Applications of recombinant DNA technology to the pulp and paper industry

New gene selection techniques (Recombinant DNA) are currently available to exploit useful properties of various biological systems hitherto regarded as interesting but of little or no immediate commercial value. The application of genetic engineering techniques to problems in the Pulp and Paper Industry are many. As a first step these techniques are being used to provide much needed fundamental information on the cellular and molecular mechanisms involved in the expression of extra-cellular enzymes that degrade lignocellulosic pulping wastes. The information gleaned from the studies on cellulolytic fungi and bacteria can be used to genetically engineer a yeast or bacterium capable of converting pulping wastes into ethanol and other useful by-products.     

Simultaneous saccharification and fermentation of amorphous cellulose to ethanol by recombinant Klebsiella oxytoca SZ21 without supplemental cellulase

A derivative of Klebsiella oxytoca M5A1 containing chromosomally integrated genes for ethanol production from Zymomonas mobilis (pdc, adhB) and endoglucanase genes from Erwinia chrysanthemi (celY, celZ) produced over 20 000 U endoglucanase l^–1 activity during fermentation. In combination with the native ability to metabolize cellobiose and cellotriose, this strain was able to ferment amorphous cellulose to ethanol (58–76% of theoretical yield) without the addition of cellulase enzymes from other organisms.     

Conversion of corn fiber to ethanol by recombinant E. coli strain FBR3

Escherichia coli strain FBR3 that is an efficient biocatalyst for converting mixed sugar streams (eg, arabinose, glucose, and xylose) into ethanol. In this report, the strain was tested for conversion of corn fiber hydrolysates into ethanol. Corn fiber hydrolysates with total sugar concentrations of 7.5% (w/v) were prepared by reacting corn fiber with dilute sulfuric acid at 145°C. Initial fermentations of the hydrolysate by strain FBR3 had lag times of approximately 30 h judged by ethanol production. Further experiments indicated that the acetate present in the hydrolysate could not solely account for the long lag. The lag phase was greatly reduced by growing the pre-seed and seed cultures on corn fiber hydrolysate. Ethanol yields for the optimized fermentations were 90% of theoretical. Maximum ethanol concentrations were 2.80% w/v, and the fermentations were completed in approximately 50 h. The optimal pH for the fermentation was 6.5. Below this pH, sugar consumption was incomplete and above it, excess base addition was required throughout the fermentation. Two alternative neutralization methods (overliming and overliming with sulfite addition) have been reported for improving the fermentability of lignocellulosic hydrolysates. These methods further reduced the lag phase of the fermentation, albeit by a minor amount. Received 29 September 1998/ Accepted in revised form 20 February 1999     

Hydrolysis of filter-paper cellulose to glucose by two recombinant endogenous glycosyl hydrolases of Coptotermes formosanus

Abstract Genes encoding for glycosyl hydrolases (GH) in multiple families were recovered from an expression sequence tag library of Coptotermes formosanus, a xylophagous lower termite species. Functional analyses of these genes not only shed light on the mechanisms the insect employs to successfully use cellulosic materials as energy sources, which may serve as strategic targets for designing molecular-based bio-pesticides, but also enrich discoveries of new cellulolytic enzymes for conversion of biomass into biofuel. Our study demonstrated that cellulose could be converted to glucose by two recombinant endogenous glycosyl hydrolases (endo-β-1,4 glucanase in GH9 and β-glucosidase in GH1). While the former cleaved cellulose to cellobiose and cellotriose, the resulting simple cellodextrins were digested to glucose. Both of the Escherichia coli-expressed recombinant proteins showed properties that could be incorporated in a glucose-based ethanol production program.     

固态发酵玉米淀粉渣生产蛋白饲料的研究

本文对同化淀粉能力强的糖化酵母菌Ｙ９６０１株在固态发酵玉米淀粉渣生产蛋白饲料中的应用进行了研究,在合适的发酵条件下,３０℃发酵３６ｈ,发酵产物（干基）酵母达到７．４５×１０＾９个／ｇ,粗蛋白从２５．５％提高到３８．２％,增加１２．７％,粗蛋白增幅为４９．８％。     

Synergistic metabolism in hybrid corn indicates bottlenecks in the carotenoid pathway and leads to the accumulation of extraordinary levels of the nutritionally important carotenoid zeaxanthin

Lutein and zeaxanthin cannot be synthesized de novo in humans, and although lutein is abundant in fruit and vegetables, good dietary sources of zeaxanthin are scarce. Certain corn varieties provide adequate amounts because the ratio of endosperm β : ε lycopene cyclase activity favours the β‐carotene/zeaxanthin branch of the carotenoid pathway. We previously described a transgenic corn line expressing the early enzymes in the pathway (including lycopene β‐cyclase) and therefore accumulating extraordinary levels of β‐carotene. Here, we demonstrate that introgressing the transgenic mini‐pathway into wild‐type yellow endosperm varieties gives rise to hybrids in which the β : ε ratio is altered additively. Where the β : ε ratio in the genetic background is high, introgression of the mini‐pathway allows zeaxanthin production at an unprecedented 56 μg/g dry weight. This result shows that metabolic synergy between endogenous and heterologous pathways can be used to enhance the levels of nutritionally important metabolites.     

High solid simultaneous saccharification and fermentation of wet oxidized corn stover to ethanol

In this study ethanol was produced from corn stover pretreated by alkaline and acidic wet oxidation (WO) (195 degrees C, 15 min, 12 bar oxygen) followed by nonisothermal simultaneous saccharification and fermentation (SSF). In the first step of the SSF, small amounts of cellulases were added at 50 degrees C, the optimal temperature of enzymes, in order to obtain better mixing condition due to some liquefaction. In the second step more cellulases were added in combination with dried baker's yeast (Saccharomyces cerevisiae) at 30 degrees C. The phenols (0.4-0.5 g/L) and carboxylic acids (4.6-5.9 g/L) were present in the hemicellulose rich hydrolyzate at subinhibitory levels, thus no detoxification was needed prior to SSF of the whole slurry. Based on the cellulose available in the WO corn stover 83% of the theoretical ethanol yield was obtained under optimized SSF conditions. This was achieved with a substrate concentration of 12% dry matter (DM) acidic WO corn stover at 30 FPU/g DM (43.5 FPU/g cellulose) enzyme loading. Even with 20 and 15 FPU/g DM (corresponding to 29 and 22 FPU/g cellulose) enzyme loading, ethanol yields of 76 and 73%, respectively, were obtained. After 120 h of SSF the highest ethanol concentration of 52 g/L (6 vol.%) was achieved, which exceeds the technical and economical limit of the industrial-scale alcohol distillation. The SSF results showed that the cellulose in pretreated corn stover can be efficiently fermented to ethanol with up to 15% DM concentration. A further increase of substrate concentration reduced the ethanol yield significant as a result of insufficient mass transfer. It was also shown that the fermentation could be followed with an easy monitoring system based on the weight loss of the produced CO2.     

A method for three-dimensional protein characterization and its application to a complex plant (corn) extract

Knowledge of host protein properties is critical for developing purification methods for recombinant proteins from a specific host, or for choosing suitable hosts and targeted expression tissues for a specific recombinant protein. A method to obtain a three-dimensional (3D) map (surface hydrophobicity (SH), isoelectric point (pI), and molecular weight (MW)), of a host's aqueous soluble protein properties was developed. The method consists of hydrophobic partitioning in a PEG 3350 (15.7%)-Na(2)SO(4) (8.9%)-NaCl (3%) aqueous two-phase (ATP) system followed by quantitative, 2D-electrophoretic characterization of the proteins of each equilibrium phase and the original extract. The pI and MW of host proteins were obtained directly through 2D electrophoresis. The partition coefficients of individual proteins were obtained by quantitative matching of protein spots in the top and bottom phase gels and calculating the protein partition coefficients from this information. Correlation of the partition coefficient to a SH scale was established by partitioning several model proteins with known surface hydrophobicities in the same ATP system. The inclusion of the extract gel provided for a spot selection criterion based on satisfactory mass balance closure. The method is illustrated by application to a mixture of model proteins and to complex mixtures, that is, corn germ proteins extracted at pH 7 and pH 4.     

Fractionation of transgenic corn seed by dry and wet milling to recover recombinant collagen‐related proteins

Corn continues to be considered an attractive transgenic host for producing recombinant therapeutic and industrial proteins because of its potential for producing recombinant proteins at large volume and low cost as coproducts of corn seed‐based biorefining. Efforts to reduce production costs have been primarily devoted to increasing accumulation level, optimizing protein extraction conditions, and simplifying the purification. In the present work, we evaluated two grain fractionation methods, dry milling and wet milling, to enrich two recombinant collagen‐related proteins; thereby, reducing the amount and type of corn‐derived impurities in subsequent protein extraction and purification steps. The two proteins were a full‐length human recombinant collagen type I alpha 1(rCIα1) chain with telopeptides and peptide foldon to effect triple helix formation and a 44‐kDa rCIα1 fragment. For each, ～60% of the rCIα1s in the seed was recovered in the dry‐milled germ‐rich fractions making up ca. 25% of the total kernel mass. For wet milling, ～60% of each was recovered in three fractions accounting for 20–25% of the total kernel mass. The rCIα1s in the dry‐milled germ‐rich fractions were enriched three to six times compared with the whole corn kernel, whereas the rCIα1s were enriched 4–10 times in selected wet‐milled fractions. The recovered starch from wet milling was almost free of rCIα1. Therefore, it was possible to generate rCIα1‐enriched fractions by both dry and wet milling along with rCIα1‐free starch using wet milling. Because of its simplicity, the dry milling procedure could be accomplished on‐farm thus minimizing the risk of inadvertent release of viable transgenic seeds. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Bt毒蛋白在转基因抗虫玉米中的表达及在亚洲玉米螟中的转移积累

以两个转Bt基因抗虫玉米品系G03-2396、G03-2739和对照玉米品种苏玉16为材料,采用室内生物测定法研究它们对亚洲玉米螟的抗性, 并采用酶联免疫技术（ELISA）检测这两个转基因玉米品系不同组织中Bt毒蛋白的表达量及亚洲玉米螟3龄与5龄幼虫取食转基因玉米后体内和粪便中的Bt毒蛋白含量.结果表明：转Bt基因抗虫玉米心叶对玉米螟幼虫的毒性较强,初孵幼虫取食6 d后的存活率不到3%,3龄幼虫取食6 d后的存活率小于70%,抗虫玉米雌穗的毒性小于心叶.两个转Bt基因玉米心叶和雌穗中均表达了一定量的Bt毒蛋白,但心叶中的毒蛋白含量高于雌穗;Bt毒蛋白表达量依次为G03-2739心叶（39.6 μg·g^-1FM）> G03-2396心叶（26.1 μg·g^-1 FM）> G03-2396雌穗（17.0 μg·g^-1 FM）> G03-2739雌穗（14.6 μg·g^-1 FM）.取食转基因玉米心叶或雌穗后,3龄幼虫体内的Bt毒蛋白含量显著高于5龄幼虫;同龄幼虫取食心叶后其体内及粪便中Bt毒蛋白含量均显著高于取食雌穗的个体.其中,取食G03-2739心叶的5龄幼虫粪便中的Bt毒蛋白含量最高,达10.4 μg·g^-1 FM;取食其雌穗的3龄幼虫粪便中的Bt毒蛋白含量最低,仅2.7 μg·g^-1 FM.     

Preservation of high phenylalanine ammonia lyase activities in roots of Japanese Striped corn: A potential oral therapeutic to treat phenylketonuria

Phenylketonuria (PKU) is an inherited metabolic disorder caused by deficient phenylalanine hydroxylase (PAH) activity, the enzyme responsible for the disposal of excess amounts of the essential amino acid phenylalanine (Phe). Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) has potential to serve as an enzyme substitution therapy for this human genetic disease. Using 7-day-old Japanese Striped corn seedlings (Japonica Striped maize, Zea mays L. cv. japonica) that contain high activities of PAL, we investigated a number of methods to preserve the roots as an intact food and for long-term storage. The cryoprotectant effects of maple syrup and other edible sugars (mono- and oligosaccharides) were evaluated. Following thawing, the preserved roots were then examined to determine whether the rigid plant cell walls could protect the PAL enzyme from proteolysis during simulated (in vitro) digestion comprised of gastric and intestinal phases. While several treatments led to retention of PAL activity during freezing, upon thawing and in vitro digestion, root tissues that had been previously frozen in the presence of maple syrup exhibited the highest residual PAL activities (∼50% of the initial enzyme activity), in marked contrast to all of the treatments using other edible sugars. The structural integrity of the root cells, and the stability of the functional PAL tetramer were also preserved with the maple syrup protocol. These results have significance for the formulation of oral enzyme/protein therapeutics. When plant tissues are adequately preserved, the rigid cell walls constitute a protective barrier even under harsh (e.g. gastrointestinal-like) conditions.     

Distribution of enzyme activities within the developing maize (Zea mays) kernel in relation to starch, oil and protein accumulation

The association of enzyme activities in developing kernels with specific storage product accumulation at maturity was analyzed in different parts of Zea mays inbred OH43 kernels. Maize kernels were harvested at 20 and 55 days post-pollination and dissected into basal region, pericarp, embryo, lower endosperm, middle endosperm and upper endosperm. Mature (55 days pos(-pollination) kernel parts were analyzed for starch, total protein, zein and oil content. Immature (20 days post-pollination) kernel parts were assayed for activities of 15 enzymes of sugar and amino acid metabolism. Statistical analyses of the data suggested that glucokinase (EC 2.7.1.2) fructokinase (EC 2.7.1.4) and phosphofructokinase (EC 2.7.1.1 11) activities were primarily associated with oil accumulation, whereas ADP'-glueose pyrophosphorylasc (EC 2.7.7.27) and sucrose synthase (EC 2.4.1.13) activities were associated with starch accumulation. The results suggest that oil biosynthesis utilizes inveitase-mediated sucrose degradation in a pathway not requiring pyrophosphatc. whereas starch biosynthesis utilizes a sucrose synthase-mediated pathway of sucrose degradation in a pathway requiring pyrophosphatc. Additional groups of enzyme activities were associated with each oilier but not with any specific storage product and appeared to be associated with general metabolic activity.     

Determination of ferrichrome binding to the FhuA outer membrane transport protein,periplasmic accumulation of ferrichrome,or transport of ferrichrome into cells using a three-layer oil technique

A new method for the determination of ferrichrome binding to the FhuA transporter in the Escherichia coli outer membrane, ferrichrome accumulation in the periplasmic space, and ferrichrome transport into the cytoplasm was developed. Cells were separated from residual, soluble, radiolabeled ferrichrome by centrifugation in a micro-test tube containing three layers of nonmixable solutions of different densities. Cells in the upper aqueous layer passed through the middle silicone oil layer, but did not enter the underlying NaI layer, thereby accumulating on top of the NaI layer; soluble compounds remained in the upper aqueous layer. Cells were then easily recovered by centrifugation, and radioactivity was determined by liquid scintillation counting. Reproducible results for all applications tested were obtained without the need for any washing steps. The method was tested by determination of receptor binding and transport of ferrichrome with various FhuA mutants which, in contrast to their transport activity, showed only a weak binding of ferrichrome to FhuA and compared with the commonly used cellulose nitrate filter method. Similar transport rates were obtained with the two methods, but binding of ferrichrome to the mutated FhuA proteins and accumulation of ferrichrome in the periplasm could be measured only with the new method.