古茶树林菌株D2的鉴定、酶学特性及基因组学分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-1053

摘要/Abstract

摘要：

目的鉴定1株百色野生古茶树林根际高产蛋白酶菌株D2，分析菌株D2的酶学特性及基因组，为进一步应用菌株D2开发农业用途的功能性肥料提供依据。方法基于16S rRNA扩增技术和菌落形态鉴定菌株D2，福林法测定蛋白酶的酶活力，二代测序技术对菌株D2的基因组测序并进行生物信息学分析。结果鉴定百色野生古茶树林根际高产蛋白酶菌株D2为Stenotrophomonas属的一个新种，与嗜麦芽寡养单胞菌（Stenotrophomonas maltophilia）的亲缘关系最近。菌株D2发酵24 h产生255.73 U/mL的中性蛋白酶以及282.12 U/mL的碱性蛋白酶，总蛋白酶酶活力为544.60 U/mL。菌株D2蛋白酶催化反应的最适温度为50 ℃、最适pH为8，高温对酶活力的影响较大，pH和有机溶剂的影响相对较小，二甲基亚砜处理会提高酶活力。菌株D2基因组的全长为4 599 465 bp，GC含量为66.77%，包含4 176个CDS，71个tRNA，5个rRNA 基因，1个ncRNA。菌株D2基因组还存在丰富的碳水化合物活性酶基因和重金属抗性基因，具有11个Saccharide合成基因簇（占总基因簇的57.89%），作用于38种植物病原菌。结论古茶树林菌株D2鉴定为Stenotrophomonas属的一个新种，其产蛋白酶的活力高且酶学性质较好。菌株D2基因组具有丰富的碳水化合物活性酶、重金属抗性、次级代谢产物合成基因簇以及植物病原菌抗病等基因资源。

关键词: 嗜麦芽寡养单胞菌, 野生古茶树林, 根际微生物, 中性蛋白酶, 碱性蛋白酶, 酶学特性, 基因组学

Abstract:

Objective Identify a high-yield protease-producing strain D2 in the rhizosphere of a wild ancient tea forest in Baise, and analyze the enzymatic characteristics and genome of strain D2, and provide a basis for further application of strain D2 in the development of functional fertilizers for agricultural purposes. Method Based on 16S rRNA amplification technology and colony morphology identification of strain D2, the enzyme activity of protease was determined by the Folin method, and the genome of strain D2 was sequenced and subjected to bioinformatics analysis using next-generation sequencing technology. Result The strain D2 with high protease production in the rhizosphere of wild ancient tea forests in Baise was identified as a new species of Stenotrophomonas genus, with the closest genetic relationship to Stenotrophomonas maltophilia. Strain D2 produced 255.73 U/mL neutral protease and 282.12 U/mL alkaline protease after 24 h of fermentation, with a total protease activity of 544.60 U/mL. The optimal temperature and pH for the protease catalyzed reaction of strain D2 are 50 ℃ and 8, respectively. High temperature has a significant impact on enzyme activity, while pH and organic solvents have relatively small effects. Treatment with organic solvent DMSO can also enhance enzyme activity. The full-length genome of strain D2 is 4 599 465 bp, with a GC content of 66.77%. It contains 4 176 CDS, 71 tRNA, 5 rRNA genes, and 1 ncRNA. The genome of strain D2 also contains abundant carbohydrate active enzyme genes and heavy metal resistance genes, with 11 Saccharide synthesis gene clusters (accounting for 57.89% of the total gene clusters), which interact with 38 plant pathogens. Conclusion The strain D2 from the ancient tea forest has been identified as a new species of Stenotrophomonas genus, with high protease activity and good enzymatic properties. The genome contains abundant gene clusters for carbohydrate active enzymes, resistance to heavy metal, secondary metabolite synthesis, and plant pathogen resistance.

Key words: Stenotrophomonas maltophilia, wild ancient tea forest, rhizosphere microorganisms, neutral protease, alkaline protease, enzymatic characteristics, genomics

叶柳健, 蒙健宗, 覃福方, 何双, 朱绮霞, 王小虎, 韦圣博, 周礼芹. 古茶树林菌株D2的鉴定、酶学特性及基因组学分析[J]. 生物技术通报, 2025, 41(5): 267-279.

YE Liu-jian, MENG Jian-zong, QIN Fu-fang, HE Shuang, ZHU Qi-xia, WANG Xiao-hu, WEI Sheng-bo, ZHOU Li-qin. Identification， Enzymatic Characteristics， and Genomic Analysis of Strain D2 from Ancient Tea Forest[J]. Biotechnology Bulletin, 2025, 41(5): 267-279.

图/表 11

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菌株名称 Strain name	ANI值 ANI value （%）	菌株名称 Strain name	ANI值 ANI value （%）
Stenotrophomonas maltophilia ISMMS5	93.31	Stenotrophomonas sepilia SM16975	92.41
Stenotrophomonas pavanii DSM 25135	92.10	Stenotrophomonas riyadhensis CFS3442	91.45
Stenotrophomonas geniculata JCM 13324	91.41	Stenotrophomonas cyclobalanopsidis TPQG1-4	87.12
Stenotrophomonas indicatrix WS40	86.65	Stenotrophomonas lactitubi M15	86.58
Stenotrophomonas chelatiphaga DSM 21508	81.35	Stenotrophomonas pennii Sa5BUN4	81.17
Stenotrophomonas lacuserhaii K32	81.16	Stenotrophomonas tumulicola JCM 30961	80.94
Stenotrophomonas rhizophila DSM14405	80.76	Stenotrophomonas aracearum A5588	80.73
Stenotrophomonas bentonitica DSM 103927	80.72	Stenotrophomonas nematodicola CPCC 101271	80.53
Stenotrophomonas oahuensis A5586	80.12	Stenotrophomonas daejeonensis JCM 16244	79.45
Stenotrophomonas acidaminiphila DSM 13117	79.19	Stenotrophomonas nitritireducens DSM 12575	79.19

菌株名称 Strain name	ANI值 ANI value （%）	菌株名称 Strain name	ANI值 ANI value （%）
Stenotrophomonas maltophilia ISMMS5	93.31	Stenotrophomonas sepilia SM16975	92.41
Stenotrophomonas pavanii DSM 25135	92.10	Stenotrophomonas riyadhensis CFS3442	91.45
Stenotrophomonas geniculata JCM 13324	91.41	Stenotrophomonas cyclobalanopsidis TPQG1-4	87.12
Stenotrophomonas indicatrix WS40	86.65	Stenotrophomonas lactitubi M15	86.58
Stenotrophomonas chelatiphaga DSM 21508	81.35	Stenotrophomonas pennii Sa5BUN4	81.17
Stenotrophomonas lacuserhaii K32	81.16	Stenotrophomonas tumulicola JCM 30961	80.94
Stenotrophomonas rhizophila DSM14405	80.76	Stenotrophomonas aracearum A5588	80.73
Stenotrophomonas bentonitica DSM 103927	80.72	Stenotrophomonas nematodicola CPCC 101271	80.53
Stenotrophomonas oahuensis A5586	80.12	Stenotrophomonas daejeonensis JCM 16244	79.45
Stenotrophomonas acidaminiphila DSM 13117	79.19	Stenotrophomonas nitritireducens DSM 12575	79.19

基因簇 Cluster	类型 Type	开始 From	结束 To	最相似的基因簇 The most similar gene cluster	相似度 Similarity （%）
Cluster1	Saccharide	74 130	97 224	-	-
Cluster2	NRP-metallophore	647 251	695 618	2，3-Dihydroxybenzoylserine	94
Cluster3	Lanthipeptide-class-iv	899 781	922 489	-	-
Cluster4	Saccharide	1 334 563	1 357 469	O-antigen	14
Cluster5	RiPP	1 703 062	1 713 937	-	-
Cluster6	RiPP	2 007 614	2 018 459	Entolysin	6
Cluster7	Saccharide	2 038 974	2 091 044	-	-
Cluster8	Fatty acid	2 161 274	2 186 839	-	-
Cluster9	Fatty acid	20 535	41 893	-	-
Cluster10	Saccharide	283 664	307 161	-	-
Cluster11	Saccharide	321 833	365 738	-	-
Cluster12	Saccharide	401 738	447 486	Lipopolysaccharide	54
Cluster13	Fatty acid	466 989	488 197	-	-
Cluster14	Saccharide	583 826	601 409	-	-
Cluster15	Saccharide	162 130	194 883	Eicoseicosapentaenoic acid	10
Cluster16	Saccharide	137 019	166 054	-	-
Cluster17	Saccharide	191 650	213 743	-	-
Cluster18	Arylpolyene	120 542	164 144	APE Vf	40
Cluster19	Saccharide	184 940	206 717	-	-

基因簇 Cluster	类型 Type	开始 From	结束 To	最相似的基因簇 The most similar gene cluster	相似度 Similarity （%）
Cluster1	Saccharide	74 130	97 224	-	-
Cluster2	NRP-metallophore	647 251	695 618	2，3-Dihydroxybenzoylserine	94
Cluster3	Lanthipeptide-class-iv	899 781	922 489	-	-
Cluster4	Saccharide	1 334 563	1 357 469	O-antigen	14
Cluster5	RiPP	1 703 062	1 713 937	-	-
Cluster6	RiPP	2 007 614	2 018 459	Entolysin	6
Cluster7	Saccharide	2 038 974	2 091 044	-	-
Cluster8	Fatty acid	2 161 274	2 186 839	-	-
Cluster9	Fatty acid	20 535	41 893	-	-
Cluster10	Saccharide	283 664	307 161	-	-
Cluster11	Saccharide	321 833	365 738	-	-
Cluster12	Saccharide	401 738	447 486	Lipopolysaccharide	54
Cluster13	Fatty acid	466 989	488 197	-	-
Cluster14	Saccharide	583 826	601 409	-	-
Cluster15	Saccharide	162 130	194 883	Eicoseicosapentaenoic acid	10
Cluster16	Saccharide	137 019	166 054	-	-
Cluster17	Saccharide	191 650	213 743	-	-
Cluster18	Arylpolyene	120 542	164 144	APE Vf	40
Cluster19	Saccharide	184 940	206 717	-	-

重金属名称 Heavy metal name	抗性基因种类 Resistance gene type	基因数 Gene count
锑Antimony （Sb）	adeJ	1
砷Arsenic （As）	adeK， aioR； aoxR， chrC， baeR， baeS， bcr	11
铋Bismuth （Bi）	corC	2
镉Cadmium （Cd）	corR， corS， crdR， cutC	7
铬Chromium （Cr）	dsbC， emhA	2
钴Cobalt （Co）	emhB， mvrC， emrE， evgA， evgS， fabL， ygaA， fabV， fbpC	20
铜Copper （Cu）	fecD， fecE， fptA， fpvA， frnE， gesA， gesB， golS， irlS， telA， kilB， klaB， cnrB， mdeA， yhiU， mdtE， yjcR， mdtN， mepA， merA， mexD， mexF， mexJ， mexT， mexV， mntH， yfeP	36
金Gold （Au）	nikE	3
铁Iron （Fe）	pstB， nrsS， oprJ， cnrX， cnr， ruvB， ostA， lptD， PA0320， pcoB， pcoR， pitA， pstA	10
铅Lead （Pb）	pstC	1
汞Mercury （Hg）	smdA， silS	4
镍Nickel （Ni）	smrA， smfY	9
银Silver （Ag）	znuC， yebM， hydH， zraR， zraS， hydG， ygiE， zupT	6
钨Tungsten （W）	modA， modB， wtpC， tupC， copS， copR， mgtA， corD， mdtB， merA	15
锌Zinc （Zn）	bcrA， sodB， chtR， arsT， copR， copS， copA， tcrY	9