Soil Chemical Analysis

A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes--factors affecting enzymes, conversion and synergy.

GH11 xylanases: Structure/function/properties relationships and applications

Instructions This exam consists of 50 questions. You may write on the exam itself, but be sure to answer all your questions on a “Scantron” sheet with a #2 pencil. For each question there is one response that is the best response. You must select the one best response to receive credit for each question. If you select more than one response for a question, then you will receive no credit for that question.

人体の解剖生理学 = Human anatomy & physiology

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Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes.

Using degenerate polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR, a 1,347-bp full-length complementary DNA fragment encompassing the gene man5A, which encodes a 429-amino acid β-mannanase with a calculated mass of 46.8 kDa, was cloned from acidophilic Bispora sp. MEY-1. The deduced amino acid sequence (catalytic domain) displayed highest identity (54.1%) with the Emericella nidulans endo-β-1,4-d-mannanase, a member of the glycoside hydrolase family 5. Recombinant MAN5A was overexpressed in Pichia pastoris, and its activity in the culture medium reached 500 U ml−1. The enzyme was acidophilic, with highest activity at pH 1.0–1.5, lower than any known mannanases, and optimal temperature for activity was 65°C. MAN5A had good pH adaptability, excellent thermal and pH stability, and high resistance to both pepsin and trypsin. The specific activity, Km, and Vmax for locust bean gum substrate was 3,373 U mg−1, 1.56 mg ml−1, and 6,587.6 μmol min−1 mg−1, respectively. The enzymatic activity was not significantly affected by ions such as Ca2+, Cr3+, Co2+, Zn2+, Na+, K+, and Mg2+ and enhanced by Ni2+, Fe3+, Mn2+ and Ag+. These favorable properties make MAN5A a potential candidate for use in various industrial applications.

A novel highly acidic β-mannanase from the acidophilic fungus Bispora sp. MEY-1: gene cloning and overexpression in Pichia pastoris

Phytate is the most abundant organic phosphorus compound in nature, and microbial mineralization of phytate by phytase is a key process for phosphorus recycling in the biosphere. In the present study, beta-propeller phytase (BPP) gene fragments were readily amplified from the intestinal contents of grass carp (Ctenopharyngodon idellus) directly or from phytate-degrading isolates from the same source, confirming the widespread occurrence of BPP in aquatic communities. The amounts of sequences collected using these two methods differed (88 distinct genes versus 10 isolates), but the sequences showed the same general topology based on phylogenetic analysis. All of the sequences fell in five clusters and were distinct from those of Anabaena, Gloeobacter, Streptomyces, Flavobacterium, Prosthecochloris, and Desulfuromonas, which have never been found in the grass carp intestine. Analysis of the microbial diversity by denaturing gradient gel electrophoresis demonstrated that unculturable bacteria were dominant bacteria in the grass carp intestine and thus the predominant phytate-degrading organisms. The predominant cultured species corresponding to the phytate-degrading isolates, Pseudomonas, Bacillus and Shewanella species, might be the main source of known BPPs. A phytase from Brevundimonas was first obtained from cultured species. Combining our results with Lim et al.'s inference that phytate-mineralizing bacteria are widely distributed and highly diverse in nature (B. L. Lim, P. Yeung, C. Cheng, and J. E. Hill, ISME J. 1:321-330, 2007), we concluded that BPP is the major phytate-degrading enzyme in nature, that most of this enzyme might originate from unculturable bacteria, and that the distribution of BPP may be related to the type of niche. To our knowledge, this is the first study to experimentally estimate BPP diversity in situ.

Diversity of Beta-Propeller Phytase Genes in the Intestinal Contents of Grass Carp Provides Insight into the Release of Major Phosphorus from Phytate in Nature

Two xylose-tolerant GH43 bifunctional β-xylosidase/α-arabinosidases and one GH11 xylanase from Humicola insolens and their synergy in the degradation of xylan.

1,3-1,4-beta-D-glucanase is an important endoglycosidase in the brewing and animal feed industries. To achieve high-level expression of recombinant glucanase in Pichia pastoris, we designed sequences encoding the alpha-factor signal peptide from Saccharomyces cerevisiae and the truncated 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes as a whole. The codons encoding the 52 amino acids of the signal peptide and 106 residues of the glucanase protein were optimized for expression in P. pastoris; 189 nucleotides were changed. The G + C content was adjusted to 48-49%, and AT-rich stretches were eliminated to avoid premature termination. The messenger ribonucleic acid secondary structure near the AUG start codon was also adjusted to ensure efficient translation; the resulting glucanase production was twofold higher compared with that achieved with gene structure optimization alone. We also propose a new fermentation strategy for the induction phase, in which 5/95% glycerol/methanol mixed feed was used in days 1-3 and 100% methanol was used on days 4-6. By comparison with methanol feed and glycerol/methanol-mixed feed alone, the yield of recombinant glucanase increased by 38.5 and 16.5%, respectively. The expressed optimized recombinant 1,3-1,4-beta-D-glucanase constituted approximately 90% of the total secreted protein, reaching up to 3 g l(-1) in the medium.

High-level expression of a truncated 1,3-1,4-β-d-glucanase from Fibrobacter succinogenes in Pichia pastoris by optimization of codons and fermentation

A new xylanase gene, xynAS9, was cloned from Streptomyces sp. S9, which was isolated from Turpan Basin, China. The full-length gene consists of 1,395 bp and encodes 465 amino acids including 38 residues of a putative signal peptide. The overall amino acid sequence shares the highest identity (50.8%) with a putative endo-1,4-β-xylanase from Streptomyces avermitilis of the glycoside hydrolase family 10. The gene fragment encoding the mature xylanase was expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to electrophoretic homogeneity and subsequently characterized. The optimal pH and temperature for the recombinant enzyme were 6.5 and 60°C, respectively. The enzyme showed broad temperature adaptability, retaining more than 65% of the maximum activity when assayed at 50–80°C. The enzyme also had good thermal and pH stability. The K
m values for oat spelt xylan and birchwood xylan substrates were 2.85 and 2.43 mg ml−1, with the V
max values of 772.20 and 490.87 μmol min−1 mg−1, respectively. The hydrolysis products of xylan were mainly xylose and xylobiose. These favorable properties should make XynAS9 a good candidate in various industrial applications.

Yaru Wang

Papers

A novel highly acidic β-mannanase from the acidophilic fungus Bispora sp. MEY-1: gene cloning and overexpression in Pichia pastoris

Diversity of Beta-Propeller Phytase Genes in the Intestinal Contents of Grass Carp Provides Insight into the Release of Major Phosphorus from Phytate in Nature

Two xylose-tolerant GH43 bifunctional β-xylosidase/α-arabinosidases and one GH11 xylanase from Humicola insolens and their synergy in the degradation of xylan.

High-level expression of a truncated 1,3-1,4-β-d-glucanase from Fibrobacter succinogenes in Pichia pastoris by optimization of codons and fermentation

Cloning, expression, and characterization of a new xylanase with broad temperature adaptability from Streptomyces sp. S9