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Lignocellulolytic activities of a novel strain of Trichoderma harzianum

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Advances in Bioscience and biotechnology 2013年第2期4卷 214-221页

作者： M. Rubeena Kannan Neethu S. Sajith S. Sreedevi Prakasan Priji K. N. Unni M. K. Sarath Josh V. N. Jisha S. Pradeep Sailas Benjamin enzyme Technology Laboratory Biotechnology Division Department of Botany University of Calicut Malappuram India

This study describes a novel dark-green spore producing strain of Trichoderma harzianum exhibiting higher activities of cellulase, hemicellulase and ligninase on specific plate assays. To assess the cellulase production in detail, basal salt medium (BSM) was supplemented with synthetic [carboxymethyl cellulose (CMC), glucose, sucrose, dextrose, lactose or maltose] and natural (flours of banana, banana peel, jack seed, potato or tapioca) carbon as well as nitrogen (yeast extract, beef extract, peptone, NaNO3 or NH4NO3) sources. Temperature and pH optima were 28?C and 4, respectively for the growth of the fungus in CMC- BSM with 146 U/ml cellulase activity. Flours of potato and banana supported comparable yields of cellulase to that of CMC (147 U/ml and 168 U/ml, respectively), while sodium nitrate was the preferred nitrogen source (150 U/ml). The water soluble yellowish-green pigment (a probable siderophore) extracted from the spores showed an absorption maximum at 414 nm. To comprise, this fungus shows the complete lignocellulolytic potential which offers great industrial significance, especially for the ethanol production from the lignocellulosic waste coupled with the production of a new pigment.

关键词： Trichoderma harzianum Morphology Submerged Fermentation Yellowish-Green Pigment

Versatility of microbial proteases

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Advances in enzyme Research 2013年第3期1卷 39-51页

作者： Veloorvalappil N. Jisha Robinson B. Smitha Selvanesan Pradeep Sasidharan Sreedevi Kizhakkepawothail N. Unni Sreedharan Sajith Prakasan Priji Moolakkariyil Sarath Josh Sailas Benjamin enzyme Technology Laboratory Biotechnology Division Department of Botany University of Calicut Kerala India

Proteases or peptidases constitute the largest group of enzymes in bio-industry with a long array of uses. They play an invincible role in industrial biotechnology, especially in detergent, food and pharmaceutical arena. This focused review encompasses an overview on alkaline proteases, mainly of microbial sources in a handy module. Following an introduction and general classification with evolutionary insight, major sources of proteases (animal, plant and microbial including fungal, bacterial), their general properties with mechanism of action and molecular masses are discussed. Proteases fromBacillusspp. have been given special attention. In addition to this, an overview on the applications of proteases in detergent, tannery, food, metal recovery and waste treatment industries is also addressed briefly.

关键词： Review Proteases Classification Sources Bacillus Industrial Uses

A monograph on amylases from Bacillusspp.

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Advances in Bioscience and biotechnology 2013年第2期4卷 227-241页

作者： Sailas Benjamin R. B. Smitha V. N. Jisha S. Pradeep S. Sajith S. Sreedevi Prakasan Priji K. N. Unni M. K. Sarath Josh enzyme Technology Laboratory Biotechnology Division Department of Botany University of Calicut Kerala India

Owing to the production of alpha, beta and gamma amylase subtypes;starch degrading microbes, especially bacteria have an invincible role in the food, fermentation, textile and paper industries. Of them, α-amylases from Bacillus spp. have contributed tremendous advancements in bio-industry, especially in starch, detergent and pharmaceutical arena. Though general reviews are seen in literature on amylases, no focused review is available yet solely on α-amylases produced by Bacillus spp. Hence, this focused review on α-amylases from the genus Bacillus is designed in such a way that it should give a vivid picture on most of the aspects on bacillial α-amylases in a handy module with an industrial perspective. With a short introduction on amylases in general, α-amylases from various species of Bacillus reviewed herein encompasses production of α-amylases by submerged and solid-state fermentations;nutrients and other factors required for maximizing production;immobilization strategies for whole cells or purified enzyme;an overview on the molecular weight of the enzyme;followed by distinct sections for purification, characterisation, stability and crystal structure;and concluded with a section on industrial applications of the α-amylases from Bacillus spp.

关键词： Bacillus Amylases Production Fermentation Crystal Immobilization Stability Molecular Weight Characteristics Applications

Cellulase Producing Bacteria from the Wood-Yards on Kallai River Bank

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Advances in Microbiology 2013年第4期3卷 326-332页

作者： Sasidharan Sreedevi Sreedharan Sajith Sailas Benjamin enzyme Technology Laboratory Biotechnology Division Department of Botany University of Calicut Malappuram India

This study evaluates the influence of growth parameters such as pH, temperature, Carboxy Methyl Cellulose (CMC) concentration and agitation on cellulase production from three bacterial strains, viz., Achromobacter xylosoxidans BSS4, Bacillus sp. BSS3 and Pseudomonas sp. BSS2 isolated from the wood-yards on Kallai river bank in Kerala. Production of cellulase by these isolates was detected using basal salt medium (BSM) with 0.5% CMC as supplement, and CMCase activity was confirmed by iodine test. Dinitrosalicylic acid method was employed for assaying the cellulase production by measuring the amount of glucose liberated in μmol/mL/min. Maximum enzyme production from Pseudomonas sp. BSS2 was at pH 8, 37℃ with 1% CMC and 150 rpm, and cellulase production increased from initial 49.84 U/mL to 91.28 U/mL after optimization. The highest enzyme activity from Bacillus sp. BSS3 was at pH 9, 37℃ with 1% CMC, 150 rpm, and cellulase production increased from initial 26.05 U/mL to 104.68 U/mL after optimization. The maximum enzyme production from A. xylosoxidans BSS4 was at pH 7, 40℃ with 0.5% CMC and 150 rpm, and cellulase production increased from initial 55.28 U/mL to 68.37 U/mL after optimization. Thus among the three isolates, Bacillus sp. BSS3 showed maximum enzyme yield which can be explored for further scale up studies with an industrial perspective.

关键词： Cellulase Carboxy Methyl Cellulose Dinitrosalicylic Acid Optimization Submerged Fermentation

Solid-State Fermentation for the Concomitant Production of δ-Endotoxin and Endospore from Bacillus thuringiensissubsp. kurstaki

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Advances in Bioscience and biotechnology 2014年第10期5卷 797-804页

作者： Veloorvalappil Narayanan Jisha Sailas Benjamin enzyme Technology Laboratory Biotechnology Division Department of Botany University of Calicut Kerala India

Water stress and limited aeration imparted by solid-state fermentation (SSF) were reported as crucial factors for the enhancement of endospore production by Bacillus thuringiensis (Bt);and thus, more δ-endotoxin could be produced concomitantly with reduced time. Therefore, Bt subsp. kurstaki (Btk) was employed in the present study to evaluate its efficiency for the concomitant production of endospores and δ-endotoxin in LB medium supplemented with various naturally available agricultural products, i.e., flours of soybean, Bengal gram or jack seed at various concentrations (10%, 20%, 30%, 40%, 50%, 60%, 80% or 100%, all w/v). After 12 h fermentation, the supernatant in it was centrifuged off aseptically to obtain solid substrate for subsequent SSF. Of them, soybean (30%) supplemented medium was the best for the enhanced production of endospore and δ-crystals. The maximum yield of endospores during solid-state fermentation was observed 48 h, i.e., compared to submerged fermentation in LB, it was 24 h less gestation period. In control sample, the endospores achieved the maximum length (1.10 ± 0.13 μm) and diameter (0.63 ± 0.07 μm) at 72 h;while in soybean supplemented medium, the maximum length (2.10 ± 0.16 μm) and diameter (1.63 ± 0.16 μm) were at 48 h and 72 h, respectively. Upon staining, acridine orange specifically stained the endospores;malachite green-saffranin stained both δ-crystals and endospores;and coomassie brilliant blue specifically stained δ-endotoxin. Briefly, normal gestation period or harvest time for Btk is 72 h, which could be reduced to 48 h, if SSF is employed as demonstrated in this study.

关键词： Bacillus thuringiensis subsp. kurstaki Spore Solid-State Fermentation δ-Endotoxin

Cloning, expression, purification, and characterization of LC-1 ScFv with GFP tag

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Journal of Zhejiang University-Science B(Biomedicine & biotechnology) 2005年第8期6卷 832-837页

作者：卢敏龚兴国于红郦剑勇 Institute of Biologic Macromolecule and enzyme Engineering School of Life Science Zhejiang UniversityHangzhou 310027 China

Total RNA was isolated from the hybridoma cell line （LC- 1 ）, which secretes anti-lung adenocarcinoma monoclonal antibody, and was transferred into cDNA. Based on the FRl （framework region l） and FR4 conserved regions of LC-1 gene, the variable regions of heavy chain （Vh） and light chain （Vl） were amplified, and the Vh and modified Vl were connected to single chain Fv （ScFv） by SOE-PCR （splice overlap extension PCR）. The modified ScFv was fused with green fluorescent protein （GFP） and introduced into E. coli JM109. The fusion protein induced by lPTG （Isopropylthiogalactoside） was about 57000 on a 10% SDS-PAGE gel （10% Sds Polyacrylamide Gel Electrophoresis）, and primarily manifested as inclusion bodies. The renatured protein purified by Ni-NTA Superflow resins showed ability to bind to antigen on SPC-A-l lung adenocarcinoma. In addition, the induced host cells fluoresced bright green under 395 nm wavelength, which indicated that the expected protein with dual activity was expressed in the prokaryotic system. The ScFv with GFP tag used in this research can be applied as a new reagent to detect immunological dye, and provide a feasible way to detect adenocarcinoma in a clinical setting.

关键词： ScFv (single chain variable fragment) GFP (green fluorescent protein) tag Protein fusion Purification

Conjugated linoleic acid isomers and their precursor fatty acids regulate peroxisome proliferator-activated receptor subtypes and major peroxisome proliferator responsive element-bearing target genes in HepG2 cell model

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Journal of Zhejiang University-Science B(Biomedicine & biotechnology) 2013年第2期14卷 115-123页

作者： Sailas BENJAMIN Silke FLOTHO Torsten BRCHERS Friedrich SPENER Department of Biochemistry University of Münster48149 MünsterGermany enzyme Technology Laboratory Biotechnology DivisionDepartment of BotanyUniversity of CalicutKerala 673 635India Department of Molecular Biosciences Lipidomics Research CenterUniversity of GrazHeinrichstrasse 318010 GrazAustria

The purpose of this study was to examine the induction profiles （as judged by quantitative reverse tran- scription polymerase chain reaction （qRT-PCR）） of peroxisome proliferator-activated receptor （PPAR） α,β, y subtypes and major PPAR-target genes bearing a functional peroxisome proliferator responsive element （PPRE） in HepG2 cell model upon feeding with cis-9,trans-11-octadecadienoic acid （9-CLA） or trans-10,cis-12-octadecadienoic acid （10-CLA） or their precursor fatty acids （FAs）. HepG2 cells were treated with 100 pmol/L 9-CLA or 10-CLA or their precursor FAs, viz., oleic, linoleic, and trans-11-vaccenic acids against bezafibrate control to evaluate the induc- tion/expression profiles of PPAR （α, β, γ subtypes and major PPAR-target genes bearing a functional PPRE, i.e., fatty acid transporter （FAT）, glucose transporter-2 （GLUT-2）, liver-type FA binding protein （L-FABP）, acyl CoA oxidase-1 （ACOX-1）, and peroxisomal bifunctional enzyme （PBE） with reference to β-actin as house keeping gene. Of the three housekeeping genes （glyceraldehyde 3-phosphate dehydrogenase （GAPDH）, β-actin, and ubiquitin）, β-actin was found to be stable. Dimethyl sulfoxide （DMSO）, the common solubilizer of agonists, showed a significantly higher induction of genes analyzed, qRT-PCR profiles of CLAs and their precursor FAs clearly showed upregulation of FAT, GLUT-2, and L-FABP （-0.5-.0-fold）. Compared to 10-CLA, 9-CLA decreased the induction of the FA metabolizing gene ACOX-1 less than did PBE, while 10-CLA decreased the induction of PBE less than did ACOX-I. Both CLAs and precursor FAs upregulated PPRE-beadng genes, but with comparatively less or marginal activation of PPAR subtypes This indicates that the binding of CLAs and their precursor FAs to PPAR subtypes results in PPAR activation, thereby induction of the target transporter genes coupled with downstream lipid metabolising genes such as ACOX-1 and PBE. To sum up, the expression profiles of these candidate genes showed that CLAs and th

关键词： Conjugated linoleic acid HepG2 cell model qRT-PCR Peroxisome proliferator-activated receptor Peroxisome proliferator responsive element-bearing genes Lipid agonists

Biosensor-assisted CRISPRi high-throughput screening to identify genetic targets in Zymomonas mobilis for high d-lactate production

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Synthetic and Systems biotechnology 2024年第2期9卷 242-249页

作者： Qiqun Peng Weiwei Bao Binan Geng Shihui Yang State Key Laboratory of Biocatalysis and enzyme Engineering and School of Life SciencesHubei UniversityWuhan430062China

Lactate is an important monomer for the synthesis of poly-lactate(PLA),which is a substitute for the petrochemical *** achieve the goal of high lactate titer,rate,and yield for commercial production,efficient lactate production pathway is needed as well as genetic targets that affect high lactate production and *** this study,an LldR-based d-lactate biosensor with a broad dynamic range was first applied into Zymomonas mobilis to select mutant strains with strong GFP fluorescence,which could be the mutant strains with increased d-lactate ***,LldR-based d-lactate biosensor was combined with a genome-wide CRISPR interference(CRISPRi)library targeting the entire genome to generate thousands of mutants with gRNA targeting different genetic targets across the whole ***,two mutant libraries were selected containing 105 and 104 mutants with different interference sites from two rounds of fluorescence-activated cell sorting(FACS),*** genetic targets of ZMO1323 and ZMO1530 were characterized and confirmed to be associated with the increased d-lactate production,further knockout of ZMO1323 and ZMO1530 resulted in a 15%and 21%increase of d-lactate production,*** work thus not only established a high-throughput approach that combines genome-scale CRISPRi and biosensor-assisted screening to identify genetic targets associated with d-lactate production in ***,but also provided a feasible high-throughput screening approach for rapid identification of genetic targets associated with strain performance for other industrial microorganisms.

关键词： D-lactate Biosensor LldR Genome-wide CRISPRi FACS Zymomonas mobilis

Recombinant human B7-H4 expressed in Escherichia coli inhibits T lym-phocyte proliferation and IL-2 secretion in vitro

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Acta Pharmacologica Sinica 2006年第6期27卷 741-746页

作者： Yi-xiang MAO~(2,3)Yong-Jing CHEN~2 Yan GE~2 Hong-bing MA~2 Jian-feng YU~2 Hong-ya WU~2 Yu-min HU~2 Qin WANG~2 Qin SHI~2 Xue-guang ZHANG~(2,4)2 Institute of biotechnology,Soochow University,Suzhou 215007,China 3 Department of Oncology,First Affiliated Hospital to Soochow University,Suzhou 215007,China Institute of biotechnology Soochow University

Aim:To explore the biofunctions of human B7-H4 generated from ***:The gene of human B7-H4 extracellular region（IgV-like andIgC-like domains）was obtained by PCR from human cDNA FLJ22418 and theninserted into the prokaryotic expression vector pGEX-5X-3 expressing glutathiones-transferase（GST）fusion *** being identified by restriction enzymedigestion and sequencing,the recombinant vector was transferred into host strainE coli BL21-RIL（DE3）.A 47 kDa fusion protein（GST/hB7-H4）was induced byisopropyl-beta-D-thiogalactopyranoside（IPTG）and purified by standard meth-ods reported in the prokaryotic *** inhibitory effect of GST/hBT-H4 onproliferation of T cells was observed in vitro by CD3mAb activated T-cell cultur-ing system and [3H]-thymidine incorporation *** concentrations ofinterleukin-2 and iterferon-g in the supernatants of T cells were determined ***:We successfully constructed the method for high-level expres-sion and purification of the hB7-H4 extracellular domain as GST fusion proteinfrom E *** GST/hB7-H4 fusion protein produced in bacteria had obviousbiological activity to inhibit T-lymphocyte proliferation and IL-2 ***:The prokaryote expression system could be used to generate hB7-H4 protein with natural spatial conformations and biological functions,whichprovided an efficient and economical way for the preparation of this target protein.

关键词： B7-H4 glutathione s-transferase fusion protein inhibition