变株（Ｋｌｕｙｖｅｒｏｍｙｃｅｓ－ＵＶ－Ｇ－４０－３）所产菊粉酶的分布为胞外酶∶胞壁酶∶胞内酶比是５．７∶１．６∶１。该酶Ｓ／Ｉ为５．３,最适温度为５０℃,最适ｐＨ为４．５,在５０℃以下、ｐＨ４．５～８的范围内比较稳定,４℃贮存稳定性好,１４ｄ后仍保持７６％活力,为外切型菊粉酶,酶解粗菊糖（洋姜提取液）活性为纯菊糖的４倍。     

马克斯克鲁维酵母菊粉酶的分离纯化和酶学性质研究

马克斯克鲁维酵母产生的孢外菊粉酶经超滤浓缩、DEAE-Cellulose阴离子交换色谱、SephadexG-100凝胶色谱分离纯化,得到两个菊粉酶组分ExoⅠ和ExoⅡ,I/S值分别为0.0249和0.0253,均属外切菊粉酶。ExoⅠ经聚丙烯酰胺凝胶鉴定为均一组分,分子量为85kD。ExoⅠ最适pH值为4.0,最适温度为60℃,Mn2+和Mg2+对酶活力有促进作用,而Cu2+、Fe2+对酶活力有抑制作用,ExoⅠ水解菊粉溶液的产物为果糖和少量葡萄糖。     

Sucrose hydrolysis by gelatin-immobilized inulinase from Kluyveromyces marxianus var. bulgaricus

The crude cell-free medium from a culture of Kluyveromyces marxianus var. bulgaricus was immobilized in a gelatin-water support, with an immobilization yield of 82.60% for inulinase activity. The optimum pH for both free and immobilized inulinase was the same (3.5) and the optimum temperatures were 55 °C for the free and 60 °C for the immobilized enzyme. The Arrhenius plots were linear and activation energies were 56.20 (free enzyme) and 20.27 kJ/mol K (immobilized enzyme). The kinetic parameters were calculated by Lineweaver–Burk plots and the V_max and K_m were 37.60 IU/mg protein and 61.83 mM for the free inulinase and 31.45 IU/mg protein and 149.28 mM for the immobilized enzyme, respectively. The operational stability of the immobilized inulinase was studied in a continuous fixed-bed column reactor for 33 days, at the end of which the sucrose conversion was 58.12%.     

Comparison of volatile compounds produced in model cheese medium deacidified by Debaryomyces hansenii or Kluyveromyces marxianus

The aroma of a deacidified cheese medium is the result of the overall perception of a large number of molecules belonging to different classes. The volatile compound composition of (60%) cheese medium (pH 5.8) deacidified by Debaryomyces hansenii (DCM(Dh)) was compared with the one deacidified by Kluyveromyces marxianus (DCM(Km)). It was determined by dynamic headspace extraction, followed by gas chromatography separation and quantification as well as by mass spectrometry identification. Whatever the media tested, a first class of volatile compounds can be represented by the ones not produced by any of the yeasts, but some of them are affected by K. marxianus or by D. hansenii. A second class of volatile compounds can be represented by the ones produced by K. marxianus, which were essentially esters. Their concentrations were generally higher than their thresholds, explaining the DCM(Km) global fruity odor. A third class can be represented by the ones generated by D. hansenii, which were essentially methyl ketones with fruity, floral (rose), moldy, cheesy, or wine odor plus 2-phenylethanol with a faded-rose odor. The impact of methyl ketones on the DCMDh global flavor was lower than the impact of 2-phenylethanol and even negligible. Therefore, the global faded-rose odor of D. hansenii DCM can be explained by a high concentration of 2-phenylethanol.