Enhanced endoxylanase production by Myceliophthora thermophila using rice straw and its synergism with phytase in improving nutrition

The goal of the present investigation was to attain the enhanced production of endoxylanase in submerged fermentation using different approaches followed by its utility in improving nutrition of wheat and rice flours along with phytase. Myceliophthora thermophila BJTLRMDU3 produced 51.70 U/mL of xylanase using rice straw as a substrate after optimization with ‘one variable at a time’ approach. After Plackett-Burman design study, sodium nitrate, K₂HPO₄ and Tween 20 were selected as critical factors and further optimized by response surface methodology. Increased xylanase production (80.15 U/mL) was attained with 2.5 % (w/v) sodium nitrate, 1.25 % (w/v) K₂HPO₄, and 2 % (v/v) Tween 20 at 40 °C. An overall 1.5-fold increase in xylanase production was achieved after statistical optimization. Applicability of M. thermophila xylanase (200 U/g flour) alone and in combination with phytase (15 U/g flour) from Aspergillus oryzae SBS50 in wheat and rice flours showed enhancement in nutritional qualities of both flours. About 45.67 %, 29.73 %, and 107.91 % increase in reducing sugars, soluble proteins and inorganic phosphate, respectively in wheat flour, while 94.16 %, 134.52 %, and 473.33 % increase in reducing sugars, soluble proteins and inorganic phosphate, respectively in rice flour was achieved at 60 °C and pH 5.0 by synergistic action of xylanase and phytase as compared to control having only xylanase.     

芽孢杆菌植酸酶分子生物学研究进展

来源于芽孢杆菌的中性植酸酶具有良好的热稳定性,适合作为消化道呈中性鲤科鱼类的饲料添加剂。该植酸酶晶体具有独特的螺旋桨样空间结构,钙离子在其稳定性和催化活性的结构功能关系中具有重要作用,并表现出特有的催化机理。该基因与已知植酸酶基因的结构有所不同,是一种新的植酸酶。目前主要采用多种原核表达系统对其进行表达。比较表明,芽孢杆菌表达系统和酵母表达系统是实现芽孢杆菌植酸酶规模化生产的有效途径。     

植酸酶及其植物基因工程

综述了植酸酶的来源、其酶学性质及植酸酶的植物摹因工程,并对其存在的问题、应用前景作了展望。     

Atomistic details of effect of disulfide bond reduction on active site of Phytase B from Aspergillus niger: A MD Study

The molecular integrity of the active site of phytases from fungi is critical for maintaining phytase function as efficient catalytic machines. In this study, the molecular dynamics (MD) of two monomers of phytase B from Aspergillus niger, the disulfide intact monomer (NAP) and a monomer with broken disulfide bonds (RAP), were simulated to explore the conformational basis of the loss of catalytic activity when disulfide bonds are broken. The simulations indicated that the overall secondary and tertiary structures of the two monomers were nearly identical but differed in some crucial secondary–structural elements in the vicinity of the disulfide bonds and catalytic site. Disulfide bonds stabilize the β-sheet that contains residue Arg66 of the active site and destabilize the α-helix that contains the catalytic residue Asp319. This stabilization and destabilization lead to changes in the shape of the active–site pocket. Functionally important hydrogen bonds and atomic fluctuations in the catalytic pocket change during the RAP simulation. None of the disulfide bonds are in or near the catalytic pocket but are most likely essential for maintaining the native conformation of the catalytic site.

Abbreviations

PhyB - 2.5 pH acid phophatese from Aspergillus niger, NAP - disulphide intact monomer of Phytase B, RAP - disulphide reduced monomer of Phytase B, Rg - radius of gyration, RMSD - root mean square deviation, MD - molecular dynamics.     

不同添加方式植酸酶处理豆粕对牙鲆生长和饲料利用率的影响

以初始平均体重(2.02±0.02)g的牙鲆(Paralichthys olivaceus)为实验对象,进行为期70d的摄食生长实验,研究不同添加方式的植酸酶对牙鲆生长和饲料利用的影响。在5000.0g豆粕中添加2.5g植酸酶,然后用产朊假丝酵母(Candidautilis)进行发酵预处理,得到植酸酶预处理豆粕。共制作4种等氮等能(粗蛋白49.7%、总能20.9kJ/g)饲料,对照饲料主要以鱼粉为蛋白源;在对照饲料的基础上,用豆粕蛋白替代45%的鱼粉蛋白配制成豆粕组饲料;在每千克豆粕组饲料中添加1000IU植酸酶,配制成植酸酶组饲料;用植酸酶预处理豆粕蛋白替代45%的鱼粉蛋白配制成植酸酶预处理豆粕组饲料。结果表明,与对照组相比较,用豆粕蛋白替代饲料中45%的鱼粉蛋白,若不添加植酸酶则显著降低牙鲆的特定生长率(P0.01)、饲料效率、蛋白质效率和氮贮积率(P0.05);直接添加植酸酶组、植酸酶预处理豆粕组牙鲆的特定生长率、饲料效率、蛋白质效率和氮贮积率与鱼粉对照组相比较没有出现显著差异(P0.05);与不添加植酸酶的豆粕组相比较,在含豆粕饲料中添加1000IU/kg饲料的植酸酶显著提高牙鲆的特定生长率(P0.01)、氮贮积率(P0.05)和磷贮积率(P0.01),显著降低氮排放率(P0.05)和磷排放率(P0.01),但饲料效率和蛋白质效率没有显著变化(P0.05);在豆粕中添加植酸酶进行发酵预处理,降低了豆粕中植酸含量,在饲料中添加植酸酶预处理豆粕显著提高牙鲆的特定生长率(P0.01)、饲料效率、蛋白质效率和氮贮积率(P0.05),显著降低氮(P0.05)、磷和钙的排放率(P0.01)。       

植酸酶及其热稳定性研究进展

植酸酶能降解植物性饲料中的植酸盐类,释放出无机磷酸,对于提高饲料中磷的利用率,减轻畜禽高磷排泄物对环境的污染以及促进单胃动物对饲料中矿质营养的吸收利用有重要作用,因此植酸酶的研究有重要的科学和实用价值。获得高活性高热稳定性的植酸酶是近年来植酸酶工业的研究热点和难点,综述了植酸酶及其热稳定性研究的现状和提高植酸酶热稳定性方法的最新进展 。     

Production,characterization and gene cloning of the extracellular enzymes from the marine-derived yeasts and their potential applications

In this review article, the extracellular enzymes production, their properties and cloning of the genes encoding the enzymes from marine yeasts are overviewed. Several yeast strains which could produce different kinds of extracellular enzymes were selected from the culture collection of marine yeasts available in this laboratory. The strains selected belong to different genera such as Yarrowia, Aureobasidium, Pichia, Metschnikowia and Cryptococcus. The extracellular enzymes include cellulase, alkaline protease, aspartic protease, amylase, inulinase, lipase and phytase, as well as killer toxin. The conditions and media for the enzyme production by the marine yeasts have been optimized and the enzymes have been purified and characterized. Some genes encoding the extracellular enzymes from the marine yeast strains have been cloned, sequenced and expressed. It was found that some properties of the enzymes from the marine yeasts are unique compared to those of the homologous enzymes from terrestrial yeasts and the genes encoding the enzymes in marine yeasts are different from those in terrestrial yeasts. Therefore, it is of very importance to further study the enzymes and their genes from the marine yeasts. This is the first review on the extracellular enzymes and their genes from the marine yeasts.     

Advantage of using PSIRB over PSRB and IRB to improve plant health of tomato

Twenty-one isolates of phosphate solubilizing-indole acetic acid producing rhizobacteria (PSIRB), 20 isolates of phosphate solubilizing rhizobacteria (PSRB) and 42 isolates of indole acetic acid producing rhizobacteria (IRB) were isolated from 49 rhizospheric soil samples of tomato (Lycopersicon esculentum Mill.) collected from tomato growing regions of Karnataka. A method combining Pikovskaya’s and Bric’s technique was developed to isolate PSRIB, PSRB and IRB’s. The selected isolates were further analyzed for their ability to solubilize calcium phytate. Based on the root colonization assays and the abilities of bacterial isolates to increase the seed germination and seedling vigor under laboratory conditions, five isolates were selected from each group for further studies. Under greenhouse conditions, all the selected rhizobacteria isolates significantly increased root length, shoot length, fresh weight, dry weight and total phosphorus content of 30-day-old-seedlings with respect to control. Isolate PSIRB1 and IRB36 significantly reduced the Fusarium wilt incidence over other isolates of same and other group, and the control. On the basis of results from laboratory and greenhouse studies, one bacterial isolate from each group was selected for plant growth and yield analysis studies. Isolate PSIRB2 showed increased plant height, fresh weight, number of fruits per plant and average weight of fruit over PSRB9, IRB36 and untreated controls. Studies on the nature of protection offered by these bacterial isolates following split-root technique revealed that the isolates PSIRB2 and PSRB9 had the ability to induce systemic resistance. One isolate, IRB36 appeared to protect the tomato seedlings through direct antagonism.     

Enhancing thermostability of <Emphasis Type="Italic">Escherichia coli</Emphasis> phytase AppA2 by error-prone PCR

Phytases are used to improve phosphorus nutrition of food animals and reduce their phosphorus excretion to the environment. Due to favorable properties, Escherichia coli AppA2 phytase is of particular interest for biotechnological applications. Directed evolution was applied in the present study to improve AppA2 phytase thermostability for lowering its heat inactivation during feed pelleting (60–80°C). After a mutant library of AppA2 was generated by error-prone polymerase chain reaction, variants were expressed initially in Saccharomyces cerevisiae for screening and then in Pichia pastoris for characterizing thermostability. Compared with the wild-type enzyme, two variants (K46E and K65E/K97M/S209G) showed over 20% improvement in thermostability (80°C for 10 min), and 6–7°C increases in melting temperatures (T _m). Structural predictions suggest that substitutions of K46E and K65E might introduce additional hydrogen bonds with adjacent residues, improving the enzyme thermostability by stabilizing local interactions. Overall catalytic efficiency (k _cat / K _m) of K46E and K65E/K97M/S209G was improved by 56% and 152% than that of wild type at pH 3.5, respectively. Thus, the catalytic efficiency of these enzymes was not inversely related to their thermostability.