Flaking and extrusion as mechanical treatments for enzyme-assisted aqueous extraction of oil from soybeans

Flaking and extruding dehulled soybeans were evaluated as a means of enhancing oil extraction efficiency during enzyme-assisted aqueous processing of soybeans. Cellulase, protease, and their combination were evaluated for effectiveness in achieving high oil extraction recovery from extruded flakes. Aqueous extraction of extruded full-fat soy flakes gave 68% recovery of the total available oil without using enzymes. A 0.5% wt/wt protease treatment after flaking and extruding dehulled soybeans increased oil extraction recovery to 88% of the total available oil. Flaking and extruding enhanced protease hydrolysis of proteins freeing more oil. Treating extruded flakes with cellulase, however, did not enhance oil extraction either alone or in combination with protease. Discrepancies in oil extraction recoveries were encountered when merely considering crude free fat because some oil became bound to denatured protein during extrusion and/or sample drying. Bound fat was unavailable for determination by using the hexane extraction method, but was accounted for by using the acid hydrolysis method for total oil determination. Oil extraction recovery from extruded soybean flakes was affected by oil determination methods, which was not the case for unextruded full-fat soy flour.     

Properties of cuticular oxidases used for sex pheromone biosynthesis byHeliothis zea

Biosynthesis of the aldehydic sex pheromone components released by females ofHeliothis zea was found to be catalyzed by primary alcohol oxidases residing in the cuticle that covers the glands. Activity, as indicated by conversion of primary alcohol to aldehyde, was as high in cell-free cuticle as it was in intact pheromone glands. Studies indicated that some activity was associated with the surface of the epicuticle and could be removed, into buffer, by sonication. However, the majority of activity lies within the inner epicuticle and exo- and endocuticular layers. The oxidase was not functional in pharate pupae that did not have mature adult cuticle but became functional just prior to adult emergence. The enzyme in individual glands was saturated at alcohol concentrations above 100 n. moles. Nonionic detergents did not affect the activity of the oxidase in the cuticle but treatment with either 7 M urea or 1% SDS resulted in total loss of activity. Studies on the effect of pH indicated an optimum at 6.4; however, activity was high throughout the range of 5–9. The oxidase was functional in both dichloromethane and hexane, suggesting that this enzyme system may have applications for organic synthesis of aldehydes.     

纤维素酶活性测定的生物传感器研究

用TiO2凝胶包埋葡萄糖氧化酶电化学传感器(GOD/TiO2)测定纤维素酶(CE)的活性,即基于电极对纤维素水解后生成葡萄糖的催化氧化。考察了各种实验条件对CE酶活性测定的影响。实验表明,GOD/TiO2电极检测电位为+0.50Vvs.SCE,稳态响应电流与纤维素酶活性在10～300U/L之间有线性关系,该方法可用于微量羧甲基纤维素酶活性测定。     

Improvement of cyclodextrin glucanotransferase as an antistaling enzyme by error-prone PCR

In an effort to improve the properties of cyclodextrin glucanotransferase (CGTase) as an antistaling enzyme, error-prone PCR was used to introduce random mutations into a CGTase cloned from alkalophilic Bacillus sp. I-5 (CGTase I-5). A mutant CGTase[3-18] with the three mutations M234T, F259I and V591A was selected by agar plate assay. Sequence alignment of various CGTases indicated that M234 and F259 are located in the vicinity of the catalytic sites of the enzyme and V591 in the starch binding domain E. The cyclization activity of CGTase[3-18] was dramatically decreased by 10-fold, while the hydrolyzing activity was increased by up to 15-fold. These mutations near subsite +1 (M234T) and at subsite +2 (F259I) are likely to alter the enzyme activity in a concerted manner, promoting hydrolysis of substrate while retarding cyclization. The addition of CGTase[3-18] reduced the retrogradation rate of bread by as much as did the commercial antistaling enzyme Novamyl during 7-day storage at 4 degrees C. No cyclodextrin (CD) was detected in bread treated with CGTase[3-18], whereas 21 mg of CD per 10 g of bread was produced in bread treated with wild-type CGTase.     

Oil and fat hydrolysis with lipase fromAspergillus sp.

Hydrolysis of olive oil, soybean oil, mink fat, lard, palm oil, coconut oil, and a hydrogenated, hardened oil with lipase from anAspergillus sp. has been studied. The lipase had high specific activity (60,000 U/g) and did not show any positional specificity. The lipase proved to be a more effective catalyst than Lipolase fromA. oryzae, with an optimal activity at 37°C and pH 6.5–7.0. It was activated by Ca²⁺ but inactivated by organic solvents such as isopropanol and propanone. All substrates examined could be hydrolyzed to corresponding fatty acids with this enzyme at concentrations of 5–30 U/meq with yields of 90–99% in 2–24 h. The degree of hydrolysis was almost logarithmically linear with reaction time and occurred in two stages. The lipase was stable and could be repeatedly recycled for hydrolysis.     

Inhibitory effect of Cr（Ⅵ） on activities of soil enzymes

To evaluate the influence of various Cr(VI) concentrations (0.05, 0.25, 0.50, 1.00 and 2.00 g/kg) on the activity of soil enzymes, the activities of catalase, polyphenol oxidase, dehydrogenase, alkaline phosphatase in soils were investigated in the incubation experiment with a period of 35 d. The results indicate that all the tested Cr(VI) concentrations significantly inhibit dehydrogenase activity by over 70% after 35 d. The activity of alkaline phosphatase is slightly inhibited during the whole experiment except for on the day 7. Cr(VI) has no obvious effect on the activity of catalase in soil. On the contrary, Cr(VI) stimulates the activity of polyphenol oxidase. The results suggest that dehydrogenase activity can be used as an indicator for assessing the severity of chromium pollution. Foundation item: Projects(2006AA06Z374, 2007AA021304) supported by the National High-Tech Research and Development Program of China; Project(2008SK2007) supported by the Key Program of Science and Technology of Hunan Province, China     

金属离子对红球菌腈水合酶活力的影响

不同金属离子对红球菌（Rhodococcus sp．）TCCC 28001的生长及其腈水合酶的酶活有着不同程度的影响．菌株TCCC 28001产生的腈水合酶是钴型,且Co^2＋对该腈水合酶诱导激活的最适浓度为10×10^-5mol／L．选择酵母粉中含有且含量波动较大的5种金属离子,考察了在添加10×10^-5 mol／L Co^2＋诱导激活下,5种金属离子对酶活的影响．结果表明：Fe^2＋,Mn^2＋,Mo^6＋,Cu^2＋4种金属离子具有促进作用,Zn^2＋产生轻微抑制作用．6×10^-5mol／L Fe^2＋的促进效果显著,使菌株TCCC 28001的酶活从453U／mL增加到1941U／mL,提高了328％．     

Fractionation of transgenic corn for recovery of recombinant enzymes

Two germ-separation methods, dry-milling and density separation by flotation, were evaluated for recovering recombinant β-glucuronidase (rGUS) that accumulated primarily in the germ of transgenic corn. The dry-milling process consisted of (i) seed tempering, (ii) degerming with a horizontal-drum degermer/dehuller, (iii) particle size fractionation with standard sieves, (iv) germ and endosperm separation by roller milling and sifting, and (v) removal of hulls by aspiration. Sieves nos. 5, 6, and 7 retained the majority of germ, and subfractions from these sieves were pooled as a germ-rich fraction. Mass balances showed that the germ-rich fraction, which constituted 17% of the total dry-milled corn weight, contained 49% of rGUS activity and 64% of the total recoverable oil. Germ fractionation by flotation was tested as a proof-of-concept method aimed at separating corn fractions based on their difference in specific gravity (sp gr). The process consisted of impact-grinding of corn kernels followed by density separation using 1.15 or 1.3 specific gravity sodium nitrate solution. The oil-containing germ fraction floated, whereas the heavier endosperm fraction sedimented. The flotation method was simpler and resulted in higher enzyme recovery, that is, the germ-rich fraction was 20% (w/w) of the initial corn weight, and accounted for 80% of rGUS activity and 77% of total oil. The sodium nitrate solution did not have an adverse effect on the enzyme activity.     

Activity and stability ofPenicillium cyclopium lipase in surfactant and detergent solutions

Activity and stability of an alkaline lipase fromPenicillium cyclopium var.album (PG 37) were studied in surfactant and detergent solutions. Three anionic surfactants [Na salts of C₁₂SO₄ ^? (sodium dodecyl sulfate), C₁₂ØSO₃/^? (linear alkyl benzene sulfonate), and C₁₁COO^? (laurate)] and four homologous series of nonionic surfactants of C_12–15 polyoxethylenated fatty alcohols (AEO₃, AEO₅, AEO₇, and AEO₉) were evaluated. At a concentration range of 3.2–40 μM, sodium dodecyl sulfate and laurate stimulated the activity of PG 37 lipase. At concentrations greater than 5.6 μM, linear alkylbenzene sulfonate inhibited PG 37 lipase activity. Nonionic surfactants, AEO₅ and AEO₇, in the concentration range of 0.25–20 mM, enhanced and stabilized the activity of PG 37 lipase. The presence of PG 37 lipase in detergent formulaton improved detergency ～20%. The mechanism of inhibition of the lipolytic activity of PG 37 lipase is proposed to be partly due to the formation of inactive (BR)_n-E complex between the hydrophobic moiety of the surfactants and the surface of the lipase. Conversely, formation of a soluble (RB)_n-E complex between the hydrophilic group of the surfactant and lipase may account for the increased lipolytic activity of PG 37 lipase.     

关于木素生物降解酶类活力测定问题的讨论

由于木素结构和生物降解过程复杂多样，全面测定一个菌株的木素降解活力也比较困难，目前已发现三类主要参与木素降解的酶类（LIP、MnP、漆酶），但至今很少有国际上公认的酶活力测定方法，也未见到能把某一菌株木素降解酶类的活力与其在木质纤维材料中降解木素的能力定量关联的报导。本文综合评述了几类主要的木素降解酶类活力测定方法的作用机制和存在的问题，指出一个菌株的木素降解活力是由它的生理特性和总体代谢能力共同决定的，仅由一类或几类酶活力的表现水平难以对其作出确切的评价，从这个角度出发进行研究才能更客现他认识菌株的木素降解活力．